anthesis meaning of

Definition of Antithesis

Antithesis is a literary device that refers to the juxtaposition of two opposing elements through the parallel grammatical structure. The word antithesis, meaning absolute opposite, is derived from Greek for “ setting opposite,” indicating when something or someone is in direct contrast or the obverse of another thing or person.

Antithesis is an effective literary and rhetorical device , as it pairs exact opposite or contrasting ideas by utilizing the parallel grammatical structure. This helps readers and audience members define concepts through contrast and develop an understanding of something through defining its opposite. In addition, through the use of parallelism , antithesis establishes a repetitive structure that makes for rhythmic writing and lyrical speech.

For example, Alexander Pope states in  An Essay on Criticism , “ To err is human ; to forgive divine.” Pope’s use of antithesis reflects the impact of this figure of speech in writing, as it creates a clear, memorable, and lyrical effect for the reader. In addition, Pope sets human error in contrast to divine forgiveness, allowing readers to understand that it is natural for people to make mistakes, and therefore worthy for others to absolve them when they do.

Examples of Antithesis in Everyday Speech

Antithesis is often used in everyday speech as a means of conveying opposing ideas in a concise and expressive way. Since antithesis is intended to be a figure of speech, such statements are not meant to be understood in a literal manner. Here are some examples of antithesis used in everyday speech:

• Go big or go home.
• Spicy food is heaven on the tongue but hell in the tummy.
• Those who can, do; those who can’t do, teach.
• Get busy living or get busy dying.
• Speech is silver but silence is gold.
• No pain, no gain.
• It’s not a show, friends; it’s show business.
• No guts, no glory.
• A moment on the lips; a lifetime on the hips.
• If you fail to plan, then you plan to fail.

Common Examples of Antithesis from Famous Speeches

Antithesis can be an effective rhetorical device in terms of calling attention to drastic differences between opposing ideas and concepts. By highlighting the contrast side-by-side with the exact same structure, the speaker is able to impact an audience in a memorable and significant way. Here are some common examples of antithesis from famous speeches:

• “I have a dream that my four little children will one day live in a nation where they will not be judged by the color of their skin but by the content of their character .” (Martin Luther King, Jr. “I Have a Dream”)
• “The world will little note, nor long remember what we say here, but it can never forget what they did here.” (Abraham Lincoln “The Gettysburg Address”)
• “‘Some men see things as they are and say why. I dream things that never were and say why not.'” (Edward Kennedy quoting Robert F. Kennedy during eulogy )
• “We observe today not a victory of party, but a celebration of freedom, symbolizing an end as well as a beginning, signifying renewal as well as change.” (John F. Kennedy “Presidential Inaugural Speech”)
• “You see, for any champion to succeed, he must have a team — a very incredible, special team; people that he can depend on, count on, and rely upon through everything — the highs and lows, the wins and losses, the victories and failures, and even the joys and heartaches that happen both on and off the court.” (Michael Chang “ Induction Speech for Tennis Hall of Fame”)

Examples of Proverbs Featuring Antithesis

Proverbs are simple and often traditional sayings that express insight into truths that are perceived, based on common sense or experience. These sayings are typically intended to be metaphorical and therefore rely on figures of speech such as antithesis. Proverbs that utilize antithetical parallelism feature an antithesis to bring together opposing ideas in defined contrast. Therefore, antithesis is effective as a literary device in proverbs by allowing the reader to consider one idea and then it’s opposite. It also makes for lyrical and easily remembered sayings.

Here are some examples of proverbs featuring antithesis:

• Cleanliness is next to godliness.
• Beggars can’t be choosers.
• Easy come, easy go.
• Hope for the best; prepare for the worst.
• Keep your friends close; keep your enemies closer.
• Like father, like son.
• Where there’s smoke, there’s fire.
• An ounce of protection is worth a pound of cure.
• Be slow in choosing, but slower in changing.
• Give them an inch and they’ll take a mile.
• If you can’t beat them, join them.
• Keep your mouth closed and your eyes open.
• One man’s junk is another man’s treasure.
• Out of sight, out of mind.
• Where there’s a will, there’s a way.

Utilizing Antithesis in Writing

As a literary device, antithesis allows authors to add contrast to their writing. This is effective in terms of comparing two contrasting ideas, such as a character’s conflicting emotions or a setting’s opposing elements. In literature, antithesis doesn’t require a pairing of exact opposites, but rather concepts that are different and distinct. In addition, since antithesis creates a lyrical quality to writing through parallel structure , the rhythm of phrasing and wording should be as similar as possible. Like most literary and rhetorical devices, overuse of antithesis will create confusion or invoke boredom in a reader as well as make the writing seem forced.

Antithesis and Parallelism

Both terms demonstrate a fundamental difference. An antithesis comprises two contradictory ideas and parallelism does not necessarily comprise opposite ideas or persons. It could have more than two ideas or persons. As the name suggests that parallelism is a condition where is an antithesis is an opposition. For example, man proposes, God disposes, has two contradictory ideas. However, it is also a parallel sentence . Furthermore, parallelism occurs mostly in structure and less in ideas. Even similar ideas could occur in parallelism, while an antithesis has only dissimilar ideas.

Antithesis and Juxtaposition

As far as juxtaposition is concerned, it means placing two ideas together that are dissimilar. They need not be opposite to each other. In the case of antithesis, they must be opposite to each other as in the case of man proposes, God disposes. Not only these two ideas are dissimilar, but also they are opposite. In the case of juxtaposition, a poet only puts two ideas together and they are not opposed to each other.

Use of Antithesis in Sentences  

• As soon he dies, he becomes a dead living.
• Most people do not understand the value of money when the poor put money ahead of them.
• Some people make money, while some waste it.
• Although they have gone leaps ahead, they have also stepped back just in the nick of time.
• The public comes forward when there is prosperity and moves back when there is adversity.

Examples of Antithesis in Literature

Antithesis is an effective literary device and figure of speech in which a writer intentionally juxtaposes two contrasting ideas or entities. Antithesis is typically achieved through parallel structure, in which opposing concepts or elements are paired in adjacent phrases , clauses , or sentences. This draws the reader’s attention to the significance or importance of the agents being contrasted, thereby adding a memorable and meaningful quality to the literary work.

Here are some examples of antithesis in well-known works of literature:

Example 1:  Hamlet (William Shakespeare)

Give every man thine ear, but few thy voice ; Take each man’s censure, but reserve thy judgment.

In Shakespeare’s well-known play , he utilizes antithesis as a literary device for Polonius to deliver fatherly advice to his son before Laertes leaves for France. In these lines, Polonius pairs contrasting ideas such as listening and speaking using parallel structure. This adds a lyrical element to the wording, in addition to having a memorable and foreboding impact on the characters and audience members with the meaning of each line.

Despite the attempt by Polonius to impart logical thinking, measured response, and wise counsel to his son through antithesis, Laertes becomes so fixated on avenging his father’s death that his actions are impulsive and imprudent. Polonius’s antithetical words are not heeded by his son, resulting in the death of several characters including Hamlet and Laertes himself.

Example 2:  Paradise Lost  (John Milton)

Here at least We shall be free; the Almighty hath not built Here for his envy, will not drive us hence: Here we may reign secure, and in my choice To reign is worth ambition though in Hell: Better to reign in Hell, than serve in Heaven.

In Milton’s epic poem , he explores the Fall of Satan as well as the temptation and subsequent Fall of Man. This passage is spoken by Satan after he has been condemned to Hell by God for attempting to assume power and authority in Heaven. Satan is unrepentant of his actions, and wants to persuade his followers that Hell is preferable to Heaven.

Satan utilizes antithesis in the last line of this passage to encourage his rebellious followers to understand that, in Hell, they are free and rule their own destiny. In this line, Milton contrasts not just the ideas of Hell and Heaven, but also of reign and servitude as concepts applied to the angels , respectively. Pairing these opposites by using this literary device has two effects for the reader. First, Satan’s claim foreshadows his ability to use his words describing independence to tempt Eve, resulting in her and Adam’s expulsion from the Garden of Eden. Second, this antithesis invites the reader to consider Satan’s thought-process and experience to gain a deeper understanding of his motives in the poem.

Example 3:  Fire and Ice  (Robert Frost)

Some say the world will end in fire, Some say in ice. From what I’ve tasted of desire I hold with those who favor fire. But if it had to perish twice, I think I know enough of hate To say that for destruction ice Is also great And would suffice.

In his poem, Frost utilizes antithesis to contrast fire and ice as elements with devastating and catastrophic potential to end the world. Frost effectively demonstrates the equal powers for the destruction of these elements, despite showcasing them as opposing forces. In this case, the poet’s antithesis has a literal as well as figurative interpretation. As the poem indicates, the world could literally end in the fire as well as ice. However, fire and ice are contrasting symbols in the poem as well. Fire represents “desire,” most likely in the form of greed, the corruption of power, domination, and control. Conversely, ice represents “hate” in the form of prejudice, oppression, neglect, and isolation.

The presence of antithesis in the poem is effective for readers in that it evokes contrasting and powerful imagery of fire and ice as opposing yet physically destructive forces. In addition, the human characteristics associated with fire and ice, and what they represent as psychologically and socially destructive symbols, impact the reader in a powerful and memorable way as well. Antithesis elevates for the reader the understanding that the source of the end of the world may not be natural causes but rather human action or behavior; and that the end of the world may not be simply the destruction of the earth, but rather the destruction of humankind.

Example 4: The Gettysburg Address by Abraham Lincoln

We have come to dedicate a portion of that field, as a final resting place for those who here gave their lives so that nation might live.

The brave men, living and dead, who struggled here, have consecrated it, far above our poor power to add or detract.

The world will little note, nor long remember what we say here, but it can never forget what they did here.

These three examples from the address of Abraham Lincoln show the use of contradictory ideas put together in one sentence. They show how he mentions living and dead putting them side by side. This antithesis has helped Lincoln as well as America to come out of the ravages of the Civil War.

Function of Antithesis

An antithesis helps make an idea distinct and prominent when it contradicts another idea in the first part of the argument . This contrastive feature helps make readers make their argument solid, cogent, and eloquent. Sentences comprising anthesis also become easy to remember, quote, and recall when required. When an antithesis occurs in a text, it creates an argumentative atmosphere where a dialectic could take place and helps writers and speakers hook their audience easily with antithetical statements.

Synonyms of Antithesis

Antithesis has no exact synonyms but several words come closer in meanings such as opposite, reverse, converse, reversal, inverse, extreme, another side of the coin, or flip side or contrast.

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Definition of antithesis

Did you know.

Writers and speechmakers use the traditional pattern known as antithesis for its resounding effect; John Kennedy's famous "ask not what your country can do for you—ask what you can do for your country" is an example. But antithesis normally means simply "opposite". Thus, war is the antithesis of peace, wealth is the antithesis of poverty, and love is the antithesis of hate. Holding two antithetical ideas in one's head at the same time—for example, that you're the sole master of your fate but also the helpless victim of your terrible upbringing—is so common as to be almost normal.

Examples of antithesis in a Sentence

These examples are programmatically compiled from various online sources to illustrate current usage of the word 'antithesis.' Any opinions expressed in the examples do not represent those of Merriam-Webster or its editors. Send us feedback about these examples.

Word History

Late Latin, from Greek, literally, opposition, from antitithenai to oppose, from anti- + tithenai to set — more at do

1529, in the meaning defined at sense 1b(1)

Dictionary Entries Near antithesis

anti-theoretical

Cite this Entry

“Antithesis.” Merriam-Webster.com Dictionary , Merriam-Webster, https://www.merriam-webster.com/dictionary/antithesis. Accessed 14 May. 2024.

Kids Definition

Kids definition of antithesis, more from merriam-webster on antithesis.

Nglish: Translation of antithesis for Spanish Speakers

Britannica English: Translation of antithesis for Arabic Speakers

Britannica.com: Encyclopedia article about antithesis

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Antithesis Definition

What is antithesis? Here’s a quick and simple definition:

Antithesis is a figure of speech that juxtaposes two contrasting or opposing ideas, usually within parallel grammatical structures. For instance, Neil Armstrong used antithesis when he stepped onto the surface of the moon in 1969 and said, "That's one small step for a man, one giant leap for mankind." This is an example of antithesis because the two halves of the sentence mirror each other in grammatical structure, while together the two halves emphasize the incredible contrast between the individual experience of taking an ordinary step, and the extraordinary progress that Armstrong's step symbolized for the human race.

Some additional key details about antithesis:

• Antithesis works best when it is used in conjunction with parallelism (successive phrases that use the same grammatical structure), since the repetition of structure makes the contrast of the content of the phrases as clear as possible.
• The word "antithesis" has another meaning, which is to describe something as being the opposite of another thing. For example, "love is the antithesis of selfishness." This guide focuses only on antithesis as a literary device.
• The word antithesis has its origins in the Greek word antithenai , meaning "to oppose." The plural of antithesis is antitheses.

How to Pronounce Antithesis

Here's how to pronounce antithesis: an- tith -uh-sis

Antithesis and Parallelism

Often, but not always, antithesis works in tandem with parallelism . In parallelism, two components of a sentence (or pair of sentences) mirror one another by repeating grammatical elements. The following is a good example of both antithesis and parallelism:

To err is human , to forgive divine .

The two clauses of the sentence are parallel because each starts off with an infinitive verb and ends with an adjective ("human" and "divine"). The mirroring of these elements then works to emphasize the contrast in their content, particularly in the very strong opposite contrast between "human" and "divine."

Antithesis Without Parallelism

In most cases, antitheses involve parallel elements of the sentence—whether a pair of nouns, verbs, adjectives, or other grammar elements. However, it is also possible to have antithesis without such clear cut parallelism. In the Temptations Song "My Girl," the singer uses antithesis when he says:

"When it's cold outside , I've got the month of May ."

Here the sentence is clearly cut into two clauses on either side of the comma, and the contrasting elements are clear enough. However, strictly speaking there isn't true parallelism here because "cold outside" and "month of May" are different types of grammatical structures (an adjective phrase and a noun phrase, respectively).

Antithesis vs. Related Terms

Three literary terms that are often mistakenly used in the place of antithesis are juxtaposition , oxymoron , and foil . Each of these three terms does have to do with establishing a relationship of difference between two ideas or characters in a text, but beyond that there are significant differences between them.

Antithesis vs. Juxtaposition

In juxtaposition , two things or ideas are placed next to one another to draw attention to their differences or similarities. In juxtaposition, the pairing of two ideas is therefore not necessarily done to create a relationship of opposition or contradiction between them, as is the case with antithesis. So, while antithesis could be a type of juxtaposition, juxtaposition is not always antithesis.

Antithesis vs. Oxymoron

In an oxymoron , two seemingly contradictory words are placed together because their unlikely combination reveals a deeper truth. Some examples of oxymorons include:

• Sweet sorrow
• Cruel kindness
• Living dead

The focus of antithesis is opposites rather than contradictions . While the words involved in oxymorons seem like they don't belong together (until you give them deeper thought), the words or ideas of antithesis do feel like they belong together even as they contrast as opposites. Further, antitheses seldom function by placing the two words or ideas right next to one another, so antitheses are usually made up of more than two words (as in, "I'd rather be among the living than among the dead").

Antithesis vs. Foil

Some Internet sources use "antithesis" to describe an author's decision to create two characters in a story that are direct opposites of one another—for instance, the protagonist and antagonist . But the correct term for this kind of opposition is a foil : a person or thing in a work of literature that contrasts with another thing in order to call attention to its qualities. While the sentence "the hare was fast, and the tortoise was slow" is an example of antithesis, if we step back and look at the story as a whole, the better term to describe the relationship between the characters of the tortoise and the hare is "foil," as in, "The character of the hare is a foil of the tortoise."

Antithesis Examples

Antithesis in literature.

Below are examples of antithesis from some of English literature's most acclaimed writers — and a comic book!

Antithesis in Charles Dickens' A Tale of Two Cities

In the famous opening lines of A Tale of Two Cities , Dickens sets out a flowing list of antitheses punctuated by the repetition of the word "it was" at the beginning of each clause (which is itself an example of the figure of speech anaphora ). By building up this list of contrasts, Dickens sets the scene of the French Revolution that will serve as the setting of his tale by emphasizing the division and confusion of the era. The overwhelming accumulation of antitheses is also purposefully overdone; Dickens is using hyperbole to make fun of the "noisiest authorities" of the day and their exaggerated claims. The passage contains many examples of antithesis, each consisting of one pair of contrasting ideas that we've highlighted to make the structure clearer.

It was the best of times , it was the worst of times , it was the age of wisdom , it was the age of foolishness , it was the epoch of belief , it was the epoch of incredulity , it was the season of Light , it was the season of Darkness , it was the spring of hope , it was the winter of despair , we had everything before us, we had nothing before us, we were all going direct to Heaven , we were all going direct the other way —in short, the period was so far like the present period, that some of its noisiest authorities insisted on its being received, for good or for evil, in the superlative degree of comparison only.

Antithesis in John Milton's Paradise Lost

In this verse from Paradise Lost , Milton's anti-hero , Satan, claims he's happier as the king of Hell than he could ever have been as a servant in Heaven. He justifies his rebellion against God with this pithy phrase, and the antithesis drives home the double contrast between Hell and Heaven, and between ruling and serving.

Better to reign in Hell than serve in Heaven.

Antithesis in William Shakespeare's Othello

As the plot of Othello nears its climax , the antagonist of the play, Iago, pauses for a moment to acknowledge the significance of what is about to happen. Iago uses antithesis to contrast the two opposite potential outcomes of his villainous plot: either events will transpire in Iago's favor and he will come out on top, or his treachery will be discovered, ruining him.

This is the night That either makes me or fordoes me quite .

In this passage, the simple word "either" functions as a cue for the reader to expect some form of parallelism, because the "either" signals that a contrast between two things is coming.

Antithesis in William Shakespeare's Hamlet

Shakespeare's plays are full of antithesis, and so is Hamlet's most well-known "To be or not to be" soliloquy . This excerpt of the soliloquy is a good example of an antithesis that is not limited to a single word or short phrase. The first instance of antithesis here, where Hamlet announces the guiding question (" to be or not to be ") is followed by an elaboration of each idea ("to be" and "not to be") into metaphors that then form their own antithesis. Both instances of antithesis hinge on an " or " that divides the two contrasting options.

To be or not to be , that is the question: Whether 'tis nobler in the mind to suffer The slings and arrows of outrageous fortune Or to take arms against a sea of troubles, And by opposing end them ...

Antithesis in T.S. Eliot's "Four Quartets"

In this excerpt from his poem "Four Quartets," T.S. Eliot uses antithesis to describe the cycle of life, which is continuously passing from beginning to end, from rise to fall, and from old to new.

In my beginning is my end . In succession Houses rise and fall , crumble, are extended, Are removed, destroyed, restored, or in their place Is an open field, or a factory, or a by-pass. Old stone to new building , old timber to new fires ...

Antithesis in Green Lantern's Oath

Comic book writers know the power of antithesis too! In this catchy oath, Green Lantern uses antithesis to emphasize that his mission to defeat evil will endure no matter the conditions.

In brightest day , in blackest night , No evil shall escape my sight. Let those who worship evil's might Beware my power—Green lantern's light!

While most instances of antithesis are built around an "or" that signals the contrast between the two parts of the sentence, the Green Lantern oath works a bit differently. It's built around an implied "and" (to be technical, that first line of the oath is an asyndeton that replaces the "and" with a comma), because members of the Green Lantern corps are expressing their willingness to fight evil in all places, even very opposite environments.

Antithesis in Speeches

Many well-known speeches contain examples of antithesis. Speakers use antithesis to drive home the stakes of what they are saying, sometimes by contrasting two distinct visions of the future.

Antithesis in Patrick Henry's Speech to the Second Virginia Convention, 1775

This speech by famous American patriot Patrick Henry includes one of the most memorable and oft-quoted phrases from the era of the American Revolution. Here, Henry uses antithesis to emphasize just how highly he prizes liberty, and how deadly serious he is about his fight to achieve it.

Is life so dear, or peace so sweet, as to be purchased at the price of chains and slavery? Forbid it, Almighty God! I know not what course others may take: but as for me, give me liberty or give me death .

Antithesis in Martin Luther King Jr.'s Oberlin Commencement Address

In this speech by one of America's most well-known orators, antithesis allows Martin Luther King Jr. to highlight the contrast between two visions of the future; in the first vision, humans rise above their differences to cooperate with one another, while in the other humanity is doomed by infighting and division.

We must all learn to live together as brothers —or we will all perish together as fools .

