Akane Kubota

Circadian rhythms regulate crucial physiological processes in plants, making the analysis of rhythmic gene expression vital for understanding how plants adapt to and anticipate daily environmental changes, and for improving agricultural traits. Foundational databases for plant circadian rhythms have been invaluable, but face challenges in keeping pace with the rapid accumulation of modern transcriptomic data, particularly for the most widely used model organism, Arabidopsis thaliana. To address this critical gap, we developed GEAR (Gene Expression Archives for Rhythm), a new database resource dedicated to A. thaliana that serves as an integrated atlas of gene expression dynamics. GEAR builds a comprehensive data archive by integrating both legacy microarray and modern RNA-seq datasets, covering 83 diverse experimental conditions. Its intuitive web interface functions as an interactive atlas, allowing users to compare expression profiles not only across different experimental treatments but also across independent studies conducted under nominally identical settings. This unique feature enables researchers to move beyond single-experiment findings and distinguish robust biological signals from interlaboratory variability. For the first time, this allows for a meta-analysis to define a high-confidence set of core clock-regulated genes, providing foundational insights into the most conserved outputs of the plant circadian network.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

Temperature changes along seasonal transitions control flowering. Under natural long-day conditions, flowering is controlled by a bimodal expression pattern of the florigen gene, FLOWERING LOCUS T (FT), in Arabidopsis. Although cool ambient temperatures delay flowering through FT repression, it is unknown how daily temperature fluctuations regulate bimodal FT profiles. Seasonal increases in nighttime temperatures are less variable than those during the daytime in spring, potentially providing more reliable timing information. By simulating daily temperature fluctuations of spring, we showed that cool night-to-morning temperatures activate multiple mechanisms to control morning and evening FT peaks separately. Lowering night temperatures regulates the CONSTANS (CO) protein function by reducing its stability from midday to dusk and increasing its interaction with a colder-night induced repressor, B-BOX DOMAIN PROTEIN 29 (BBX29), in the morning. Our results demonstrated the dynamics of time-specific regulatory mechanisms in temperature signaling during spring when cooler temperatures determine the timing of flowering.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T ( FT ) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT -expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT -expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT -expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT -expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

In order to flower in the appropriate season, plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT). In Arabidopsis, FT messenger RNA levels peak in the morning and evening under natural long‐day conditions (LDs). However, the regulatory mechanisms governing morning FT induction remain poorly understood. The morning FT peak is absent in typical laboratory LDs characterized by high red:far‐red light (R:FR) ratios and constant temperatures. Here, we demonstrate that ZEITLUPE (ZTL) interacts with the FT repressors TARGET OF EATs (TOEs), thereby repressing morning FT expression in natural environments. Under LDs with simulated sunlight (R:FR = 1.0) and daily temperature cycles, which are natural LD‐mimicking environmental conditions, FT transcript levels in the ztl mutant were high specifically in the morning, a pattern that was mirrored in the toe1 toe2 double mutant. Low night‐to‐morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning. Far‐red light counteracted ZTL activity by decreasing its abundance (possibly via phytochrome A (phyA)) while increasing GIGANTEA (GI) levels and negatively affecting the formation of the ZTL–GI complex in the morning. Therefore, the phyA‐mediated high‐irradiance response and GI play pivotal roles in morning FT induction. Our findings suggest that the delicate balance between low temperature‐mediated ZTL activity and the far‐red light‐mediated functions of phyA and GI offers plants flexibility in fine‐tuning their flowering time by controlling FT expression in the morning.

Plants adapt to periodic environmental changes, such as day and night, by using circadian clocks. Cell division and elongation are primary steps to adjust plant development according to their environments. In Arabidopsis, hypocotyl elongation has been studied as a representative model to understand how the circadian clock regulates cell elongation. However, it remains unknown whether similar phenomena exist in other organs, such as roots, where circadian clocks regulate physiological responses. Here, we show that root hair elongation is controlled by both light and the circadian clock. By developing machine-learning models to automatically analyze the images of root hairs, we found that genes encoding major components of the central oscillator, such as TIMING OF CAB EXPRESSION1 (TOC1) or CIRCADIAN CLOCK ASSOCIATED1 (CCA1), regulate the rhythmicity of root hair length. The partial illumination of light to either shoots or roots suggested that light received in shoots is mainly responsible for the generation of root hair rhythmicity. Furthermore, grafting experiments between wild-type (WT) and toc1 plants demonstrated that TOC1 in shoots is responsible for the generation of root hair rhythmicity. Our results illustrate the combinational effects of long-distance signaling and the circadian clock on the regulation of root hair length.

Summary: In natural long days, the florigen gene FLOWERING LOCUS T (FT) shows a bimodal expression pattern with morning and dusk peaks in Arabidopsis. This pattern differs from the one observed in the laboratory, and little is known about underlying mechanisms. A red : far‐red (R : FR) ratio difference between sunlight and fluorescent light causes this FT pattern mismatch. We showed that bimodal FT expression patterns were induced in a day longer than 14 h with sunlight R : FR (= c. 1) conditions. By circadian gating experiments, we found that cumulative exposure of R : FR‐adjusted light (R : FR ratio was adjusted to 1 with FR supplement) spanning from the afternoon to the next morning required full induction of FT in the morning. Conversely, only 2 h of R : FR adjustment in the late afternoon was sufficient for FT induction at dusk. We identified that phytochrome A (phyA) is required for the morning FT expression in response to the R : FR adjustment on the previous day. As a part of this mechanism, we showed that PHYTOCHROME‐INTERACTING FACTOR 7 contributes to FT regulation. Our results suggest that phyA‐mediated high‐irradiance response and the external coincidence mechanism contribute to morning FT induction under natural long‐day conditions.

The circadian clock allows plants to anticipate and adapt to periodic environmental changes. Organ- and tissue-specific properties of the circadian clock as well as shoot-to-root circadian signaling have been reported. While this long-distance signaling is thought to coordinate physiological functions across tissues, little is known about the feedback regulation of root clock on the shoot clock in the hierarchical circadian network. Here, we show that the plant circadian clock conveys circadian information between shoots and roots through sucrose and K⁺. We also demonstrate that K⁺ transport from roots suppress the variance of period length in shoots and then improve the accuracy of shoot circadian clock. Sucrose measurements and qPCR showed that root sucrose accumulation was regulated by the circadian clock. Furthermore, root circadian clock genes, including PSEUDO-RESPONSE REGULATOR7 (PRR7), were regulated by sucrose, suggesting the involvement of sucrose from the shoot in the regulation of root clock gene expression. Therefore, we performed time-series measurements of xylem sap and micrografting experiments using prr7 mutants and showed that root PRR7 regulates K⁺ transport and suppresses variance of period length in the shoot. Our modeling analysis supports the idea that root-to-shoot signaling contributes to the precision of the shoot circadian clock. We performed micrografting experiments that illustrated how root PRR7 plays key roles in maintaining the accuracy of shoot circadian rhythms. We thus present a novel directional signaling pathway for circadian information from roots to shoots and propose that plants modulate physiological events in a timely manner through various timekeeping mechanisms.