Figure 8.7 Radial patterning of the ground tissue in the root meristem. (A) SHR mRNA localization (left), SHR protein (middle) and SCR mRNA (right). (B) Intercellular signalling mechanism in radial pattern formation of wild type (left) and pSCR::SHR transgenic plants (right). In wild type, SHR protein moves from the stele to a single adjacent layer. In the adjacent layer it activates SCR transcription, which is essential for the asymmetric cell divisions that form the cortex and endodermis. In the mature region SHR presence in the adjacent layer confers endodermal cell fate. How SHR protein movement is restricted to a single cell layer is as yet unknown. In pSCR::SHR transgenic plants (right) the SCR promoter is repeatedly activated in adjacent layers by the production of SHR protein from the pSCR::SHR transgene, resulting in supernumerary layers due to cortex-endodermis initial daughter cell (Ceid) divisions and the acquisition of endodermal fate in these new layers. Abbreviations for the cell types are given in Figure 8.4A. Adapted from Nakajima et al. (2001).
2000). SCR expression is down-regulated in the shr background, indicating that SHR is upstream of SCR. On the other hand, ectopic SHR expression under the 35S promoter results in supernumerary cell divisions and altered cell specification including ectopic SCR expression, indicating that SHR is both necessary and sufficient to regulate cell division and cell specification in the root meristem.
Surprisingly, SHR RNA is not expressed in the ground tissue cell lineage but in the stele (pericycle and vascular cylinder) located immediately adjacent to it, suggesting a non-cell autonomous mode of action of SHR (Fig. 8.7A) (Helariutta et al., 2000). In order to approach this non-cell autonomous mechanism, Nakajima et al. (2001) compared the protein accumulation to the RNA accumulation pattern and found that the SHR protein is transported from the stele to the adjacent endodermal layer, probably through plasmodesmata. This indicates that SHR acts as a mobile signal in exerting its functions as a transcriptional regulator. Although movement of transcription factors had been observed before (Lucas et al., 1995), this was the first time a functional significance for such movement was demonstrated. Nakajima et al. (2001) also explored the outcome of introducing SHR ectopically to the endodermal layer under the SCR promoter. In this case a highly specific increase of ground tissue layers having endodermal characteristics was observed (Fig. 8.7B). Based on the morphological analysis of the scr and shr mutants as well as on a detailed spatio-temporal analysis of SCR and SHR gene expression, it is evident that the patterning of ground tissue is established already during early embryogenesis.
The formation of the vascular network of a plant takes place continuously at the meristematic regions of a plant during both shoot and root development. Even though under normal conditions vascular development is highly predictable, when the situation arises it can also react and adapt to either localized or environmental stimuli. As described in the context of distal patterning of the root, the establishment of the vascular network has also been shown to involve auxin transport and auxin signalling (Aloni, 1987; Sachs, 1991; Ye, 2002).
The vascular network in plants consists of transport tissues: xylem, which transports water and nutrients; and phloem, which transports photosynthates. Between them there is a third vascular tissue type, the (pro)cambium, that consists of the stem cells from which the xylem and phloem elements originate. There are several distinct patterns in which these three tissues are organized in different plant species (Ye, 2002).
Although xylem and phloem both are formed from cambial initial cells, their fate is quite different. The phloem is a system of several cell types: sieve elements (SE), companion cells (CC), phloem fibres and phloem parenchyma cells. The SE are the actual transport cells. They undergo a partial autolysis (involving disintegration of the nucleus) and in some regions of the plant deposit callose on its cell walls. The CC support SE with macromolecules (Esau, 1977; Kuhn et al., 1997; Oparka and Turgeon, 1999). Xylem consists of tracheary elements (TE), xylem parenchyma cells and xylem fibres. The differentiation of TE involves deposition of elaborate cell wall thickenings (containing cellulose and lignin) and programmed cell death.
In contrast to other tissue types, the vascular pattern is established only during the last stages of embryonic development after the vascular initials have divided to first form a pattern of four near-identical poles, followed by a set of tangential and periclinal cell divisions associated with the formation of phloem poles (Plate 8.1) (Bonke et al., 2003). Recently, a couple of informative Arabidopsis pattern formation mutants have been able to shed some light on the underlying mechanisms of vascular patterning in roots. In the wooden leg (wol) mutant cell divisions in the vascular bundle are reduced, leading to a phenotype in which only approximately 9-11 cells occupy the vascular cylinder and after germination all vascular cells differentiate into protoxylem. This phenotype is first seen during the torpedo stage of embryogenesis (Scheres etal., 1995; Mahonen etal., 2000). When wol was introduced into the fass background, phloem was restored. Thus, WOL does not appear to directly influence cell fate within the vascular bundle but has a more indirect influence by controlling the number of cells in the vascular cylinder.
WOL encodes a two-component hybrid-type receptor molecule (Mahonen et al., 2000) and is identical to CRE1/AHK4 (Inoue et al., 2001; Suzuki et al., 2001), a cytokinin receptor. It is expressed in the vascular initials already during the globular stage of embryogenesis, linking cytokinin signalling to vascular embryonic development.
Recently, Bonke et al. (2003) identified the gene ALTERED PHLOEM DEVELOPMENT (APL) as a MYB-CC transcription factor that is required for phloem development throughout the plant. In the apl mutant, phloem patterning is affected; the phloem-specific cell divisions occur less frequently and the cells in the phloem pole area take on a xylem identity.
APL has a complex gene expression pattern mirroring the dynamic nature of phloem development. It is expressed in developing protophloem and also further up in the companion cells and metaphloem. APL expression can first be detected during embryogenesis. Even though the phloem-specific asymmetric divisions are delayed in the apl mutant, APL expression is initiated only after these have occurred. This could indicate that APL acts as a cell non-autonomous factor to control these divisions. However, a GFP-APL protein fusion appears to be expressed in a spatially similar manner as APL RNA, indicating that APL itself probably does not act as the cell non-autonomous factor. It is also possible that metabolic defects resulting from loss of functional phloem may be the cause for the delay in the phloem-specific cell divisions (Bonke et al., 2003).
APL expression driven by the procambium-specific WOL promoter showed that ectopic APL expression is able to fully suppress xylem differentiation in the pro-toxylem pole position and to some extent also in the metaxylem position. Importantly, the affected protoxylem cells retained their nucleus, which indicates that they did not change fate to phloem identity, meaning that APL is necessary, but not sufficient, for phloem identity. It has recently been established that in the aerial part of the plant, class III HD-ZIP and KANADI family transcription factors regulate the distribution pattern of xylem and phloem in stems and leaves (Bowman et al., 2002; Emery et al., 2003). These genes however do not lead to phloem defects. It remains to be studied if the class III HD-ZIP and KANADI genes regulate APL.
The exact mechanisms of vascular patterning are still relatively poorly understood, but the results so far seem to point to a temporal model where auxin is required for establishment of vascular tissue, cytokinin signalling is necessary for cell proliferation and the 'prepatterning' of four poles on which the vascular tissue identity (consisting of the two xylem poles and two phloem poles) is established. APL is necessary for this last phase of the vascular development.
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