Antithesis in Songs

In songs, contrasting two opposite ideas using antithesis can heighten the dramatic tension of a difficult decision, or express the singer's intense emotion—but whatever the context, antithesis is a useful tool for songwriters mainly because opposites are always easy to remember, so lyrics that use antithesis tend to stick in the head.

Antithesis in "Should I Stay or Should I Go" by The Clash (1981)

In this song by The Clash, the speaker is caught at a crossroads between two choices, and antithesis serves as the perfect tool to express just how confused and conflicted he is. The rhetorical question —whether to stay or to go—presents two opposing options, and the contrast between his lover's mood from one day (when everything is "fine") to the next (when it's all "black") explains the difficulty of his choice.

One day it's fine and next it's black So if you want me off your back Well, come on and let me know Should I stay or should I go ? Should I stay or should I go now? Should I stay or should I go now? If I go, there will be trouble If I stay it will be double ...

Antithesis in "My Girl" by the Temptations (1965)

In this song, the singer uses a pair of metaphors to describe the feeling of joy that his lover brings him. This joy is expressed through antithesis, since the singer uses the miserable weather of a cloudy, cold day as the setting for the sunshine-filled month of May that "his girl" makes him feel inside, emphasizing the power of his emotions by contrasting them with the bleak weather.

I've got sunshine on a cloudy day When it's cold outside I've got the month of May Well I guess you'd say, What can make me feel this way? My girl, my girl, my girl Talkin' bout my girl.

Why Do Writers Use Antithesis?

Fundamentally, writers of all types use antithesis for its ability to create a clear contrast. This contrast can serve a number of purposes, as shown in the examples above. It can:

• Present a stark choice between two alternatives.
• Convey magnitude or range (i.e. "in brightest day, in darkest night" or "from the highest mountain, to the deepest valley").
• Express strong emotions.
• Create a relationship of opposition between two separate ideas.
• Accentuate the qualities and characteristics of one thing by placing it in opposition to another.

Whatever the case, antithesis almost always has the added benefit of making language more memorable to listeners and readers. The use of parallelism and other simple grammatical constructions like "either/or" help to establish opposition between concepts—and opposites have a way of sticking in the memory.

Other Helpful Antithesis Resources

• The Wikipedia page on Antithesis : A useful summary with associated examples, along with an extensive account of antithesis in the Gospel of Matthew.
• Sound bites from history : A list of examples of antithesis in famous political speeches from United States history — with audio clips!
• A blog post on antithesis : This quick rundown of antithesis focuses on a quote you may know from Muhammad Ali's philosophy of boxing: "Float like a butterfly, sting like a bee."

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an•the•sis

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Definition of antithesis noun from the Oxford Advanced Learner's Dictionary

• Love is the antithesis of selfishness.
• Students finishing their education at 16 is the very antithesis of what society needs.
• The current establishment is the antithesis of democracy.
• antithesis between
• antithesis of

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• There is an antithesis between the needs of the state and the needs of the people.
• the sharp antithesis between their views

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anthesis noun

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Earlier version

• anthesis in OED Second Edition (1989)

What does the noun anthesis mean?

There is one meaning in OED's entry for the noun anthesis . See ‘Meaning & use’ for definition, usage, and quotation evidence.

How common is the noun anthesis ?

How is the noun anthesis pronounced, british english, u.s. english, where does the noun anthesis come from.

Earliest known use

The earliest known use of the noun anthesis is in the late 1700s.

OED's earliest evidence for anthesis is from 1783, in C. Linnaeus' Syst. Veg.

anthesis is a borrowing from Latin.

Etymons: Latin anthesis .

Nearby entries

• antheridium, n. 1818–
• antheriferous, adj. 1799–
• antheriform, adj. 1802–
• antherine, n. 1689–
• antherless, adj. 1798–
• antherogenous, adj. 1847
• antheroid, adj. 1818–
• antherozoid, n. 1853–
• antherozoidal, adj. 1865–
• anther valve, n. 1839–
• anthesis, n. 1783–
• anthias, n. 1601–
• anthill, n. Old English–
• ant-hillock, n. 1656–
• ant-hilly, adj. 1796–
• anthine, n. & adj. 1601–1768
• ant-hive, n. 1817–
• antho-, comb. form
• anthobian, n. & adj. 1835–
• anthocarpous, adj. 1835–
• anthocephalous, adj. 1847

Meaning & use

The Anthesis [Latin Anthesis ] takes place, when the burnt Anthers scatter their bags of Dust upon the Stigma.

Bractea of the female flowers very much enlarged after anthesis , when the spike presents the appearance of a pine-apple; bright yellow, with red apices.

The term anthesis is sometimes used to indicate the period at which the flower-bud opens.

There were both delayed and extended antheses and most of the time the flowers were semi-open.

Histologically the ovary and style are relatively simple at anthesis .

From the time of anthesis , when the floral parts open to receive pollen, the developing grain becomes the dominant sink.

A later planting date reduced pre-anthesis moisture stress by reducing the number of days..for the crop to reach anthesis .

• efflorescence 1626– The process of producing flowers, or bursting into flower; the period of flowering.
• blow 1748– Manner, style, or time of blossoming. Also figurative .
• anthesis 1783– The stage at which a flower is open, allowing fertilization to occur. Also: an instance of this.
• florescence 1793– The process of producing flowers or bursting into flower; the period or state of flowering. Also concrete . Flowers collectively.

Pronunciation

Plural: antheses.

• ð th ee
• ɬ rhingy ll

Some consonants can take the function of the vowel in unstressed syllables. Where necessary, a syllabic marker diacritic is used, hence <petal> /ˈpɛtl/ but <petally> /ˈpɛtl̩i/.

• a trap, bath
• ɑː start, palm, bath
• ɔː thought, force
• ᵻ (/ɪ/-/ə/)
• ᵿ (/ʊ/-/ə/)

Other symbols

• The symbol ˈ at the beginning of a syllable indicates that that syllable is pronounced with primary stress.
• The symbol ˌ at the beginning of a syllable indicates that that syllable is pronounced with secondary stress.
• Round brackets ( ) in a transcription indicate that the symbol within the brackets is optional.

View the pronunciation model here .

* /d/ also represents a 'tapped' /t/ as in <bitter>

Some consonants can take the function of the vowel in unstressed syllables. Where necessary, a syllabic marker diacritic is used, hence <petal> /ˈpɛd(ə)l/ but <petally> /ˈpɛdl̩i/.

• i fleece, happ y
• æ trap, bath
• ɑ lot, palm, cloth, thought
• ɔ cloth, thought
• ɔr north, force
• ə strut, comm a
• ər nurse, lett er
• ɛ(ə)r square
• æ̃ sal on

Simple Text Respell

Simple text respell breaks words into syllables, separated by a hyphen. The syllable which carries the primary stress is written in capital letters. This key covers both British and U.S. English Simple Text Respell.

b, d, f, h, k, l, m, n, p, r, s, t, v, w and z have their standard English values

• arr carry (British only)
• a(ng) gratin
• o lot (British only)
• orr sorry (British only)
• o(ng) salon

Inflections

anthesis typically occurs about 0.2 times per million words in modern written English.

anthesis is in frequency band 4, which contains words occurring between 0.1 and 1 times per million words in modern written English. More about OED's frequency bands

Frequency of anthesis, n. , 1810–2010

* Occurrences per million words in written English

Historical frequency series are derived from Google Books Ngrams (version 2), a data set based on the Google Books corpus of several million books printed in English between 1500 and 2010.

The overall frequency for a given word is calculated by summing frequencies for the main form of the word, any plural or inflected forms, and any major spelling variations.

For sets of homographs (distinct entries that share the same word-form, e.g. mole , n.¹, mole , n.², mole , n.³, etc.), we have estimated the frequency of each homograph entry as a fraction of the total Ngrams frequency for the word-form. This may result in inaccuracies.

Smoothing has been applied to series for lower-frequency words, using a moving-average algorithm. This reduces short-term fluctuations, which may be produced by variability in the content of the Google Books corpus.

Frequency of anthesis, n. , 2017–2023

Modern frequency series are derived from a corpus of 20 billion words, covering the period from 2017 to the present. The corpus is mainly compiled from online news sources, and covers all major varieties of World English.

Smoothing has been applied to series for lower-frequency words, using a moving-average algorithm. This reduces short-term fluctuations, which may be produced by variability in the content of the corpus.

Compounds & derived words

• synanthesis , n. 1880– Simultaneous ripening of the stamens and pistils in a flower.

Entry history for anthesis, n.

anthesis, n. was revised in March 2016.

anthesis, n. was last modified in July 2023.

oed.com is a living text, updated every three months. Modifications may include:

• further revisions to definitions, pronunciation, etymology, headwords, variant spellings, quotations, and dates;
• new senses, phrases, and quotations.

Revisions and additions of this kind were last incorporated into anthesis, n. in July 2023.

Earlier versions of this entry were published in:

OED First Edition (1885)

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Citation details

Factsheet for anthesis, n., browse entry.

• Literary Terms
• Definition & Examples
• How to Use Antithesis

I. What is an Antithesis?

“Antithesis” literally means “opposite” – it is usually the opposite of a statement, concept, or idea. In literary analysis, an antithesis is a pair of statements or images in which the one reverses the other. The pair is written with similar grammatical structures to show more contrast. Antithesis (pronounced an-TITH-eh-sis) is used to emphasize a concept, idea, or conclusion.

II. Examples of Antithesis

That’s one small step for a man – one giant leap for mankind .  (Neil Armstrong, 1969)

In this example, Armstrong is referring to man walking on the moon. Although taking a step is an ordinary activity for most people, taking a step on the moon, in outer space, is a major achievement for all humanity.

To err is human ; to forgive , divine . (Alexander Pope)

This example is used to point out that humans possess both worldly and godly qualities; they can all make mistakes, but they also have the power to free others from blame.

The world will little note , nor long remember , what we say here, but it can never forget what they did  (Abraham Lincoln, The Gettysburg Address )

In his speech, Lincoln points out that the details of that moment may not be memorable, but the actions would make history, and therefore, never entirely forgotten.

Antithesis can be a little tricky to see at first. To start, notice how each of these examples is separated into two parts . The parts are separated either by a dash, a semicolon, or the word “but.” Antithesis always has this multi-part structure (usually there are two parts, but sometimes it can be more, as we’ll see in later examples). The parts are not always as obvious as they are in these examples, but they will always be there.

Next, notice how the second part of each example contains terms that reverse or invert terms in the first part: small step vs. giant leap; human vs. divine; we say vs. they do. In each of the examples, there are several pairs of contrasted terms between the first part and the second, which is quite common in antithesis.

Finally, notice that each of the examples contains some parallel structures and ideas in addition to the opposites. This is key! The two parts are not simply contradictory statements. They are a matched pair that have many grammatical structures or concepts in common; in the details, however, they are opposites.

For example, look at the parallel grammar of Example 1: the word “one,” followed by an adjective, a noun, and then the word “for.” This accentuates the opposites by setting them against a backdrop of sameness – in other words, two very different ideas are being expressed with very, very similar grammatical structures.

To recap: antithesis has three things:

• Two or more parts
• Reversed or inverted ideas
• (usually) parallel grammatical structure

III. The Importance of Verisimilitude

Antithesis is basically a complex form of juxtaposition . So its effects are fairly similar – by contrasting one thing against its opposite, a writer or speaker can emphasize the key attributes of whatever they’re talking about. In the Neil Armstrong quote, for example, the tremendous significance of the first step on the moon is made more vivid by contrasting it with the smallness and ordinariness of the motion that brought it about.

Antithesis can also be used to express curious contradictions or paradoxes. Again, the Neil Armstrong quote is a good example: Armstrong is inviting his listeners to puzzle over the fact that a tiny, ordinary step – not so different from the millions of steps we take each day – can represent so massive a technological accomplishment as the moon landing.

Paradoxically, an antithesis can also be used to show how two seeming opposites might in fact be similar.

IV. Examples of Verisimilitude in Literature

(adsbygoogle = window.adsbygoogle || []).push({}); Forgive us this day our trespasses as we forgive those who trespass against us . (The Lord’s Prayer)

The antithesis is doing a lot of work here. First, it shows the parallel between committing an evil act and being the victim of one. On the surface, these are opposites, and this is part of the antithesis, but at the same time they are, in the end, the same act from different perspectives. This part of the antithesis is basically just an expression of the Golden Rule.

Second, the antithesis displays a parallel between the speaker (a human) and the one being spoken to (God). The prayer is a request for divine mercy, and at the same time a reminder that human beings should also be merciful.

All the joy the world contains has come through wanting happiness for others . All the misery the world contains has come through wanting pleasure for yourself . (Shantideva, The Way of the Bodhisattva )

The antithesis here comes with some pretty intense parallel structure. Most of the words in each sentence are exactly the same as those in the other sentence. (“All the ___ the world contains has come through wanting ____ for ____.”) This close parallel structure makes the antithesis all the more striking, since the words that differ become much more visible.

Another interesting feature of this antithesis is that it makes “pleasure” and “happiness” seem like opposites, when most of us might think of them as more or less synonymous. The quote makes happiness seem noble and exalted, whereas pleasure is portrayed as selfish and worthless.

The proper function of man is to live , not to exist . I shall not waste my days in trying to prolong  (Jack London, Credo )

The opening antithesis here gets its punch from the fact that we think of living and existing as pretty similar terms. But for London, they are opposites. Living is about having vivid experiences, learning, and being bold; simply existing is a dull, pointless thing. These two apparently similar words are used in this antithesis to emphasize the importance of living as opposed to mere existing.

The second antithesis, on the other hand, is just the opposite – in this case, London is taking two words that seem somewhat opposed (waste and prolong), and telling us that they are in fact the same . Prolonging something is making it last; wasting something is letting it run out too soon. But, says London, when it comes to life, they are the same. If you try too hard to prolong your days (that is, if you’re so worried about dying that you never face your fears and live your life), then you will end up wasting them because you will never do anything worthwhile.

V. Examples of Verisimilitude in Pop Culture

Everybody doesn’t like something, but nobody doesn’t like Sara Lee. (Sara Lee pastry advertisement)

This classic ad uses antithesis to set up a deliberate grammatical error. This is a common technique in advertising, since people are more likely to remember a slogan that is grammatically incorrect. (Even if they only remember it because they found it irritating, it still sticks in their brain, which is all that an ad needs to do.) The antithesis helps make the meaning clear, and throws the grammatical error into sharper relief.

What men must know , a boy must learn . (The Lookouts)

Here’s another example of how parallel structure can turn into antithesis fairly easily. (The structure is noun-“must”-verb. ) The antithesis also expresses the basic narrative of The Lookouts , which is all about kids learning to fend for themselves and become full-fledged adults.

Shut Your Mouth and Open Your Eyes (the band “AFI” – album title)

The antithesis here is a juxtaposition of two different actions (opening and shutting) that are actually part of the same sort of behavior – the behavior of somebody who wants to understand the world rather than be the center of attention. It’s basically a restatement of the old adage that “those who speak the most often have the least to say.”

VI. Related Terms

• Juxtaposition

Antithesis is basically a form of juxtaposition . Juxtaposition, though, is a much broader device that encompasses any deliberate use of contrast or contradiction by an author. So, in addition to antithesis, it might include:

• The scene in “The Godfather” where a series of brutal murders is intercut with shots of a baptism, juxtaposing birth and death.
• “A Song of Ice and Fire” (George R. R. Martin book series)
• Heaven and Hell
• Mountains and the sea
• Dead or alive
• “In sickness and in health”

Antithesis performs a very similar function, but does so in a more complicated way by using full sentences (rather than single words or images) to express the two halves of the juxtaposition.

Here is an antithesis built around some of the common expressions from above

• “ Sheep go to Heaven ; goats go to Hell .”
• “Beethoven’s music is as mighty as the mountains and as timeless as the sea .”
• “In sickness he loved me; in health he abandoned ”

Notice how the antithesis builds an entire statement around the much simpler juxtaposition. And, crucially, notice that each of those statements exhibits parallel grammatical structure . In this way, both Juxtaposition and parallel structures can be used to transform a simple comparison, into antithesis.

List of Terms

• Alliteration
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• APA Citation
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• Deuteragonist
• Doppelganger
• Double Entendre
• Dramatic irony
• Equivocation
• Extended Metaphor
• Figures of Speech
• Flash-forward
• Foreshadowing
• Intertextuality
• Literary Device
• Malapropism
• Onomatopoeia
• Parallelism
• Pathetic Fallacy
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• Protagonist
• Red Herring
• Rhetorical Device
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[ an- tith - uh -sis ]

the antithesis of right and wrong.

Her behavior was the very antithesis of cowardly.

Synonyms: reverse , opposite

• the placing of a sentence or one of its parts against another to which it is opposed to form a balanced contrast of ideas, as in “Give me liberty or give me death.”
• the second sentence or part thus set in opposition, as “or give me death.”
• Philosophy. Hegelian dialectic

/ ænˈtɪθɪsɪs /

• the exact opposite
• contrast or opposition
• rhetoric the juxtaposition of contrasting ideas, phrases, or words so as to produce an effect of balance, such as my words fly up, my thoughts remain below
• philosophy the second stage in the Hegelian dialectic contradicting the thesis before resolution by the synthesis

Discover More

Other words from.

• self-an·tithe·sis noun

Word History and Origins

Origin of antithesis 1

Example Sentences

In the Democratic primary in his home state, he was humiliated by his old antithesis Wallace, who beat him decisively.

It cannot be stressed enough that the behavior of the sitting president is the antithesis of the ideals of American democracy, institutions or peaceful transitions.

American Christians may have chosen cynicism in 2016, but cynicism is the antithesis of the Christian faith, and cynicism won’t have the final word in America, either.

The transhuman cannot exist outside of ubuntu, of course, which is the antithesis of the colonial order for a number of reasons.

Tesla’s being touted as a go-go player in the antithesis of a go-go sector.

Belle Knox is the antithesis of Jenna Jameson—and not just in looks.

To me this is the antithesis of what travel should be about.

Married at First Sight is the antithesis of The Bachelor and The Bachelorette.

Yet its sound is the musical antithesis of a blended Frappuccino.

Now Joffrey, the Starks' black-hearted antithesis, has met a similar fate.

If you did fail, you would try Exclusion, and you would find nothing which is the antithesis of the area of New York.

Thus seen, socialism appeared as the very antithesis of law and order, of love and chastity, and of religion itself.

There is, however, but little danger of overdoing the parallel construction where there is no antithesis.

Nor is it to be wondered at, if we consider the antithesis which is presented to their usual mode of life.

He is a sentimental Classicist, and his subjects the antithesis of the Grco-Roman ideal to which he does homage in his technique.

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Meaning of antithesis in English

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• antithetical
• anything but idiom
• diametrical
• dichotomist
• dichotomous
• or otherwise idiom
• poles apart idiom

You can also find related words, phrases, and synonyms in the topics:

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Antithesis | american dictionary, examples of antithesis, translations of antithesis.

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Literary Devices

Literary devices, terms, and elements, definition of antithesis.

Antithesis is the use of contrasting concepts, words, or sentences within parallel grammatical structures. This combination of a balanced structure with opposite ideas serves to highlight the contrast between them. For example, the following famous Muhammad Ali quote is an example of antithesis: “Float like a butterfly, sting like a bee.” This is an antithesis example because there is the contrast between the animals and their actions (the peaceful floating butterfly versus the aggressive stinging bee) combined with the parallel grammatical structure of similes indicated by “like a.” Ali is indicating the contrasting skills necessary to be a good boxer.

Difference Between Antithesis and Juxtaposition

Antithesis is very similar to juxtaposition, as juxtaposition also sets two different things close to each other to emphasize the difference between them. However, juxtaposition does not necessarily deal with completely opposite ideas—sometimes the juxtaposition may be between two similar things so that the reader will notice the subtle differences. Juxtaposition also does not necessitate a parallel grammatical structure. The definition of antithesis requires this balanced grammatical structure.

Common Examples of Antithesis

The use of antithesis is very popular in speeches and common idioms, as the inherent contrasts often make antithesis quite memorable. Here are some examples of antithesis from famous speeches:

• “We must learn to live together as brothers or perish together as fools.” – Martin Luther King, Jr.
• “And so, my fellow Americans: ask not what your country can do for you — ask what you can do for your country. My fellow citizens of the world: ask not what America will do for you, but what together we can do for the freedom of man.” –John F. Kennedy Jr.
• “We will extend a hand if you are willing to unclench your fist.” –Barack Obama
• “Decided only to be undecided, resolved to be irresolute, adamant for drift, solid for fluidity, all-powerful to be impotent.” –Winston Churchill
• “The world will little note, nor long remember what we say here, but it can never forget what they did here.” –Abraham Lincoln

Significance of Antithesis in Literature

Antithesis can be a helpful tool for the author both to show a character’s mindset and to set up an argument. If the antithesis is something that the character is thinking, the audience can better understand the full scope of that character’s thoughts. While antithesis is not the most ubiquitous of literary devices, some authors use antithesis quite extensively, such as William Shakespeare. Many of his sonnets and plays include examples of antithesis.

Examples of Antithesis in Literature

HAMLET: To be, or not to be, that is the question— Whether ’tis Nobler in the mind to suffer The Slings and Arrows of outrageous Fortune, Or to take Arms against a Sea of troubles, And by opposing, end them?

( Hamlet by William Shakespeare)

Arguably the most famous six words in all of Shakespeare’s work are an example of antithesis. Hamlet considers the important question of “to be, or not to be.” In this line, he is considering the very nature of existence itself. Though the line is quite simple in form it contrasts these very important opposite states. Hamlet sets up his soliloquy with this antithesis and continues with others, including the contrast between suffering whatever fortune has to offer or opposing his troubles. This is a good example of Shakespeare using antithesis to present to the audience or readers Hamlet’s inner life and the range of his thinking.

It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity, it was the season of Light, it was the season of Darkness, it was the spring of hope, it was the winter of despair, we had everything before us, we had nothing before us, we were all going direct to Heaven, we were all going direct the other way…

( A Tale of Two Cities by Charles Dickens)

The opening paragraph of Charles Dickens’s A Tale of Two Cities employs many different literary devices all at once. There are many examples of antithesis back-to-back, starting with the first contrast between “the best of times” and “the worst of times.” Each pair of contrasting opposites uses a parallel structure to emphasize their differences. Dickens uses these antithetical pairs to show what a tumultuous time it was during the setting of his book. In this case, the use of antithesis is a rhetorical device that foreshadows the conflicts that will be central to the novel.

There was only one catch and that was Catch-22, which specified that a concern for one’s own safety in the face of dangers that were real and immediate was the process of a rational mind. Orr was crazy and could be grounded. All he had to do was ask; and as soon as he did, he would no longer be crazy and would have to fly more missions. Orr would be crazy to fly more missions and sane if he didn’t, but if he was sane he had to fly them. If he flew them he was crazy and didn’t have to; but if he didn’t want to he was sane and had to. Yossarian was moved very deeply by the absolute simplicity of this clause of Catch-22 and let out a respectful whistle.

( Catch-22 by Joseph Heller)

In Joseph Heller’s classic anti-war novel Catch-22 , Heller uses a specific type of humor in which antithetical statements show the true absurdity of war. This very famous quote explains the concept of the “Catch-22,” which became a popular idiomatic expression because of the book. In fact, this example is not so much an antithetical statement but instead an antithetical situation. That is to say, the two possible outcomes for Orr are opposite: either he’s deemed crazy and would thus not be forced to fly any more combat missions, or he’s sane and then would indeed have to fly them. However, the one situation negates the possibility of the other, as only a sane man would be clear-headed enough to ask not to fly more missions.

This case is not a difficult one, it requires no minute sifting of complicated facts, but it does require you to be sure beyond all reasonable doubt as to the guilt of the defendant.

( To Kill a Mockingbird by Harper Lee)

In Harper Lee’s novel To Kill a Mockingbird , Atticus Finch is a lawyer representing Tom Robinson. Atticus presents the above statement to the jury, setting up an antithesis. He asserts that the case is not difficult and yet requires the jury to be absolutely sure of their decision. Atticus believes the case to have a very obvious conclusion, and hopes that the jury will agree with him, but he is also aware of the societal tensions at work that will complicate the case.

Test Your Knowledge of Antithesis

1. What is the correct antithesis definition? A. Using two very similar concepts and showing their subtle differences. B. Setting up a contrast between two opposite ideas or phrases in a balanced grammatical structure. C. Using words to convey an opposite meaning to their literal sense. [spoiler title=”Answer to Question #1″] Answer: B is the correct answer. A is one possible definition of juxtaposition, while C is one possible definition of irony.[/spoiler]

2. What is the difference between antithesis and juxtaposition? A. They are exactly the same device. B. They are completely different literary devices. C. Antithesis parallels opposite concepts, while juxtaposition sets up a comparison and contrast between two concepts that can be either similar or different. [spoiler title=”Answer to Question #2″] Answer: C is the correct answer.[/spoiler]

3. Which of the following quotes from Shakespeare’s Macbeth contains an example of antithesis? A. 

WITCHES: Fair is foul, and foul is fair: Hover through the fog and filthy air.

MACBETH: Is this a dagger which I see before me, The handle toward my hand?

WITCHES: Something wicked this way comes.

[spoiler title=”Answer to Question #3″] Answer: A is the correct answer.[/spoiler]

4. Which of the following quotes from Heller’s Catch-22 contains an example of antithesis? A. There are now fifty or sixty countries fighting in this war. Surely so many counties can’t all be worth dying for. B. He had decided to live forever or die in the attempt, and his only mission each time he went up was to come down alive. C. You’re inches away from death every time you go on a mission. How much older can you be at your age? [spoiler title=”Answer to Question #4″] Answer: B is the correct answer.[/spoiler]

What is Antithesis? Definition, Usage, and Literary Examples

Antithesis definition.

Antithesis  (ann-TIH-thuh-suhs), put simply, means the absolute opposite of something. As a literary term, it refers to the juxtaposition of two opposing entities in parallel structure. Antithesis is an effective literary device because humans tend to define through contrast. Therefore, antithesis can help readers understand something by defining its opposite.

Antithesis  comes from the Latin word, via Greek, for “to place against.” It was first used in English in the 1520s as rhetorical term, but the concept goes back at least as far as Aristotle, who believed an argument could be strengthened by illustrating it with contrast.

Examples of Antithesis

• “Spicy food is heaven on the tongue but hell in the tummy.” The concepts of heaven and hell are opposites—the former being very pleasant and ideal, the latter being highly undesirable. This antithetical statement is using these concepts to convey that spicy food is delicious, but it can lead to an unfortunate digestive reaction.
• “I’m either an impressive vegetarian or a disappointing vegan.” On the scale between vegetarianism and the stricter veganism, the speaker’s current diet lies somewhere in the middle. So, while a vegetarian might applaud their efforts, a vegan might berate them for being so lax.
• “Psychiatrists write prescriptions, therapists prescribe writing.” This example includes a humorous inversion to explain the difference between psychiatrists and therapists. The former prescribes medicine to address mental issues on a biological level, while the latter might suggest a more psychologically focused approach, like journaling, as a way of easing mental stress.

Antithesis vs. Other Comparative Terms

There are several literary terms that, like antithesis, make comparisons between two things or concepts that are opposites or contrast in some way. Three such terms are  dichotomy ,  oxymoron , and foil.

Dichotomy is a division between two entities, whereas antithesis pits two opposing entities against each other. For example, the colors black and white are considered opposites, but they are not in opposition; they can’t be in conflict nor do they cancel each other out. The concepts of war and peace, on the other hand, are at odds and can’t be reconciled.

An oxymoron is a contradiction in terms, but unlike antithesis, these terms are working together. The basic oxymoron construction is a word + an antonymic modifier, and the two essentially function as a single unit. For example, calling something a “minor crisis” is an oxymoron because  minor  implies something insignificant, while  crisis  means it requires immediate attention. Based on this, an oxymoron can’t be a component of antithesis because the point of the latter is to pit two things against each other.

Where antithesis is a verbal or written opposition, a foil is a literary opposition, usually embodied by a character in a narrative. For example, Draco Malfoy can be considered Harry Potter’s foil in the  Harry Potter  series because where Harry is honorable and loyal, Draco is somewhat corrupt and unfaithful.

Antithesis Outside of Literature

A common theme in American popular music is the difference between the middle and lower classes. In “Men of Good Fortune” by Lou Reed, the singer describes all the things rich men can do that poor men cannot:

Men of good fortune

Often cause empires to fall

While men of poor beginnings

Often can’t do anything at all

Antithesis is common in political speeches, particularly when it comes to the underrepresented pushing for equitable policies. In Malcolm X’s famous “ Ballot or the Bullet ”  speech, he discusses how America was built by Black and indigenous people for white people’s benefit, saying, “We didn’t land on Plymouth Rock; the rock was landed on us.”

Examples of Antithesis in Literature

1. Charles Dickens,  A Tale of Two Cities

This classic tale of love and sacrifice features the French Revolution as its backdrop. In this tumultuous era, where the differences between the haves and the have-nots was at its starkest, Dickens illustrates the antithetical concepts that existed simultaneously:

It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity, it was the season of Light, it was the season of Darkness, it was the spring of hope, it was the winter of despair, we had everything before us, we had nothing before us, we were all going direct to Heaven, we were all going direct the other way […]

2. William Shakespeare,  The Merchant of Venice

At the beginning of this romantic comedy, chatty lout Gratiano wants to understand why his friend Antonio is so down—and why anyone would ever be down at all:

And let my liver rather heat with wine

Than my heart cool with mortifying groans.

Gratiano is implying that he’d rather experience life through a chemically altered (and therefore unreliable) lens than face any ordeals, even though they would be truer to reality.

3. John Milton,  Paradise Lost

Milton’s epic poem explores many facets of the Christian bible and belief systems—including the concept of free will. When Lucifer, once one of God’s brightest angels, is cast into Hell, he says, “Better to reign in Hell than serve in Heaven.” This leads the reader to question whether anyone is truly free, though ultimately the Bible’s core argument is that willingly giving over control to God is what will leads to a happy life.

Further Resources on Antithesis

MasterClass’s  How to Use Antithesis in Your Writing  course is a concise guide on the mechanics of antithesis and when to use it.

This excerpt from  Hegel for Beginners  by Lloyd Spencer is a handy introduction to antithesis as a component of dialectics (a system for pursuing truth by way of logical argument).

Related Terms

• Juxtaposition

• English Grammar
• Figures Of Speech

Antithesis: Meaning, Definition and Examples

Figures of speech , otherwise known as rhetorical devices, are used in the English language to beautify and make your language look and sound a lot more effective rather than a literal presentation of information. Each figure of speech has its function and is meant to perform its roles giving the context a unique effect. In this article, you will learn about one such figure of speech called antithesis. Read through the article to learn more about what antithesis is, its definition and how it differs from an oxymoron. You can also check out the examples and analyse how it is written for an in-depth understanding of the same.

Table of Contents

What is antithesis – meaning and definition, what differentiates an antithesis from an oxymoron, some common examples of antithesis, frequently asked questions on antithesis.

An antithesis is a figure of speech that states strongly contrasting ideas placed in juxtaposition. They contain compound sentences with the two independent clauses separated by a comma or a semicolon , in most cases. However, there are also instances where the antithesis is a compound sentence with a conjunction . An antithesis is mainly used to portray the stark difference between the two opposing ideas.

Antithesis, according to the Oxford Learner’s Dictionary, is defined as “a contrast between two things”, and according to the Cambridge Dictionary, “a difference or opposition between two things”. The Merriam-Webster Dictionary gives a more explanatory definition. According to it, antithesis is “the rhetorical contrast of ideas by means of parallel arrangements of words, clauses, or sentences”.

Knowing the difference between an antithesis and an oxymoron will help you comprehend and use both the rhetorical devices effectively. Take a look at the table given below to learn more.

Here are some of the most common examples of antithesis for your reference.

• Hope for the best; prepare for the worst.
• Keep your mouth closed and your eyes open.
• “It was the best of times, it was the worst of times. It was the age of wisdom, it was the age of foolishness.” – Charles Dickens
• “That’s one small step for a man, one giant leap for mankind.” – Neil Armstrong
• “Better to reign in Hell, than to serve in Heaven.” – John Milton
• Speech is silver, but silence is gold.
• “Give every man thy ear, but few thy voice.” – William Shakespeare
• Keep your friends close; keep your enemies closer.
• “To err is human; to forgive divine.” – Alexander Pope
• Money is the root of all evil: poverty is the fruit of all goodness.

What is antithesis?

An antithesis is a figure of speech that states strongly contrasting ideas placed in juxtaposition. They contain compound sentences with the two independent clauses separated by a comma or a semicolon, in most cases. However, there are also instances where the antithesis is a compound sentence with a conjunction.

What is the definition of antithesis?

What is the difference between antithesis and oxymoron.

The main difference between an antithesis and an oxymoron is that antithesis refers to the use of two contrasting ideas or thoughts conveyed in two independent clauses placed in juxtaposition, separated by a comma, a semicolon or a conjunction; whereas, the term ‘oxymoron’ refers to the use of two opposite words within a phrase to create an effect.

Give some examples of antithesis.

Here are a few examples of antithesis for your reference.

• “Love is an ideal thing, marriage a real thing.” – Goethe
• “Folks who have no vices have very few virtues.” – Abraham Lincoln
• “Man proposes, God disposes.”
• Beggars can’t be choosers.
• Be slow in choosing, but slower in changing.

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What is Antithesis — Definition & Examples in Literature & Film

I f you’ve ever heard sentence structure, met characters, or witnessed ideas that seem diametrically opposed, you’re actually pretty familiar with the idea of the antithesis. But there is more to it than just juxtaposing ideas. Read on to learn exactly what is antithesis, how this tool is used, and how you can include an antithesis in your next project. 

Antithesis Definition

First, let’s define antithesis.

There are a number of terms often confused for antithesis (like paradox or oxymoron ). But an antithesis has a particular grammatical structure that helps differentiate it from the rest. So, here’s the antithesis definition and then we'll look at specific examples:

ANTITHESIS DEFINITION

What is antithesis.

An antithesis is a rhetorical and literary device with parallel grammar structure but which establishes a nearly complete or exact opposition in ideas or characters. It can be effective in emphasizing drastic differences between opposing concepts.

How to pronounce antithesis: [an-TITH-uh-sis]  

Familiar antithesis examples:

• “That’s one small step for man, one giant leap for mankind.”
• “No pain, no gain.”
• “Out of sight, out of mind.”

The word “antithesis” comes from the Greek word meaning “setting opposite,” which is an idea that has been used in various forms. Let’s look at those various forms in more antithesis examples.

Antitheses Examples

How do we use antithesis today.

The purpose of antithetical language is not just mentioning the existence of opposing ideas, but rather emphasizing the stark differences between them.  The often lyrical and rhythmic nature of this device helps accentuate the parallel grammatical structure.

Watch the video below to learn more about how we use antitheses today. 

Antithesis Definition, Examples and Techniques

We use this device in that pure form today (see the examples above) in everyday turns of phrase. But there are more in-depth ways (in actions and story in general) that fit the antithesis definition. 

People and characters can act in an antithetical manner to their beliefs.

Antithesis Examples in Behavior: 

• A character who says they love animals but wears real fur coats.
• Someone who says they are vegetarian but eats a big steak for dinner.
• A person who uses a “Shop Small” tote bag but does their holiday shopping at Walmart. 

In addition, characters in literary or scripted works, much like people, can be antitheses to each other in and of themselves. In fact, this is often how great villains are created.

Check out the video below to see more on writing great villains , and how antagonists can mirror or juxtapose protagonists . 

Page to Picture: How to Write a Villain  •   Subscribe on YouTube

Protagonists can be an “antihero,” or the villain of a story can be portrayed separately as a parallel to the protagonist; therefore, the protagonist and antagonist highlight each other’s strengths and weaknesses, and evil and benevolent qualities. Some classic examples of this pseudo-mirrored antagonist concept are: 

Snow White and the Queen

Batman and Joker

Dumbledore and Voldemort

As you can see, the antithesis is typically the ultimate antagonist, even if the character they are meant to parallel isn’t the protagonist, as is the case in the Harry Potter series. 

Both a strong example of antithesis and nuanced portrayal of complicated character relationships, the Harry Potter series showcases a number of moral ambiguities as they pertain to Dumbledore and Voldemort.

We imported the script into StudioBinder’s screenwriting software to see exactly how this juxtaposition is first established.

Read Full Harry Potter and the Sorcerer's Stone Script

Harry Potter and the Sorcerer’s Stone masterfully establishes the characteristics that Harry and Voldemort share, but it also establishes similarities between Dumbledore and Voldemort.

Throughout the series, these shared traits influence Dumbledore and Voldemort in their objectives and decisions. 

For instance, in the Sorcerer’s Stone , we are introduced to how Voldemort and Dumbledore move through the world. Hagrid tells Harry early on about Voldemort’s rise to power: he was a wizard “who went as bad as you can go [...] anyone who stood up to him ended up dead.” Hagrid frames Voldemort as a powerful wizard, capable of massive destruction. 

Antithesis Example in Harry Potter  •   Read Full Scene

Alternatively, towards the end of the first film Dumbledore explains his tactics in his work with Nicolas Flamel on the Sorcerer’s Stone . “Only a person who wanted to find the stone—find it, but not use it—would be able to get it.” Dumbledore respects power and the laws of magic, and his actions reflect that. 

Further into the series, in Order of the Phoenix , we see what happens when Dumbledore’s tremendous abilities collide with Voldemort’s formidable power in their epic Ministry of Magic duel.

Voldemort’s spells all aim to destruct, whereas Dumbledore’s are equally amazing, but meant to disarm, distinguish, or defend. 

The series is a remarkable example of how antitheses can be essential to a story, and the respective backstories, unique abilities, and of course choices of Voldemort and Dumbledore prove it.  

Implementing Antitheses

How to use antithesis.

With all the ways you can implement and define antithesis, it’s good to have a number of tricks or rules of thumb to keep in your back pocket. Whether you’re writing a short story or your next feature screenplay, here are some things you can keep in mind. 

1. Aim for Moderation

If you’re using antithesis in the form of a rhetorical device, try to keep the number of antitheses to a minimum unless it’s a crucial character trait of the speaker. Using a similar literary device too often can leave your writing predictable or even annoying. The more you use a tool, like antithetical language, the less meaning it can have. 

2. Similar Structure

Keep the structure of your antithesis as similar as possible if you want to highlight the differences more intensely. And try to keep the phrasing itself balanced. Both variables of the equation don’t have to be exact, but the lyrical phrasing can help your antithesis shine and stick long after the read. 

3. Focus on Differences

Focus on contrast but remember to find ways to draw the parallels. How can the characters be compared to the point where their differences become obvious? How can their differences lead to conversation about how the characters may actually be similar? 

Antitheses via characters and sentence structure can assist in not just interesting writing, but memorable writing. They can make your message more understandable and retainable, which should be a top goal in any written work. You never want to give you reader a reason to stop reading.

So, now that you’ve learned more about how an antithesis can strengthen your work, you can implement it  into your next project like a pro.

After all, no guts, no glory. 

What is Irony?

Antithesis is a rhetorical device you can use in everyday speech. Much like an antithesis, we encounter several types of irony in everyday life, too. Keep reading to learn about the types of irony and how they’re used in TV and Film. 

Up Next: Irony Explained →

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Photosynthesis in newly developed leaves of heat-tolerant wheat acclimates to long-term nocturnal warming

Helen Bramley Present address: Hobart, Tasmania, Australia

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Onoriode Coast, Andrew P Scafaro, Helen Bramley, Nicolas L Taylor, Owen K Atkin, Photosynthesis in newly developed leaves of heat-tolerant wheat acclimates to long-term nocturnal warming, Journal of Experimental Botany , Volume 75, Issue 3, 2 February 2024, Pages 962–978, https://doi.org/10.1093/jxb/erad437

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We examined photosynthetic traits of pre-existing and newly developed flag leaves of four wheat genotypes grown in controlled-environment experiments. In newly developed leaves, acclimation of the maximum rate of net CO 2 assimilation ( A n ) to warm nights (i.e. increased A n ) was associated with increased capacity of Rubisco carboxylation and photosynthetic electron transport, with Rubisco activation state probably contributing to increased Rubisco activity. Metabolite profiling linked acclimation of A n to greater accumulation of monosaccharides and saturated fatty acids in leaves; these changes suggest roles for osmotic adjustment of leaf turgor pressure and maintenance of cell membrane integrity. By contrast, where A n decreased under warm nights, the decline was related to lower stomatal conductance and rates of photosynthetic electron transport. Decreases in A n occurred despite higher basal PSII thermal stability in all genotypes exposed to warm nights: T crit of 45–46.5 °C in non-acclimated versus 43.8–45 °C in acclimated leaves. Pre-existing leaves showed no change in A n –temperature response curves, except for an elite heat-tolerant genotype. These findings illustrate the impact of night-time warming on the ability of wheat plants to photosynthesize during the day, thereby contributing to explain the impact of global warming on crop productivity.

