A double-negative feedback loop between miR319c and JAW-TCPs establishes growth pattern in incipient leaf primordia in Arabidopsis thaliana.

The microRNA miR319 and its target JAW-TCP transcription factors regulate the proliferation-to-differentiation transition of leaf pavement cells in diverse plant species. In young Arabidopsis leaf primordia, JAW-TCPs are detected towards the distal region whereas the major mRNA319-encoding gene MIR319C, is expressed at the base. Little is known about how this complementary expression pattern of MIR319C and JAW-TCPs is generated. Here, we show that MIR319C is initially expressed uniformly throughout the incipient primordia and is later abruptly down-regulated at the distal region, with concomitant distal appearance of JAW-TCPs, when leaves grow to ~100 µm long. Loss of JAW-TCPs causes distal extension of the MIR319C expression domain, whereas ectopic TCP activity restricts MIR319C more proximally. JAW-TCPs are recruited to and are capable of depositing histone H3K27me3 repressive marks on the MIR319C chromatin. JAW-TCPs fail to repress MIR319C in transgenic seedlings where the TCP-binding cis-elements on MIR319C are mutated, causing miR319 gain-of-function-like phenotype in the embryonic leaves. Based on these results, we propose a model for growth patterning in leaf primordia wherein MIR319C and JAW-TCPs repress each other and divide the uniformly growing primordia into distal differentiation zone and proximal proliferation domain.

The CIN-TCP transcription factors promote commitment to differentiation in Arabidopsis leaf pavement cells via both auxin-dependent and independent pathways.

Cells in organ primordia undergo active proliferation at an early stage to generate sufficient number, before exiting proliferation and entering differentiation. However, how the actively proliferating cells are developmentally reprogrammed to acquire differentiation potential during organ maturation is unclear. Here, we induced a microRNA-resistant form of TCP4 at various developmental stages of Arabidopsis leaf primordium that lacked the activity of TCP4 and its homologues and followed its effect on growth kinematics. By combining this with spatio-temporal gene expression analysis, we show that TCP4 commits leaf cells within the transition zone to exit proliferation and enter differentiation. A 24-hour pulse of TCP4 activity was sufficient to impart irreversible differentiation competence to the actively dividing cells. A combination of biochemical and genetic analyses revealed that TCP4 imparts differentiation competence by promoting auxin response as well as by directly activating HAT2, a HD-ZIP II transcription factor-encoding gene that also acts downstream to auxin response. Our study offers a molecular link between the two major organ maturation factors, CIN-like TCPs and HD-ZIP II transcription factors and explains how TCP activity restricts the cell number and final size in a leaf.

Lysinibacillus macroides-mediated control of cellulose-producing morphotype of Salmonella.

BACKGROUND: Soil-dwelling human pathogens like Salmonella are transmitted by fresh produce such as tomato, spinach, onion and cabbage. With >2600 serovars, it is difficult to classify the good plant colonizers from the non-colonizers. Generally, soil microbiota are classified as autochthonous or zymogenous organisms, based on their ability to survive in soil. However, such information for soil-dwelling human pathogens is not available Thus there is a need to classify these organisms for designing a strategy to prevent their outbreak. Moreover, soil harbours a plethora of microbes, which can be screened for competitive organisms to control such human pathogens. RESULTS: In this study, we examined whether the morphotype based on the attachment factors (e.g., cellulose and curli fimbri) of Salmonella was important for its colonization of roots. Secondly, we tracked the location of the bacteria in the plant cell. Interestingly, most of the epidermal cells occupied by Salmonella showed propidium iodide-positive nuclei. As an extension of the study, a screening of competitive rhizospheric bacteria was performed. One isolate, identified as Lysinibacillus macroides, was able to inhibit the biofilm of Salmonella and subsequently reduced its colonization on roots. CONCLUSION: Based on this study, we classified the Rdar (red, dry and rough) morphotypes as good plant colonists. The ability to colonize and subsequent kill the live plant cell throws light on the zymogenous life cycle of soil-dwelling Salmonella. Additionally, Lysinibacillus macroides served as a biocontrol agent by reducing the burden of Salmonella in various vegetables. Such organisms can further be explored to prevent contamination of the food chain. © 2022 Society of Chemical Industry.

