Regulation of Vascular Development by CLE Peptide-receptor Systems

Cell division and differentiation of stem cells are controlled by non-cell-autonomous signals in higher organisms. The plant vascular meristem is a stem-cell tissue comprising procambial cells that produce xylem cells on one side and phloem cells on the other side. Recent studies have revealed that TDIF (tracheary element differentiation inhibitory factor)/CLE41/CLE44 peptide signal controls the procambial cell fate in a non-cell-autonomous manner. TDIF produced in and secreted from phloem cells is perceived by TDR/PXY, a leucine-rich repeat receptor kinase located in the plasma membrane of procambial cells. This signal suppresses xylem cell differentiation of procambial cells and promotes their proliferation. In addition to TDIF, some other CLE peptides play roles in vascular development. Here, we summarize recent advances in CLE signaling governing vascular development.     

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex

The phloem, located within the vascular system, is critical for delivery of nutrients and signaling molecules throughout the plant body. Although the morphological process and several factors regulating phloem differentiation have been reported, the molecular mechanism underlying its initiation remains largely unknown. Here, we report that the small peptide‐coding gene, CLAVATA 3 (CLV3)/EMBEYO SURROUNDING REGION 25 (CLE25), the expression of which begins in provascular initial cells of 64‐cell‐staged embryos, and continues in sieve element‐procambium stem cells and phloem lineage cells, during post‐embryonic root development, facilitates phloem initiation in Arabidopsis. Knockout of CLE25 led to delayed protophloem formation, and in situ expression of an antagonistic CLE25_G6T peptide compromised the fate‐determining periclinal division of the sieve element precursor cell and the continuity of the phloem in roots. In stems of CLE25_G6T plants the phloem formation was also compromised, and procambial cells were over‐accumulated. Genetic and biochemical analyses indicated that a complex, consisting of the CLE‐RESISTANT RECEPTOR KINASE (CLERK) leucine‐rich repeat (LRR) receptor kinase and the CLV2 LRR receptor‐like protein, is involved in perceiving the CLE25 peptide. Similar to CLE25, CLERK was also expressed during early embryogenesis. Taken together, our findings suggest that CLE25 regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex.     

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the gall constriction hypothesis is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.We thank Dr. Zs. Koncz (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) for Agrobacterium strains and the Deutsche Forschungsgemeinschaft (SFB 199) for financial support to C.I.U.     

Polycentric vascular rays in Suaeda monoica and the control of ray initiation and spacing

Summary The early-formed xylem of Suaeda monoica Forssk. ex J. F. Gmel (Chenopodiaceae) is temporarily rayless. Vascular rays differentiate during later stages of its xylem ontogeny. The rays in Suaeda are heterogeneous, and some of them are aggregated. The mature xylem of this species is characterized by two unique types of vascular rays: (1) rays with several inside initiation centres of small cells formed by local frequent cell divisions in the cambium, and (2) huge xylem rays with radial phloem strands that are connected to the axial phloem. The spacing of the xylem rays is not even, and possible mechanisms controlling ray spacing are discussed. Our observations indicate that rays do not have an inhibitory zone around them in which ray initiation is prevented. The initiation of radial patterns of small cells which appear like inside rays within a large vascular ray suggests that initiation and spacing of rays is controlled by radial signal flows in relationship with axial signal fluxes.     