Wheat ( Triticum aestivum L) is the most widely cultivated crop, with >220 Mha cultivated worldwide. Wheat yield must increase by 0.83% per annum to meet the projected 44% increase in demand for it by 2050 ( CGIAR Advisory Services, 2020 ). To achieve an environmentally sustainable increase in production of this magnitude, while also coping with rising global surface temperatures, represents a major challenge. Moreover, most research has focused on effects of elevated temperatures during the day on wheat, but rising temperatures have been more pronounced for daily minimums (i.e. night warming) than for daily maximums ( Easterling et al. , 1997 ; Alexander et al. , 2006 ; Sillmann et al. , 2013 ). Night warming is linked with a reduction in wheat yield in many regions including the USA, Mexico, and Argentina ( Lobell et al. , 2005 ; Prasad et al. , 2008 ; García et al. , 2015 , 2016 , 2018 ; Russell and Van Sanford, 2020 ; Fisher et al. , 2022 ). Given the importance of wheat for global food production, it is vital that we develop a more complete understanding of the physiological basis for why nocturnal warming reduces wheat yields. Reflecting this, interest in wheat physiological responses to warm nights has increased, with reports on steady-state photosynthesis, acclimation of leaf and root respiration, and altered source–sink capacity at anthesis and grain filling ( Fan et al. , 2015 , 2017 ; Impa et al. , 2019 , 2020 ; Bahuguna et al. , 2022 ; Posch et al. , 2022a ). However, there remain key gaps in knowledge, especially in relation to the effect of warm nights on overall photosynthetic capacity, and whether key photosynthetic processes such as the critical temperature above which incipient damage to photosystem II (PSII) occurs ( T crit ) acclimate to warm nights. PSII is considered the most heat-sensitive component of electron transport in the photosynthetic machinery, with heat stress resulting in the unfolding of protein complexes and loss of manganese from the oxygen-evolving complex ( Enami et al. , 1994 ).

According to the Farquhar, von Caemmerer, and Berry (FvCB model; Farquhar et al. , 1980 ; von Caemmerer, 2000 ) biochemical model of photosynthesis, light-saturated A n at a given temperature can be limited by: (i) the maximum capacity of Rubisco carboxylation ( V cmax ) and internal CO 2 concentration ( C i ); or (ii) the regeneration of ribulose 1,5-bisphosphate (RuBP) regeneration which relies on photosynthetic electron transport capacity ( J ) and irradiance. A n can also be limited if triose phosphate use (TPU) restricts the recycling of phosphate within the chloroplast ( Sharkey, 1985 ). V cmax , J , and TPU can be estimated by fitting the FvCB model ( Farqhuar et al. , 1980 ) to plots of light-saturated A n versus C i . Light-saturated rates of A n respond to short- and long-term changes in growth environment temperature ( Atkin and Tjoelker, 2003 ; Way and Yamori, 2014 ). The thermal response of light-saturated A n to short-term changes in growth environment temperature is curvilinear—increasing with a rise in temperature at suboptimal temperatures to a maximum rate ( A max ) at the optimum temperature ( T opt ), then decreasing at temperatures beyond T opt . Longer term changes in the thermal environment cause either positive or detractive adjustments of light-saturated A n . Positive adjustments increase A max and/or shift T opt towards the new, higher growth temperature, indicating acclimation, whereas detractive adjustments reduce A max and/or T opt ( Sage and Kubien, 2007 ; Way and Yamori, 2014 ; Posch et al. , 2019 ). Changes in the A n –temperature response are controlled by different factors, including the temperature sensitivities of V cmax and J ( Hikosaka et al. , 2005 ; Sage and Kubien, 2007 ; Lin et al. , 2012 ), with alterations in V cmax reflecting changes in the abundance and/or activation state of Rubisco ( Scafaro et al. , 2023 ). Another controlling factor, albeit indirectly, is stomatal conductance ( g s ). At high temperatures, stomatal closure reduces water loss when the atmospheric vapour pressure deficit (VPD) rises, but this simultaneously reduces CO 2 uptake. In wheat, the A n –temperature response to elevated daily temperatures varies from positive acclimation to detractive adjustments ( Yamasaki et al. , 2002 ; Cai et al. , 2018 , 2020 ; Coast et al ., 2021a ). Importantly, information on thermal acclimation of wheat photosynthesis to specifically warm nights is scarce (but see Turnbull et al. , 2002 for work on a broadleaved tree species). It is likely that under night warming, wheat photosynthesis and acclimation capacity may be affected directly by constraining leaf biochemical components underlying photosynthesis (i.e. V cmax and J ), or indirectly by stomatal closure, or both. These suggestions remain untested.

The degree of physiological acclimation in leaves to warming varies substantially with the duration and intensity of warming, as well as the plant’s developmental stage. Leaves that develop under a new warming regime exhibit a higher degree of acclimation than pre-existing leaves that were formed under an earlier thermal regime and which then experience a sustained change in growth temperature ( Loveys et al. , 2003 ; Yamori et al. , 2005 ; Rashid et al. , 2020 ; Slot et al. , 2021 ). Varying acclimation capacity with leaf development has also been shown in cold regimes ( Hurry et al. , 2005 ; Campbell et al. , 2007 ). Limited reports exist about leaf development under warm conditions and the magnitude of photosynthetic acclimation in newly developed and pre-existing leaves. Determining these responses could be relevant in managing crop performance and useful in parameterizing carbon fluxes in Earth system models ( Rogers et al. 2017 ).

T crit , a common measure of photosynthetic heat tolerance ( Geange et al. , 2020 ; Lancaster and Humphreys, 2020 ), is estimated from temperature-dependent changes in minimum Chl a fluorescence ( F 0 ) ( Schreiber et al. , 1975 ; Schreiber and Berry, 1977 ). T crit correlates with the maximum temperature at which A n approaches zero ( Downton et al ., 1984 ). Under warmer growth conditions, an increase in T crit is considered evidence of acclimation and indicative of higher temperature tolerance. Acclimation of T crit to warmer growth conditions has been shown in many non-crop, mostly woody species. For example, 62 Australian species acclimated seasonally with T crit , increasing on average from winter to summer by 0.34 °C per °C increase in growth temperature ( Zhu et al. , 2018 ). Similar reports exist for Mediterranean and temperate European oak species ( Quercus spp., Daas et al. , 2008 ), and at a global scale for 218 plant species spanning seven biomes ( O’Sullivan et al. , 2017 ). Acclimation of T crit is assumed to increase the upper thermal threshold of photosynthesis by relaxing the limits of photosynthetic electron transport. As such, the capacity to acclimate T crit might underlie photosynthetic performance under warm nights. However, it is not known whether T crit acclimates in response to warm nights.

Metabolites function as regulatory agents, compatible solutes, antioxidants, and reductants in adaptation to thermal stress. Warming alters the abundance of primary metabolites [sugars, sugar alcohols, organic acids, amino acids, or tricarboxylic acid (TCA) cycle intermediates] in leaves of stress-sensitive cereals ( Glaubitz et al. , 2014 ; Impa et al. , 2019 ; Schaarschmidt et al. , 2020 ; Rashid et al. , 2021 ). Increases in the abundance of organic acids and TCA cycle components in heat-sensitive genotypes under heat stress often reflect impairment to CO 2 assimilation, regulation of the TCA cycle, and amino acid biosynthesis, and these processes are central to plant photosynthesis. Deciphering changes in the metabolic phenotype of wheat in response to warm nights could aid our understanding of plant responses to climate change and provide input for developing adaptation tools for crop production in a warmer world.

To address some of the above issues, we compared four wheat genotypes (including a commercial Australian cultivar and an elite heat-tolerant genotype) under night temperatures of 15, 20, or 25 °C for different durations (5–7 d at anthesis or 9–13 weeks prior to and including anthesis). Our primary objectives were to: (i) quantify the acclimation capacity of photosynthesis and T crit to warm nights; and (ii) assess changes in the wheat metabolic phenotype under warm nights. In addition, we set out to compare effects of warm nights on the temperature dependencies of photosynthetic parameters in newly developed versus pre-existing leaves. The results indicate that plants stressed by warm nights reduced photosynthetic performance via down-regulation of J even while exhibiting high T crit (i.e. high PSII thermal stability). By contrast, tolerance of warm nights (in terms of photosynthesis) was marked by improved A n linked to acclimation in V cmax , PSII thermal stability, and an increase in leaf metabolic signatures for monosaccharides.

To assess the extent to which photosynthetic capacity of pre-existing and newly developed leaves of wheat respond to warm nights, two experiments were conducted. In Experiment I, acclimation of T crit , instantaneous temperature response functions of photosynthetic capacity [light-saturated A n , V cmax , J 1500 ( J measured at a photosynthetic photon flux of 1500 µmol m –2 s –1 ), and TPU], and changes in the metabolite profile of plants that developed from seedling to anthesis (9–13 weeks of night warming) were compared at three night temperatures (15, 20, and 25 °C) and a common day temperature of 26 °C. These temperature treatments are reflective of conditions in some wheat-growing regions including parts of the USA ( Hein et al. , 2020 ) and India ( Bahuguna et al. , 2022 ). In Geraldton and Northampton on the northern tip of the West Australian wheatbelt, the mean minimum temperatures between October and December (i.e. during reproductive development to harvest) range from 12 °C to 17 °C. Similar temperatures, in controlled-environment settings, enable dissection of the physiological mechanism of tolerance and application of high-throughput phenotyping platforms to screen for stress tolerance; for example, see Wang et al. (2022) with control nights of 21 °C and high nights of 28 °C. Experiment II was a repetition of Experiment I except plants with mature (pre-existing) flag leaves which had grown under 15 °C nights were exposed to a shorter period of warming (5–7 nights at 20 °C and 25 °C environments) at anthesis. As such Experiment I focused on newly developed leaves and Experiment II on pre-existing leaves. For both experiments, all measurements were taken when plants were at the same developmental stage—anthesis (i.e. between Zadok scale ZS60 and ZS69; Zadoks et al. , 1974 ).

Plant materials, management, and temperature treatments

Four wheat ( T. aestivum L.) genotypes were used for this study: Mace (pedigree Wyalkatchem/Stylet/Wyalkatchem), a well-adapted, commercial, Australian cultivar; ACIAR09PBI C38-150C-DH9 (pedigree PBW343+L24+LR28/LANG; henceforth 1704), a heat-susceptible genotype; ACIAR09PBI C27-0C-0N-3N (pedigree DBW16/ANNUELLO; henceforth 1898), also a heat-susceptible genotype; and 8:ZW11 [pedigree D67.2/P66.270//AE.SQUARROSA(320)/3/CUNNINGHAM/4/VORB; henceforth 2254], a heat-tolerant elite genotype. Mace was a benchmark variety for yield in Western Australia where it was widely grown. It accounted for 66% of plantings between 2015 and 2016 ( Zaicou-Kunesch et al. , 2017 ), although this reduced to 31% by 2018. Mace can cope with high temperatures ( Bokshi et al. , 2022 ). The two heat-susceptible genotypes (1704 and 1898) exhibit low PSII thermal stability (i.e. low basal T crit ) when grown under non-heat-stressed conditions but also the capacity to acclimate T crit to warmer growth regimes in field conditions ( Posch et al. , 2022b ). Genotype 2254 was developed by the International Maize and Wheat Improvement Centre (CIMMYT) and, like Mace, it is considered heat tolerant ( Posch et al. , 2022a ). Unpublished yield data from field experiments conducted under heat stress conditions in Ciudad Obregon, Mexico and Narrabri, Australia by Professor Richard Trethowan of the University of Sydney, Australia, support the heat-tolerant classification of genotype 2254. These were chosen for their varied yield, and agronomic and physiological performance under heat stress conditions in fields across wheat-growing regions of Australia.

Seeds were germinated on moist filter papers in Petri dishes. One-week-old seedlings were sown into 6 litre plastic pots (one seedling per pot) filled with Martins mix (Martins Fertilizers Ltd, Yass, NSW Australia). The Martins mix was enriched with 4 g l –1 Osmocote® OSEX34 EXACT slow‐release fertilizer (Scotts Australia, Bella Vista, NSW, Australia) and treated at 63 °C for 1 h prior to filling pots. Potted seedlings were transferred into temperature-controlled growth chambers (Thermoline, Wetherhill Park, Australia) at the Controlled Environment Facilities of the Research School of Biology, The Australian National University, Canberra, Australia (ANU). Genotypes were arranged randomly in each chamber. Growth chambers were maintained at day/night temperatures of 26/15, 26/20. and 26/25 °C either throughout plant growth (lasting 9–13 weeks; Experiment I) or only at anthesis (lasting 5–7 d; Experiment II). For the latter conditions, day/night temperature prior to anthesis was maintained at 26/15 °C. In all growth chambers, relative humidity varied from 30% during the light period to 70% during the dark, and [CO 2 ] was at ambient ~400 μmol mol –1 (38.4 Pa, considering a mean atmospheric pressure of 96 kPa at ANU). Lighting was supplied by 1000 W metal halide lamps (Multi-Vapor®; GE Lighting, Derrimut, Australia) producing photosynthetically active radiation at a plant height of 720–750 µmol m –2 s –1 . A 12 h photothermal regime was maintained throughout plant growth. General plant management followed the established protocol of the Controlled Environment Facilities at ANU. Eight replicate plants of each genotype were assigned to each night temperature treatment.

Determination of flag leaf photosynthetic heat tolerance

T crit was estimated according to the method of Schreiber and Berry (1977) and recently applied by Zhu et al. (2018) , Arnold et al. (2021) , and Coast et al. (2021b) . A detailed description of the method is given in Coast et al. (2021b) ; briefly, discs excised during the day from the middle section of detached dark-adapted leaves were exposed to a temperature ramp at a constant rate of 1 °C min −1 from 20 °C to 65 °C with simultaneous continuous measurement of F 0 taken. T crit was calculated as the intersection point of two regression lines extrapolated from the flat and steep portion of the F 0 –temperature response curve. T crit was determined for all four genotypes in Experiment I, and for two genotypes (Mace and 1704) in Experiment II.

Gas exchange measurements

Plants in growth chambers were moved into a temperature-controlled cabinet (Thermoline Model‐1175-SD-1SL, Thermoline Scientific, Smithfield, NSW, Australia) for all gas exchange measurements. Gas exchange measurements were conducted on intact flag leaves of the main tiller identified as the first tiller to reach anthesis. Five LI-COR portable photosynthesis systems (LI-6400XT, LI-COR Inc., Lincoln, NE, USA) were used for gas exchange measurements. The LI-COR units were fitted with 6 cm 2 leaf chambers with a red–blue light source (6400-18 RGB Light Source, LI-COR). Leaves were exposed to saturating irradiance of 1500 μmol photons m −2 s −1 within the LI-COR leaf chamber, with both the LI-COR leaf chamber/block and the whole plant placed within the temperature-controlled cabinet. The LI-COR leaf chamber was initially set to 20 °C, reference line atmospheric [CO 2 ] of 400 ppm, a flow rate of 500 μmol s −1 , and relative humidity maintained between 40% and 75%. After flag leaves had been exposed to these conditions in the leaf chamber for at least 5 min and following equilibrium (stable readings for at least 1 min), A n was determined. Thereafter photosynthetic [CO 2 ] response curves ( A : C i curves) were generated, at a constant irradiance of 1500 μmol photons m −2 s −1 , by varying the [CO 2 ] inside the LI-COR leaf chambers as follows: 30, 50, 100, 150, 250, 400, 400, 600, 800, 1000, 1200, 1400, and 400 μmol mol −1 . The A : C i curves were repeated with the leaves exposed to measurement temperatures of 25, 30, 35, 40, and 50 °C. The temperature setting of the cabinet was adjusted to enable the LI-COR leaf chamber/block to achieve the desired measurement temperatures. Plants were kept well irrigated throughout the measurement period to avoid water stress. In both Experiment I and II, four replicate plants per night temperature treatment were used. All gas measurements were taken between 08.00 h and 17.00 h during weeks 9–13 for Experiment I or days 5–7 for Experiment II. The ranges (9–13 weeks, and 5–7 d) in period reflect differences in time to anthesis of the different genotypes.

Modelling photosynthetic capacity

Model parameters for each growth and measurement temperature were estimated following the FvCB model and using the Plantecowrap package ( Stinziano et al. , 2018 ) in the R computing environment ( R Development Core Team, 2021 ). Plantecowrap eliminates potential bias associated with manually determining the concentration of intercellular CO 2 ( C i ) where one limitation transitions to another, as is the case with the commonly used Sharkey Excel spreadsheet ( Sharkey et al. , 2007 ), and allows for species-specific kinetic parameters to be pre-defined. The kinetic parameters used in modelling photosynthetic capacity for wheat were: mesophyll conductance at 25 °C ( g m =5.5 μmol m −2 s −1 Pa –1 ); activation energy of mesophyll conductance ( E a =47.65 kJ mol –1 ); apparent Michaelis–Menten constant for Rubisco carboxylation in 21% oxygen ( K air =772 µmol mol –1 ); activation energy of K c (93.72 kJ mol –1 ); photorespiratory CO 2 compensation point or Gamma star at 25 °C (Γ*=37.74 µmol mol –1 , equivalent to µbar bar –1 ); and Gamma star activation energy (24.42 kJ mol –1 ). The temperature responses of V cmax , J 1500 , and TPU were modelled using non-linear least squares fit of the Arrhenius temperature response function accounting for deactivation ( Medlyn et al. , 2002 ; Kattge and Knorr, 2007 ). The deactivation energy ( E d ) was assumed to be 200 kJ mol –1 ; the activation energy ( E a ) and entropy factor (ΔS) were estimated from iterative fits of the model.

Gas chromatography–mass spectrometry metabolite analysis

All leaf samples used for metabolite analysis were collected during the day (between 08.00 h and 17.00 h) and within an hour of completing gas exchange measurements. Metabolite extraction was conducted using a GC-MS procedure described in Rashid et al. (2021) . Derivatization was performed using the MPS2 XL-Twister autosampler (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany), and metabolite samples were analysed on an Agilent GC/MSD system composed of an Agilent GC 6890N gas chromatograph (Agilent Technologies) fitted with a 7683B Automatic Liquid Sampler (Agilent Technologies) and 5975B Inert MSD quadrupole MS detector (Agilent Technologies). The gas chromatograph was fitted with a 0.25 mm (i.d.), 0.25 μm film thickness, 30 m Agilent FactorFour VF-5ms capillary column with a 10 m integrated guard column (Agilent Technologies). Raw GC-MS data were converted using the GC/MS Translator (ver 1.0, Agilent Technologies, Inc., Santa Clara, CA, USA) then processed (peak detection, retention time alignment, and relative quantitation) with MS-DIAL (ver 4.60; Tsugawa et al. , 2020 ). Metabolites were identified by comparing mass spectral features against available spectral libraries from the Golm Metabolome Database ( Kopka et al. , 2004 ). Metabolite abundance values were normalized against the averaged signal of an internal standard ([ 13 C 5 ]valine) and the sample fresh mass, followed by weighting against the average measured signal across all samples for each compound.

Statistical analysis

Data preparation, analysis, and visualization were performed with R ( R Development Core Team, 2021 ) using the packages tidyverse ( Wickham et al. , 2019 ), tidyr ( Wickham, 2021 ), dplyr ( Wickham et al. , 2021 ), ggplot2 ( Wickham, 2016 ), FactoMineR ( Lê et al. , 2008 ), factoextra ( Kassambara and Mundt, 2020 ), ade4 ( Chessel et al. , 2004 ; Dray and Dufour, 2007 ; Dray et al. , 2007 ; Bougeard and Dray, 2018 ; Thioulouse et al. , 2018 ), nls2 ( Grothendieck, 2013 ), minpack.lm ( Elzhov et al. , 2016 ), vegan ( Oksanen et al. , 2020 ), lattice ( Sarkar, 2008 ), latticeExtra ( Sarkar and Andrews, 2022 ), car ( Fox and Weisberg, 2019 ), AICcmodavg ( Mazerolle, 2020 ), and agricolae ( de Mendiburu, 2021 ).

Experiments I and II were analysed separately. Gas exchange data were checked for outliers prior to analysis. Differences in treatment means for T crit , gas exchange parameters ( A max and g s ), and modelled photosynthetic capacities ( V cmax and J 1500 at a standardized temperature of 25 °C; T opt of A n , V cmax , J 1500 , and TPU; and V cmax , J 1500 , and TPU at T opt ) were tested using ANOVA and treatments separated using Fisher’s LSD or Tukey’s HSD. Comparative regressions were used to test the effects of night temperature and genotype on the temperature response curves of A n , V c , J , and TPU. The regression models tested fits of a common line (the simplest model) and separate lines (the advanced model) for the genotype and night temperature. The simplest model assumed a common intercept and curvature, whereas the advanced model assumed differences in intercept or curvature, or both. F -tests were used to select the model with the best fit. The A max (i.e. A n at T opt ) and T opt of A n were derived from equations of the second-order polynomials that best described the instantaneous A n –temperature relationships. Metabolite abundance data were analysed using principal component analysis (PCA) and a non-parametric, permutation tests-based, multivariate ANOVA ( Anderson, 2001 ). This form of analysis generates test statistics analogous to Fisher’s F -ratio, and P -values are obtained using permutations.

T crit of newly developed leaves (i.e. leaves that developed entirely under conditions where the prevailing night temperature was elevated) varied between genotypes ( P <0.001) but did not significantly respond to night warming ( Fig. 1A ). Across night temperatures, mean T crit of the heat-susceptible genotype 1704 (45.7 °C) was higher ( P <0.05) than the T crit of Mace (43.8 °C) and the elite genotype 2254 (44.6 °C), but not of genotype 1898 (45.2 °C, P =0.503). In pre-existing leaves (i.e. leaves that had developed under controlled-environment conditions and then exposed to 5–7 consecutive warm nights), responses of both genotypes to night warming differed: T crit of Mace increased with warm nights whereas T crit of 1704 did not significantly change, resulting in a significant genotype by night temperature interaction ( P =0.008, Fig. 1B ). Across night temperatures, Mace and 1704 differed in T crit ( P =0.015, for main effect of genotype). However, the effect of night temperature alone was not significant ( P =0.107).

Photosynthetic high temperature tolerance ( T crit ) of newly developed leaves of four wheat genotypes (top panel) and pre-existing leaves of two wheat genotypes (bottom panel) at night temperatures of 15, 20, and 25 °C. The four genotypes were the heat-susceptible 1704 and 1898, and the heat-tolerant Mace and 2254. Means are of 3–8 plants.

When measured at ambient CO 2 , instantaneous temperature responses of light-saturated rates of A n in newly developed wheat leaves responded to night warming ( Fig. 2A ; Table 1 ). The responses were characterized by maintaining or increasing the maximum rate of A n (i.e. A max at T opt ) or the optimum temperature of A n ( T opt of A n ). The largest increase with night warming from 15 °C to 25 °C in A max at T opt and T opt of A n were in genotype 2254 and 1898, respectively. However, for genotype 1704, estimates of A max at T opt and T opt of A n for plants grown at 20 °C and 25 °C nights could not be determined due to the linear declines in A n within the limited measurement temperature range studied. For genotype 1704 alone, comparison of regressions for the different night temperatures showed variation [ F =65.07 (4, 68) , P <0.001, and R 2 =0.79], but this was due to a lower A n –temperature response at 20 °C compared with both 15 °C and 25 °C nights. Stomatal conductance ( g s ) also responded to night warming, and was generally curvilinear with increasing measurement temperature ( Fig. 2B ). There was a clear interaction effect of genotype by night temperature on g s , with lower g s for genotypes 1704 and 1898 under warmer nights, and the opposite for Mace and genotype 2254. The short-term leaf temperature response of g s and A n were curvilinear. Stomatal conductance was marked by an initial decline as temperature increased from 20 °C to ~30 °C, followed by an uptick at higher temperatures. There were minimal changes in photosynthesis for leaves that pre-existed when night temperatures were altered ( Fig. 2C ).

Estimated optimum temperature ( T opt ) of light-saturated net assimilation at ambient CO 2 ( A n ) in leaves of four wheat genotypes, and A n at T opt (i.e. maximum A n or A max ) derived from regression models of instantaneous temperature responses of A n

a The heat-susceptible genotypes are 1704 and 1898, and the heat-tolerant genotypes are 2254 and Mace.

b The final models for newly developed leaves of each genotype were based on separate regression fits for the three different night temperatures, whereas for pre-existing leaves models were common regressions across the night temperature. --- The best fit was linear, T opt could not be determined within the limits of temperature range tested.

At ambient CO 2 : instantaneous temperature response curves of light-saturated net assimilation ( A n ; top panels) and stomatal conductance ( g s ; bottom panels) in wheat flag leaves that developed (newly developed) at a day temperature of 20 °C and night temperatures of 15, 20, or 25 °C. Individual panels are presented for the heat-susceptible genotypes 1704 (A) and 1898 (B), and the heat-tolerant genotypes 2254 (C) and Mace (D). For pre-existing leaves that only experienced night warming after spikes emerged and flowered, the instantaneous A n –temperature responses were unaffected by night warming ( Supplementary Fig. S1 ). Lines are polynomial regression curves fitted to the data. The shaded regions indicate confidence intervals for the fitted polynomial curves. Means are of 2–4 plants.

V cmax of newly developed leaves increased exponentially with rising leaf temperatures before peaking then declining at measurement temperatures above 36–47 °C for V cmax and 29–34 °C for J 1500 ( Fig. 3 ; Table 2 ), which enabled a classical Arrhenius temperature response with a deactivation component to be fit. T opt of V cmax was unresponsive to night warming ( P =0.112, Table 2 ) but modelled rates of V cmax at T opt increased significantly, between 24% and 43% ( Fig. 3 ; Table 2 ). Below leaf temperatures of 36 °C, V cmax and its response to temperature were similar between the genotypes and not affected by night temperature. Thus, the impact of night warming was most evident as an increase in Rubisco carboxylation capacity of leaves developed under the warmest nights.

Mean estimates (±SD) of the optimum temperature ( T opt ) and maximum capacity of V cmax , J 1500 , and TPU in newly developed leaves of four wheat genotypes at three different night temperatures

a The heat-susceptible genotypes are 1704 and 1898, and the heat-tolerant genotypes are 2254 and Mace. --- The model fit could not be resolved. Highlighted in bold are P -values <0.05.

Temperature response curves of the maximum CO 2 carboxylation capacity ( V cmax ), photosynthetic electron transport capacity ( J 1500 ), and triphosphate utilization (TPU) of leaves that developed (newly developed) at a common day temperature of 20 °C and night temperatures of 15, 20, or 25 °C for four wheat genotypes. The genotype name/ID are indicated in the panels for both the heat-susceptible genotypes 1704 (A) and 1898 (B), and the heat-tolerant genotypes 2254 (C) and Mace (D). V cmax was iteratively fit with an Arrhenius equation with parameters given in Table 2 . J 1500 and TPU were fit with quadratic functions. Plots for pre-existing leaves of Mace and 1704 are presented in Supplementary Figure S2 . Means are of 3–4 plants. Error bars represent the standard error of the mean.

J 1500 and TPU displayed a curvilinear response to leaf temperature ( Fig. 3E , F ). The genotype by night temperature effect on J 1500 was significant ( P =0.047, Table 2 ). This was due in part to J 1500 decreasing with night warming for genotypes 1704 and 1898 but not being detrimentally affected by night warming in Mace and genotype 2254. J 1500 at T opt for Mace and 2254 increased by 5–30% with warmer nights ( Table 2 ). By contrast, night warming reduced J 1500 in 1704 and 1898 by 7–28% ( Table 2 ). TPU was largely unresponsive to night warming and did not differ between genotypes. Like T opt of V cmax , T opt of J 1500 and TPU were not significantly changed under warmer nights. However, T opt values of photosynthetic capacity were high for V cmax (range 37–47 °C) relative to J 1500 (range 29–34 °C) and least for TPU (range 11–16 °C). As with net assimilation rates, photosynthetic capacity in terms of V cmax , J 1500 , and TPU were not influenced by night warming in pre-existing leaves of both genotypes ( P >0.05, Supplementary Fig. S1 ; Supplementary Table S1 ).

To determine whether light-saturated A n becomes RuBP limited in newly developed leaves, as suggested by the decline in J 1500 , A n was plotted against C i for the different night temperatures. The curvilinear responses were fitted using the modelled carboxylation and RuBP regeneration limitation rates ( Fig. 4 ). At C i corresponding to an ambient atmospheric CO 2 concentration of 400 µmol mol –1 , light-saturated A n was reduced in 1704 and 1898 in response to a greater RuBP regeneration limitation at 20 °C and 25 °C nights (see black outlined boxes in Fig. 4 ). Genotypes 2254 and Mace either maintained or increased their RuBP regeneration capacity with night warming and consequently A n at 400 µmol mol –1 across night treatments.

A – C i curves and corresponding C 3 photosynthesis model fits for newly developed leaves of wheat genotypes 1704 (A, heat-susceptible), 1898 (B, heat-susceptible), 2254 (C, heat-tolerant), and Mace (D, heat-tolerant). Plants were treated to a common day temperature of 20 °C and night temperatures of 15 (purple lines and shapes), 20 (green lines and shapes), or 25 °C (yellow lines and shapes). Solid, dashed, and dotted curves represent predicted carboxylation-limited A n rates, RuBP regeneration-limited A n rates, and triphosphate utilization- (TPU) limited A n rates, respectively. The black outlined box encapsulates the values corresponding to ambient CO 2 concentration of 400 μmol mol –1 . Values are the means of pooled measurements at leaf temperatures of 25 °C and 30 °C and model parameters standardized to 28 °C. Means are of 4–6 plants.

Across the four genotypes and night temperatures of Experiment I, a total of 162 metabolites were identified in newly developed leaves. PCA determined relationships among individual metabolites and across treatments. Metabolites were assigned to classes as listed in Fig. 5 (metabolite relative abundance and classification are presented in Supplementary Table S2 ). The response of metabolites generally followed a similar pattern for a particular class, and often a class responded independently of other classes. Most notably, organic acids and amino acids clustered in the right half of the PCA (positive Dim1), while many carbohydrates clustered in the opposite left half. Thus, organic and amino acid abundance were in general positively related to one another and negatively correlated with many carbohydrates. In terms of night treatment, there was a shift to a more distinct separation of metabolite abundance in genotypes that were more physiologically responsive and tolerant to the warmer nights ( Fig. 5 ). For example, the most sensitive and non-responsive genotype 1704 had little separation of metabolite profiles among night temperatures—evident in overlapping night temperature-dependent metabolite distribution clouds in Fig. 5 . By contrast, the two most heat-responsive and tolerant genotypes (Mace and 2254) had a distinct special separation of the 25 °C night treatment metabolites, represented by special separation at negative Dim1 and Dim2 values (i.e. the lower left corner of PCA graphs). Genotype 1898 had intermediate separation of metabolite profiles in response to night warming, shifting to negative Dim1 values. Interestingly, many of the same metabolites positively correlated with warmer nights for the three genotypes (1898, 2254, and Mace) that responded to warmer nights ( Fig. 6 ). Of these metabolites that responded positively, most were monosaccharides, namely 3,6-anhydro- d -hexose, fructose, mannose, mannose-6-phosphate, and tagatose (metabolites 14, 55, 113, 114, and 149, respectively). The other metabolites that consistently responded to the warmest night of 25 °C were the saturated 18:0 and 16:0 fatty acids octadecanoic acid and palmitic acid (metabolites 124 and 129).

Principal component analysis (PCA) decomposition of metabolite responses in newly developed leaves of wheat genotypes to three different night temperature. The genotype name/ID are indicated in the panels for both the heat-susceptible genotypes 1704 and 1898, and the heat-tolerant genotypes 2254 and Mace. Metabolites are colour-coded by functional class. The direction and length of vector arrows provide information on the contribution each metabolite makes to dimensions 1 (Dim 1) and 2 (Dim 2), and the inter-relatedness of metabolites among themselves (opposing directions represent strong negative correlations while arrows of similar direction represent positive correlations), as well as in relation to night temperatures (the latter represented by blue, green, and red shaded areas). Arrow numbers correspond to individual metabolites which are listed in Supplementary Table S1 , along with their relative abundance.

Individual metabolites that consistently responded positively to the warmest night of 25 °C in the genotypes 1898 (open circles; heat-susceptible), 2254 (open squares; heat-tolerant), and Mace (open triangles; heat tolerant). Metabolites presented are monosaccharides (A–D) and fatty acids (E, F). The genotype 1704 was excluded from this analysis as its metabolite profile showed no significant response to night temperature, evident in the PCA plots presented in Fig. 5 . The metabolite numbers refer to their corresponding labels in the PCA plots.

In this study, we showed that wheat photosynthesis and acclimation capacity are directly affected by night warming, with varying genotypic responses and differences between newly developed and pre-existing leaves. For most genotypes, metabolites for monosaccharides and saturated fatty acids had the strongest positive correlation to warmer nights. Osmotic regulation and changes in fatty acid saturation with warming, which is consistent with changing membrane structural integrity ( Rudolph and Goins, 1991 ; Harishchandra et al. , 2010 ; Marček et al. , 2019 ; Siddiqui et al. , 2020 ), may be linked to the heat tolerance potential of wheat to night warming. Difference between newly developed and pre-existing leaves were consistent with previous physiological reports of plants demonstrating a far greater acclimation potential in tissue that developed under a particular thermal regime rather than simply experiencing thermal change post-development ( Armstrong et al. , 2006 ; Campbell et al. , 2007 ; Rashid et al. , 2020 ). However, we note that our results are based on a small number of genotypes, with a focus on photosynthesis-related traits measured at a single growth stage (anthesis) in controlled-environment conditions. Thus, possible conclusions about mechanisms of high night temperature tolerance are limited and deserve further attention in future studies.

Heat-tolerant wheat genotypes can sustain V cmax at higher leaf temperatures when grown at warmer nights

Two heat-tolerant genotypes (Mace, a locally adapted commercial cultivar; and 2254, an elite heat-tolerant genotype) were able to either maintain or increase CO 2 fixing capacity at hotter leaf temperatures in response to warmer nights ( Fig. 3 ). This was most evident in the significantly greater V cmax reached at its T opt when grown under warmer nights ( Table 2 ; Fig. 3 ). An increase in photosynthetic capacity through an increase in V cmax and its T opt , in response to both day and night warming, is evident across a range of plant species ( Kattge and Knorr, 2007 ; Smith and Dukes, 2017 ). The fact that we observed these acclimation responses in wheat genotypes due to increases in night temperature alone is interesting considering that the night temperature was not expected to have a direct influence on photosynthesis due to the temporal separation of the night warming from photosynthesis in C 3 species. Night-time respiration is more likely to be a major contributor to acclimation than photosynthesis. Although we have focused on photosynthesis and not respiration, we expect that night warming will alter the ratio of dark respiration to A n , similar to previous studies ( Turnbull et al. , 2002 ; Bahuguna et al. , 2017 ; Impa et al. , 2019 ).

The stable to higher V cmax at hotter leaf temperature in 2254 and Mace with warming nights was probably a result of more active Rubisco rather than a change in its abundance or kinetics ( Scafaro et al. , 2023 ). It is well established, including in wheat, that a proportion of Rubisco is inactive, and this inactivity increases with leaf temperature ( Law and Crafts-Brandner, 1999 ; Sharwood et al. , 2016 ; Perdomo et al. , 2017 ). The decline in V cmax at the hotter leaf temperatures is consistent with Rubisco becoming inactive. Declines in Rubisco activity with heat are not due to Rubisco per se which is a thermally stable protein ( Salvucci et al. , 2001 ), but rather due to the heat-labile nature of Rubisco activase (Rca), its accessory protein ( Salvucci and Crafts-Brandner, 2004 ). The stable to higher V cmax of the heat-tolerant genotypes at hot leaf temperatures—and only when grown at warmer nights—suggests that night warming is inducing changes in Rca and its control over Rubisco. Recently, a thermally stable isoform of Rca (Rca1β) was identified in wheat, and its expression and abundance increase with exposure of wheat to heat stress ( Scafaro et al. , 2019 ; Degen et al. , 2021 ; Perdomo et al. , 2021 ). Night warming-induced expression of Rca1β in 2254 and Mace but not in 1704 and 1898 would confirm this hypothesis.

The shift in V cmax with night temperature could potentially be due to temperature-dependent shifts in the kinetic properties of Rubisco. However, there are limited indications that Rubisco kinetics may alter in response to growth conditions. One example is spinach ( Spinacia oleracea ), when grown at the hotter day/night temperature of 30/25 °C compared with 15/10 °C ( Yamori et al. , 2006 ). A small but significant increase in the thermal stability of spinach Rubisco implied acclimation-induced alterations in Rubisco functionality. A wider study, which included wheat, demonstrated that Rubisco properties such as its CO 2 substrate affinity can differ depending on growth temperature ( Orr et al. , 2016 ). More studies are needed to conclusively determine if Rubisco isoforms and associated kinetics within a species can shift in response to growth temperature. What is most unlikely is that night warming increased Rubisco abundance, as this would manifest as an increase in V cmax across all measured leaf temperatures, but night warming did not influence V cmax at leaf temperatures below 36 °C ( Fig. 3 ). Furthermore, growth of wheat under both hotter days and nights does not appear to change Rubisco abundance ( Perdomo et al. , 2017 ).

CO 2 conductance and electron transport limit photosynthesis in the heat-susceptible genotypes with night warming

Newly developed leaves of the genotype 1898 and the heat-susceptible genotype 1704 (known to be susceptible in terms of yield and growth in the field under hotter growth conditions) exhibited reduced photosynthesis rates when night temperature was warmer than 15 °C. The detrimental effects of night warming on the photosynthetic activity could be attributed to a decline in CO 2 substrate availability, electron transport capacity, and TPU under the warmer nights ( Figs 2 – 4 ). Declines in g s with night warming are associated with a decline in the C i / C a ratio. A decline in C i / C a (as a result of decreased g s ) without an increase in A n is a strong indication of water stress and CO 2 substrate limitations on photosynthetic rates ( Morison and Gifford, 1983 ; Condon et al. , 2004 ). Thus, it is likely that these wheat genotypes had a sensitive response to perceived limitations in water availability triggered by night warming, or adopted reduced g s by increasing abscisic acid in direct response to heat stress ( Rodriguez and Davies, 1982 ; Li et al. , 2020 ). It may also have been due to the link between temperature or VPD and stomatal conductance. For the well-adapted Mace and heat-tolerant elite genotype 2254, the strong coupling of g s with A n ( Lawson et al. , 2011 ), and other mechanisms linked to water status (e.g. increase in xylem and mesophyll hydraulic conductance; Urban et al. , 2017a ), would explain the parallel increases in g s and A n at higher nocturnal temperature. The assumption of a fixed g s – A n relationship is central to many models of stomatal control of photosynthesis at different scales: leaf, plant, ecosystem, and global circulation models ( Farquhar and Wong, 1984 ; Leuning 1995 ; Buckley et al. , 2003 ; Verhoef and Egea, 2014 ). However, this g s – A n relationship can be decoupled under extreme conditions such as heatwaves ( Ameye et al. , 2012 ; von Caemmerer and Evans, 2015 ) or at extremely high leaf temperatures ( Urban et al. , 2017b ).

Night warming above 15 °C reduced the photosynthetic electron capacity and/or the T opt of J 1500 of genotypes 1704 and 1898 ( Fig. 3 ; Table 2 ). This reduction in J 1500 led to a modelled RuBP regeneration limitation in A n at current ambient CO 2 concentrations ( Fig. 4 ). Despite the reductions in J 1500 and/or the T opt of J 1500 with night warming, PSII was found to be thermally stable (higher T crit ) than the heat-tolerant Mace and 2254 ( Fig. 1A ). The mismatch between T crit and J 1500 demonstrates that the upper temperature limit of electron transport is not necessarily aligned with susceptibility of electron transport rates under the prevailing growth temperature. In other words, photosynthesis of 1898 and 1704 may have an ability to withstand a more severe heat shock despite being more susceptible to loss of function under milder but more sustained heating. Genotype 2254 was able to increase J 1500 and had relatively larger increases in T crit with night warming than 1704 and 1898, possibly meaning that the extent of change in T crit is more informative than the upper limit of T crit when assessing the potential for electron capacity to adjust to temperature. Pre-existing leaves of Mace, but not 1704, responded strongly to warmer nights. This suggests a greater role for older leaves in heat stress resilience, even though these leaves are assumed to have less capacity to acclimate to high temperatures.