The Ubiquitin-Specific Protease TNI/UBP14 Functions in Ubiquitin Recycling and Affects Auxin Response.

The ubiquitin-mediated proteasomal pathway regulates diverse cellular processes in plants by rapidly degrading target proteins, including the repressors of hormone signaling. Though ubiquitin proteases play a key role in this process by cleaving polyubiquitin chains to monomers, their function has not been studied in detail by mutational analysis. Here, we show that mutation in TARANI/UBIQUITIN-SPECIFIC PROTEASE14 (TNI/UBP14) leads to reduced auxin response and widespread auxin-related phenotypic defects in Arabidopsis (Arabidopsis thaliana). In a tni partial loss-of-function mutant that was originally isolated based on altered leaf shape, activity of the auxin-responsive reporters DR5::GUS, DR5::nYFP, and IAA2::GUS was reduced. Genetic interaction studies suggest that TNI is involved in auxin signaling and acts alongside TIR1, ARF7, and AUX1 Map-based cloning identified TNI as UBP14 Inefficient splicing of the mutant TNI transcript resulted in the formation of an inactive UBP14 protein, which led to accumulation of polyubiquitin chains and excess polyubiquitinated proteins in the mutant. In addition to the reduced auxin response, increased levels of DII:VENUS, IAA18:GUS, and HS::AXR3-NT:GUS were also observed in tni, perhaps due to inefficient polyubiquitin hydrolysis and proteasome-mediated degradation. Together, our study identifies a function for TNI/UBP14 in the auxin response through ubiquitin recycling.

The TCP4 Transcription Factor Directly Activates TRICHOMELESS1 and 2 and Suppresses Trichome Initiation.

Trichomes are the first line of defense on the outer surface of plants against biotic and abiotic stresses. Because trichomes on leaf surfaces originate from the common epidermal progenitor cells that also give rise to pavement cells and stomata, their density and distribution are under strict genetic control. Regulators of trichome initiation have been identified and incorporated into a biochemical pathway wherein an initiator complex promotes trichome fate in an epidermal progenitor cell, while an inhibitor complex suppresses it in the neighboring cells. However, it is unclear how these regulator proteins, especially the negative regulators, are induced by upstream transcription factors and integrated with leaf morphogenesis. Here, we show that the Arabidopsis (Arabidopsis thaliana) class II TCP proteins activate TRICHOMELESS1 (TCL1) and TCL2, the two established negative regulators of trichome initiation, and reduce trichome density on leaves. Loss-of-function of these TCP proteins increased trichome density whereas TCP4 gain-of-function reduced trichome number. TCP4 binds to the upstream regulatory elements of both TCL1 and TCL 2 and directly promotes their transcription. Further, the TCP-induced trichome suppression is independent of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE family of transcription factors, proteins that also reduce trichome density at later stages of plant development. Our work demonstrates that the class II TCP proteins couple leaf morphogenesis with epidermal cell fate determination.

The TCP4 transcription factor regulates trichome cell differentiation by directly activating GLABROUS INFLORESCENCE STEMS in Arabidopsis thaliana.

Trichomes are the first cell type to be differentiated during the morphogenesis of leaf epidermis and serve as an ideal model to study cellular differentiation. Many genes involved in the patterning and differentiation of trichome cells have been studied over the past decades, and the majority of these genes encode transcription factors that specifically regulate epidermal cell development. However, the upstream regulators of these genes that link early leaf morphogenesis with cell type differentiation are less studied. The TCP proteins are the plant-specific transcription factors involved in regulating diverse aspects of plant development including lateral organ morphogenesis by modulating cell proliferation and differentiation. Here, we show that the miR319-regulated class II TCP proteins, notably TCP4, suppress trichome branching in Arabidopsis leaves and inflorescence stem by direct transcriptional activation of GLABROUS INFLORESCENCE STEMS (GIS), a known negative regulator of trichome branching. The trichome branch number is increased in plants with reduced TCP activity and decreased in the gain-of-function lines of TCP4. Biochemical analyses show that TCP4 binds to the upstream regulatory region of GIS and activates its expression. Detailed genetic analyses show that GIS and TCP4 work in same pathway and GIS function is required for TCP4-mediated regulation of trichome differentiation. Taken together, these results identify a role for the class II TCP genes in trichome differentiation, thus providing a connection between organ morphogenesis and cellular differentiation.

VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2 Interact with CONSTANS and Promote Photoperiodic Flowering Transition.

In plants, endogenous and environmental signals such as light control the timing of the transition to flowering. Two phytochrome B-interacting transcription factors, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2, redundantly promote flowering in Arabidopsis (Arabidopsis thaliana). In the voz1 voz2 mutant, the expression of FLOWERING LOCUS C (FLC) was up-regulated and that of FLOWERING LOCUS T (FT) was down-regulated, which was proposed to be the cause of late flowering in voz1 voz2 However, the detailed mechanism by which the VOZ genes promote flowering is not well understood. Here, we show that neither the reduced FT expression nor the late-flowering phenotype of voz1 voz2 is suppressed in the voz1 voz2 flc triple mutant. Genetic interaction experiments between voz1 voz2 and constans-2 (co-2) mutants reveal that the VOZs and CO work in the same genetic pathway. Using in vitro pull-down, electrophoretic mobility shift, and bimolecular fluorescence complementation assays, we show that VOZ1 and VOZ2 interact with CO. The voz1 voz2 35S::CO:YFP plants show suppression of the early-flowering phenotype induced by CO overexpression, suggesting that CO requires VOZ for the induction of flowering. Determination of the VOZ consensus-binding site followed by genome-wide sequence analysis failed to identify any VOZ-binding sites near known flowering time genes. Together, these results indicate that the VOZ genes regulate flowering primarily through the photoperiod pathway, independent of FLC, and suggest that VOZs modulate CO function to promote flowering.

Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis.

Cell expansion is an essential process in plant morphogenesis and is regulated by the coordinated action of environmental stimuli and endogenous factors, such as the phytohormones auxin and brassinosteroid. Although the biosynthetic pathways that generate these hormones and their downstream signaling mechanisms have been extensively studied, the upstream transcriptional network that modulates their levels and connects their action to cell morphogenesis is less clear. Here, we show that the miR319-regulated TCP (TEOSINTE BRANCHED1, CYCLODEA, PROLIFERATING CELL FACTORS) transcription factors, notably TCP4, directly activate YUCCA5 transcription and integrate the auxin response to a brassinosteroid-dependent molecular circuit that promotes cell elongation in Arabidopsis thaliana hypocotyls. Furthermore, TCP4 modulates the common transcriptional network downstream to auxin-brassinosteroid signaling, which is also triggered by environmental cues, such as light, to promote cell expansion. Our study links TCP function with the hormone response during cell morphogenesis and shows that developmental and environmental signals converge on a common transcriptional network to promote cell elongation.

Root mediated uptake of Salmonella is different from phyto-pathogen and associated with the colonization of edible organs.

BACKGROUND: Pre-harvest contamination of fruits and vegetables by Salmonella in fields is one of the causes of food-borne outbreaks. Natural openings like stomata, hydathodes and fruit cracks are known to serve as entry points. While there are reports indicating that Salmonella colonize and enter root through lateral root emerging area, further investigations regarding how the accessibility of Salmonella to lateral root is different from phyto-pathogenic bacteria, the efficacy of lateral root to facilitate entry have remained unexplored. In this study we attempted to investigate the lateral root mediated entry of Salmonella, and to bridge this gap in knowledge. RESULTS: Unlike phytopathogens, Salmonella cannot utilize cellulose as the sole carbon source. This negates the fact of active entry by degrading plant cellulose and pectin. Endophytic Salmonella colonization showed a high correlation with number of lateral roots. When given equal opportunity to colonize the plants with high or low lateral roots, Salmonella internalization was found higher in the plants with more lateral roots. However, the epiphytic colonization in both these plants remained unaltered. To understand the ecological significance, we induced lateral root production by increasing soil salinity which made the plants susceptible to Salmonella invasion and the plants showed higher Salmonella burden in the aerial organs. CONCLUSION: Salmonella, being unable to degrade plant cell wall material relies heavily on natural openings. Therefore, its invasion is highly dependent on the number of lateral roots which provides an entry point because of the epidermis remodeling. Thus, when number of lateral root was enhanced by increasing the soil salinity, plants became susceptible to Salmonella invasion in roots and its transmission to aerial organs.