Changes in cell length and mitotic index in vascular pattern-related pericycle cell types along the apical meristem and elongation zone of the onion root

Summary Three pericycle cell types (opposite xylem, opposite phloem and intervening) distinguished by their location in relation to different elements of the vascular system were studied in the adventitious root ofAllium cepa L. Changes in cell length and mitotic index were analysed in these cells along the apical meristem and elongation zone of the root. The opposite phloem and intervening pericycle cells are significantly shorter than the opposite xylem pericycle cells in the apical half of the meristem. Between 1,200 and 1,400 m behind the tip, length became similar in all three pericycle cell types, while in more proximal zones the opposite phloem cells were significantly longer. These results suggest that the number of transverse divisions is different in the three types of pericycle cells. In the apical half of the meristem, mitotic index increased in intervening and opposite xylem cells but remained unchanged in opposite phloem cells, a fact likely to account for the relative lengthening of the latter. In the proximal half of the meristem, mitotic index fell in all three cell types until cell division had ceased. However, mitotic index in opposite xylem cells remained high for longer than in the other two cell types, implying that increase of the mean cell length in the former was slower. These results suggest that differences in mean cell length between the three pericycle cell types are due to different rates of proliferation.     

Peptide signaling in vascular development

In plants and animals, putative small peptide ligands have been suggested to play crucial roles in development as signal molecules of cell-cell communication. Recent studies of CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) genes and their products have revealed that distinctive dodeca-CLE peptide ligands function in various developmental processes. In particular, the finding and characterization of TDIF, a dodeca-CLE peptide suppressing tracheary element differentiation, indicates regulation of vascular organization by cell-cell communication through CLE peptides. In addition, other extracellular peptides such as phytosulfokine, proteins such as xylogen, and phytohormones all participate in the ordered formation of vascular tissues.     

Cover Caption: Plant Vascular System

This issue focuses on the plant vascular system, with a comprehensive review article written by Lucas et al. (pp. 294–388). The cover drawing illustrates the phosphate‐stress signaling and response network (pp. 347–351). A Pi deficiency signal is generated in roots and transported to shoots via the xylem (blue lines). This signal is recognized by source leaves to activate the Pi stress response pathway and then to load the subsequent signals into the phloem (red lines). Phloemmobile RNAs move to roots to increase Pi uptake and alter root architecture . Different phloem‐mobile RNAs are also delivered from source leaves to developing leaves and the shoot apex where they regulate development under Pi‐stress conditions.     

Morphological changes and transversal growth kinetics along the apical meristem in the pericycle cell types of the onion adventitious root

Summary In the apical meristem of the adventitious root ofAllium cepa, all pericycle cells show a marked increase in cross-sectional area between 400 and 800 m behind the tip, this transversal growth ceasing in the 1,200–1,400 m interval. However, different pericycle cell types (opposite xylem, intervening and opposite phloem) show different transversal growth kinetics. Along the meristem, the opposite xylem cells are narrower than both the intervening and opposite phloem cells, and these latter are similar in cross-sectional area. Another relevant difference is in the polarity of the transversal expansion, which in turn gives rise to changes in cell shape. In fact, in apical most portions of the meristem, the opposite phloem cells mainly expand tangentially, while the intervening cells do so radially, and the opposite xylem cells undergo a similar tangential and radial expansion. By contrast, in basal most portions of the meristem, radial expansion continues in the opposite phloem cells when it has ceased in the intervening cells. These latter expand tangentially once again when tangential expansion has ceased in the opposite phloem cells. As a consequence of this transversal growth, the opposite xylem cells, which can initiate lateral root primordia, retain their isodiametric transversal shape along the meristem, whereas the transversal shape of the opposite phloem and intervening cells initially changes from isodiametric to markedly enlarged tangentially (opposite phloem) or radially (intervening), after which both cell types tend to become more rounded in shape.     

Role of CLE41 Peptide in the Development of Root Storage Parenchyma in Species of the Genus <Emphasis Type="Italic">Raphanus</Emphasis> L.