In contrast to V cmax , the response of J 1500 and TPU to leaf measuring temperatures did not follow an expected exponential rise that could be fit with an Arrhenius equation ( Fig. 3 ). A similar finding has previously been observed in wheat for J ( Silva-Perez et al. , 2017 ). This is likely to be linked to V cmax being driven by the kinetics of Rubisco enzyme and its exponential response to temperature (apart from the aforementioned decline at hotter leaf temperatures due to Rca loss of function). J 1500 is set by more complex protein- and membrane-dependent interactions, some of which may not respond as dynamically to short-term rises in leaf temperature. TPU, on the other hand, largely comes into play at high CO 2 levels, beyond the ambient conditions under which these experiments were conducted.

Monosaccharide and saturated fatty acid accumulation are key responses of metabolite to warm nights

The most striking change in metabolites was a build-up of monosaccharide soluble sugars in newly developed leaves of the more heat-tolerant genotypes when grown at warmer nights. The build-up of monosaccharides may be related to maintenance of the osmotic potential of cells ( Marček et al. , 2019 ) and regulation of stomatal conductance, which is also linked to photosynthesis ( Flütsch et al. , 2020 ). Soluble sugars are potent contributors to cell osmotic potential and regulation ( Moinuddin et al. , 2005 ; Blum, 2017 ). It is noted that osmolyte adjustments contribute to drought and heat stress avoidance in wheat ( Blum and Pnuel, 1990 ). Specific to night warming, the disaccharide trehalose accumulated in response to night warming in wheat spike tissue ( Impa et al. , 2019 ). Thus, the accumulation of soluble sugars in the heat-responsive genotypes but not in 1704 under warmer nights may have contributed to greater osmotic protection during warmer nights. The assumed decline in osmotic potential of the heat-responsive 2254 and Mace leaves may have contributed to its increase in g s with warmer nights, as opposed to 1704 which had a reduction in g s . Solutes, including soluble sugars, have also been shown to provide protection to membrane structural integrity during stress through greater hydration of the lipid surfaces ( Rudolph and Goins, 1991 ; Harishchandra et al. , 2010 ). The higher abundance of simple soluble carbohydrates together with night warming may have contributed to the maintenance of electron transport (maintained and even increased J 1500 ) and ability to acclimate T crit to a greater extent than 1704. In agreement with our postulation that osmolytes are protecting J 1500 in 2254 and Mace but not 1704, supplementing Populus tremula L. leaves with increasing amounts of the monosaccharide sucrose substantially reduced leaf osmotic potential and led to an increase in stability of photosynthetic electron transport at higher T leaf ( Hüve et al. , 2006 ). That we observed a consistent increase in the saturated fatty acids octadecanoic acid and palmitic acid in the more photosynthetically heat-tolerant genotypes with night warming is a further indication of membranes adjusting to be more stable in response to warmer temperatures. Saturation of fatty acids in membranes is known to be a key heat tolerance response of plants ( Larkindale et al. , 2005 ; Zhu et al. , 2018 ). Our results suggest that soluble sugar and saturated fatty acid contents in wheat leaves are key metabolites linking night warming and alterations in day processes. The connection between night temperature, carbohydrate and saturated fatty acid accumulation, and CO 2 conductance and electron transport capacity needs further exploration. Having identified a specific heat tolerance metabolic profile, high-throughput metabolic approaches such as hyperspectral imaging could be used to build models to predict this metabolic profile as has been achieved for amino acids in maize ( Shu et al. , 2022 ). These high-throughput measures could then be deployed by wheat populations through both ground and aerial sensing applications to determine heat tolerance.

Conclusions

Night warming has significant implications on the photosynthetic performance of wheat leaves that have developed under the prevailing night temperature. The heat-tolerant genotype (2254 and Mace) had higher light-saturated A n when grown at warmer nights, in contrast to the other genotypes (1704 and 1898) which had reduced A n . These night temperature-dependent differences in A n between genotypes of wheat could in part be attributed to how night warming influenced photosynthetic capacity and CO 2 conductance. The thermally tolerant genotypes had a more stable V cmax at higher leaf temperatures when grown at warmer nights, which is likely to be due to night-dependent alteration to the temperature sensitivity of the activation state of Rubisco. The thermally susceptible genotypes had reduced g s and J 1500 when grown at warmer nights, which accounts for the corresponding reduction in A n . The stability of electron transport and CO 2 conductance in the heat-tolerant genotypes with night warming may be linked to the greater accumulation of monosaccharides and saturated fatty acids in its leaves, balancing osmotic pressure alterations and cell membrane integrity on exposure to warm nights. The lack of a night warming response in pre-existing leaves, as well as differences between the two wheat genotypes, demonstrates the divergence of strategies needed to improve wheat performance under future climate trends. Improving CO 2 conductance and photosynthetic capacity may be beneficial in some genotypes and potentially achieved through altering monosaccharide and saturated fatty acid contents of cells. In other genotypes with no detrimental impact of night warming on photosynthetic performance (exemplified by the heat-tolerant 2254 and Mace), it might be best to focus on sink tissue limitations and other physiological processes detrimentally affected by heat and not photosynthetic capacity. An important physiological process that clearly deserves investigating is mitochondrial respiration in the dark.

The following supplementary data are available at JXB online.

Fig. S1. Instantaneous temperature response curves of light-saturated net assimilation ( A n ) and stomatal conductance ( g s ) in pre-existing flag leaves of four wheat genotypes at ambient CO 2 .

Fig. S2. Temperature response curves of the maximum CO 2 carboxylation capacity ( V cmax ), photosynthetic electron transport capacity ( J 1500 ), and triphosphate utilization (TPU) of pre-existing wheat leaves at night temperatures of 15, 20, or 25 °C.

Table S1. Mean estimates (±SD) of the optimum temperature ( T opt ) and maximum capacity of V cmax , J 1500 , and TPU in pre-existing leaves of two wheat genotypes at three different night temperatures.

Table S2. Metabolite relative abundance and classification.

We acknowledge and celebrate the First Australians on whose traditional land this research was undertaken, and pay our respect to their elders past, present, and emerging. We thank the ANU Research School of Biology Plant Services team, especially Christine Larsen, Jenny Rath, Gavin Pritchard, and Steven Dempsey, for maintaining the plants in the controlled environments; and Australian Grain Technologies, Narrabri and Professor Richard Trethowan of the University of Sydney, Australia for providing the seeds. The GC-MS analysis for this work was performed by the Centre for Microscopy, Characterisation and Analysis (Metabolomics Australia), UWA and was supported by infrastructure funding from the Western Australian State Government in partnership with the Australian Federal Government, through the National Collaborative Research Infrastructure Strategy (NCRIS).

OC and OKA: conceptualization; OC and APS: data curation, formal analysis, visualization, and writing—original draft: OC, OKA, and HB: funding acquisition; OC: investigation; OC, APS, and NT: methodology; OC, APS, HB, and OKA: project administration; OKA, resources and supervision; OC, APS, HB, NT, and OKA: writing—review and editing.

No conflict of interest declared.

This work was supported by grants from the ARC Centre of Excellence in Plant Energy Biology (CE140100008), the Australian Grains Research and Development Corporation (GRDC) Postdoctoral Fellowship: ‘Photosynthetic acclimation to high temperature in wheat’ (US1904-003RTX–9177346), and project ‘A national approach to improving heat tolerance in wheat through more efficient carbon allocation’ (US00080). OC was supported by Research England’s Expanding Excellence in England (E3)-funded Food and Nutrition Security Initiative of the Natural Resources Institute.

All primary data to support the findings of this study are openly available in the Dryad Digital Repository at doi: 10.5061/dryad.fqz612jx7 ( Coast et al. , 2023 ).

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In Focus: Singapore Climate-related Disclosures and the ESG Landscape in APAC

Rising investor expectations have spurred a global push for publicly listed companies (PLCs) to disclose comprehensive sustainability information. As a result, regulatory bodies worldwide are either mandating or strongly recommending the issuance of sustainability reports by PLCs, focusing on environmental, social, and governance (ESG) factors. The integration of ESG disclosures with financial reporting enables investors…

Mandatory Climate Risk Reporting Australia – What’s on the horizon?

Australian financial regulators have recommended that companies disclose their climate-related financial risks against the framework established by the Task Force on Climate-related Financial Disclosures (TCFD). Currently, a significant proportion of the ASX-200 report voluntarily against this framework, with the number growing steadily year-on-year. Despite Australian businesses generally being proactive in the (voluntary) climate disclosure space…

The Australian Treasury has just released the draft legislative framework that will introduce mandatory requirements for large listed and unlisted entities as well as financial institutions to disclose their climate-related risks and opportunities. In this article we take a closer look at the exposure draft legislation on climate-related financial disclosures. Context – Current climate-related reporting…

Exposure Draft Legislation on Climate-related Financial Disclosures in Australia

The Australian Treasury has just released the draft legislative framework that will introduce mandatory climate-related financial disclosures for large listed and unlisted entities as well as financial institutions to disclose their climate-related risks and opportunities. In this article, our experts take a closer look at the exposure draft legislation on climate-related financial disclosures. Context –…

What Does the Government’s Climate-related Financial Disclosure Consultation Paper Mean for Business?

Since 2023 started with highly anticipated releases of the review into Australia’s carbon market and the proposed Safeguard Mechanism reforms it’s easy to forget that at the end of 2022, the Treasury released its consultation paper into the design of a mandatory climate-related financial disclosure framework. It marked another big shift in Australia’s regulatory environment, with reporting intended to support…

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'Anatomy of a Fall' review: A riveting courtroom drama where there's more than meets the eye

J ustine Triet's thoroughly engaging Anatomy of a Fall examines the way information reveals character, and vice versa, during an unfolding murder trial. Sandra, a German novelist played  by Sandra Hüller, is accused of killing her husband, Samuel (Samuel Theis), at their isolated French cabin. A year later in the courtroom, their relationship is placed under a microscope, carefully scrutinized and picked apart for all the world to see. This includes their blind adolescent son, Daniel (Milo Machado Graner), the only witness to his father's death; Daniel also found Samuel's body after he seemingly fell from a high window, leaving odd blood-spatter patterns nearby and dying on impact.

However, before any of the plot unfolds, Triet and co-writer Arthur Harari lure us in with mementos and pieces of music, including perhaps the most amusing and absurd use of a 50 Cent song in recent memory; an instrumental cover of his upbeat, energetic 2003 single "P.I.M.P" is an important plot point! Alongside the courtroom dialogue, these details play an equally vital part in unearthing the characters' identities, insecurities, and — if such a thing is possible — their truest selves.

The film features riveting performances and courtroom scenes.

In response to the French ministère public and its ruthless public prosecutor (Antoine Reinartz), Sandra is forced to undertake legal strategies that conflict with her own outlook on Samuel. This causes crises of conscience for both her and Daniel, who grows increasingly unsure of what to believe.

When Sandra stands trial, she's forced to speak broken French — her husband's mother tongue but her third language; she occasionally slips into English — which keeps her on the back foot during most of the movie. Her attorney, Vincent (Swann Arlaud), is a personal friend to them both, and so he's left with little choice but to approach Samuel's death as a suicide, since an accidental fall from his attic workshop seems hard to prove to a jury. With her freedom on the line, she has little choice but to go along with this narrative; it leaves a sour taste in her mouth, but it soon helps her see Samuel in a new light as well.

These unexpected layers to each character aren't plot twists per se since they don't follow the typical set-up/pay-off structure, but rather, they're narrative zigzags that help reframe existing events and the way the audience sees the couple's marriage. In fact, all we ever see of Samuel are reconstructions; he isn't presented to us on screen until after he dies, and all we're let in on are other people's memories of him, and Triet's visualization of covert recordings he took of some of his arguments with Sandra. 

With their marriage having been on the rocks for some time, perhaps more than either the audience or Daniel realized, Sandra comes increasingly under attack, as Reinartz slithers his way through each accusatory monologue, fighting a righteous battle that still makes him come off as utterly detestable. It also helps that Hüller, in her role as Sandra, attempts to strike a cautious balance between the character's public and private selves, though she often slips up and cracks under pressure. It's a deeply vulnerable performance, one that doesn't just stew in the grief of a spouse's death and the anxiety of being accused of his murder, but one that makes this an ongoing process, leaving her at the end of her rope. When the character, in a private moment, says, "I'm so tired of crying. It's really ridiculous, it's so exhausting," you fully believe that the movie's premise — to which we've just been introduced — has been chipping away at her for a year.

However, the film's secret weapon is arguably Machado Graner, a child performer who takes on a monumentally mature task, embodying not only the uncontrollable agony of loss but the character's confusion and suspicion surrounding what's left of his family. Nobody wants to believe their mother is a murderer, let alone that their parents' marriage was far from perfect. But Daniel is so desperate to have some kind of solid ground beneath his feet that he's willing to embrace any possibility, even the most monstrous one, if it means having some kind of certainty again. Machado Graner, therefore, grasps at all possible outcomes as soon as they arise, though each new piece of evidence only seems to further confuse Daniel, forcing the young actor into a constant state of searching — that, too, without the use of the character's sight.

Triet keeps the camera transfixed on Daniel during several court scenes, using only his reactions and the dialogue of the other characters just off-screen to guide the movement of her camera, which swoops around him as new information is thrown his way. This departure from the film's otherwise traditional cinematic form not only adds a sense of realism to his experience but disorients the viewer in the process. It also keeps Machado Graner in the frame as much as possible, tethering us to his fragility.

However, while the performances and dialogue help make explicit the various characters' conundrums and internal mindsets, many of the design details, and the implications of certain questions, tend to shift the subtext of the trial itself, which becomes its own fluid character of sorts. The longer it goes on, the more all-encompassing the prosecution gets, targeting not only Sandra and Samuel's marriage but their respective literary careers, their relationship with their son, and even Sandra's femininity and sexuality.

Anatomy of a Fall puts Sandra's femininity on trial.

At the outset, the trial follows the physical evidence of Samuel's death, but when things don't add up conclusively either way, it takes on a more pointed narrative. Sandra, the film casually reveals, is bisexual, something of which her family has always been aware, but it's an element of her background used to poison the court of public opinion against her when questions of her infidelity arise. Earlier in the day of Samuel's death, she's interviewed by a young female reporter, and their interactions — which seem flirtatious, but only casually so — become evidence as well, of implied impropriety and potential arguments between the couple. (Samuel also seemingly sabotages this interview by playing "P.I.M.P." at an unreasonable volume.)

This is the more explicit way Sandra's femininity is put on trial, suggesting a sort of scheming duplicity, though there are some more implicit ways too, which are equally if not more sinister. For one thing, her success is held against her. She thrived at a time when Samuel found himself in professional limbo. This subplot certainly picks up more nuance along the way, revealing Samuel to be a dynamic if troubled character, but it can't help but play at times like the centerpiece of the entire trial, forcing us to read even the prosecutor's viciousness as some form of overcompensation. No statement he makes directly hints at this, but editor Laurent Sénéchal's rhythmic, whip-smart assembly of the courtroom scenes leaves room for the possibility, given where and how his reaction shots seem to manifest.

Despite a completely self-assured performance from Reinartz, the cinematic language on display pokes the tiniest of holes in what seems like a known quantity — a perspective and motivation that feels entirely certain. We know what the prosecutor wants, just as we know what every character wants, but subtle elements of doubt creep their way in from the edges of the screen. Similarly, the way Sandra presents herself (which is to say, the way she is designed and presented) seems poised to introduce similar doubts, though these are likely to be held by the audience rather than any specific character. Her short haircut and gray pantsuits are the antithesis of an archetypical image of Western femininity, the kind that, in a courtroom narrative, might seek to please a more conservative jury.

The film doesn't get into the jurors as individuals, but it becomes hard not to wonder whether someone on the bench might view Sandra more disdainfully for her queerness, or might even subconsciously read her, thanks to her subtly masculine appearance, as someone inherently more capable of brutality. Ultimately, every bit of evidence comes down to this and similar biases, and even as an audience, it's hard not to reckon with some of the notions the film puts forth, whether or not it plants seeds of doubt in the viewers' minds.

Justine Triet's use of sound is sensational.

Anatomy of a Fall doesn't depend on Sandra's guilt or innocence to be effective. In fact, its narrative perspective seems to endorse one version of facts and events early on, while its lingering questions tend to surround the way various interpretations of those facts and events will inevitably impact the case. The film is, in a way, an anthropological guessing game as much as it is a winding drama, with clues in the form of physical details and bits of information that seem to ask, "How does this make Sandra look to the jury? And what about to her son?"

One particularly tongue-in-cheek way it achieves this is through music, and more specifically, through frequent instrumental repetition of 50 Cent's "P.I.M.P." The first time it shows up, it's an annoyance, or perhaps even an instigator, designed to interrupt or sabotage. The song choice doesn't really matter at first, when Samuel plays it in the opening scene during Sandra's interview — it's an absurd happenstance that plays subtly on early-2000s nostalgia — but when the track becomes a piece of evidence in the case, all its potential meanings must be parsed by necessity, in case some sort of insight can be gleamed, no matter how minor.

At first, doing so seems like an exercise in absurdity, but meeting the movie on its own terms seems to open up a whole world of possible interpretations, no matter how bizarre. There is, as the prosecutor notes, the song's sexually provocative nature, which immediately reframes her interview in a specific context that may not have previously existed, and there are numerous such possible readings that could shed new light on the case while being equally ridiculous.

Do the song's absent lyrics , like "I don't know what you heard about me" or "But a bitch can't get a dollar out of me," inadvertently inform the movie's themes of innuendo and speculation, or its suspicions surrounding Samuel's life insurance? Do its Caribbean steel drums — an immediate contrast with Sandra and Samuel's frigid surroundings — speak to some kind of desire to escape? And is the title of the album it belongs to, Get Rich or Die Tryin' , just an unfortunate irony given Samuel's dwindling career, or does it, too, gesture at some sort of cryptic intent on his behalf? All of this feels like too much inquiry into a single, amusing detail, but it's a detail that recurs constantly, in a film whose plot is overflowing with characters analyzing any and every possibility if it means coming a step closer to some kind of truth, whether legal or spiritual. 

Perhaps the meaning behind the song's use is as unknowable as the question of whether it's something we ought to be looking at in the first place. The only way to make sense of all the contradictory evidence is to accept this contradiction because otherwise, its unknowability can drive one mad — as it nearly does Daniel, a young character desperate for the reprieve of a familiar pattern that makes sense. As much as Sandra's world is turned upside down, his is thrown into similar disarray, as evidenced by another musical cue he tries to recall on the piano — Isaac Albéniz's entrancing Asturias (Leyenda) — but fails to play completely, as if some of its notes have grown just beyond his reach.

It's an external arrhythmia that represents Daniel's innermost thoughts, which he can barely form or share with those around him. He hasn't yet learned to parse information the way the adults around him have, but watching him slowly learn to do so is among the most gripping parts of the Anatomy of a Fall — alongside the growing possibility that he might come to conclusions that damage his relationship with Sandra. She knows this too, and as she glances over to him constantly during the trial, the question of what will become of mother and son becomes just as pressing and emotionally intriguing as who killed the father.

Anatomy of a Fall is now streaming on Hulu.

UPDATE: Mar. 21, 2024, 6:04 PM EDT Anatomy of a Fall was reviewed out of Cannes, where it won the Palme d'Or. The film has since won the Academy Award for Best Original Screenplay. This review has been republished to celebrate its debut on streaming.

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The Drake/Kendrick Lamar Beef Has a Winner. Where Do We Go From Here?

By Lawrence Burney

This past weekend, after a month of flirting with open warfare, the battle Drake and Kendrick Lamar finally tipped over into three days of exhilarating back-and-forth, and we’re still feeling the aftershocks. 2024’s biggest rap beef reached its presumed conclusion Sunday night with Drake’s despondent “The Heart Part 6,” but the online world hasn’t stopped reacting. The West Coast is treating “Not Like Us” like a triumphant battle cry . Educators are dissecting the lyrical complexities of the disses. Superfans of both artists appear to be spiraling.

Kendrick and Drake are the two most prominent rap superstars of the millennial generation, and over the years they’ve been framed as each other’s antithesis— Kendrick, the tastefully restrained storyteller who pops out every few years when he really has something on his heart to share, versus Drake, the decadent, ever-present workaholic who’s never let more than a few months of his 17-year career pass without releasing new material. Their struggle to upstage each other—culminating in the release of six tracks between them just in the last week alone— offered no shortage of spectacle. Drake used A.I. to troll Kendrick in the voices of his West Coast heroes to strike a nerve on “Taylor Made Freestyle”; Kendrick responded, on “Euphoria,” by paraphrasing the late DMX’s anti-Drake sentiments , drawing blood in a lower-tech way. “6:16” samples “What A Wonderful Thing Love Is,” a 1972 Al Green song that features Drake’s uncle Teenie Hodges on guitar; Drake promoted “Family Matters” on his Instagram by sharing “Buried Alive Interlude, Pt. 2,” named for a song from his 2011 Take Care album that featured Kendrick back when things were copacetic. Both rappers also swiped each other’s trademarks: Kendrick’s “6:16 in LA” played on Drake’s timestamp-in-a-specific-city song-titling convention, while Drake pulled a Young Thug Barter 6 -esque move by naming his final entry “The Heart Part 6,” after Kendrick’s career-spanning series of the same name.

