LONGIFOLIA1 and LONGIFOLIA2, two homologous genes, regulate longitudinal cell elongation in Arabidopsis.

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2,in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis.     

拟南芥YUAN1基因调控器官形状和大小

器官形状和大小的控制是一个基本的发育生物学过程, 受细胞分裂和细胞扩展的影响。到目前为止, 人们对植物器官形状和大小的调控机制知之甚少。本实验室前期研究发现了一个种子和器官大小的调控基因DA1, 其编码一个泛素受体。在拟南芥(Arabidopsis thaliana)中, DA1通过抑制细胞的分裂来限制种子和器官的大小。本研究通过激活标签的方法在da1-1突变体背景下筛选到一个叶子形状发生改变的半显性突变体(yuan1-1D)。yuan1-1D形成短而圆的叶片和短的叶柄, 细胞学分析显示, 叶片和叶柄变短的主要原因是细胞的长向扩展降低导致的。YUAN1编码一个含有PHD锌指结构域的蛋白。GFP-YUAN1融合蛋白定位在细胞核内。过量表达YUAN1基因导致叶片和叶柄变短。遗传学分析显示, YUAN1和DA1、ROT3以及ROT4在控制叶片形状和大小方面作用于不同的遗传途径中。因此, 本研究鉴定了一个新的控制器官形状和大小的基因YUAN1, 为阐明植物器官形状和大小调控的分子机制提供了重要线索。     

miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.     

Characterization of the AE7 gene in Arabidopsis suggests that normal cell proliferation is essential for leaf polarity establishment

The formation of leaf polarity is critical for leaf morphogenesis. In this study, we characterized and cloned an Arabidopsis gene, AS1/2 ENHANCER7 (AE7), which is required for both leaf adaxial-abaxial polarity formation and normal cell proliferation. The ae7 mutant exhibited leaf adaxial-abaxial polarity defects and double mutants combining ae7 with the leaf polarity mutants as1 (asymmetric leaves1), as2, rdr6 (RNA-dependent RNA polymerase6) or ago7/zip (argonaute7/zippy) all resulted in plants with an apparently enhanced loss of adaxial leaf identity. In addition, ae7 also showed decreased cell proliferation in both leaves and roots, compensated by increased cell sizes in leaves. AE7 encodes a protein conserved in many eukaryotic organisms, ranging from unicellular yeasts to humans; however, the functions of AE7 family members from other species have not been reported. In situ hybridization revealed that AE7 is expressed in a spotted pattern in plant tissues, similar to cell-cycle marker genes such as HISTONE4. Moreover, the ae7 endoploidy and expression analysis of several cell-cycle marker genes in ae7 suggest that the AE7 gene is required for cell cycle progression. As the previously characterized 26S proteasome and ribosome mutants also affect both leaf adaxial-abaxial polarity and cell proliferation, similar to the defects in ae7, we propose that normal cell proliferation may be essential for leaf polarity establishment. Possible models for how cell proliferation influences leaf adaxial-abaxial polarity establishment are discussed.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.     

Targeted manipulation of leaf form via local growth repression

A classical view is that leaf shape is the result of local promotion of growth linked to cell proliferation. However, an alternative hypothesis is that leaf form is the result of local repression of growth in an otherwise growing system. Here we show that leaf form can indeed be manipulated in a directed fashion by local repression of growth. We show that targeting expression of an inhibitor of a cyclin-dependent kinase (KRP1) to the sinus area of developing leaves of Arabidopsis leads to local growth repression and the formation of organs with extreme lobing, including generation of leaflet-like organs. Directing KRP1 expression to other regions of the leaf using an miRNA target sequence tagging approach also leads to predictable novel leaf forms, and repression of growth in the leaf margin blocks the outgrowth of lobes, leading to a smoother perimeter. In addition, we show that decreased growth around the perimeter and across the leaf abaxial surface leads to a change in 3D form, as predicted by mechanical models of leaf growth. Our analysis provides experimental evidence that local repression of growth influences leaf shape, suggesting that it could be part of the mechanism of morphogenesis in plants in the context of an otherwise growing system.     

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells.     

AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development

The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.     

