Chemical synthesis of the EPF-family of plant cysteine-rich proteins and late-stage dye attachment by chemoselective amide-forming ligations.

Chemical protein synthesis can provide well-defined modified proteins. Herein, we report the chemical synthesis of plant-derived cysteine-rich secretory proteins and late-stage derivatization of the synthetic proteins. The syntheses were achieved with distinct chemoselective amide bond forming reactions - EPF2 by native chemical ligation (NCL), epidermal patterning factor (EPF) 1 by the α-ketoacid-hydroxylamine (KAHA) ligation, and fluorescent functionalization of their folded variants by potassium acyltrifluoroborate (KAT) ligation. The chemically synthesized EPFs exhibit bioactivity on stomatal development in Arabidopsis thaliana. Comprehensive synthesis of EPF derivatives allowed us to identify suitable fluorescent variants for bioimaging of the subcellar localization of EPFs.

Illuminating the molecular mechanisms underlying shoot apical meristem homeostasis in plants.

Unlike animals, terrestrial plants are sessile and able to give rise to new organs throughout their lifetime. In the most extreme cases, they can survive for over a thousand years. With such protracted life cycles, plants have evolved sophisticated strategies to adapt to variable environments by coordinating their morphology as well as their growth, and have consequently acquired a high degree of developmental plasticity, which is supported by small groups of long-lived stem cells found in proliferative centers called meristems. Shoot apical meristems (SAMs) contain multipotent stem cells and provide a microenvironment that ensures both a self-renewable reservoir, to produce primordia and sustain growth, and a differentiating population that develops into all of the above-ground organs of land plants. The homeodomain transcription factor WUSCHEL (WUS) is expressed in the organizing center and acts as a master regulator to govern shoot stem cell homeostasis. In this review, I highlight recent advances in our understanding of the molecular mechanisms and signaling networks that underlie SAM maintenance, and discuss how plants utilize WUS to integrate intrinsic and extrinsic cues.

Regulation of the Plant Cell Cycle in Response to Hormones and the Environment.

Developmental and environmental signals converge on cell cycle machinery to achieve proper and flexible organogenesis under changing environments. Studies on the plant cell cycle began 30 years ago, and accumulated research has revealed many links between internal and external factors and the cell cycle. In this review, we focus on how phytohormones and environmental signals regulate the cell cycle to enable plants to cope with a fluctuating environment. After introducing key cell cycle regulators, we first discuss how phytohormones and their synergy are important for regulating cell cycle progression and how environmental factors positively and negatively affect cell division. We then focus on the well-studied example of stress-induced G2 arrest and view the current model from an evolutionary perspective. Finally, we discuss the mechanisms controlling the transition from the mitotic cycle to the endocycle, which greatly contributes to cell enlargement and resultant organ growth in plants.

CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots.

KEY MESSAGE: This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots. Plants sense environmental stimuli and convert them into cellular signals, which are transmitted to distinct cells and tissues to induce adequate responses. Plant hormones and small secretory peptides often function as environmental stress mediators. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED proteins, CLE1-CLE7, which share closely related CLE domains, mediate environmental stimuli in Arabidopsis thaliana. Expression analysis of CLE1-CLE7 revealed that these genes respond to different environmental stimuli, such as nitrogen deprivation, nitrogen replenishment, cold, salt, dark, and sugar starvation, in a sophisticated manner. To further investigate the function of CLE2, we generated transgenic Arabidopsis lines expressing the ß-glucuronidase gene under the control of the CLE2 promoter or expressing the CLE2 gene under the control of an estradiol-inducible promoter. We also generated cle2-1 and cle2-2 mutants using the CRISPR/Cas9 technology. In these transgenic lines, dark induced the expression of CLE2 in the root vasculature. Additionally, induction of CLE2 in roots induced the expression of various genes not only in roots but also in shoots, and genes related to light-dependent carbohydrate metabolism were particularly induced in shoots. In addition, cle2 mutant plants showed chlorosis when subjected to a shade treatment. These results suggest that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots.

Topology of regulatory networks that guide plant meristem activity: similarities and differences.

Plants adapt their morphology in response to variable environmental conditions such as nitrate availability, drought, and temperature shifts. Three crucial aspects to this developmental plasticity are the control of initiation, identity and activity of meristems. At the cellular level, the activity of meristems is controlled by balancing self-renewal in stem cells, amplifying divisions in their daughter cells, and cell differentiation. Recent studies in plants have uncovered transcription factors regulating meristem activity at cellular resolution, and regulatory networks that couple these factors with phytohormone signalling for global plant growth regulation. Here, we highlight selected recent advances in our understanding of the multidimensional transcriptional networks that regulate meristem activity and discuss emerging insights on how a selection of environmental cues impinges on these networks.

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules.

Continuous formation of somatic tissues in plants requires functional stem cell niches where undifferentiated cells are maintained. In Arabidopsis thaliana, PLETHORA (PLT) and SCARECROW (SCR) genes are outputs of apical-basal and radial patterning systems, and both are required for root stem cell specification and maintenance. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene is specifically expressed in and required for functions of a small group of root stem cell organizer cells, also called the quiescent center (QC). PLT and SCR are required for QC function, and their expression overlaps in the QC; however, how they specify the organizer has remained unknown. We show that PLT and SCR genetically and physically interact with plant-specific teosinte-branched cycloidea PCNA (TCP) transcription factors to specify the stem cell niche during embryogenesis and maintain organizer cells post-embryonically. PLT-TCP-SCR complexes converge on PLT-binding sites in the WOX5 promoter to induce expression.