CIN-TCP transcription factors: Transiting cell proliferation in plants.

Leaves are the most conspicuous planar organs in plants, designed for efficient capture of sunlight and its conversion to energy that is channeled into sustaining the entire biosphere. How a few founder cells derived from the shoot apical meristem give rise to diverse leaf forms has interested naturalists and developmental biologists alike. At the heart of leaf morphogenesis lie two simple cellular processes, division and expansion, that are spatially and temporally segregated in a developing leaf. In leaves of dicot model species, cell division occurs predominantly at the base, concomitant with the expansion and differentiation of cells at the tip of the lamina that drives increase in leaf surface area. The timing of the transition from one cell fate (division) to the other (expansion) within a growing leaf lamina is a critical determinant of final leaf shape, size, complexity and flatness. The TCP proteins, unique to plant kingdom, are sequence-specific DNA-binding transcription factors that control several developmental and physiological traits. A sub-group of class II TCPs, called CINCINNATA-like TCPs (CIN-TCPs henceforth), are key regulators of the timing of the transition from division to expansion in dicot leaves. The current review highlights recent advances in our understanding of how the pattern of CIN-TCP activity is translated to the dynamic spatio-temporal control of cell-fate transition through the transactivation of cell-cycle regulators, growth-repressing microRNAs, and interactions with the chromatin remodeling machinery to modulate phytohormone responses. Unravelling how environmental inputs influence CIN-TCP-mediated growth control is a challenge for future studies. © 2018 IUBMB Life, 70(8):718-731, 2018.

Divergence in Patterns of Leaf Growth Polarity Is Associated with the Expression Divergence of miR396.

Lateral appendages often show allometric growth with a specific growth polarity along the proximo-distal axis. Studies on leaf growth in model plants have identified a basipetal growth direction with the highest growth rate at the proximal end and progressively lower rates toward the distal end. Although the molecular mechanisms governing such a growth pattern have been studied recently, variation in leaf growth polarity and, therefore, its evolutionary origin remain unknown. By surveying 75 eudicot species, here we report that leaf growth polarity is divergent. Leaf growth in the proximo-distal axis is polar, with more growth arising from either the proximal or the distal end; dispersed with no apparent polarity; or bidirectional, with more growth contributed by the central region and less growth at either end. We further demonstrate that the expression gradient of the miR396-GROWTH-REGULATING FACTOR module strongly correlates with the polarity of leaf growth. Altering the endogenous pattern of miR396 expression in transgenic Arabidopsis thaliana leaves only partially modified the spatial pattern of cell expansion, suggesting that the diverse growth polarities might have evolved via concerted changes in multiple gene regulatory networks.

Organocatalytic Asymmetric Tamura Cycloaddition with α- Branched Nitroolefins: Synthesis of Functionalized 1-Tetralones.

A new catalytic asymmetric Tamura cycloaddition with nitroolefins was developed. This demonstration of the reaction of α-branched nitroolefins with homophthalic anhydrides delivers highly functionalized 1-tetralone compounds. With bifunctional squaramide catalyst, the desired tetralone products are obtained with high enantioselectivity and good diastereoselectivity.

The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division.

The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs.

Organocatalytic asymmetric Michael addition of 1-acetylcyclohexene and 1-acetylcyclopentene to nitroolefins.

Enantioselective organocatalytic Michael addition reactions of 1-acetylcyclohexene, 1-acetylcyclopentene and 1-acetylcyclobutene to nitroolefins have been developed. This is the first report where an α-branched enone has been activated by an amine catalyst for the asymmetric Michael addition reaction to an electrophile. The Michael products have also been cyclized to bicyclic compounds.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling.

