Cytokinin signaling is involved in root hair elongation in response to phosphate starvation.

Root hair, single-celled tubular structures originating from the epidermis, plays a vital role in the uptake of nutrients from the soil by increasing the root surface area. Therefore, optimizing root hair growth is crucial for plants to survive in fluctuating environments. Root hair length is determined by the action of various plant hormones, among which the roles of auxin and ethylene have been extensively studied. However, evidence for the involvement of cytokinins has remained elusive. We recently reported that the cytokinin-activated B-type response regulators, ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12 directly upregulate the expression of ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4), which encodes a key transcription factor that controls root hair elongation. However, depending on the nutrient availability, it is unknown whether the ARR1/12-RSL4 pathway controls root hair elongation. This study shows that phosphate deficiency induced the expression of RSL4 and increased the root hair length through ARR1/12, though the transcript and protein levels of ARR1/12 did not change. These results indicate that cytokinins, together with other hormones, regulate root hair growth under phosphate starvation conditions.

The canonical E2Fs together with RETINOBLASTOMA-RELATED are required to establish quiescence during plant development.

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth.

Arabidopsis thaliana Subclass I ACTIN DEPOLYMERIZING FACTORs Regulate Nuclear Organization and Gene Expression.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs is downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. Arabidopsis thaliana possesses eight ACTIN (ACT) genes, among which ACT2, -7 and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACT regulate nuclear organization and gene expression is discussed.

Cytokinin signaling promotes root hair growth by directly regulating RSL4 expression.

Root hairs are single-celled tubular structures produced from the epidermis, which play an essential role in water and nutrient uptake from the soil. Therefore, root hair formation and elongation are controlled not only by developmental programs but also by environmental factors, enabling plants to survive under fluctuating conditions. Phytohormones are key signals that link environmental cues to developmental programs; indeed, root hair elongation is known to be controlled by auxin and ethylene. Another phytohormone, cytokinin, also affects root hair growth, while whether cytokinin is actively involved in root hair growth and, if so, how it regulates the signaling pathway governing root hair development have remained unknown. In this study, we show that the two-component system of cytokinin, which involves the B-type response regulators ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12, promotes the elongation process of root hairs. They directly up-regulate ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) encoding a basic helix-loop-helix (bHLH) transcription factor that plays a central role in root hair growth, whereas the ARR1/12-RSL4 pathway does not crosstalk with auxin or ethylene signaling. These results indicate that cytokinin signaling constitutes another input onto the regulatory module governed by RSL4, making it possible to fine-tune root hair growth in changing environments.

At the Nexus between Cytoskeleton and Vacuole: How Plant Cytoskeletons Govern the Dynamics of Large Vacuoles.

Large vacuoles are a predominant cell organelle throughout the plant body. They maximally account for over 90% of cell volume and generate turgor pressure that acts as a driving force of cell growth, which is essential for plant development. The plant vacuole also acts as a reservoir for sequestering waste products and apoptotic enzymes, thereby enabling plants to rapidly respond to fluctuating environments. Vacuoles undergo dynamic transformation through repeated enlargement, fusion, fragmentation, invagination, and constriction, eventually resulting in the typical 3-dimensional complex structure in each cell type. Previous studies have indicated that such dynamic transformations of plant vacuoles are governed by the plant cytoskeletons, which consist of F-actin and microtubules. However, the molecular mechanism of cytoskeleton-mediated vacuolar modifications remains largely unclear. Here we first review the behavior of cytoskeletons and vacuoles during plant development and in response to environmental stresses, and then introduce candidates that potentially play pivotal roles in the vacuole-cytoskeleton nexus. Finally, we discuss factors hampering the advances in this research field and their possible solutions using the currently available cutting-edge technologies.

MYB3R-mediated and cell cycle-dependent transcriptional regulation of a tobacco ortholog of SCARECROW-LIKE28 in synchronized cultures of BY-2 cells.

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.

