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The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge on the APC/C by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis (Arabidopsis thaliana) plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits the growth of ccs52a2-1 plants, indicating the need for control of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its systemic overexpression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and the phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionarily conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific protein with a role in cell division that is likely proteolytically controlled by the CCS52A2-activated APC/C., Competing Interests: Conflict of interest statement. The authors have no conflict of interest., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the main photosynthetic instruments of vascular plants, leaves are crucial and complex plant organs. A strict organization of leaf mesophyll and epidermal cell layers orchestrates photosynthesis and gas exchange. In addition, water and nutrients for leaf growth are transported through the vascular tissue. To establish the single-cell transcriptomic landscape of these different leaf tissues, we performed high-throughput transcriptome sequencing of individual cells isolated from young leaves of Arabidopsis (Arabidopsis thaliana) seedlings grown in two different environmental conditions. The detection of approximately 19,000 different transcripts in over 1,800 high-quality leaf cells revealed 14 cell populations composing the young, differentiating leaf. Besides the cell populations comprising the core leaf tissues, we identified subpopulations with a distinct identity or metabolic activity. In addition, we proposed cell-type-specific markers for each of these populations. Finally, an intuitive web tool allows for browsing the presented dataset. Our data present insights on how the different cell populations constituting a developing leaf are connected via developmental, metabolic, or stress-related trajectories., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The rapid appearance of herbicide-resistant weeds combined with a lack of novel herbicides being brought to market reduces crop production, thereby threatening food security worldwide. Here, we report on the use of the previously identified cellulose biosynthesis-inhibiting chemical compound C17 as a potential herbicide. Toxicity tests showed that C17 efficiently inhibits the growth of various weeds and widely cultivated dicotyledonous crops, whereas only slight or no growth inhibition was observed for monocotyledonous crops. Surprisingly, when exposed to a mixture of C17 and one of two well-known cellulose biosynthesis inhibitors (CBIs), isoxaben and indaziflam, an additive growth inhibition was observed, demonstrating that C17 has a different mode of action that can be used to sensitize plants toward known CBIs. Moreover, we demonstrate that a C17-resistant CESA3 allele can be used as a positive transformation selection marker and that C17 resistance can be obtained through genome engineering of the wild-type CESA3 allele using clustered regularly interspaced short palindromic repeats-mediated base editing. This editing system allowed us to engineer C17 tolerance in an isoxaben-resistant line, resulting in double herbicide-resistant plants., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Wounding is a primary trigger of organ regeneration, but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study, we combined transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis ( Arabidopsis thaliana ). Our time course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes, starting from rapid stress responses followed by the activation of metabolic processes and protein synthesis and subsequent activation of cell cycle regulators. Gene ontology analyses further uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signaling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin is critical for wound-induced callus formation. We further demonstrate that type-B ARABIDOPSIS RESPONSE REGULATOR-mediated cytokinin signaling regulates the expression of CYCLIN D3;1 ( CYCD3;1 ) and that mutations in CYCD3;1 and its homologs CYCD3;2 and 3 cause defects in callus formation. In addition to these hormone-mediated changes, our transcriptome data uncovered that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 and PLETHORA3 (PLT3), PLT5, and PLT7 in callus generation. All together, these results provide novel mechanistic insights into how wounding reactivates cell proliferation during callus formation., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis ( Arabidopsis thaliana ), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/C CCS52A1 and APC/C CCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/C CCS52A1 and APC/C CCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/C CCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The stem cell niche of the Arabidopsis (Arabidopsis thaliana) primary root apical meristem is composed of the quiescent (or organizing) center surrounded by stem (initial) cells for the different tissues. Initial cells generate a population of transit-amplifying cells that undergo a limited number of cell divisions before elongating and differentiating. It is unclear whether these divisions occur stochastically or in an orderly manner. Using the thymidine analog 5-ethynyl-2'-deoxyuridine to monitor DNA replication of cells of Arabidopsis root meristems, we identified a pattern of two, four, and eight neighboring cells with synchronized replication along the cortical, epidermal, and endodermal cell files, suggested to be daughters, granddaughters, and great-granddaughters of the direct progeny of each stem cell. Markers of mitosis and cytokinesis were not present in the region closest to the transition zone where the cells start to elongate, suggesting that great-granddaughter cells switch synchronously from the mitotic cell cycle to endoreduplication. Mutations in the stem cell niche-expressed ASH1-RELATED3 (ASHR3) gene, encoding a SET-domain protein conferring histone H3 lysine-36 methylation, disrupted this pattern of coordinated DNA replication and cell division and increased the cell division rate in the quiescent center. E2Fa/E2Fb transcription factors controlling the G1-to-S-phase transition regulate ASHR3 expression and bind to the ASHR3 promoter, substantiating a role for ASHR3 in cell division control. The reduced length of the root apical meristem and primary root of the mutant ashr3-1 indicate that synchronization of replication and cell divisions is required for normal root growth and development., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation.