CLE peptides (CLAVATA3/ENDOSPERM SURROUNDING REGION) are signal molecules or plant peptide hormones that play an important role in regulation of development of various meristems governing the expression of WOX (WUSCHEL-RELATED HOMEOBOX) genes. In particular, CLE peptides belonging to a small TDIF (Tracheary Element Differentiation Inhibitory Factor) group are responsible for the operation of gene WOX4 controlling the development of cambium and the conducting system. We looked into the role of CLE41 peptide from the TDIF group in the development of storage root in two species of the genus Raphanus: cultivated radish (Raphanus sativus var. radicula Pers.) that is a popular root crop with a storage root and its ancestor wild radish (Raphanus raphanistrum L.) where storage parenchyma of the root is poorly developed. It was shown that overexpression of gene RsCLE41 and plant treatment with exogenous peptide CLE41 influenced the development of cambium and xylem in the roots of R. sativus and R. raphanistrum and affected expression of the genes from different groups. One could say that peptide CLE41 activates expression of the genes whose homologues in arabidopsis play a key role in the maintenance of cambium (RsWOX4, RsWOX14, RsHAM4, and RsCYCD3). In the storage root of radish, peptide CLE41 activates proliferation of cambium cells reducing the amount of one of the xylem’s elements (lignified parenchyma). The obtained results point to an important role of CLE41 in the development of storage root in radish.     

Light,temperature and tocopherol status influence foliar vascular anatomy and leaf function in Arabidopsis thaliana

This study addressed whether the winter annual Arabidopsis thaliana can adjust foliar phloem and xylem anatomy both differentially and in parallel. In plants acclimated to hot vs cool temperature, foliar minor vein xylem‐to‐phloem ratio was greater, whereas xylem and phloem responded concomitantly to growth light intensity. Across all growth conditions, xylem anatomy correlated with transpiration rate, while phloem anatomy correlated with photosynthetic capacity for two plant lines (wild‐type Col‐0 and tocopherol‐deficient vte1 mutant) irrespective of tocopherol status. A high foliar vein density (VD) was associated with greater numbers and cross‐sectional areas of both xylem and phloem cells per vein as well as higher rates of both photosynthesis and transpiration under high vs low light intensities. Under hot vs cool temperature, high foliar VD was associated with a higher xylem‐to‐phloem ratio and greater relative rates of transpiration to photosynthesis. Tocopherol status affected development of foliar vasculature as dependent on growth environment. The most notable impact of tocopherol deficiency was seen under hot growth temperature, where the vte1 mutant exhibited greater numbers of tracheary elements (TEs) per vein, a greater ratio of TEs to sieve elements, with smaller individual sizes of TEs, and resulting similar total areas of TEs per vein and transpiration rates compared with Col‐0 wild‐type. These findings illustrate the plasticity of foliar vascular anatomy acclimation to growth environment resulting from independent adjustments of the vasculature's components.     

Orientation of vascular cell divisions in Arabidopsis

Orientation of cell division is essential for plant development as the direction of growth is determined by the direction of cell expansion and orientation of cell division. We have demonstrated that cell division orientation in vascular tissue is regulated by the interactions between a receptor kinase (PXY) expressed in dividing cells and its peptide ligand (CLE41) that is localized to adjacent phloem cells. Given that other receptor kinases have been identified as orienting the cell division plane in several developmental processes, we suggest that localized signaling from adjacent cells may be a general mechanism for defining the plane of cell division.Key words: xylem, phloem, cell division orientation, procambium, cambiumThroughout the life of plants, new organs are generated from meristems which contain stem cells at their center. Meristematic cells divide in regulated processes resulting in displacement of daughter cells to the periphery of the meristem where they differentiate, taking on new cell identities.¹ Vascular meristems (cambium and procambium) are responsible for radial growth and are the main source of plant biomass.² Their regulation has a come under increasing scrutiny as biomass is likely to play an increasing role in generation of renewable energy.³Arabidopsis vascular tissue is organized into discrete collateral bundles in stems,⁴ whereas in hypocotyls, vasculature forms in a continuous ring, much like that of trees.⁵ In both cases spatially separated xylem and phloem are formed along the stem mediolateral axis and are populated with cells derived from the procambium or cambium (Fig. 1). Vascular initials displaced from the meristematic zone towards the center of the stem differentiate into xylem whereas those displaced towards the outside of the stem differentiate into phloem. This organization occurs because vascular meristematic cells are long and thin and divide periclinally down their long axis, perpendicular to the mediolateral axis. Because these are highly ordered divisions, vascular tissue is characterized by long files of cells. Until recently regulatory factors which influence the highly ordered nature of these divisions—and therefore plant vascular tissue organization were entirely unknown.Open in a separate windowFigure 1Arabidopsis vascular tissue at the base of inflorescence stems (A) and hypocotyls (B). The mediolateral axes are marked with arrows, x is xylem, ph is phloem, pc is procambium, c is cambium. Scale bars are 50 µm.     

