Polarization of the zygote defines the body axis during plant development. In Arabidopsis (Arabidopsis thaliana), the zygote becomes polarized and elongates in the longitudinal direction, ultimately forming the apical-basal axis of the mature plant. Despite its importance, the mechanism for this elongation remains poorly understood. Based on live-cell imaging of the zygote, we developed new image analysis methods, referred to as coordinate normalization, that appropriately fix and align positions in an image, preventing fluctuation across a temporal sequence of images. Using these methods, we discovered that the zygote elongates only at its apical tip region, similar to tip-growing cells such as pollen tubes and root hairs. We also investigated the spatiotemporal dynamics of the apical tip contour of the zygote and observed that the zygote tip retains its isotropic, hemispherical apical shape during cell elongation. By looking at the elliptical fitting of the contour over time, we further discovered that the apical cell tip becomes thinner at first and then thickens, with a transient increase in growth speed that is followed by the first cell division. We performed the same series of analyses using root hairs and established that the hemispherical tip shape and the changes in growth rate associated with changes in tip size are both specific to the zygote. In summary, the Arabidopsis zygote undergoes directional elongation as a tip-growing cell, but its tip retains an unusual isotropic shape, and the manner of growth changes with the developmental stage.

The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias, but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from brightfield microscopy images containing mesophyll tissue as noisy backgrounds. By combining a YOLOX-based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved 0.875 mAP50 (mean average precision; stomata detection performance) and 0.745 IoU (intersection of union; pore segmentation performance) against images of leaf discs taken with a brightfield microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots.

Plants can exert remarkable capacity for cell reprogramming even from differentiated cells. This ability allows plants to regenerate tissues/organs and even individuals in nature and in vitro. In recent decades, Arabidopsis research has uncovered molecular mechanisms of plant regeneration; however, our understanding of how plant cells retain both differentiated status and developmental plasticity is still obscure. In this review, we first provide a brief outlook of the representative modes of plant regeneration and key factors revealed by Arabidopsis research. We then re-examine historical tissue culture systems that enable us to investigate the molecular details of cell reprogramming in differentiated cells and discuss the different approaches, specifically highlighting our recent progress in shoot regeneration from the epidermal cell of Torenia fournieri.

Shoot stem cells act as the source of the aboveground parts of flowering plants. A precise regulatory basis is required to ensure that plant stem cells show the right status during the stages of proliferation, senescence and cell death. Over the past few decades, the genetic circuits controlling stem cell fate, including the regulatory pathways of establishment, maintenance and differentiation, have been largely revealed. However, the morphological changes and molecular mechanisms of the final stages of stem cells, which are represented by senescence and cell death, have been less studied. The senescence and death of shoot stem cells are under the control of a complex series of pathways that integrate multiple internal and external signals. Given the crucial roles of shoot stem cells in influencing plant longevity and crop yields, researchers have attempted to uncover details of stem cell senescence and death. Recent studies indicate that stem cell activity arrest is controlled by the FRUITFULL-APETALA2 pathway and the plant hormones auxin and cytokinin, while the features of senescent and dead shoot apical stem cells have also been described, with dynamic changes in reactive oxygen species implicated in stem cell death. In this review, we highlight the recent breakthroughs that have enriched our understanding of senescence and cell death processes in plant stem cells.

During their postembryonic development, plants continuously form branches to conquer more space and adapt to changing environments. In seed plants, this is achieved by lateral branching, in which axillary meristems (AMs) initiate at the leaf axils to form axillary buds. The developmental potential of AMs to form shoot branches is the same as that of embryonic shoot apical meristems (SAMs). Recent studies in Arabidopsis thaliana have revealed the cellular origin of AMs and have identified transcription factors and phytohormones that regulate sequential steps leading to AM initiation. In particular, a group of meristematic cells detached from the SAM are key to AM initiation, which constitutes an excellent system for understanding stem cell fate and de novo meristem formation.

The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type-specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1, which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth.

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

During organogenesis, a key step toward the development of a functional organ is the separation of cells into specific domains with different activities. Mutual inhibition of gene expression has been shown to be sufficient to establish and maintain these domains during organogenesis in several multicellular organisms. Here, we show that the mutual inhibition between the PLETHORA transcription factors (PLTs) and the ARABIDOPSIS RESPONSE REGULATORs (ARRs) transcription factors is sufficient to separate cell division and cell differentiation during root organogenesis. In particular, we show that ARR1 suppresses PLT activities and that PLTs suppress ARR1 and ARR12 by targeting their proteins for degradation via the KISS ME DEADLY 2 F-box protein. These findings reveal new important aspects of the complex process of root zonation and development.

