Chimeric identities and reduced stiffness characterise the shoot apex of Arabidopsis stem cell mutants

Abstract Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3, produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. Compromised CLAVATA activity leads to massive meristem overgrowth, which is thought to be caused by stem cell overproliferation. However, it is unknown how uncontrolled stem cell divisions lead to the specific changes observed in clavata mutants. Here we first quantitatively characterise these mutants, to reveal underlying tissue curvature defects. We use analytical models to show how perturbed mechanical properties and/or growth rates may contribute to altered meristem morphology. Indeed, we find that clavata meristems are softer than the wild type, and that stereotypical meristem organisation is lost, with cells instead simultaneously expressing multiple domain markers. Furthermore, we show that mutant meristematic cells are auxin-responsive, suggesting that they are functionally different from wild-type stem cells. We propose that the clavata phenotype is not caused by stem cell overproliferation, but rather by the disruption of a more complex regulatory framework that is key to maintaining distinct genetic and functional domains at the shoot apex. Summary statement Mechanical, genetic and functional evidence supported by theoretical models call into question the current definition of stem cells in the shoot apex

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants

International audienceABSTRACT Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis

International audienceThe shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition

Evidence for the Regulation of Gynoecium Morphogenesis by ETTIN via Cell Wall Dynamics

International audienceBackground and Aims Plant stature and shape are largely determined by cell elongation, a process that is strongly controlled at the level of the cell wall. This is associated with the presence of many cell wall proteins implicated in the elongation process. Several proteins and enzyme families have been suggested to be involved in the controlled weakening of the cell wall, and these include xyloglucan endotransglucosylases/hydrolases (XTHs), yieldins, lipid transfer proteins and expansins. Although expansins have been the subject of much research, the role and involvement of expansin-like genes/proteins remain mostly unclear. This study investigates the expression and function of AtEXLA2 (At4g38400), a member of the expansin-like A (EXLA) family in arabidposis, and considers its possible role in cell wall metabolism and growth. Methods Transgenic plants of Arabidopsis thaliana were grown, and lines over-expressing AtEXLA2 were identified. Plants were grown in the dark, on media containing growth hormones or precursors, or were gravistimulated. Hypocotyls were studied using transmission electron microscopy and extensiometry. Histochemical GUS (beta-glucuronidase) stainings were performed. Key Results AtEXLA2 is one of the three EXLA members in arabidopsis. The protein lacks the typical domain responsible for expansin activity, but contains a presumed cellulose-interacting domain. Using promoter::GUS lines, the expression of AtEXLA2 was seen in germinating seedlings, hypocotyls, lateral root cap cells, columella cells and the central cylinder basally to the elongation zone of the root, and during different stages of lateral root development. Furthermore, promoter activity was detected in petioles, veins of leaves and filaments, and also in the peduncle of the flowers and in a zone just beneath the papillae. Over-expression of AtEXLA2 resulted in an increase of > 10 % in the length of dark-grown hypocotyls and in slightly thicker walls in non-rapidly elongating etiolated hypocotyl cells. Biomechanical analysis by creep tests showed that AtEXLA2 over-expression may decrease the wall strength in arabidopsis hypocotyls. Conclusions It is concluded that AtEXLA2 may function as a positive regulator of cell elongation in the dark-grown hypocotyl of arabidopsis by possible interference with cellulose metabolism, deposition or its organization

Expression of cell-wall related genes is highly variable and correlates with sepal morphology

Control of organ morphology is a fundamental feature of living organisms. There is, however, observable variation in organ size and shape within a given genotype. Taking the sepal of Arabidopsis as a model, we investigated whether we can use variability of gene expression alongside variability of organ morphology to identify gene regulatory networks potentially involved in organ size and shape determination. We produced a dataset composed of morphological parameters and genome-wide transcriptome obtained from 27 individual sepals from wild-type plants with nearly identical genetic backgrounds, environment, and developmental stage. Sepals exhibited appreciable variability in both morphology and transcriptome, with response to stimulus genes and cell-wall related genes displaying high variability in expression. We additionally identified five modules of co-expressed genes which correlated significantly with morphology, revealing biologically relevant gene regulatory networks. Interestingly, cell-wall related genes were overrepresented in two of the top three modules. Overall, our work highlights the benefit of using coupled variation in gene expression and phenotype in wild-type plants to shed light on the mechanisms underlying organ size and shape determination. Although causality between gene expression and sepal morphology has not been established, our approach opens the way to informed analysis for mutant characterization and functional studies

Spatial consistency of cell growth direction during organ morphogenesis requires CELLULOSE SYNTHASE INTERACTIVE1

International audienceExtracellular matrices contain fibril-like polymers often organized in parallel arrays. Although their role in morphogenesis has been long recognized, it remains unclear how the subcellular control of fibril synthesis translates into organ shape. We address this question using the Arabidopsis sepal as a model organ. In plants, cell growth is restrained by the cell wall (extracellular matrix). Cellulose microfibrils are the main load-bearing wall component, thought to channel growth perpendicularly to their main orientation. Given the key function of CELLULOSE SYNTHASE INTERACTIVE1 (CSI1) in guidance of cellulose synthesis, we investigate the role of CSI1 in sepal morphogenesis. We observe that sepals from csi1 mutants are shorter, although their newest cellulose microfibrils are more aligned compared to wild-type. Surprisingly, cell growth anisotropy is similar in csi1 and wild-type plants. We resolve this apparent paradox by showing that CSI1 is required for spatial consistency of growth direction across the sepal

Robustness of organ morphology is associated with modules of co-expressed genes related to plant cell wall

Reproducibility in organ size and shape is a fundamental trait of living organisms. Themechanisms underlying such robustness remain, however, to be elucidated. Taking the sepal ofArabidopsis as a model, we investigated whether variability of gene expression plays a role invariation of organ morphology. To address this question, we produced a dataset composed ofboth transcriptomic and morphological information obtained from 27 individual sepals fromwild-type plants. Although nearly identical in their genetic background, environment, anddevelopmental stage, these sepals exhibited appreciable variability in both morphology andtranscriptome. We identified modules of co-expressed genes in sepals, three of whichcorrelated significantly with morphology, revealing biologically relevant gene regulatorynetworks. Interestingly, cell-wall related genes were overrepresented in two of these threemodules. Additionally, we found that highly variable genes were unexpectedly enriched incell-wall related processes. We then analyzed sepal morphology from 16 cell-wall mutants andfound that the more a gene is expressed in wild-type, the more variable the morphology of thecorresponding mutant. Altogether, our work unravels the contribution of cell-wall related genesto the robustness of sepal morphology. More generally, we propose that canalizing traits duringdevelopment could rely on the modulation of highly expressed genes

Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation

International audienceAbstract Pectin methylesterases (PMEs) modify homogalacturonan’s chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55–70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties