Alexis Peaucelle

MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems.

We have analysed the role of a microRNA, miR164, in boundary formation during organ initiation from Arabidopsis meristems. The establishment and maintenance of the boundary domain are controlled by three partially redundant genes, CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3. We show that miR164 overexpression phenocopies the cuc1 cuc2 double mutant by inducing post-transcriptional downregulation of CUC1 and CUC2 but not CUC3 mRNA levels. Disruption of CUC2 regulation by miR164, either by making CUC2 resistant to the miRNA or by reducing miRNA levels leads to similar enlarged boundary domains. We relate this enlargement to the division patterns of the boundary cells. We propose that miR164 constrains the expansion of the boundary domain, by degrading CUC1 and CUC2 mRNAs.

The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis.

CUP-SHAPED COTYLEDON1 (CUC1), CUC2, and CUC3 define the boundary domain around organs in the Arabidopsis thaliana meristem. CUC1 and CUC2 transcripts are targeted by a microRNA (miRNA), miR164, encoded by MIR164A, B, and C. We show that each MIR164 is transcribed to generate a large population of primary miRNAs of variable size with a locally conserved secondary structure around the pre-miRNA. We identified mutations in the MIR164A gene that deepen serration of the leaf margin. By contrast, leaves of plants overexpressing miR164 have smooth margins. Enhanced leaf serration was observed following the expression of an miR164-resistant CUC2 but not of an miR164-resistant CUC1. Furthermore, CUC2 inactivation abolished serration in mir164a mutants and the wild type, whereas CUC1 inactivation did not. Thus, CUC2 specifically controls leaf margin development. CUC2 and MIR164A are transcribed in overlapping domains at the margins of young leaf primordia, with transcription gradually restricted to the sinus, where the leaf margins become serrated. We suggest that leaf margin development is controlled by a two-step process in Arabidopsis. The pattern of serration is determined first, independently of CUC2 and miR164. The balance between coexpressed CUC2 and MIR164A then determines the extent of serration.

Pectin-Induced Changes in Cell Wall Mechanics Underlie Organ Initiation in Arabidopsis

Tissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1-4] and may have an equally important role in plants [5-9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers-the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.

The presence of diffuse morphogen gradients in tissues supports a view in which growth is locally homogenous. Here we challenge this view: we used a high-resolution quantitative approach to reveal significant growth variability among neighboring cells in the shoot apical meristem, the plant stem cell niche. This variability was strongly decreased in a mutant impaired in the microtubule-severing protein katanin. Major shape defects in the mutant could be related to a local decrease in growth heterogeneity. We show that katanin is required for the cells competence to respond to the mechanical forces generated by growth. This provides the basis for a model in which microtubule dynamics allow the cell to respond efficiently to mechanical forces. This in turn can amplify local growth-rate gradients, yielding more heterogeneous growth and supporting morphogenesis.

Arabidopsis Phyllotaxis Is Controlled by the Methyl-Esterification Status of Cell-Wall Pectins

Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. HG is deposited in the cell wall in a highly (70%-80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

Interplay of miR164, CUP‐SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana

Aerial architecture in higher plants is established post-embryonically by the inception of new meristems in the axils of leaves. These axillary meristems develop into side shoots or flowers. In Arabidopsis, the NAC domain transcription factors CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 function redundantly in initiating the shoot apical meristem and establishing organ boundaries. Transcripts of CUC1 and CUC2 are targeted for degradation by miR164. In this study, we show that cuc3-2 mutants are impaired in axillary meristem initiation. Overexpression of miR164 in the cuc3-2 mutant caused an almost complete block of axillary meristem formation. Conversely, mir164 mutants and plants harbouring miR164-resistant alleles of CUC1 or CUC2 developed accessory buds in leaf axils. Collectively, these experiments reveal that, in addition to CUC3, redundant functions of CUC1 and CUC2 as well as miR164 regulation are required for the establishment of axillary meristems. Studies on LAS transcript accumulation in mir164 triple mutants and cuc3-2 plants overexpressing miR164 suggest that regulation of axillary meristem formation by miR164 is mediated through CUC1 and CUC2, which in turn regulate LAS.

Cell wall mechanics and growth control in plants: the role of pectins revisited

How is the extensibility of growing plant cell walls regulated? In the past, most studies have focused on the role of the cellulose/xyloglucan network and the enigmatic wall-loosening agents expansins. Here we review first how in the closest relatives of the land plants, the Charophycean algae, cell wall synthesis is coupled to cell wall extensibility by a chemical Ca2+-exchange mechanism between Ca2+–pectate complexes. We next discuss evidence for the existence in terrestrial plants of a similar “primitive” Ca2+–pectate-based growth control mechanism in parallel to the more recent, land plant-specific, expansin-dependent process.

Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin.

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.

Plants expressing a miR164-resistant CUC2 gene reveal the importance of post-meristematic maintenance of phyllotaxy in Arabidopsis

In plants, the arrangement of organs along the stem (phyllotaxy) follows a predictable pattern. Recent studies have shown that primordium position at the meristem is governed by local auxin gradients, but little is known about the subsequent events leading to the phyllotaxy along the mature stem. We show here that plants expressing a miR164-resistant CUP-SHAPED COTYLEDON2 (CUC2) gene have an abnormal phyllotactic pattern in the fully grown stem, despite the pattern of organ initiation by the meristem being normal. This implies that abnormal phyllotaxy is generated during stem growth. These plants ectopically express CUC2 in the stem, suggesting that the proper timing of CUC2 expression is required to maintain the pattern initiated in the meristem. Furthermore, by carefully comparing the phyllotaxy in the meristem and along the mature inflorescence in wild types, we show that such deviation also occurs during wild-type development, although to a smaller extent. We therefore suggest that the phyllotactic pattern in a fully grown stem results not only from the organogenetic activity of the meristem, but also from the subsequent growth pattern during stem development.

Cell differentiation and organ initiation at the shoot apical meristem

Plants continuously generate organs at the flanks of their shoot apical meristems (SAMs). The patterns in which these organs are initiated, also called patterns of phyllotaxis, are highly stereotypic and characteristic for a particular species or developmental stage. This stable, predictable behaviour of the meristem has led to the idea that organ initiation must be based on simple and robust mechanisms. This conclusion is less evident, however, if we consider the very dynamic behaviour of the individual cells. How dynamic cellular events are coordinated and how they are linked to the regular patterns of organ initiation is a major issue in plant developmental biology.