Plants have the regenerative ability to reconnect cut organs, which is physiologically important to survive severe tissue damage. The ability to reconnect organs is utilized as grafting to combine two different individuals. Callus formation at the graft junction facilitates organ attachment and vascular reconnection. While it is well documented that local wounding signals provoke callus formation, how callus formation is differentially regulated at each cut end remains elusive. Here, we report that callus formation activity is asymmetrical between the top and bottom cut ends and is regulated by differential auxin accumulation. Gene expression analyses revealed that cellular auxin response is preferentially upregulated in the top part of the graft. Disruption of polar auxin transport inhibited callus formation from the top, while external application of auxin was sufficient to induce callus formation from the bottom, suggesting that asymmetric auxin accumulation is responsible for active callus formation from the top end. We further found that the expression of a key regulator of callus formation, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), is induced by auxin. The ectopic callus formation from the bottom end, which is triggered by locally supplemented auxin, requires WOX13 function, demonstrating that WOX13 plays a pivotal role in auxin-dependent callus formation. The asymmetric WOX13 expression is observed both in grafted petioles and incised inflorescence stems, underscoring the generality of our findings. We propose that efficient organ reconnection is achieved by a combination of local wounding stimuli and disrupted long-distance signaling.

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 glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

Lysin motif receptor-like kinases (LysM-RLKs) are a plant-specific receptor protein family that sense components from soil microorganisms, regulating innate immunity and symbiosis. Every plant species possesses multiple LysM-RLKs in order to interact with a variety of soil microorganisms; however, most receptors have not been characterized yet. Therefore, we tried to identify LysM-RLKs from diverse plant species and proposed a new classification to indicate their evolution and characteristics, as well as to predict new functions. In this study, we have attempted to explore and update LysM-RLKs in Lotus japonicus using the latest genome sequencing and divided 20 LysM-RLKs into 11 clades based on homolog identity and phylogenetic analysis. We further identified 193 LysM-RLKs from 16 Spermatophyta species including L. japonicus and divided these receptors into 14 clades and one out-group special receptor based on the classification of L. japonicus LysM-RLKs. All plant species not only have clade I receptors such as Nod factor or chitin receptors but also have clade III receptors where most of the receptors are uncharacterized. We also identified dicotyledon- and monocotyledon-specific clades and predicted evolutionary trends in LysM-RLKs. In addition, we found a strong correlation between plant species that did not possess clade II receptors and those that lost symbiosis with arbuscular mycorrhizal fungi. A clade II receptor in L. japonicus Lys8 was predicted to express during arbuscular mycorrhizal symbiosis. Our proposed new inventory classification suggests the evolutionary pattern of LysM-RLKs and might help in elucidating novel receptor functions in various plant species.

We show that the knockout of a sugar transporter gene OsSWEET15 led to a significant drop in rice fertility with around half of the knockout mutant's spikelets bearing blighted or empty grains. The rest of the spikelets bore fertile grains with a slightly reduced weight. Notably, the ovaries in the blighted grains of the ossweet15 mutants expanded after flowering but terminated their development before the endosperm cellularization stage and subsequently aborted. β- glucuronidase (GUS) and Green Fluorescent Protein (GFP) reporter lines representing the OsSWEET15 expression showed that the gene was expressed in the endosperm tissues surrounding the embryo, which supposedly supplies nutrients to sustain embryo development. These results together with the protein's demonstrated sucrose transport capacity and plasma membrane localization suggest that OsSWEET15 plays a prominent role during the caryopsis formation stage, probably by releasing sucrose from the endosperm to support embryo development. By contrast, the empty grains were probably caused by the reduced pollen viability of the ossweet15 mutants. Investigation of ossweet11 mutant grains revealed similar phenotypes to those observed in the ossweet15 mutants. These results indicate that both OsSWEET15 and OsSWEET11 play important and similar roles in rice pollen development, caryopsis formation and seed-setting, in addition to their function in seed-filling that was demonstrated previously.

Nicotiana benthamiana is widely used as a model plant for dicotyledonous angiosperms. In fact, the strains used in research are highly susceptible to a wide range of viruses. Accordingly, these strains are subject to plant pathology and plant-microbe interactions. In terms of plant-plant interactions, N. benthamiana is one of the plants that exhibit grafting affinity with plants from different families. Thus, N. benthamiana is a good model for plant biology and has been the subject of genome sequencing analyses for many years. However, N. benthamiana has a complex allopolyploid genome, and its previous reference genome is fragmented into 141,000 scaffolds. As a result, molecular genetic analysis is difficult to perform. To improve this effort, de novo whole-genome assembly was performed in N. benthamiana with Hifi reads, and 1,668 contigs were generated with a total length of 3.1 Gb. The 21 longest scaffolds, regarded as pseudomolecules, contained a 2.8-Gb sequence, occupying 95.6% of the assembled genome. A total of 57,583 high-confidence gene sequences were predicted. Based on a comparison of the genome structures between N. benthamiana and N. tabacum, N. benthamiana was found to have more complex chromosomal rearrangements, reflecting the age of interspecific hybridization. To verify the accuracy of the annotations, the cell wall modification genes involved in grafting were analyzed, which revealed not only the previously indeterminate untranslated region, intron and open reading frame sequences but also the genomic locations of their family genes. Owing to improved genome assembly and annotation, N. benthamiana would increasingly be more widely accessible.