Developmental genetics of leaf morphogenesis in dicotyledonous plants

A full understanding of the leaf is essential for a full understanding of plant morphology. However, leaf morphogenesis is still poorly understood, in particular in dicotyledonous plants, because of the complex nature of the development of leaves. Mutational analysis seems to be the most suitable strategy for investigations of such processes, and should allow us to dissect the developmental pathways into genetically programmed unit processes. The techniques of developmental genetics have been applied to the study of leaf morphogenesis in model plants, such asArabidopsis thaliana, and several key processes in leaf morphogenesis have been identified. The fundamental processes in leaf morphogenesis include the identification of leaf organs, determination of leaf primordia (occurrence of marginal meristem), and the polar or non-polar elongation of leaf cells. This review will focus on the genes that are essential for these processes and have been identified in mutational analyses. Mutational analyses of the photomorphogenesis is also briefly summarized from the perspective of the plasticity of leaf morphogenesis.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

The COBRA family of putative GPI-anchored proteins in Arabidopsis. A new fellowship in expansion

Identification of regulatory molecules that determine the extent and direction of expansion is necessary to understand how cell morphogenesis is controlled in plants. We recently identified COB (COBRA) as a key regulator of the orientation of cell expansion in the root. Analysis of the Arabidopsis genome sequence indicated that COB belongs to a multigene family consisting of 12 members, all predicted to encode glycosylphosphatidylinositol-anchored proteins. All but two of the COBL (COB-like) genes are expressed in most organs examined, suggesting possible redundancy. Sequence comparisons, phylogenetic analyses, and exon-intron positions revealed that the COB family is composed of two main subgroups sharing a common architecture, one subgroup being characterized by an additional N-terminal domain. Identification of expressed sequence tags corresponding to potential orthologs in other plant species suggested that COB-related functions are required in all vascular plants. Together, these results indicate that COB family members are likely to be important new players at the plasma membrane-cell wall interface.     

Myrosin Cell Development Is Regulated by Endocytosis Machinery and PIN1 Polarity in Leaf Primordia of Arabidopsis thaliana

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.     

SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana [corrected

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22 degrees C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.     

Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of Arabidopsis cotyledon pavement cells

In most dicotyledonous plants, leaf pavement cells exhibit complex jigsaw puzzle-like cell morphogenesis during leaf expansion. Although detailed molecular biological information and mathematical modeling of this jigsaw puzzle-like cell morphogenesis are now available, a full understanding of this process remains elusive. Recent reports have highlighted the importance of three-dimensional (3D) structures (i.e., anticlinal and periclinal cell wall) in understanding the mechanical models that describe this morphogenetic process. We believe that it is important to acquire 3D shapes of pavement cells over time, i.e., acquire and analyze four-dimensional (4D) information when studying the relationship between mechanical modeling and simulations and the actual cell shape. In this report, we have developed a framework to capture and analyze 4D morphological information of Arabidopsis thaliana cotyledon pavement cells by using both direct water immersion observations and computational image analyses, including segmentation, surface modeling, virtual reality and morphometry. The 4D cell models allowed us to perform time-lapse 3D morphometrical analysis, providing detailed quantitative information about changes in cell growth rate and shape, with cellular complexity observed to increase during cell growth. The framework should enable analysis of various phenotypes (e.g., mutants) in greater detail, especially in the 3D deformation of the cotyledon surface, and evaluation of theoretical models that describe pavement cell morphogenesis using computational simulations. Additionally, our accurate and high-throughput acquisition of growing cell structures should be suitable for use in generating in silico model cell structures.     

Genetic control of petiole length in Arabidopsis thaliana

Shade-avoidance syndrome is characterized by the formation of elongated petioles and unexpanded leaf blades under low-intensity light, but the genetic basis for these responses is unknown. In this study, two-dimensional mutational analysis revealed that the gene for phytochrome B, PHYB, had opposing effects in the leaf petioles and leaf blades of Arabidopsis, while the ROT3, ACL2, and GAI genes influenced the length of leaf petioles more significantly than the length of leaf blades. Anatomical analysis revealed that the PHYB and ACL2 genes control the length of leaf petioles exclusively via control of the length of individual cells, while the GAI, GA1 and ROT3 genes appeared to control both the elongation and proliferation of petiole cells, in particular, under strong light. By contrast, both the size and the number of cells were affected by the mutations examined in leaf blades. The differential control of leaf petiole length and leaf blade expansion is discussed.