MYB3R-SCL28-SMR module with a role in cell size control negatively regulates G2 progression in Arabidopsis.

Cell size control is one of the prerequisites for plant growth and development. Recently, a GRAS family transcription factor, SCARECROW-LIKE28 (SCL28), was identified as a critical regulator for both mitotic and postmitotic cell-size control. Here, we show that SCL28 is specifically expressed in proliferating cells and exerts its function to delay G2 progression during mitotic cell cycle in Arabidopsis thaliana. Overexpression of SCL28 provokes a significant enlargement of cells in various organs and tissues, such as leaves, flowers and seeds, to different extents depending on the type of cells. The increased cell size is most likely due to a delayed G2 progression and accelerated onset of endoreplication, an atypical cell cycle repeating DNA replication without cytokinesis or mitosis. Unlike DWARF AND LOW-TILLERING, a rice ortholog of SCL28, SCL28 may not have a role in brassinosteroid (BR) signaling because sensitivity against brassinazole, a BR biosynthesis inhibitor, was not dramatically altered in scl28 mutant and SCL28-overexpressing plants. Collectively, our findings strengthen a recently proposed model of cell size control by SCL28 and suggest the presence of diversified evolutionary mechanisms for the regulation and action of SCL28.

Members of SIAMESE-RELATED Class Inhibitor Proteins of Cyclin-Dependent Kinase Retard G2 Progression and Increase Cell Size in Arabidopsis thaliana.

Cell size requires strict and flexible control as it significantly impacts plant growth and development. Unveiling the molecular mechanism underlying cell size control would provide fundamental insights into plants' nature as sessile organisms. Recently, a GRAS family transcription factor SCARECROW-LIKE28 (SCL28) was identified as a determinant of cell size in plants; specifically, SCL28 directly induces a subset of SIAMESE-RELATED (SMR) family genes encoding plant-specific inhibitors of cyclin-dependent kinases (i.e., SMR1, SMR2, SMR6, SMR8, SMR9, SMR13, and SMR14), thereby slowing down G2 phase progression to provide the time to increase cell volume. Of the SMR genes regulated by SCL28, genetic analysis has demonstrated that SMR1, SMR2, and SMR13 cooperatively regulate the cell size downstream of SCL28 in roots and leaves, whereas other SMR members' contribution remains unexplored. This study shows that in root meristematic cells, SMR9 redundantly participates in cell size control with SMR1, SMR2, and SMR13. Moreover, our cell cycle analysis provides the first experimental evidence that SMR proteins inhibit the G2 progression of proliferating cells. Overall, these findings illuminate the diverse yet overlapping roles of SMR proteins in cell cycle regulation while reinforcing that SMRs are essential downstream effectors of SCL28 to modulate G2 progression and cell size.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis.

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Genome Maintenance Mechanisms at the Chromatin Level.

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure "chromatin". Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.

Effectiveness of Create ML in microscopy image classifications: a simple and inexpensive deep learning pipeline for non-data scientists.

Observing chromosomes is a time-consuming and labor-intensive process, and chromosomes have been analyzed manually for many years. In the last decade, automated acquisition systems for microscopic images have advanced dramatically due to advances in their controlling computer systems, and nowadays, it is possible to automatically acquire sets of tiling-images consisting of large number, more than 1000, of images from large areas of specimens. However, there has been no simple and inexpensive system to efficiently select images containing mitotic cells among these images. In this paper, a classification system of chromosomal images by deep learning artificial intelligence (AI) that can be easily handled by non-data scientists was applied. With this system, models suitable for our own samples could be easily built on a Macintosh computer with Create ML. As examples, models constructed by learning using chromosome images derived from various plant species were able to classify images containing mitotic cells among samples from plant species not used for learning in addition to samples from the species used. The system also worked for cells in tissue sections and tetrads. Since this system is inexpensive and can be easily trained via deep learning using scientists' own samples, it can be used not only for chromosomal image analysis but also for analysis of other biology-related images.