Mung bean is an important grain-legume crop and its sprout is an economical and nutrient vegetable for the public, but the genetic regulation of anthocyanin production, which is an antioxidant in mung bean, remains elusive. In our study, we characterized a subgroup (SG) 6 R2R3-MYB anthocyanin activator VrMYB90 and a SG 4 R2R3-MYB anthocyanin repressor VrMYB3, which synergistically function in regulating anthocyanin synthesis with VrbHLHA transcription factor. The overexpressed VrMYB90 protein activates the expression of VrMYB3 and VrbHLHA in mung bean hair roots, and also promotes VrDFR and VrANS transcript levels by directly binding to the corresponding promoters at specific motifs (CAACTG and CCGTTG). VrMYB90 interacts with VrbHLHA to enhance its regulatory activities on VrDFR and VrANS. Furthermore, the interaction between VrMYB3 with VrMYB90 and VrbHLHA could result in the restriction of anthocyanin synthesis to prevent excessive anthocyanin accumulation. Our results demonstrate that the VrMYB90 protein, in conjunction with VrMYB3 and VrbHLHA, forms a key regulatory module to fine-tune anthocyanin synthesis in mung bean.

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming.

A comparative investigation was conducted to evaluate transcriptional changes in guard cells (GCs) of closely related halophytic (Chenopodium quinoa) and glycophytic (Spinacia oleracea) species. Plants were exposed to 3 weeks of 250 mM sodium chloride treatment, and GC-enriched epidermal fragments were mechanically prepared. In both species, salt-responsive genes were mainly related to categories of protein metabolism, secondary metabolites, signal transduction and transport systems. Genes related to abscisic acid (ABA) signaling and ABA biosynthesis were strongly induced in quinoa but not in spinach GCs. Also, expression of the genes encoding transporters of amino acids, proline, sugars, sucrose and potassium increased in quinoa GCs under salinity stress. Analysis of cell-wall-related genes suggests that genes involved in lignin synthesis (e.g. lignin biosynthesis LACCASE 4) were highly upregulated by salt in spinach GCs. In contrast, transcripts related to cell wall plasticity Pectin methylesterase3 (PME3) were highly induced in quinoa. Faster stomatal response to light and dark measured by observing kinetics of changes in stomatal conductance in quinoa might be associated with higher plasticity of the cell wall regulated by PME3 Furthermore, genes involved in the inhibition of stomatal development and differentiation were highly expressed by salt in quinoa, but not in spinach. These changes correlated with reduced stomatal density and index in quinoa, thus improving its water use efficiency. The fine modulation of transporters, cell wall modification and controlling stomatal development in GCs of quinoa may have resulted in high K+/Na+ ratio, lower stomatal conductance and higher stomatal speed for better adaptation to salinity stress in quinoa.

Most vegetative axes remain quiescent as dormant axillary buds until metabolic and hormonal signals, driven by environmental changes, trigger bud outgrowth. While the resumption of growth activity is well documented, the establishment and maintenance of quiescence is comparatively poorly understood, despite its major importance in the adaptation of plants to the seasonal cycle or in the establishment of their shape. Here, using the rosebush Rosa hybrida 'Radrazz' as a plant model, we highlighted that the quiescent state was the consequence of an internal and active energy control of buds, under the influence of hormonal factors previously identified in the bud outgrowth process. We found that the quiescent state in the non-growing vegetative axis of dormant axillary buds displayed a low energy state along with a high expression of the ALTERNATIVE OXIDASE 2 (AOX2) and the accumulation of the corresponding protein. Conversely, AOX2 expression and protein amount strongly decreased during bud burst as energy status shifted to a high state, allowing growth. Since AOX2 can deviate electrons from the cytochrome pathway in the mitochondrial respiratory chain, it could drastically reduce the formation of ATP, which would result in a low energy status unfavorable for growth activities. We provide evidence that the presence/absence of AOX2 in quiescent/growing vegetative axes of buds was under hormonal control and thus may constitute the mechanistic basis of both quiescence and sink strength manifestation, two important aspects of budbreak.