Chemical and Physical Ablation of Epidermal Cells

Cell ablation has been widely used to study plant development, particularly development of the accessible epidermal cell layer. Although these techniques are often extreme and cause obvious physical damage and stress, they have nonetheless provided interesting information about the roles of epidermal tissues at various stages in development. Genetic ablation of proto-dermal cells in young Arabidopsis embryos by expression of Diphtheria Toxin A under an epidermis-specific promoter leads to developmental arrest of the embryo proper at the early globular stage, suggesting a role for the protoderm in promoting continuing embryogenesis (Weijers et al. 2003). Ablation of pro-todermal cells in cotyledons during later embryogenesis seriously disrupts cotyledon development, consistent with a role in cotyledon morphogenesis and expansion. Likewise post-embryonic ablation of L1 cells in leaves using BARNASE expression causes major disruption in leaf development, characterised by the production of small unexpanded organs which tend to be thicker (in terms of cell numbers) than wild-type organs (Baroux et al. 2001). Thus, in agreement with the findings of chimera studies, it appears possible that the L1 is important for expressing information required for the correct outgrowth of the lamina as well as for organ expansion. However, when expressing toxins within developing tissues it is always possible that they may move to adjacent cells causing them to behave aberrantly. In addition the presence of the protoderm might be needed for the correct specification of underlying cell-layers, and thus growth defects could be a secondary effect of cell-specification defects.

Physical ablation of the epidermal cells of plant embryos is technically very challenging, although studies using cultured embryos of Citrus jamb-hiri (Bruck 1985a,b) have provided several interesting observations which will be discussed in more depth later. In general, physical ablation of epidermal cells in organ primordia is difficult to carry out over large enough areas to allow the examination of epidermal roles in growth control, and the physical properties of the scar tissues which form at wound sites also complicate interpretation. However, laser ablation studies in meristems of tomato have suggested a role of the meristematic L1 layer in controlling meristem growth. For example, ablating the meristematic L1 layer causes a dramatic change in the behaviour of underlying (L2) cells which cease to divide anti-

clinally and commence periclinal divisions, suggesting that the presence of the L1 cell layer restricts division planes in underlying cell layers (Reinhardt et al. 2003a). This observation is interesting in light of the models proposed by Wegner, although the assumption that epidermal cells all have develop-mentally similar properties becomes important when interpreting this type of study. Differences between concentric zones of the meristem might also be relevant. In the centre of the angiosperm meristem (central zone) a group of self-maintaining, slowly dividing stem cells produce progeny which enter a more rapidly proliferating peripheral zone, prior to being incorporated into organ primordia or stem tissues (Grandjean et al. 2004; Carraro et al. 2006). The molecular mechanism underlying the maintenance of the stem cell population in the central zone is relatively well characterised and hinges on expression of a putative peptide ligand (CLAVATA3) in the central zone tunica cells in response to the activity of the transcription factor WUSCHEL in the lower corpus. Perception of CLV3 is thought to negatively regulate WUS expression via the Receptor-Like Kinase CLV1 and associated factors, restricting the size of the central zone (reviewed in Doerner 2003; Williams and Fletcher 2005). Bearing this in mind, the question then arises whether the phenotype caused by ablating the L1 of the CZ is specifically due to removal of the epidermal layer, or to loss of CLV3 expressing cells which could cause a developmental reprogramming of underlying cells. Interestingly, changes in the orientation of cell divisions from anticlinal to periclinal in underlying cell layers of root meristems upon chemical ablation of the root epidermis have also been noted (Baroux et al. 2001), suggesting that one of the intrinsic properties of the L1 is indeed to restrict cell division planes in underlying cell layers. Additionally, loss of L1 cells either through ablation or in mutants with decreased epidermal specification leads to premature differentiation of meristem tissue, thus, although CLV3 signalling from the tunica acts to repress the size of the stem cell population, the presence of the L1 appears to be intrinsically necessary for the very existence of stem cells (Baroux et al. 2001; Abe et al. 2003; Johnson et al. 2005).

In addition to its role in meristem activity, the L1 appears to be necessary for the inception of organ primordia at the shoot apex. Thus, physical removal of the L1 layer causes an immediate cessation in organ initiation at the site of removal, even though underlying cells are still able to divide and expand normally (Reinhardt et al. 2003a). Reinhard et al. argue that this is unlikely to be due to an immediate loss of meristem identity as meristematic markers are lost only slowly from "skinned" meristems. One possible explanation is that removing the epidermis perturbs auxin transport. Polar auxin transport, which is now widely accepted to cause localised increases in auxin activity that promote organ formation, appears to be largely restricted to the L1 cell layer in the shoot meristem (discussed below). Another possible explanation, is that the L1 cell layer is key in the perception of signals required for organ initiation (again, possibly auxin, but equally possibly other signals).

The role of the L1-specific WOX proteins in recruiting subsets of meristematic cells to organ primordia (again discussed below) may support this view.

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