Enormous progress has been achieved understanding the molecular mechanisms regulating endoreduplication. By contrast, how this process is coordinated with the cell cycle or cell expansion and contributes to overall growth in multicellular systems remains unclear. A holistic approach was used here to give insight into the functional links between endoreduplication, cell division, cell expansion, and whole growth in the Arabidopsis (Arabidopsis thaliana) leaf. Correlative analyses, quantitative genetics, and structural equation modeling were applied to a large data set issued from the multiscale phenotyping of 200 genotypes, including both genetically modified lines and recombinant inbred lines. All results support the conclusion that endoreduplication in leaf cells could be controlled by leaf growth itself. More generally, leaf growth could act as a "hub" that drives cell division, cell expansion, and endoreduplication in parallel. In many cases, this strategy allows compensations that stabilize leaf area even when one of the underlying cellular processes is limiting.

Endoreduplication represents a variation on the cell cycle in which multiple rounds of DNA replication occur without subsequent chromosome separation and cytokinesis, thereby increasing the cellular DNA content. It is known that the DNA ploidy level of cells is controlled by external stimuli such as light; however, limited knowledge is available on how environmental signals regulate the endoreduplication cycle at the molecular level. Previously, we had demonstrated that the conversion from a mitotic cell cycle into an endoreduplication cycle is controlled by the atypical E2F transcription factor, DP-E2F-LIKE1 (DEL1), that represses the endocycle onset. Here, the Arabidopsis (Arabidopsis thaliana) DEL1 gene was identified as a transcriptional target of the classical E2Fb and E2Fc transcription factors that antagonistically control its transcript levels through competition for a single E2F cis-acting binding site. In accordance with the reported opposite effects of light on the protein levels of E2Fb and E2Fc, DEL1 transcription depended on the light regime. Strikingly, modified DEL1 expression levels uncoupled the link between light and endoreduplication in hypocotyls, implying that DEL1 acts as a regulatory connection between endocycle control and the photomorphogenic response.

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the vacuolar soluble N-ethylmaleimide-sensitive factor attachment receptor protein VAMP2. However, little is known about PRA1 family members in plants. Sequence analysis revealed that higher plants, compared with animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis (Arabidopsis thaliana) PRA1 (AtPRA1) proteins were found to homodimerize and heterodimerize in a manner corresponding to their phylogenetic distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly coexpressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins, probably dealing with the various demands of the complex trafficking system.

Organ growth results from the progression of component cells through subsequent phases of proliferation and expansion before reaching maturity. We combined kinematic analysis, flowcytometry, and microarray analysis to characterize cell cycle regulation during the growth process of leaves 1 and 2 of Arabidopsis (Arabidopsis thaliana). Kinematic analysis showed that the epidermis proliferates until day 12; thereafter, cells expand until day 19 when leaves reach maturity. Flowcytometry revealed that endoreduplication occurs from the time cell division rates decline until the end of cell expansion. Analysis of 10 time points with a 6k-cDNA microarray showed that transitions between the growth stages were closely reflected in the mRNA expression data. Subsequent genome-wide microarray analysis on the three main stages allowed us to categorize known cell cycle genes into three major classes: constitutively expressed, proliferative, and inhibitory. Comparison with published expression data obtained from root zones corresponding to similar developmental stages and from synchronized cell cultures supported this categorization and enabled us to identify a high confidence set of 131 proliferation genes. Most of those had an M phase-dependent expression pattern and, in addition to many known cell cycle-related genes, there were at least 90 that were unknown or previously not associated with proliferation.