The DREAM complex represses growth in response to DNA damage in Arabidopsis.

The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage.

Identification of two tobacco genes encoding MYB3R proteins with repressor function and showing cell cycle-regulated transcript accumulation.

MYB3R family transcription factors play a central role in the regulation of G2/M-specific gene transcription in Arabidopsis thaliana. Among the members of this family, MYB3R3 and MYB3R5 are structurally closely related and are involved in the transcriptional repression of target genes in both proliferating and quiescent cells. This type of MYB3R repressor is widespread in plants; however, apart from the studies on MYB3Rs in Arabidopsis thaliana, little information about them is available. Here we isolated tobacco cDNA clones encoding two closely related MYB3R proteins designated as NtmybC1 and NtmybC2 and determined the nucleotide sequences of the entire coding regions. Phylogenetic analysis suggested that NtmybC1 and NtmybC2 can be grouped into a conserved subfamily of plant MYB3Rs that also contains MYB3R3 and MYB3R5. When transiently expressed in protoplasts prepared from tobacco BY-2 cells, NtmybC1 and NtmybC2 repressed the activity of target promoters and blocked promoter activation mediated by NtmybA2, a MYB3R activator from tobacco. Unlike MYB3R3 and MYB3R5, NtmybC1 and NtmybC2 showed cell cycle-regulated transcript accumulation. In synchronized cultures of BY-2 cells, mRNAs for both NtmybC1 and NtmybC2 were preferentially expressed during the G2 and M phases, coinciding with the expression of NtmybA2 and G2/M-specific target genes. These results not only broadly confirm our fundamental view that this type of MYB3R protein acts as transcriptional repressor of G2/M-specific genes but also suggest a possible divergence of MYB3R repressors in terms of the mechanisms of their action and regulation.

Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roots.

This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2'-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.

MYB3R-mediated active repression of cell cycle and growth under salt stress in Arabidopsis thaliana.

Under environmental stress, plants are believed to actively repress their growth to save resource and alter its allocation to acquire tolerance against the stress. Although a lot of studies have uncovered precise mechanisms for responding to stress and acquiring tolerance, the mechanisms for regulating growth repression under stress are not as well understood. It is especially unclear which particular genes related to cell cycle control are involved in active growth repression. Here, we showed that decreased growth in plants exposed to moderate salt stress is mediated by MYB3R transcription factors that have been known to positively and negatively regulate the transcription of G2/M-specific genes. Our genome-wide gene expression analysis revealed occurrences of general downregulation of G2/M-specific genes in Arabidopsis under salt stress. Importantly, this downregulation is significantly and universally mitigated by the loss of MYB3R repressors by mutations. Accordingly, the growth performance of Arabidopsis plants under salt stress is significantly recovered in mutants lacking MYB3R repressors. This growth recovery involves improved cell proliferation that is possibly due to prolonging and accelerating cell proliferation, which were partly suggested by enlarged root meristem and increased number of cells positive for CYCB1;1-GUS. Our ploidy analysis further suggested that cell cycle progression at the G2 phase was delayed under salt stress, and this delay was recovered by loss of MYB3R repressors. Under salt stress, the changes in expression of MYB3R activators and repressors at both the mRNA and protein levels were not significant. This observation suggests novel mechanisms underlying MYB3R-mediated growth repression under salt stress that are different from the mechanisms operating under other stress conditions such as DNA damage and high temperature.

CDKD-dependent activation of CDKA;1 controls microtubule dynamics and cytokinesis during meiosis.

