Circadian Clock Controls Root Hair Elongation through Long-Distance Communication.

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.

Root PRR7 Improves the Accuracy of the Shoot Circadian Clock through Nutrient Transport.

The circadian clock allows plants to anticipate and adapt to periodic environmental changes. Organ- and tissue-specific properties of the circadian clock and 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 the 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 suppresses the variance of period length in shoots and then improves the accuracy of the 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.

The FLOWERING LOCUS T gene expression is controlled by high-irradiance response and external coincidence mechanism in long days in Arabidopsis.

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.

Spatially specific mechanisms and functions of the plant circadian clock.

Like many organisms, plants have evolved a genetic network, the circadian clock, to coordinate processes with day/night cycles. In plants, the clock is a pervasive regulator of development and modulates many aspects of physiology. Clock-regulated processes range from the correct timing of growth and cell division to interactions with the root microbiome. Recently developed techniques, such as single-cell time-lapse microscopy and single-cell RNA-seq, are beginning to revolutionize our understanding of this clock regulation, revealing a surprising degree of organ, tissue, and cell-type specificity. In this review, we highlight recent advances in our spatial view of the clock across the plant, both in terms of how it is regulated and how it regulates a diversity of output processes. We outline how understanding these spatially specific functions will help reveal the range of ways that the clock provides a fitness benefit for the plant.

Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling.

Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

Sulfanilamide Regulates Flowering Time through Expression of the Circadian Clock Gene LUX.

Flowering time is an agriculturally important trait that can be manipulated by various approaches such as breeding, growth control and genetic modifications. Despite its potential advantages, including fine-tuning the regulation of flowering time, few reports have explored the use of chemical compounds to manipulate flowering. Here, we report that sulfanilamide, an inhibitor of folate biosynthesis, delays flowering by repressing the expression of florigen FLOWERING LOCUS T (FT) in Arabidopsis thaliana. Transcriptome deep sequencing and quantitative polymerase chain reaction analyses showed that the expression of the circadian clock gene LUX ARRYTHMO/PHYTOCLOCK1 (LUX/PCL1) is altered by sulfanilamide treatment. Furthermore, in the lux nox mutant harboring loss of function in both LUX and its homolog BROTHER OF LUX ARRHYTHMO (BOA, also named NOX), the inhibitory effect of sulfanilamide treatment on FT expression was weak and the flowering time was similar to that of the wild type, suggesting that the circadian clock may contribute to the FT-mediated regulation of flowering by sulfanilamide. Sulfanilamide also delayed flowering time in arugula (Eruca sativa), suggesting that it is involved in the regulation of flowering across Brassicaceae. We propose that sulfanilamide is a novel modulator of flowering.

Oscillator networks with tissue-specific circadian clocks in plants.

Many organisms rely on circadian clocks to synchronize their biological processes with the 24-h rotation of the earth. In mammals, the circadian clock consists of a central clock in the suprachiasmatic nucleus and peripheral clocks in other tissues. The central clock is tightly coupled to synchronize rhythmicity and can organize peripheral clocks through neural and hormonal signals. In contrast to mammals, it has long been assumed that the circadian clocks in each plant cell is able to be entrained by external light, and they are only weakly coupled to each other. Recently, however, several reports have demonstrated that plants have unique oscillator networks with tissue-specific circadian clocks. Here, we introduce our current view regarding tissue-specific properties and oscillator networks of plant circadian clocks. Accumulating evidence suggests that plants have multiple oscillators, which show distinct properties and reside in different tissues. A direct tissue-isolation technique and micrografting have clearly demonstrated that plants have hierarchical oscillator networks consisting of multiple tissue-specific clocks.

Time-Series Single-Cell RNA-Seq Data Reveal Auxin Fluctuation during Endocycle.