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth.

Identification of specific DNA binding residues in the TCP family of transcription factors in Arabidopsis.

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an approximately 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix (bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Role of lactoyl-glutathione lyase of Salmonella in the colonization of plants under salinity stress.

Salmonella, a foodborne human pathogen, can colonize the members of the kingdom Plantae. However, the basis of the persistence of Salmonella in plants is largely unknown. Plants encounter various biotic and abiotic stress agents in soil. We conjectured that methylglyoxal (MG), one of the common metabolites that accumulate in plants during both biotic and abiotic stress, plays a role in regulating the plant-Salmonella interaction. The interaction of Salmonella Typhimurium with plants under salinity stress was investigated. It was observed that wild-type Salmonella Typhimurium can efficiently colonize the root, but mutant bacteria lacking MG detoxifying enzyme, lactoyl-glutathione lyase (Lgl), showed lower colonization in roots exclusively under salinity stress. This colonization defect is due to the poor viability of the mutated bacterial strains under these conditions. This is the first report to prove the role of MG-detoxification genes in the colonization of stressed plants and highlights the possible involvement of metabolic genes in the evolution of the plant-associated life of Salmonella.

Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation.

Plant organs are initiated as primordial outgrowths, and require controlled cell division and differentiation to achieve their final size and shape. Superimposed on this is another developmental program that orchestrates the switch from vegetative to reproductive to senescence stages in the life cycle. These require sequential function of heterochronic regulators. Little is known regarding the coordination between organ and organismal growth in plants. The TCP gene family encodes transcription factors that control diverse developmental traits, and a subgroup of class II TCP genes regulate leaf morphogenesis. Absence of these genes results in large, crinkly leaves due to excess division, mainly at margins. It has been suggested that these class II TCPs modulate the spatio-temporal control of differentiation in a growing leaf, rather than regulating cell proliferation per se. However, the link between class II TCP action and cell growth has not been established. As loss-of-function mutants of individual TCP genes in Arabidopsis are not very informative due to gene redundancy, we generated a transgenic line that expressed a hyper-activated form of TCP4 in its endogenous expression domain. This resulted in premature onset of maturation and decreased cell proliferation, leading to much smaller leaves, with cup-shaped lamina in extreme cases. Further, the transgenic line initiated leaves faster than wild-type and underwent precocious reproductive maturation due to a shortened adult vegetative phase. Early senescence and severe fertility defects were also observed. Thus, hyper-activation of TCP4 revealed its role in determining the timing of crucial developmental events, both at the organ and organism level.

CINCINNATA-Like TCP Transcription Factors in Cell Growth - An Expanding Portfolio.

Post-mitotic cell growth is a key process in plant growth and development. Cell expansion drives major growth during morphogenesis and is influenced by both endogenous factors and environmental stimuli. Though both isotropic and anisotropic cell growth can contribute to organ size and shape at different degrees, anisotropic cell growth is more likely to contribute to shape change. While much is known about the mechanisms that increase cellular turgor and cell-wall biomass during expansion, the genetic factors that regulate these processes are less studied. In the past quarter of a century, the role of the CINCINNATA-like TCP (CIN-TCP) transcription factors has been well documented in regulating diverse aspects of plant growth and development including flower asymmetry, plant architecture, leaf morphogenesis, and plant maturation. The molecular activity of the CIN-TCP proteins common to these biological processes has been identified as their ability to suppress cell proliferation. However, reports on their role regulating post-mitotic cell growth have been scanty, partly because of functional redundancy among them. In addition, it is difficult to tease out the effect of gene activity on cell division and expansion since these two processes are linked by compensation, a phenomenon where perturbation in proliferation is compensated by an opposite effect on cell growth to keep the final organ size relatively unaltered. Despite these technical limitations, recent genetic and growth kinematic studies have shown a distinct role of CIN-TCPs in promoting cellular growth in cotyledons and hypocotyls, the embryonic organs that grow solely by cell expansion. In this review, we highlight these recent advances in our understanding of how CIN-TCPs promote cell growth.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed.