The research, led by the team of Liu Xiaoqin, combined careful observation of leaf behavior under different lighting regimes, time-series transcriptome profiling across a full day, and a series of molecular assays to connect clock components with downstream transporter genes. The central question was deceptively simple: how do light and clock signals reach the genes responsible for moving water and sugar into and out of the motor cells that power leaf folding? The answer, the researchers propose, runs through a transcriptional network in which the clock protein CCA1 and a pulvinus-enriched MYB transcription factor regulate SWEET sugar transporters and PIP aquaporins, the very proteins that could link timekeeping to turgor pressure changes in the leaf pillow, or pulvinus.

The team began by establishing that peanut leaf movement genuinely depends on the light-dark cycle. Under a normal photoperiod of 16 hours of light and 8 hours of darkness, the leaves closed progressively after the lights went off at 21:30 and were fully closed by 23:00. When the lights came back on at 5:30 the next morning, the leaves gradually unfolded and resumed their stretched daytime posture by around 7:00. Under continuous illumination, however, this characteristic rhythm was suppressed: the leaves remained largely unfolded, showing only slight up-and-down movements. The comparison demonstrated that, in this experimental system, an alternating light-dark cycle is essential for maintaining the visible opening and closing behavior, underscoring the importance of photoperiodic entrainment for nyctinasty.

To trace the temporal signals behind the movement, the researchers sampled plants at 14 time points across the day and analyzed gene expression in both creeping and upright peanut types. Under normal light-dark cycles, the expression patterns followed a clear temporal order. Clock-related genes such as LHY/CCA1 were expressed mainly in the early morning, certain PRR genes peaked in the afternoon, and TOC1 and related genes were active in the evening or at night. When plants were kept under continuous light, the expression fluctuations of some clock genes weakened, yet the overall morning and evening ordering of their expression was largely preserved. This dissociation was informative: visible leaf movement was inhibited by constant light, but the molecular timekeeping apparatus had not simply shut down, suggesting that the photoperiod acts at a level between the clock and the physical movement of the leaves.

With the rhythmic framework in place, the team searched for candidate genes whose expression tracked the light and clock signals. Two functional classes stood out. The first comprised SWEET sugar transporter genes, members of a family that mediates the movement of sugars across membranes and could therefore influence the distribution of carbohydrates between the leaf blade and the pulvinus. The second comprised PIP aquaporin genes, which encode water channels that govern the transmembrane flow of water. Both classes are plausible entry points for connecting time signals to turgor pressure, since leaf opening and closing in legumes depends on rapid, coordinated changes in water content and solute concentration within the pulvinus motor cells. Notably, the candidate genes showed distinct tissue-specific expression patterns: the two PIP genes detected were expressed mainly in the pulvinus region of compound leaves, one MYB transcription factor gene was enriched in the pulvini of both compound leaves and leaflets, and the two SWEET candidates were more inclined toward leaf expression, hinting at collaboration between leaf and pulvinus tissues.

To test whether regulatory relationships actually connect the clock to these transporter genes, the researchers turned to yeast one-hybrid screening and dual-luciferase reporter assays. The yeast one-hybrid analysis identified candidate transcription factors capable of binding the promoters of the transporter genes, and the reporter assays measured the functional consequences of those interactions on promoter activity. The results converged on a specific connection: the clock protein CCA1 was able to recognize evening element (EE) motifs in the promoters of certain SWEET genes and of the pulvinus-enriched MYB transcription factor gene, placing a core circadian component directly upstream of the candidate regulatory network.

The sequence specificity of this binding was verified by electrophoretic mobility shift assays, or EMSA. When CCA1 was added to DNA probes containing intact EE sequences, protein-DNA complexes formed that migrated more slowly through the gel, a hallmark of specific binding. The signal weakened when the EE sequence was mutated, and it was also reduced by the addition of an excess of unlabeled competitive probe, which outcompeted the labeled probe for CCA1 binding. Together, these controls supported the conclusion that CCA1 binds the corresponding promoter fragments in a sequence-specific manner, providing direct molecular evidence that a clock factor is wired into the downstream regulatory network governing leaf movement.

The dual-luciferase reporter assays added a functional dimension to the picture. CCA1 and some of the MYB factors tested were able to enhance the activity of the promoter of a PIP aquaporin gene, indicating that clock and clock-associated transcription factors do not merely occupy these promoters but can actively modulate their output. Taken together with the tissue expression data, the results support a working model in which photoreceptors and the circadian clock coordinate the expression of transporter and regulatory genes: CCA1 and related MYB factors regulate SWEET sugar transporters and aquaporins, which may in turn participate in leaf opening and closing by affecting sugar distribution, water transport, and ultimately cell turgor pressure in the pulvinus. In this model, the upstream time signal and the downstream transport processes are linked in a single regulatory chain.

The authors are careful to note that some of the transcriptional relationships in the model remain to be functionally validated, and that further work is needed to determine exactly how changes in sugar and water transport translate into the turgor shifts that physically drive leaf movement. Even so, the study provides interconnected evidence spanning phenotype, temporal expression patterns, and molecular regulatory relationships. The direct binding of CCA1 to candidate downstream gene promoters, and the regulation of aquaporin promoter activity by CCA1 and MYB factors, offer concrete research targets for dissecting how plant time signals are transmitted to transport-related processes. Beyond peanut, the findings lay a foundation for exploring how leaf posture regulation connects to water use and environmental adaptation in legumes more broadly, and they bring researchers closer to understanding one of the plant world’s most visible daily rhythms at the level of its underlying genes.

Subject of Research: Circadian and photoperiodic regulation of nyctinastic leaf movement in peanut

Article Title: Peking University Institute of Modern Agriculture reveals molecular regulation mechanism of day opening and night closing in peanut leaves

Article References: Peking University Institute of Modern Agriculture reveals molecular regulation mechanism of day opening and night closing in peanut leaves. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: peanut, nyctinasty, circadian clock, CCA1, MYB transcription factor, SWEET sugar transporters, aquaporins, pulvinus, photoperiod, turgor pressure, legumes, transcriptome