Appropriate cell cycle regulation is crucial for achieving coordinated development and cell differentiation in multicellular organisms. In Arabidopsis, endoreduplication is often observed in terminally differentiated cells and several reports have shown its molecular mechanisms. Auxin is a key factor for the mode transition from mitotic cell cycle to endocycle; however, it remains unclear if and how auxin maintains the endocycle mode. In this study, we reanalyzed root single-cell transcriptome data and reconstructed cell cycle trajectories of the mitotic cell cycle and endocycle. With progression of the endocycle, genes involved in auxin synthesis, influx and efflux were induced at the specific cell phase, suggesting that auxin concentration fluctuated dynamically. Such induction of auxin-related genes was not observed in the mitotic cell cycle, suggesting that the auxin fluctuation plays some roles in maintaining the endocycle stage. In addition, the expression level of CYCB1;1, which is required for cell division in the M phase, coincided with the expected amount of auxin and cell division. Our analysis also provided a set of genes expressed in specific phases of the cell cycle. Taking these findings together, reconstruction of single-cell transcriptome data enables us to identify properties of the cell cycle more accurately.

Tissue-specific clocks in Arabidopsis show asymmetric coupling.

Many organisms rely on a circadian clock system to adapt to daily and seasonal environmental changes. The mammalian circadian clock consists of a central clock in the suprachiasmatic nucleus that has tightly coupled neurons and synchronizes other clocks in peripheral tissues. Plants also have a circadian clock, but plant circadian clock function has long been assumed to be uncoupled. Only a few studies have been able to show weak, local coupling among cells. Here, by implementing two novel techniques, we have performed a comprehensive tissue-specific analysis of leaf tissues, and show that the vasculature and mesophyll clocks asymmetrically regulate each other in Arabidopsis. The circadian clock in the vasculature has characteristics distinct from other tissues, cycles robustly without environmental cues, and affects circadian clock regulation in other tissues. Furthermore, we found that vasculature-enriched genes that are rhythmically expressed are preferentially expressed in the evening, whereas rhythmic mesophyll-enriched genes tend to be expressed in the morning. Our results set the stage for a deeper understanding of how the vasculature circadian clock in plants regulates key physiological responses such as flowering time.

SODIUM POTASSIUM ROOT DEFECTIVE1 regulates FLOWERING LOCUS T expression via the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module in response to potassium conditions.

To determine flowering time, plants perceive multiple environmental stimuli and integrate these signals in the regulation of a florigen gene, FLOWERING LOCUS T (FT). It has been known that nutrient availability affects flowering time in both laboratories and fields. Nitrogen (N), phosphorus (P) and potassium (K) are the three major macronutrients which are important for plant growth and development. Although N and P stimuli can alter the expression of regulators of FT including microRNA156 (miR156) and miR156-targeted transcription factors of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family, how K+ conditions affect flowering is still unclear. We focused on SODIUM POTASSUIM ROOT DEFECTIVE1 (NaKR1) whose mutant plants showed Na+ and K+ overaccumulation and late flowering. It was reported that NaKR1 is involved in the phloem transport of FT protein. Here we report that NaKR1 is also required for the promotion of FT expression in long-day conditions. NaKR1 affects the accumulation of miR156 and SPL3 expression, suggesting that NaKR1 regulates FT expression in part through the miR156-SPL3 module. The late-flowering phenotype of the nakr1-1 mutant was partially suppressed under low K+ conditions, and miR156 abundance and SPL3 expression in the nakr1-1 mutant and, to a lesser extent, in wild-type plants responded to K+ conditions. Taken together, our findings suggest that the miR156-SPL3 module mediates regulation of FT expression by NaKR1 in response to K+ conditions. Finally, we propose a model in which NaKR1 plays dual roles in regulation of flowering, one in the regulation of florigen production, the other in that of florigen transport.

Re-Evaluation of Florigen Transport Kinetics with Separation of Functions by Mutations That Uncouple Flowering Initiation and Long-Distance Transport.

In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8-12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport.

Correction to: Circadian clock during plant development.

The article" Circadian clock during plant development".

Circadian clock during plant development.

Plants have endogenous biological clocks that allow organisms to anticipate and prepare for daily and seasonal environmental changes and increase their fitness in changing environments. The circadian clock in plants, as in animals and insects, mainly consists of multiple interlocking transcriptional/translational feedback loops. The circadian clock can be entrained by environmental cues such as light, temperature and nutrient status to synchronize internal biological rhythms with surrounding environments. Output pathways link the circadian oscillator to various physiological, developmental, and reproductive processes for adjusting the timing of these biological processes to an appropriate time of day or a suitable season. Recent genomic studies have demonstrated that polymorphism in circadian clock genes may contribute to local adaptations over a wide range of latitudes in many plant species. In the present review, we summarize the circadian regulation of biological processes throughout the life cycle of plants, and describe the contribution of the circadian clock to local adaptation.

Tissue-specific regulation of flowering by photoreceptors.

Plants use various kinds of environmental signals to adjust the timing of the transition from the vegetative to reproductive phase (flowering). Since flowering at the appropriate time is crucial for plant reproductive strategy, several kinds of photoreceptors are deployed to sense environmental light conditions. In this review, we will update our current understanding of light signaling pathways in flowering regulation, especially, in which tissue do photoreceptors regulate flowering in response to light quality and photoperiod. Since light signaling is also integrated into other flowering pathways, we also introduce recent progress on how photoreceptors are involved in tissue-specific thermosensation and the gibberellin pathway. Finally, we discuss the importance of cell-type-specific analyses for future plant studies.

Transcriptional Framework of Male Gametogenesis in the Liverwort Marchantia polymorpha L.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.

BRANCHED1 interacts with FLOWERING LOCUS T to repress the floral transition of the axillary meristems in Arabidopsis.

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.

PHYTOCHROME-DEPENDENT LATE-FLOWERING accelerates flowering through physical interactions with phytochrome B and CONSTANS.

In flowering plants, light is one of the major environmental stimuli that determine the timing of the transition from the vegetative to reproductive phase. In Arabidopsis, phytochrome B (phyB); phyA; cryptochrome 2; and flavin-binding, KELCH repeat, F-BOX 1 are major photoreceptors that regulate flowering. Unlike phyA; cryptochrome 2; and flavin-binding, KELCH repeat, F-BOX 1, phyB delays flowering mainly by destabilizing the CONSTANS (CO) protein, whose reduction leads to decreased expression of a florigen gene, flowering locus T. However, it remains unclear how the phyB-mediated CO destabilization is mechanistically regulated. Here, we identify a unique phytochrome-dependent late-flowering (PHL) gene, which is mainly involved in the phyB-dependent regulation of flowering. Plants with mutant phl exhibited a late-flowering phenotype, especially under long-day conditions. The late-flowering phenotype of the phl mutant was completely overridden by a phyB mutation, indicating that PHL normally accelerates flowering by countering the inhibitory effect of phyB on flowering. Accordingly, PHL physically interacted with phyB both in vitro and in vivo in a red light-dependent manner. Furthermore, in the presence of phyB under red light, PHL interacted with CO as well. Taken together, we propose that PHL regulates photoperiodic flowering by forming a phyB-PHL-CO tripartite complex.

A guiding role of the Arabidopsis circadian clock in cell differentiation revealed by time-series single-cell RNA sequencing.

Circadian rhythms and progression of cell differentiation are closely coupled in multicellular organisms. However, whether establishment of circadian rhythms regulates cell differentiation or vice versa has not been elucidated due to technical limitations. Here, we exploit high cell fate plasticity of plant cells to perform single-cell RNA sequencing during the entire process of cell differentiation. By analyzing reconstructed actual time series of the differentiation processes at single-cell resolution using a method we developed (PeakMatch), we find that the expression profile of clock genes is changed prior to cell differentiation, including induction of the clock gene LUX ARRYTHMO (LUX). ChIP sequencing analysis reveals that LUX induction in early differentiating cells directly targets genes involved in cell-cycle progression to regulate cell differentiation. Taken together, these results not only reveal a guiding role of the plant circadian clock in cell differentiation but also provide an approach for time-series analysis at single-cell resolution.

Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

Isolation of Arabidopsis Palisade and Spongy Mesophyll Cells.

Cell-type-specific transcription factors are key to deducing the distinct functions of specialized cells from gene expression profiles. Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. Palisade and spongy mesophyll cells are sub-tissues that have different morphologies and physiologies. Thus, determining the palisade and spongy mesophyll-specific transcription factors from the respective sub-tissue-specific transcriptomes is vital to understanding or verifying functions of major plant tissues. One way in which gene expression profiles can be addressed is through direct isolation. Here, we present rapid and simple methods to isolate palisade and spongy mesophyll cells mechanically and enzymatically. This method provides a good yield of each isolated cell type, and the isolated cells can be used for various downstream applications.