Precise control of cytoskeleton dynamics and its tight coordination with chromosomal events are key to cell division. This is exemplified by formation of the spindle and execution of cytokinesis after nuclear division. Here, we reveal that the central cell cycle regulator CYCLIN DEPENDENT KINASE A;1 (CDKA;1), the Arabidopsis homologue of Cdk1 and Cdk2, partially in conjunction with CYCLIN B3;1 (CYCB3;1), is a key regulator of the microtubule cytoskeleton in meiosis. For full CDKA;1 activity, the function of three redundantly acting CDK-activating kinases (CAKs), CDKD;1, CDKD;2, and CDKD;3, is necessary. Progressive loss of these genes in combination with a weak loss-of-function mutant in CDKA;1 allowed a fine-grained dissection of the requirement of cell-cycle kinase activity for meiosis. Notably, a moderate reduction of CDKA;1 activity converts the simultaneous cytokinesis in Arabidopsis, i.e., one cytokinesis separating all four meiotic products concurrently into two successive cytokineses with cell wall formation after the first and second meiotic division, as found in many monocotyledonous species.

ABA inhibits root cell elongation through repressing the cytokinin signaling.

Cell elongation, which plays an important role in root penetration into the soil, responds to a variety of environmental factors. A previous study demonstrated that abscisic acid, a phytohormone involved in stress responses, inhibits root growth by delaying the onset of cell elongation. In contrast, we recently reported that cytokinins promote elongation of root cells by enhancing actin bundling. However, the control of root cell elongation through the interaction between abscisic acid and cytokinin signaling has not yet been uncovered. Here, we show that abscisic acid-induced delay in cell elongation requires inhibition of cytokinin signaling; further, stress is signaled to cell elongation by the pathway mediated by B-type ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), which retards root growth.

Actin Reorganization Triggers Rapid Cell Elongation in Roots.

Root growth is controlled by mechanisms underlying cell division and cell elongation, which respond to various internal and external factors. In Arabidopsis (Arabidopsis thaliana), cells produced in the proximal meristem (PM) elongate and differentiate in the transition zone (TZ) and the elongation/differentiation zone (EDZ). Previous studies have demonstrated that endoreplication is involved in root cell elongation; however, the manner by which cells increase in length by more than 2-fold remains unknown. Here, we show that epidermal and cortical cells in Arabidopsis roots undergo two modes of rapid cell elongation: the first rapid cell elongation occurs at the border of the proximal meristem and the TZ, and the second mode occurs during the transition from the TZ to the EDZ. Our previous study showed that cytokinin signaling promotes endoreplication, which triggers the first rapid cell elongation. Our cytological and genetic data revealed that the second rapid cell elongation involves dynamic actin reorganization independent of endoreplication. Cytokinins promote actin bundling and the resultant second rapid cell elongation through activating the signaling pathway involving the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE3 (AHK3) and AHK4 and the B-type transcription factor ARABIDOPSIS RESPONSE REGULATOR2. Our results suggest that cytokinins promote the two modes of rapid cell elongation by controlling distinct cellular events: endoreplication and actin reorganization.

Arabidopsis R1R2R3-Myb proteins are essential for inhibiting cell division in response to DNA damage.

Inhibition of cell division is an active response to DNA damage that enables cells to maintain genome integrity. However, how DNA damage arrests the plant cell cycle is largely unknown. Here, we show that the repressor-type R1R2R3-Myb transcription factors (Rep-MYBs), which suppress G2/M-specific genes, are required to inhibit cell division in response to DNA damage. Knockout mutants are resistant to agents that cause DNA double-strand breaks and replication stress. Cyclin-dependent kinases (CDKs) can phosphorylate Rep-MYBs in vitro and are involved in their proteasomal degradation. DNA damage reduces CDK activities and causes accumulation of Rep-MYBs and cytological changes consistent with cell cycle arrest. Our results suggest that CDK suppressors such as CDK inhibitors are not sufficient to arrest the cell cycle in response to DNA damage but that Rep-MYB-dependent repression of G2/M-specific genes is crucial, indicating an essential function for Rep-MYBs in the DNA damage response.Inhibition of cell division maintains genome integrity in response to DNA damage. Here Chen et al. propose that DNA damage causes cell cycle arrest in the Arabidopsis root via Rep-MYB transcription factor-mediated repression of G2/M-specific gene expression in response to reduced cyclin-dependent kinase activity.