Rice Pollen Fertility Hinges on a Daily Rhythm Written by Light and the Circadian Clock

Every rice leaf keeps time. As the sun rises and sets, thousands of genes inside the leaf surge and subside in a carefully choreographed daily cycle, and for decades plant biologists have understood that this internal clock helps rice decide when to flower. A new study published in Plant Cell Reports by Renhe Dong, Jingbin Li and colleagues at Shanghai Jiao Tong University now shows that the clock’s influence reaches much deeper than flowering time. The team demonstrates that the rhythmic, light-regulated expression of a single gene in rice leaves, called CSA for carbon starved anther, is essential for producing fertile pollen, revealing an unexpected link between the daily rhythms of foliage and the reproductive fate of anthers that develop far away on the same plant.

CSA is not an obscure player. It encodes a MYB domain transcription factor that was first identified because a mutation in it starves developing anthers of sugar, leaving plants male sterile. That earlier work, published in 2010, established CSA as a sugar-partitioning gene: it helps route carbohydrates from photosynthetic tissues into the anther, where developing pollen grains are voracious consumers of energy. Later studies delivered a striking twist. The csa mutant is completely male sterile when plants are grown under short-day conditions, but it regains partial fertility under long days. This photoperiod-sensitive male sterility is precisely the property that breeders prize in hybrid rice seed production, because it allows sterile lines to be maintained under one day length and multiplied for hybrid crosses under another. Yet the molecular logic behind that day-length sensitivity has remained murky.

The new study attacks that gap by asking a deceptively simple question: when and where is CSA actually switched on? The researchers measured CSA expression in rice leaves across 24-hour cycles under both short-day and long-day conditions and found that it does not hold steady. Instead, CSA transcript levels oscillate, rising and falling in a regular rhythm under both photoperiods. This oscillatory pattern is the signature of circadian regulation, the roughly 24-hour biological clock that plants, like animals, use to anticipate daily environmental changes. The finding immediately reframed CSA: rather than being a static anther-specific gene, it is a clock-controlled gene whose leaf expression pulses with the day-night cycle.

Crucially, the rhythm is not driven by the clock alone. Light signals, particularly those perceived through the red and far-red photoreceptor phytochromes, also shape the oscillation. This dual control matters because the circadian clock and light signaling pathways are the two great inputs that plants use to measure day length. By showing that both converge on the CSA promoter in leaves, the study provides a mechanistic bridge between photoperiod sensing and male reproductive development. In effect, the leaf reads the calendar and the clock, and the output of that computation is written into the oscillating expression of CSA.

To test whether the leaf rhythm actually matters for fertility, the team used tissue-specific RNA interference to knock down CSA expression specifically in leaves while leaving the anthers untouched. The result was unambiguous: reducing CSA in leaves lowered pollen viability. This experiment overturned the assumption that CSA only needs to function inside the anther itself. The gene must also be expressed, and expressed rhythmically, in leaves for pollen to develop normally. The implication is that the leaf is not merely a sugar factory passively feeding the anther; it is an active regulatory organ whose daily expression program is part of the fertility machinery.

The researchers then dissected the CSA promoter, the stretch of DNA upstream of the gene that hosts the binding sites for regulatory proteins. By generating a series of promoter truncations, they showed that different regions of the promoter carry different responsibilities. Some segments drive expression in anthers, while others are required for the rhythmic pattern in leaves. Both features proved important: the level of CSA expression in anthers and the rhythmic pattern of CSA expression in leaves were each associated with the restoration of male fertility. This promoter-level division of labor explains how a single gene can serve two tissues with distinct regulatory demands, and it suggests that the photoperiod-sensitive sterility of the csa mutant arises from disruption of the leaf-side rhythm rather than from a simple loss of anther expression.

Having localized the regulatory regions, the team went hunting for the proteins that bind them. Using a trio of complementary techniques, dual-luciferase reporter assays in plant protoplasts, yeast one-hybrid screens, and electrophoretic mobility shift assays with purified proteins and labeled DNA probes, they identified two transcription factors that directly target the CSA promoter truncations. The first, PIL11, is a phytochrome-interacting factor-like bHLH protein that binds an E-box motif, a canonical binding site for light-regulated bHLH factors. The second, DOF5, is a member of the plant-specific Dof family of zinc-finger transcription factors and binds a T/AAAAG motif. Both proteins act as direct regulators of CSA transcription, providing the molecular handholds through which light and clock signals grip the gene.

The identity of these regulators is telling. Phytochrome-interacting factors are well known as molecular switches that relay red and far-red light information from phytochromes to the genome, and they also serve as bridges between environmental signals and the circadian clock in other plant systems. Dof factors, meanwhile, have established roles in photoperiodic responses, including the regulation of CONSTANS and flowering time in Arabidopsis, and some rice Dof genes are themselves clock-regulated. By recruiting one factor from each family to the CSA promoter, rice appears to have wired a photoperiod-sensing module, structurally similar to the one that controls flowering, directly into a gene that controls sugar delivery to pollen. The study’s authors describe this as a novel mechanism linking light sensing to circadian-controlled gene expression and thereby connecting photoperiod with male reproductive development.

The agricultural implications are considerable. Hybrid rice, one of China’s great agricultural successes, depends on male-sterile lines to enable large-scale cross-pollination without laborious hand emasculation. Photoperiod-sensitive genic male sterility, the phenomenon in which the csa mutation participates, is especially valuable because sterility can be reversed by changing day length, giving breeders seasonal control over fertility. The new work supplies the first detailed regulatory circuit for that reversal: light quality and the circadian clock converge through PIL11 and DOF5 on the CSA promoter in leaves, generating the rhythmic expression that pollen development requires. Understanding this circuit opens the door to engineering sterility-fertility switches with defined molecular parts, potentially allowing breeders to tune the day-length threshold at which a sterile line becomes fertile, a critical variable for adapting hybrid seed production to different latitudes and climates.

Beyond rice, the study adds to a growing body of evidence that the circadian clock governs reproduction not only by timing the floral transition but by regulating the physiology of the reproductive organs themselves. Previous work has implicated phytochrome-interacting factors in tapetal cell death in tomato anthers and in pollen mitotic division through auxin and sugar metabolism, and rice clock components such as OsLHY have been shown to set critical day lengths for photoperiodic flowering. The new findings extend this theme to the leaf-anther axis, showing that a clock-controlled gene expressed in vegetative tissue is a required component of male fertility. For a crop that feeds roughly half the world’s population, and for a research community racing to redesign photosynthesis and reproductive resilience under climate change, the message is clear: the daily rhythm in the leaf is not background noise. It is part of the score, and pollen fertility dances to it.

Subject of Research: Circadian and light regulation of the CSA gene in rice leaves and its role in photoperiod-sensitive pollen fertility

Article Title: Circadian- and light-regulated oscillatory expression of CSA in rice leaves is required for pollen fertility

Article References: Circadian- and light-regulated oscillatory expression of CSA in rice leaves is required for pollen fertility. (n.d.). https://doi.org/10.1007/s00299-026-03976-6

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03976-6

Keywords: rice, circadian clock, CSA, photoperiod-sensitive male sterility, pollen fertility, PIL11, DOF5, phytochrome, anther development, sugar partitioning, hybrid rice, transcription factors