“Not Like Us,” meanwhile, has been read as a message to Drake fans who claim Kendrick isn’t capable of making club-ready anthems. But if you ask the right self-appointed internet rap analyst, every move that’s been made in this conflict has had a quadruple meaning. What we can say for sure: Regardless of their rivalry, these are men who’ve been acutely dialed into each other’s careers from the very beginning, which meant this standoff was always destined to be about more than deciding who was the best at rapping. It was destined to become what it became—a high-stakes battle to see who could obliterate whose integrity first.

Truth be told, the apparent end of the beef feels like the closing of a chapter in hip-hop’s story, where the teachings of the old guard met the interconnectedness of the new age. When’s the last time rappers born in the 80’s commanded this much of our attention? Save for Drake, none of the people who came in during the blog era’s latter years still mean something to any generation other than their own. Drake and Kendrick’s places within the genre’s pantheon of legends are pretty much established already. Anything that we get from them now is ornamental, which is why the sheer effort they both put in over the last few weeks has been something to marvel at. It also probably represents the end of an era: There’s so little material return for Gen Z’s biggest rappers to go toe-to-toe with one another outside of trading insults on livestreams and in interviews that this will likely not happen again. Not like this. This is for people who spent their childhoods watching rap superstars freestyle at radio stations and on BET’s after school television lineup in the early-to-mid 2000s. People who remember a time—even if it was their early childhood— when an MC was only as good as their staunchest competition, and calling yourself a rapper meant being ready to prove your superiority at any second.

In that respect, the feud has been a joy to witness. But it’s also been a reminder that, at its worst, hip-hop incentivizes the infantilization of grown men who need to be able to hold the attention of people in their teens and early 20s to remain powerful. In their late 30s, these brothers should be receptive and responsible enough to refrain from dangling abuse of women and children as a means to demonstrate dominance. That kind of decency is apparently too much to ask of men fighting desperately for a generational crown. And the consequences of those failures are already revealing themselves.

Before Drake’s “Family Matters,” he and Kendrick’s insults for one another drew on career and character deficiencies. In his “Like That” verse, K Dot shot down the notion of him being part of a three-man generational upper crust that also included J. Cole and Drake. In response, Drake came back with “Push Ups,” where he suggests Kendrick’s been getting screwed over by his former label boss Anthony “Top Dog” Tiffith and actually doesn’t even measure up to the likes of Travis Scott , SZA, or 21 Savage. On “Euphoria” Kendrick centers Drake’s obsession with fame and questions his commitment as a father— somewhat of a low blow, yet still within bounds of not mentioning anyone who wasn’t in the feud. “Family Matters” took a turn for the worst when Drake, in an attempt to quell attacks on his cultural and fatherly credibility, shifted the focus onto K Dot’s partner Whitney Alford , alleging that she’s suffered physical abuse at the Compton rapper’s hands and suggesting that one of her children with Kendrick is actually the child of his business partner Dave Free.

All hell broke loose from there. Each song that followed, from both parties, surfaced more rumors of domestic abuse, sexual assault, pedophilia, and hidden children, none of which has been substantiated. Now, as the dust is settling on the battlefield, Kendrick’s “Not Like Us,” which explicitly calls Drake and his camp “certified pedophiles,” is on track to debut at No.1 on the Billboard Hot 100, while “Family Matters” will likely not be far behind. What does it say about the state of hip-hop that an intoxicatingly catchy song that weaponizes child abuse is trending on TikTok where young people are targeted, being played at stadiums for pro sports teams, and shaping up to be one of the summer’s biggest hits?

For one, it says Kendrick and Drake are each, in their own way, determined if not desperate to fortify their respective legacies before it’s too late. In the next few years, they’ll both be 40, their tenure as pop-culture priorities behind them. You get the sense that their refusal to lose is tied to a midlife-crisis-informed, legacy-driven paranoia; Kendrick just seemed more willing to go the extra mile to secure a victory, regardless of how damaging it could be. Perhaps Drake didn’t anticipate him taking the feud so seriously. He couldn’t have, or he would have been prepared to throw more than I’m richer and more relevant than you bars K Dot’s way when the beef first started. On the other hand, Kendrick’s apparent IRL disdain for Drake seemed to drive him throughout the whole ordeal. He had a counter to everything the Toronto rapper threw at him, including what appeared to be ready-made disses with questionable assertions just in case things escalated to a personal level, as they eventually did. And unfortunately for both parties, even though “The Heart Part 6” feels like the waving of a white flag, the current social media landscape doesn’t allow for clear-cut winners in these kinds of ordeals.

By Abe Beame

By Frazier Tharpe

Drake might lose some credibility in the eyes of hip-hop purists or people that were never comfortable with his dominance in the space, but even days after the showdown, there’s a sizable audience of folks defending him and trying to piece together evidence that will tip the scales back in his favor. In a worst case scenario, he’ll take his lumps, go away for a while to regroup, and return with low-stakes, danceable anthems. In the best case, he’ll come back angrier and with an even huger chip on his shoulder to guide him until his time runs out. But one thing he did succeed in was debunking Kendrick’s claim to the moral high ground as an artist. The heart of Drake’s argument in “Family Matters” is a valid one. He’s saying to Kendrick: You, too, have compromised your integrity to be as successful as you are because that’s what this type of relentless ubiquity — via corporate backing — requires, and your politics, when put under the magnifying glass, are shaky at best .

If anything, Kendrick’s willingness to engage in real mudslinging to come out as the victor confirms that accusation. Maybe that’s what Kenny has been trying to tell us this whole time, that he’ll blow all this shit up (the wholesome framing of his career included) before he’ll let a privileged Canadian ride off into the sunset unscathed. Every move Kendrick made was designed to strip Drake down to an insincere and insecure dweeb with no real foundation to stand on outside of an outsized ego. It’s why “Not Like Us” is so potent. Not only does it mock his opponent’s fans for only seeing the value in club-ready disses (the pacification he accused him of in the beginning of “Euphoria”), the signature West Coast production doubles down on Kendrick having a homegrown culture to lean on, something he says Drake doesn’t. He’s throwing a celebration for the Toronto native’s demise and everyone’s dancing along to it.

But where does this all leave us as fans? This is the first rounds-long heavyweight rap fight of the social media era and will likely be the last. The constant online chatter, desperate attempts from casuals to seem like insiders, and the tabloid-esque misinformation that made its way into the battle have made it into an intense stan-controlled disaster, draining so much of the fun out within a matter of days. As in all facets of our society, there’s no middle ground anymore. The post-truth algorithm craves conflict and extreme conviction by any means necessary. Just from this beef alone, fictional leaked screenshots of emails from UMG meetings have been circulating, a shooting outside Drake’s house in Toronto is being attributed to Kendrick by YouTube conspiracy theorists thirsty for engagement, and photos of Kendrick’s children are being unearthed and scrutinized to prove skin-tone-related points.

It’s all so very corny. What used to make rap feuds exciting was their insulation from the broader mainstream — hip-hop culture gradually constructed folklore around the content over time, amongst ourselves, while artists threw shots for months or years on end. The rumors that came out of those battles weren’t any less harmful, but at least they could eventually fizzle out. Hip-hop is so commodified now that even diss tracks are eligible for chart placement and fair game for television syncs and brand deals. (Joe Biden's campaign has already appropriated “Euphoria” for a video throwing shots at Donald Trump .) And the victims of alleged abuse who were used as pawns in the feud will now be bombarded with heightened visibility as soon as they open their phones, including children whose brains aren’t capable of processing what they see. History will remember this as one of the most significant battles hip-hop has ever seen, if not the most— but we might regret the precedent it sets sooner than we expect.

Someone go hand him a Grammy right now!

Kendrick and Drake diss each other multiple times in one weekend, A.I. shenanigans, shots fired at and from Future, Metro Boomin, Rick Ross, Weeknd and more in a new chapter in rap geopolitics.

• Open access
• Published: 13 May 2024

Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress

• Yang Cao 1   na1 ,
• Kexin Wang 1   na1 ,
• Fengzhong Lu 1 ,
• Qingqing Yang 1 ,
• Bingliang Liu 2 ,
• Hayderbinkhalid Muhammad 3 ,
• Yingge Wang 1 ,
• Fengling Fu 1 ,
• Wanchen Li 1 &
• Haoqiang Yu 1  

BMC Genomics volume  25 , Article number:  465 ( 2024 ) Cite this article

Metrics details

The early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed.

In this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription.

Conclusions

This study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Peer Review reports

Environmental stimuli, including drought, salinity, and high temperature, have frequently occurred in recent decades and led to yield loss of crops in agricultural production. Drought stress is a main misfortune in these abiotic stressors and will be a more severe challenge and threat to agriculture and humanity by 2050 [ 1 ]. In adaption to drought, plants activate a series of physiological, morphological, and molecular changes [ 1 , 2 , 3 ]. Among them, many transcription factors (TFs) can be dominated by stress to regulate gene expression and coordinate plant antagonism to adverse factors [ 4 , 5 , 6 ].

The early E2 factor (E2F) family proteins are initially discovered as TFs of the E2 gene and play key roles in cell proliferation control in adenoviruses [ 7 ]. E2Fs can be classified into typical and atypical E2Fs according to protein structure. Typically, E2Fs have only one DNA-binding domain (DBD) and form heterodimeric complexes with dimerization proteins (DPs) to bind the promoters of downstream genes, but atypical E2Fs possess duplicated two DBD [ 8 ]. In higher plants, E2Fs are also categorized into three subclades, including E2F, DP, and DEL (DP-E2F-like), due to the difference in the composition of conserved domains [ 9 , 10 ]. Over the past decade, E2Fs have well been revealed to play significant roles in the cell cycle and DNA damage repair [ 11 , 12 , 13 , 14 , 15 , 16 ]. For instance, ectopic expression of DcE2F1 of Daucus carota promotes cell proliferation in Arabidopsis seedlings [ 14 ]. In Arabidopsis , eight E2F members exhibit antagonistic roles in cell proliferation, such as E2Fa/b, which acts as a positive regulator, but E2Fc is a negative regulator [ 17 , 18 , 19 ]. E2Fa/b can also activate the DNA damage response and cell cycle progression by differentially regulating the expression of genes [ 11 ]. Additionally, AtE2Fa/DPa also inhibits growth, and AtE2Fa/DPa -overexpressing plants show an abnormal phenotype owing to ectopic cell division or enhanced DNA endoreduplication [ 12 , 20 ].

In addition to the crucial role in the cell cycle, few reports show that atypical E2F inhibits the accumulation of salicylic acid to balance growth and defense [ 21 , 22 ]. Furthermore, AtE2Fa acts downstream of ERECTA kinase, which is involved in cell size and stomatal density [ 23 ]. Via expression analysis, it is suggested that TaE2F-DP of wheat, PheE2F/DPs of Moso bamboo, E2F/DP genes of Medicago truncatula , and PvE2F/DPs of Phaseolus vulgaris respond to drought or salt stress, suggesting their potential roles in regulating stress tolerance [ 9 , 24 , 25 , 26 ]. To date, however, the role of E2F in plant stress tolerance remains obscure.

Maize is a crucial crop worldwide and is widely used as food and livestock feed. During its growth, maize plants show sensitivity to water deficit due to its high water demand, leading to maize yield being greatly affected by drought stress [ 27 , 28 , 29 , 30 ]. Therefore, it becomes imperative to identify and explore drought tolerance-related genes that can be used to enhance maize resilience through molecular breeding [ 31 , 32 , 33 , 34 , 35 ]. In our previous study, we found that ZmPP2C26 regulated drought tolerance [ 36 ] and targeted maize ZmE2F (Zm00001d048412, data not shown), indicating that ZmE2F might be involved in drought response. Hence, in this study, we comprehensively investigated ZmE2F genes in the maize genome. Thereafter, phylogenetic relationships, conserved motifs and domains, gene structures and duplication, and protein-protein interaction networks were analyzed. Additionally, the ZmE2F6 (Zm00001d048412) was functionally verified by performing subcellular localization, expression patterns in drought treatment, and ectopic expression in Arabidopsis under drought stress. This study will significantly contribute to a better understanding of E2Fs in stress response.

Identification of ZmE2Fs in the maize genome

The genome and amino acid data of maize B73 were downloaded from the MaizeGDB database ( https://download.maizegdb.org/Zm-B73-REFERENCE-GRAMENE-4.0/ ). Meanwhile, the coding sequences and amino acid sequences of 8 AtE2Fs and 9 OsE2Fs of Arabidopsis and rice were downloaded from the Arabidopsis Information Resource (TAIR) ( https://www.arabidopsis.org/ ) and the Rice Genome Annotation Project (RGAP) database ( http://rice.uga.edu/ ) and were used as queries to perform local BLASTp with an E-value of 1e − 10 in the maize protein database for maize E2F searching, respectively. After removing the redundant sequences manually, the candidate sequences were further analyzed for the presence of the E2F_DP domain (PF02319) by using PFAM ( http://pfam.xfam.org/ ). The candidates possessing the E2F domain were identified as maize ZmE2F members. The secondary structure and physicochemical properties, including molecular weights, isoelectric point (PI), stability coefficient, and grand average of hydropathicity (GRAVY), of ZmE2Fs were analyzed using SOPMA ( https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html ) and EXPASY ( https://www.expasy.org/ ). The subcellular localization of ZmE2Fs was predicted using cNLS Mapper ( https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi ).

Conserved motifs, domains, and phylogenetic analysis

To further identify the conserved motifs and domains, the amino acid sequences of ZmE2Fs were analyzed using MEME ( http://meme-suite.org/tools/meme ) and NCBI-CDD ( https://www.ncbi.nlm.nih.gov/cdd ), respectively. The motif and domain composition of each ZmE2F was visualized by TBtools [ 37 ]. The protein sequences of all ZmE2F, AtE2F, and OsE2F were multiple-aligned using ClustalW with default parameters. The maximum likelihood tree was built with 1000 bootstrap replications by MEGA11 ( https://www.megasoftware.net/ ). Meanwhile, protein-protein interaction (PPI) analysis among ZmE2F members was performed using the STRING tool [ 38 ].

Gene structure, promoter, duplication, and synteny analyses

The chromosomal location of each ZmE2F gene was obtained from the maizeGDB database. The coding sequences and genomic DNA sequences of every ZmE2F were downloaded and used to analyze exon-intron composition using Gene Structure Display Server 2.0 (GSDS) ( http://gsds.gao-lab.org/ ). The 2000 bp upstream sequence of the transcription start site of each ZmE2F gene was retrieved from maizeGDB and used for cis -acting element analysis using PlantCARE ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Meanwhile, the gene duplication events and the synteny relationship between ZmE2F , AtE2F , and OsE2F gene members were analyzed using MCScanX with default parameters. The chromosomal location, duplications, and synteny relationships of the ZmE2F , AtE2F , and OsE2F genes were visualized using TBtools [ 37 ]. The non-synonymous (Ka) and synonymous (Ks) substitution rates per site of the duplicated gene pairs were calculated using TBtools [ 37 ]. Then, the divergence time in millions of years (Mya) was also calculated using the following formula: T = Ks/2λ × 10 − 6 Mya (λ = 6.5 × 10 − 9 for grasses) [ 39 ].

Cloning and subcellular localization of the ZmE2F6 gene

The specific primers (Table S1 ) were designed by Primer 5.0, synthesized at Tsingke Biotech (Beijing, China), and used to amplify the sequence of the ZmE2F6 gene from maize B73 cDNA using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing). After amplification, the PCR product was purified by a gel recovery kit subcloned, and inserted into the pMD19-T vector to generate pMD19-T- ZmE2F6 and verified by sequencing. The sequencing result was aligned with the candidate sequence of the ZmE2F6 gene using DNAMAN. The open reading frame (ORF) sequence of ZmE2F6 without stop codon was amplified from the pMD19-T- ZmE2F6 plasmid using the specific primers designed by CE Design V1.04 (Table S1 ) with the Xba I and Spe I recognition sites. The PCR products and pCAMBIA2300- 35 S-eGFP plasmids were digested using Xba I and Spe I. Subsequently, they were inserted into the Xba I and Spe I sites of pCAMBIA2300- 35 S-eGFP to produce the fusion expression vector 35 S-ZmE2F6-eGFP using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing). Each construct was introduced into Agrobacterium tumefaciens strain GV3101 and then used for transient expression in the leaves of Nicotiana benthamiana . As described by Sun et al. [ 40 ], the constructs were infiltrated into the leaves of five-week-old N. benthamiana . The GFP fluorescence was observed and imaged using a confocal laser scanning microscope (Zeiss 800). The empty vector 35 S-eGFP was served as the positive control.

Plant treatment, RNA extraction, and quantitative real-time PCR (qRT‒PCR) analysis

The seeds of maize B73 lines were soaked in 10% H 2 O 2 for 15 min, rinsed twice using sterile water, soaked in distilled water for 8 h, then wrapped in filter paper and cultured at 28 °C until germination. Subsequently, the seedlings of the same size were transferred to the hydroponic cassette containing hoagland nutrient solution and cultured at 16 h light at 28 °C /8 h dark at 24 °C. Three-leaf-stage seedlings were subjected to 16% PEG-6000 treatment mimicking drought stress, and then shoots containing leaf, stem and leaf sheath, and roots were sampled at 0, 3, 6, 12, and 24 h of treatment, respectively. The total RNA of each sample was extracted using an RNAison plus kit (Takara, Dalian), examined for quality using NanoDrop OneC (ThermoFisher Scientific), treated with DNase to remove DNA contamination, reverse-transcribed into cDNA using a PrimeScript™ RT Regent Kit (Takara, Dalian), and used to perform qRT-PCR. The specific primers of ZmE2F6 were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome ), synthesized at TsingkeBiotech (Beijing, China), and listed in Table S1 . The ZmGAPDH gene was amplified using specific primers (Table S1 ) and used as an internal reference. The qRT-PCR was performed in the Bio-Rad CFX96™ Real-Time PCR system using 2 × Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan). The 20 µL reaction mixture contained 10 µL of 2 × Universal SYBR Green Fast qPCR Mix, 0.4 µL of each forward and reverse primer, 1.0 µL of each cDNA as template, and 8.2 µL of ddH 2 O. The reaction protocol was set as a two-step temperature cycle including 95 °C for 3 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The relative expression level of ZmE2Fs was calculated and normalized using the 2 −ΔΔCt method [ 41 ].

Y2H and GST pull-down analysis

The ORF of ZmE2F6 was amplified using specific primers designed by CE Design V1.04 with the Nde I and EcoR I recognition sites (Table S1 ) and inserted into the pGADT7 plasmid to generate AD -ZmE2F6 as described above. The BD- ZmPP2C26 plasmid was constructed in our previous study [ 36 ]. The AD- ZmE2F6 and BD- ZmPP2C26 plasmids were cotransformed into the yeast strain Y2H Gold using a yeast transformation kit (Coolaber, Beijing). Subsequently, yeast cells were cultured on synthetic dropout (SD) medium without Trp and Leu (SD/-Trp/-Leu) at 30℃ for 2 days, and then positive clones were transferred onto SD/-Trp/-Leu/-His/-Ade plates with X-α-gal and cultured at 30℃ for 2 days. Meanwhile, the ORF of ZmE2F6 was amplified and inserted into the EcoR I and Xho I sites of pGEX-6P-1 to generate GST - ZmE2F6 and used for the GST pull-down assay. The His - ZmPP2C26 plasmids were produced in our previous study. The GST pull-down was performed as described by Lu et al. [ 36 ].

Plant transformation, phenotyping, and RNA-seq

The ORF of ZmE2F6 was amplified without a stop codon and inserted into the Xba I and Nde I sites of pRI201- 35 S - GUS to produce 35 S - ZmE2F6 - GUS as described above. The 35 S - ZmE2F6 - GUS construct was transformed into Agrobacterium tumefaciens strain GV3101 and then used to transform Arabidopsis thaliana (Col-0) by the floral-dip method [ 42 ]. According to the method of Sun et al. [ 40 ], the positive transformants were screened on 1/2 MS plates with 50 mg/L kanamycin, used for harvesting seeds individually. The homozygous lines without segregation on 1/2 MS plates with 50 mg/L kanamycin were screened and used for PCR detection. Meanwhile, the leaves of homozygous lines were sampled and used to perform GUS staining using the GUS Staining Kit (Coolaber, Beijing).