Effect of cytokinins on expression of radish CLE genes

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) peptides are peptide phytohormones playing the important role in the regulation of various type meristem development and also in the interaction between plants and parasites and symbionts. At the same time, the interaction of CLE peptides with other phytohormones, participating in these processes, is essentially unstudied. Using real-time RT-PCR, we analyzed expression of some genes encoding CLE peptides in radish (Raphanus sativus var. radicula Pers.) under normal conditions and after treatment with the cytokinin 6-benzyladenine. Even after short-term (30 min) action of cytokinin, expression of group A CLE genes was manifold suppressed, whereas cytokinin did not affect expression of group B CLE genes. Radish lines contrasting in the trait “spontaneous tumor formation” demonstrated similar dynamics of CLE gene expression in response to cytokinin treatment but differed in the levels of their expression. A possibility of interaction between CLE peptides and cytokinins in the spontaneous development of tumors in radish lines is discussed. The important part of the work was the selection of optimal reference genes for the analysis of radish gene expression by real-time RT-PCR method. Most stable expression was observed for the genes of ubiquitin (RsUBQ) and glyceraldehydes-3-phosphate dehydrogenase (RsGAPDH).     

Regulation of Vascular Development by CLE Peptide-receptor Systems

Cell division and differentiation of stem cells are controlled by non-cell-autonomous signals in higher organisms. The plant vascular meristem is a stem-cell tissue comprising procambial cells that produce xylem cells on one side and phloem cells on the other side. Recent studies have revealed that TDIF (tracheary element differentiation inhibitory factor)/CLE41/CLE44 peptide signal controls the procambial cell fate in a non-cell-autonomous manner. TDIF produced in and secreted from phloem cells is perceive...     

Structural and ultrastructural characteristics of the vascular apparatus of the sensitive plant (Mimosa pudica L.)

Summary 1. In motor organs ofMimosa pudica xylem contains living fibriform elements limited by a thick lignified highly pitted wall, whereas in other parts of the plant (stem, petiole, rachis), xylem and protoxylem vessels are closely associated with parenchyma cells which possess wall ingrowths. These ingrowths, at the apex of which the plasmalemma and the tonoplast touch, are localized like those of transfer cells of C type described byGunning andPate. Nevertheless, xylem parenchyma cells differ from cells of C type in several characteristics. Moreover, in motor organs, phloem contains cells characterized by wall ingrowths, less abundant on the parts adjacent to the sieve tubes; these cells which are localized near collenchyma cells of primary phloem, look like transfer cells of A type defined byGunning andPate; they are absent from internodes, petioles and rachides. 2. In motor organs, three types of vascular cells (companion cells, living xylem fibriform elements and protoxylem parenchyma cells) are characterized by reduced vacuolar volumes and well developed membrane systems, as compared with homologuous cells belonging to other parts of the plant. 3. A symplastic continuity holds from the middle of motor organs to their cortex: it is provided by the presence, in xylem and phloem respectively, of living fibriform elements and collenchyma cells bearing numerous pit fields containing large numbers of plasmodesmata. Several ultrastructural features suggest that the vascular apparatus ofMimosa pudica would be the site of intensive lateral transfer at different levels, specially in motor organs. Possible functions of certain structures observed are discussed in relation to some hypotheses relative to excitatory conduction pathways.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.