The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement.

Intercellular signaling through trafficking of regulatory proteins is a widespread phenomenon in plants and can deliver positional information for the determination of cell fate. In the Arabidopsis root meristem, the cell fate determinant SHORT-ROOT (SHR), a GRAS domain transcription factor, acts as a signaling molecule from the stele to the adjacent layer to specify endodermal cell fate. Upon exiting the stele, SHR activates another GRAS domain transcription factor, SCARCROW (SCR), which, together with several BIRD/INDETERMINATE DOMAIN proteins, restricts movement of SHR to define a single cell layer of endodermis. Here we report that endodermal cell fate also requires the joint activity of both SCR and its closest homologue SCARECROW-LIKE23 (SCL23). We show that SCL23 protein moves with zonation-dependent directionality. Within the meristem, SCL23 exhibits short-ranged movement from ground tissue to vasculature. Away from the meristem, SCL23 displays long-range rootward movement into meristematic vasculature and a bidirectional radial spread, respectively. As a known target of SHR and SCR, SCL23 also interacts with SCR and SHR and can restrict intercellular outspread of SHR without relying on nuclear retention as SCR does. Collectively, our data show that SCL23 is a mobile protein that controls movement of SHR and acts redundantly with SCR to specify endodermal fate in the root meristem.

Mathematical modeling and experimental validation of the spatial distribution of boron in the root of Arabidopsis thaliana identify high boron accumulation in the tip and predict a distinct root tip uptake function.

Boron, an essential micronutrient, is transported in roots of Arabidopsis thaliana mainly by two different types of transporters, BORs and NIPs (nodulin26-like intrinsic proteins). Both are plasma membrane localized, but have distinct transport properties and patterns of cell type-specific accumulation with different polar localizations, which are likely to affect boron distribution. Here, we used mathematical modeling and an experimental determination to address boron distributions in the root. A computational model of the root is created at the cellular level, describing the boron transporters as observed experimentally. Boron is allowed to diffuse into roots, in cells and cell walls, and to be transported over plasma membranes, reflecting the properties of the different transporters. The model predicts that a region around the quiescent center has a higher concentration of soluble boron than other portions. To evaluate this prediction experimentally, we determined the boron distribution in roots using laser ablation-inductivity coupled plasma-mass spectrometry. The analysis indicated that the boron concentration is highest near the tip and is lower in the more proximal region of the meristem zone, similar to the pattern of soluble boron distribution predicted by the model. Our model also predicts that upward boron flux does not continuously increase from the root tip toward the mature region, indicating that boron taken up in the root tip is not efficiently transported to shoots. This suggests that root tip-absorbed boron is probably used for local root growth, and that instead it is the more mature root regions which have a greater role in transporting boron toward the shoots.

Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis.

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

Control of cell division and transcription by cyclin-dependent kinase-activating kinases in plants.

Cyclin-dependent protein kinases (CDKs) play key roles in the progression of the cell cycle in eukaryotes. A CDK-activating kinase (CAK) catalyzes the phosphorylation of CDKs to activate their enzyme activity; thus, it is involved in activation of cell proliferation. In plants, two distinct classes of CAK have been identified; CDKD is functionally related to vertebrate-type CAKs, while CDKF is a plant-specific CAK having unique enzymatic characteristics. Recently, CDKF was shown to phosphorylate and activate CDKDs in Arabidopsis. This led to a proposal that CDKD and CDKF constitute a phosphorylation cascade that mediates environmental or hormonal signals to molecular machineries that control the cell cycle and transcription. In this review, we have summarized the biochemical features of plant CAKs and discussed the manner in which they diverge from animal and yeast orthologs. We have introduced several transgenic studies in which CAK genes were used as a tool to modify the CDK activity and to analyze cell division and differentiation during organ development.

The plant-specific kinase CDKF;1 is involved in activating phosphorylation of cyclin-dependent kinase-activating kinases in Arabidopsis.

Cyclin-dependent kinases (CDKs) play essential roles in coordinate control of cell cycle progression. Activation of CDKs requires interaction with specific cyclin partners and phosphorylation of their T-loops by CDK-activating kinases (CAKs). The Arabidopsis thaliana genome encodes four potential CAKs. CAK2At (CDKD;3) and CAK4At (CDKD;2) are closely related to the vertebrate CAK, CDK7/p40MO15; they interact with cyclin H and phosphorylate CDKs, as well as the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. CAK1At (CDKF;1) shows cyclin H-independent CDK-kinase activity and can activate a heterologous CAK, Mcs6, in fission yeast. In Arabidopsis, CAK1At is a subunit of a protein complex of 130 kD, which phosphorylates the T-loop of CAK2At and CAK4At and activates the CTD-kinase activity of CAK4At in vitro and in root protoplasts. These results suggest that CAK1At is a novel CAK-activating kinase that modulates the activity of CAK2At and CAK4At, thereby controlling CDK activities and basal transcription in Arabidopsis.

Differential phosphorylation activities of CDK-activating kinases in Arabidopsis thaliana.

Activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). Here we isolated an Arabidopsis cDNA (CAK4At) whose predicted product shows a high similarity to vertebrate CDK7/p40(MO15). Northern blot analysis showed that expressions of the four Arabidopsis CAKs (CAK1At-CAK4At) were not dependent on cell division. CAK2At- and CAK4At-immunoprecipitates of Arabidopsis crude extract phosphorylated CDK and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II with different preferences. These results suggest the existence of differential mechanisms in Arabidopsis that control CDK and CTD phosphorylation by multiple CAKs.