According to the methods described by Sun et al. [ 40 ] with minor modifications, for drought stress, the seeds of homozygous lines and wild type (WT) were surface-sterilized, planted on 1/2 MS plates supplemented with 0 (control), 150, and 250 mM mannitol, vernalized for 2 days in the dark at 4 °C, and vertically cultured in a chamber under 10 h light/14 h dark at 22 °C with 60–70% humidity. At 14 days of treatment, the seedlings were photographed and measured for root length. Moreover, another batch of overexpressed lines and WT were sown in soil and incubated in a greenhouse under the same conditions. After 2 weeks, the seedlings were subjected to withholding water for two weeks, then rewatered for at least 2 days, and monitored for phenotyping. Subsequently, the survival number of each line was counted and used to calculate the survival rate. The leaf of each line was sampled, dried at 80 °C for 3 days, and used for biomass measurement.

Meanwhile, three-week-old seedlings of homozygous lines and WT were sampled and used for RNA sequencing at Sanshubio Company (Jiangsu, China). As described by Sun et al. [ 40 ], the total RNA of each sample was extracted, qualified for quality and integrity, and used to construct a sequencing library. Then, library sequencing was conducted using the NovaSeq 6000 system. The sequencing adapters and low-quality reads of raw data were removed to generate clean data, which were mapped to the Arabidopsis genome by hisat2 [ 43 ] and used for assembling transcripts of every gene using StringTie [ 44 ]. The differentially expressed genes (DEGs) were confirmed with a p-value < 0.05 and |FoldChange| > 2 using DESeq2 [ 45 ]. The GO analysis of DEGs was performed using KOBAS [ 46 ].

Data analysis

All assays were performed with three replicates. The data are shown as the mean values ± standard error (SE). The significance was analyzed by Student’s t-test at the p  < 0.05 or p  < 0.01 level.

ZmE2F members in maize

To identify the maize ZmE2F family, a local BLASTp search against the maize protein database was performed using the amino acid sequences of 8 AtE2F and 9 OsE2F members as query references [ 10 , 47 ]. As shown in Table  1 , a total of 21 ZmE2F members were identified and designated as ZmE2F1 to ZmE2F21 . The CDS length of the ZmE2F genes ranged from 648 ( ZmE2F18 ) to 1608 bp ( ZmE2F14 ), encoding 215 to 535 amino acids with molecular weight varying from 23.39 to 59.21 kDa. The theoretical isoelectric point (pI) of ZmE2F proteins ranged from 4.71 (ZmE2F11) to 9.41 (ZmE2F13), and the grand average hydropathicity (GRAVY) of all ZmE2F proteins was less than 0 and ranged from − 0.062 (ZmE2F1) to -0.603 (ZmE2F5). The instability indices of ZmE2F proteins varied from 31.45 (ZmE2F20) to 62.39 (ZmE2F5), and 19 ZmE2F members contained the highest random coil in their secondary structure, implying that they were unstable hydrophilic proteins. All ZmE2F proteins were predicted to be localized in the nucleus.

Phylogenetic analysis

To investigate the evolutionary relationships of ZmE2Fs and model plants, a total of 38 amino acid sequences, including 21, 8, and 9 E2Fs from maize, Arabidopsis , and rice, respectively, were used to construct a phylogenetic tree (Fig.  1 ). It showed that the ZmE2F proteins could be classified into three subfamilies, including E2F, DP, and DEL clades [ 9 ]. Eight members, including ZmE2F2, 4, 5, 11, 14, 16, 17, and 21, were clustered into the E2F subscale with AtE2Fs and OsE2Fs. Seven proteins containing ZmE2F1, 7, 10, 12, 13, 15, and 20 were branched into the DEL subfamily with AtDELs and OsDELs. The other 6 ZmE2Fs (ZmE2F3, 6, 8, 9, 18, and 19) belonged to the DP subclade with AtDP and OsDP members. In addition, ZmE2Fs showed a closer phylogenetic relationship with OsE2Fs than with AtE2Fs, indicating more sequence similarity with OsE2Fs.

Phylogenetic tree of the E2F family. The E2Fs of Arabidopsis , rice, and maize are marked in blue, red, and black, respectively

Conserved motif and domain

Five conserved motifs were identified in the amino acid sequences of ZmE2Fs using the MEME online program (Fig.  2 A). Among them, motif 1 was highly conserved in all ZmE2F members and characterized by the RRIYD sequence that was a DNA-binding motif followed by dimerization residues DNVLE sequence [ 48 ]. Conserved domain analysis revealed that all ZmE2F members contained at least one DNA-binding domain (DBD) (Pfam ID PF02319, Table S2 ). ZmE2F7, 12, and 13 possessed two DBDs (Fig.  2 B). In addition, ZmE2F5, 6, 9, 18, and 19 also contained a dimerization domain (DD). The coiled coil (CC)-marked box (MB) domain (CC-MB) was found in ZmE2F4, 11, 16, and 17. Furthermore, all ZmE2F members contained a nuclear localization signal.

Conserved motif and domain of ZmE2Fs

ZmE2Fs protein-protein Interaction Prediction

Due to the presence of dimerization residues DNVLE sequence within motif 1 in every ZmE2F and DD or CC-MB in some ZmE2Fs, to explore the potential interactions among ZmE2F members, protein-protein interaction (PPI) analysis was performed. As shown in Fig.  3 , ten ZmE2Fs were predicted to interact with each other, which generated 23 PPI combinations. Among them, ZmE2F6, 9, 18 and 19 had the largest number of PPIs (6 interactions), while ZmE2F4, 5, 11, 14, 16, and 17 had 4 PPIs. These results imply that the DNVLE sequence, DD, and CC-MB play crucial roles during ZmE2F dimerization.

ZmE2F protein-protein interaction prediction

Gene structure, duplication, synteny, and cis -acting elements

Gene structure analysis revealed that the ZmE2F gene family showed diversity in exon and intron composition (Fig.  4 A). The number of exons among ZmE2Fs ranged from six to fourteen. Except for ZmE2F18 and ZmE2F20 , the other 19 ZmE2Fs contained 5ʹ or 3ʹ terminal untranslated regions. The ZmE2Fs were unevenly distributed on eight maize chromosomes, excluding chromosomes 3 and 7. Gene duplication analysis showed that there were eight paralogous pairs of ZmE2Fs in the maize genome, including ZmE2F4 and ZmE2F17 , ZmE2F6 and ZmE2F18 , ZmE2F6 and ZmE2F19 , ZmE2F7 and ZmE2F12 , ZmE2F7 and ZmE2F13 , ZmE2F11 and ZmE2F17 , ZmE2F12 and ZmE2F13 , and ZmE2F18 and ZmE2F19 . Likewise, gene synteny analysis revealed one and twenty-two orthologous pairs between ZmE2F and AtE2F and ZmE2F and OsE2F , respectively (Fig.  4 B; Table S3 ). Meanwhile, the Ka/Ks rates among ZmE2Fs paralogous pairs ranged from 0.19 to 0.35 and their duplication time was estimated to be 16.54–139.30 million years ago (Table S4 ). The results suggest that gene duplication contributed to E2F expansion during the evolutionary process.

Gene structure ( A ) and duplication ( B ) of ZmE2Fs. Yellow and blue boxes represent exons and untranslated regions, respectively. Black lines represent introns. Yellow, blue, and red boxes with a number represent chromosomes of maize, Arabidopsis , and rice, respectively. The green, blue, and red lines indicate duplicated E2F gene pairs among maize, as well as between maize and Arabidopsis and rice, respectively

Furthermore, cis -acting element analysis revealed that abundant stress- or hormone-responsive elements were found in the promoter regions of ZmE2Fs , such as ABREs, MBSs, AREs, and LTR elements (Table S5 ), which were responsive to abscisic acid, drought, anaerobic induction, and low temperature. For instance, ABREs were found in all ZmE2F promoters. MBS elements, the MYB binding site involved in drought response, were found in 15 ZmE2F promoters. This finding implies that the ZmE2F genes may be involved in abiotic stress responses.

ZmE2F6 interacts with ZmPP2C26

In our previous study, ZmE2F6 was identified as a potential target of two ZmPP2C26 splicing variants (ZmPP2C26L/S) via Y2H library screening. Hence, to verify whether ZmE2F6 interacts with ZmPP2C26, Y2H, and GST pull-down assays were performed. As shown in Fig.  5 , the yeast cells transformed with AD- ZmE2F6 and BD- ZmPP2C26L/S exhibited normal growth and turned blue on SD/-Leu/-Trp/-His/-Ade plates containing X-α-gal. Moreover, the GST-ZmE2F6 protein could be pulled down by His-ZmPP2C26 L/S in the GST pull-down assay. These findings confirmed the interaction between ZmE2F6 and ZmPP2C26L/S. Likewise, there were 7 potential phosphorylated sites in ZmE2F6 amino acid sequences (Table S6 ) were predicted using NetPhos-3.1 ( https://services.healthtech.dtu.dk/services/NetPhos-3.1/ ).

ZmE2F6 interacts with ZmPP2C26. ( A ) Yeast two-hybrid (Y2H). ( B ) GST pull-down. ZmPP2C26L and ZmPP2C26S represent two splicing variants of ZmPP2C26. The combination of AD-T with BD-53 and AD-T with BD-lam were used as negative and positive controls, respectively

ZmE2F6 localized to the nucleus

To further validate the cellular localization of the ZmE2F6 protein, the ORF of ZmE2F6 was cloned and inserted into the 35 S - eGFP plasmid to fuse with eGFP . The results of transient expression in tobacco leaves revealed that fluorescent signals were observed in whole cells, including the nucleus and cytoplasm, transformed by the 35 S - eGFP empty vector. However, in tobacco leaves transformed with 35 S - ZmE2F6 - eGFP , fluorescent signals were exclusively detected in the nucleus (Fig.  6 ). This observation is consistent with the bioinformatic prediction, confirming that the ZmE2F6 transcription factor is solely localized in the nucleus.

Subcellular localization of ZmE2Fs

The expression of ZmE2F6 was induced by drought stress

The qRT-PCR results demonstrated that the expression of the ZmE2F6 gene was responsive to drought stress (Fig.  7 ). After drought treatment, the expression of ZmE2F6 in maize shoots was significantly upregulated and reached approximately 11-, 8-, 100-, and 47-fold that of the control at 3, 6, 12, and 24 h of treatment. In roots, the expression of ZmE2F6 was significantly upregulated after 3 h of drought treatment and then downregulated at 6, 12, and 24 h of treatment. These results suggest that ZmE2F6 responds to drought stress.

The relative expression level of the ZmE2F gene under drought stress

Expression of ZmE2F6 enhanced drought tolerance in Arabidopsis thaliana

To assess the function of ZmE2F6 in regulating drought tolerance, we generated transgenic Arabidopsis lines overexpressing ZmE2F6 . In the T 1 generation, ten positive transgenic lines were screened by kanamycin on 1/2 MS plates, identified by PCR, and harvested to produce the next generation. Finally, in the T 3 generation, two homozygous lines (OE6-5 and OE6-10) were selected, identified by GUS staining, and used for phenotyping (Fig.  8 A). It was found that the root length of the OE6-5 and OE6-10 lines was significantly longer than that of the WT under 1/2 MS plates or supplemented with 150 mM mannitol (Fig.  8 B, C). Furthermore, natural drought treatment in soil was performed to monitor the drought tolerance of the OE6-5 and OE6-10 lines. The results showed that there was no difference between the transgenic lines and WT before treatment. Subsequently, after two weeks of drought treatment, WT plants were seriously wilting, but OE6-5 and OE6-10 showed slightly inhibited phenotypes. After 3 days of rewatering, the OE-65 and OE6-10 lines exhibited significantly higher survival rates and biomass, indicating that overexpression of ZmE2F6 contributes to enhancing drought tolerance in transgenic Arabidopsis and that ZmE2F6 positively regulates drought tolerance.

The phenotype of transgenic Arabidopsis under drought stress. ( A ) PCR detection and GUS staining of transgenic lines. ( B ) The phenotype of transgenic lines on 1/2 MS plates with mannitol. ( C ) Root length. ( D ) The phenotype of transgenic lines in soil. ( E ) Survival rate and biomass of each line. OE6-1 to OE6-10 represent transgenic lines overexpressing ZmE2F6 . WT, wild type

To investigate the impact of ZmE2F6 overexpression on endogenous genes in Arabidopsis thaliana , RNA-Seq was conducted on four transgenic lines, OE6-5, OE6-10, and WT. The results showed that there were 657 DEGs in transgenic lines compared to WT. Totally, 19 DEGs were identified in two transgenic lines. Among them, 15 DEGs were upregulated in transgenic lines, including AtMYB44 (AT5G67300), AtB1L (AT1G18740), AtJAZ7 (AT2G34600), AtEXS (AT1G35350), AtIP5PII (AT4G18010), AtPATL2 (AT1G22530), AtAZI1 (AT4G12470), AtXTH23 (AT4G25810), and AtEXL5 (AT2G17230) (Fig.  9 ). GO analysis showed that these DEGs were associated with stress responses (Figure S1 ).

The expression level (Log2 Fold change) of differentially expressed genes (DEGs) in each line

In eukaryotes, the ZmE2F family is characterized as a TF that plays crucial roles in cell division, DNA repair, and differentiation [ 11 , 13 , 14 , 15 , 16 ]. However, the E2F family is only genome-wide identified in a few plants, including Arabidopsis , rice, wheat, Moso bamboo, Medicago truncatula , and Phaseolus vulgaris [ 9 , 24 , 25 , 26 ]. In the present study, 21 ZmE2F members were identified in the maize genome (Table  1 ) and classified into E2F, DP, and DEL subclades (Fig.  1 ), which was higher than the number of E2F members identified in Arabidopsis (8), rice (9), Medicago truncatula (5), and Phaseolus vulgaris (7) but close to the number in wheat (27) and Moso bamboo (23), owing to their comparable genome size and gene duplication being a major driving force of gene families [ 25 , 49 ]. Likewise, eight paralogous pairs of ZmE2Fs and twenty-three orthologous pairs of E2Fs among maize, Arabidopsis , and rice were found (Fig.  4 ). All the Ka/Ks ratios of the ZmE2Fs paralogous pairs were < 1 (Table S4 ), indicating their duplication evolved under purifying selection [ 50 ]. A similar phenomenon was consistently observed in the PheE2F/DP gene family [ 25 ]. A previous study showed that there are 12 ZmE2F genes in maize [ 51 ], which is much lower than the number identified in this study. This may be due to their preliminary BLAST search using maize genome release 5b.60 and not a comprehensive analysis [ 51 ].

All ZmE2F proteins possess at least one DBD characterized by motif 1 containing the RRIYD sequence and dimerization residue DNVLE sequence (Fig.  2 ), which contributes to the binding of E2F and DNA as a homodimer or as a heterodimer with its dimerization partner DP. In addition, some ZmE2F proteins also have DD and CC-MB domains, which promotes their formation of heterodimers to regulate downstream genes [ 52 ]. As a result, ten ZmE2Fs are predicted to interact with each other and generate 23 PPI combinations (Fig.  3 ), owing to the presence of the DNVLE residues, DD, or CC-MB domain in these proteins.

To date, the function of E2F in regulating plant stress tolerance remains unknown, although few reports have shown that the expression of some E2F genes is responsive to stress [ 9 , 24 , 25 , 26 ]. In our study, abundant stress-responsive acting elements were found in ZmE2F promoter regions, such as ABREs and MBSs, suggesting the response of ZmE2Fs to stress and the potential roles of ZmE2Fs . In our previous study, the ZmPP2C26 gene, a B clade of maize PP2C members, was found to be responsive to drought stress and negatively regulated drought tolerance in Arabidopsis , rice, and maize [ 36 ] and targeted on maize ZmE2F6 (Zm00001d048412), indicating its function in the drought response. Hence, the ZmE2F6 gene was cloned and functionally validated. It is found that the ZmE2F6 interacts with two splicing variants of ZmPP2C26 but localized in the nucleus (Figs.  5 and 6 ), suggesting that ZmPP2C26 physically targets ZmE2F6. In Arabidopsis , it has been previously reported that B clade PP2C (AP2C1) dephosphorylates the autophosphorylated form of CBL-interacting protein kinase 9 (CIPK9) to regulate root growth, seedling development, and stress tolerance (low-K + ) [ 53 ]. Previous studies showed that BES1/BZR1 TFs are phosphorylated and degraded but moved to the nucleus after dephosphorylation in the cytoplasm [ 54 , 55 ]. It is proposed that ZmPP2C26 might dephosphorylate ZmE2F6 in the cytoplasm, which needs to be further revealed in our next study.

The qRT-PCR results showed that the ZmE2F6 gene was induced by drought stress (Fig.  7 ), which could be explained by the presence of three MBS and two ABREs in the ZmE2F6 promoter (Table S5 ). It’s confirmed that MYB transcription factors bind to the MYB binding sites (MBS) of nuclear gene promoters to adjust their transcription and regulate drought tolerance in plants [ 56 , 57 ]. Likewise, ABRE (ABA-responsive element), the major cis -element for ABA-responsive gene expression, is targeted by ABRE-binding protein (AREB) or ABRE-binding factor (ABF) TFs to regulate the drought response via the ABA signaling pathway [ 58 ]. These findings further imply the regulation of ZmE2F6 in drought tolerance.

After overexpressing ZmE2F6 in Arabidopsis , the transgenic lines showed longer root lengths than the WT (Fig.  8 B, C), which is consistent with the fact that E2Fs regulate root growth in Arabidopsis [ 19 , 22 , 59 ]. The elevated root growth of ZmE2F6 -overexpressing lines may confer osmotic tolerance because roots can respond to moisture and coordinate responses to drought [ 1 ]. The ectopic expression of ZmE2F6 enhances drought tolerance in transgenic Arabidopsis (Fig.  8 ). We found that some stress-related genes were significantly upregulated in the transgenic lines, such as AtMYB44 , AtB1L , AtJAZ7 , AtEXS , AtIP5PII , AtPATL2 , AtAZI1 , AtXTH23 , and AtEXL5 (Fig.  9 ), which are well known to regulate abiotic stresses [ 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. The ZmE2F6 protein acts as a TF and localizes in the nucleus to regulate the expression of these genes (Fig.  6 ). However, the molecular mechanism regulated by ZmE2F6 in maize remains unknown.

Overall, in this study, a comprehensive analysis of the ZmE2F gene family was performed. In total, 21 ZmE2F TFs were identified in the maize genome and divided into three subclades. All ZmE2F proteins possessed at least one DBD characterized by the RRIYD (DNA binding motif) and DNVLE (dimerization residues) sequences. The ZmE2F genes showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Then, the ZmE2F6 gene was cloned and functionally verified in the drought response. The ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis by upregulating stress-related gene transcription. This study sheds light on the role of ZmE2F in the drought response and provides novel insights into a greater understanding of the E2F family in crops.

Data availability

The RNA-seq datasets generated during the current study are available in the NCBI repository under BioProject accession number PRJNA1028664.

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Acknowledgements

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This study was supported by the National Key R&D Program of China (2021YFF1000303), the Sichuan Science and Technology Program (2022YFH0067) and the National Natural Science Foundation of China (32102226).

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Yang Cao and Kexin Wang contributed equally to this work.

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Maize Research Institute, Sichuan Agricultural University, Chengdu, 611130, China

Yang Cao, Kexin Wang, Fengzhong Lu, Qi Li, Qingqing Yang, Yingge Wang, Fengling Fu, Wanchen Li & Haoqiang Yu

College of Food and Biological Engineering, Chengdu University, Chengdu, 610106, China

Bingliang Liu

National Research Centre of Intercropping, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan

Hayderbinkhalid Muhammad

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Y.C., K.W. and F.L. designed and carried out the experiments; Y.C., K.W., B.L., H.M. and Y.W. carried out all bioinformatics analysis and wrote the manuscript; Q. Y. provided technical support. F.F., W.L. and H.Y supervised the experiments; H.Y. directed and revised the manuscript. All authors reviewed and approved the final manuscript.

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Correspondence to Haoqiang Yu .

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Cao, Y., Wang, K., Lu, F. et al. Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress. BMC Genomics 25 , 465 (2024). https://doi.org/10.1186/s12864-024-10369-0

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Received : 10 October 2023

Accepted : 02 May 2024

Published : 13 May 2024

DOI : https://doi.org/10.1186/s12864-024-10369-0

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