VH1/BRL2 receptor-like kinase interacts with vascular-specific adaptor proteins VIT and VIK to influence leaf venation

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.     

Tissue-specific localization of mRNA for carrot homeobox genes, CHBs, in carrot somatic embryos

We examined spatial and temporal expression patterns of four carrot HD-Zip I homeobox genes in somatic embryos. The mRNAs for CHB3, CHB4 and CHB5 were accumulated preferentially in the innermost cortical cell layers of the embryo axis in the torpedo-shaped embryo. In contrast, the accumulation of CHB6 mRNA was restricted to procambial cells of the heart- and torpedo-shaped embryos. In the embryonic cotyledons and the hypocotyl of the seedlings, all of the mRNAs for the four genes were located in the vascular tissues. These findings indicate that different HD-Zip I homeobox genes may be involved in the differentiation of specific tissues during somatic embryogenesis.     

Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis

The maize abscisic acid (ABA) responsive gene rab 28, has been shown to be ABA-inducible in embryos and vegetative tissues. A polyclonal antiserum was raised against Rab28 protein. Using immunoblotting and immunoprecipitation, the antiserum specifically recognized a protein of about 30 kDa and pl 6 which is in close agreement with the molecular weight and pl predicted by the deduced amino acid sequence. The rab 28 gene product accumulated during late embryogenesis. In vegetative tissues, dehydration stress induced rab 28 gene expression both in the light and in the dark. The spatial and temporal pattern of rab 28 mRNA expression during embryogenesis was investigated by in situ hybridization using digoxigenin-labelled rab 28 probes, and the immunochemical localization of Rab28 protein using anti-Rab28 antibodies. Expression of rab 28 mRNA is restricted to provascular tissues in young embryos, and at later stages of development the most prevalent accumulation occurred in meristem and in the vascular elements of the plumule, root and scutellum. Using immunoelectron microscopy the Rab28 protein has been located in the nucleolus of different cell types. In light of these results the stress regulation of rab 28 and a likely role for this protein during late embryogenesis are discussed.     

Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.     

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture

Summary The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidneyin vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidneyin vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.     

Expression pattern of cell cycle genes suggests the developmental arrangement of vascular cells in sugarcane strand

Unlike the ordered multiplication of vascular cells deriving from a row of initials in dicotyledons, vascular growth in monocotyledonous vascular strands does not show the procambial pattern but leads to a complex organization of the vascular bundle. Establishment of the bundle should have a specific developmental pattern. The cell cycle conferring cell proliferation represents a active state of growth and development of tissues. Here, we cloned an A-type CDK gene (Sacof;CDKA;1) from sugarcane (Saccharum officinarum cv. ROC16) and confirmed that its encoding protein interacted physically with two sugarcane CYCD4s (Sacof;CYCD4;1 and Sacof;CYCD4;2), which shared only 47% amino acid sequence similarity. The three genes were expressed concurrently in meristems of root tip, stem tip, and young leaf but not in mature leaves. More importantly, they were predominantly expressed in vascular strands of stem tips and young leaves. In stem-tip strands, the expression region extends deep basipetally to where the sieve tube increases in number in the metaphloem and the vessels are produced in the metaxylem showing a pattern of cell division occurring among differentiating or differentiated cells. This pattern suggests a positional determination of vascular cell arrangement in strands during vascular development.     

Coordination of apical and basal embryo development revealed by tissue-specific GNOM functions

Flowering-plant embryogenesis generates the basic body organization, including the apical and basal stem cell niches, i.e. shoot and root meristems, the major tissue layers and the cotyledon(s). gnom mutant embryos fail to initiate the root meristem at the early-globular stage and the cotyledon primordia at the late globular/transition stage. Tissue-specific GNOM expression in the gnom mutant embryo revealed that both apical and basal embryo organization depend on GNOM provascular expression and a functioning apical-basal auxin flux: GNOM provascular expression in gnom mutant background resulted in non-cell-autonomous reconstitution of apical and basal tissues which could be linked to changes in auxin responses in those tissues, stressing the importance of apical-basal auxin flow for overall embryo organization. Although reconstitution of apical-basal auxin flux in gnom results in the formation of single cotyledons (monocots), only additional GNOM epidermal expression is able to induce wild-type apical patterning. We conclude that provascular expression of GNOM is vital for both apical and basal tissue organization, and that epidermal GNOM expression is required for radial-to-bilateral symmetry transition of the embryo. We propose GNOM-dependent auxin sinks as a means to generate auxin gradients across tissues.     

Genetic regulation of vascular tissue patterning in Arabidopsis

Plants transport water and nutrients through a complex vascular network comprised of interconnected, specialized cell types organized in discrete bundles. To identify genetic determinants of vascular tissue patterning, we conducted a screen for mutants with altered vascular bundle organization in Arabidopsis cotyledons. Mutations in two genes, CVP1 and CVP2 (for cotyledon vascular pattern), specifically disrupt the normal pattern of vascular bundles in cotyledons, mature leaves, and inflorescence stems. The spatial distribution of the procambium, the precursor to mature vascular tissue, is altered in cvp1 and cvp2 embryos, suggesting that CVP1 and CVP2 act at a very early step in vascular patterning. Similarly, in developing stems of cvp1 and leaves of cvp2, the pattern of vascular differentiation is defective, but the maturation of individual vascular cells appears to be normal. There are no discernible alterations in cell morphology in cvp2 mutants. In contrast, cvp1 mutants are defective in directional orientation of the provascular strand, resulting in a failure to establish uniformly aligned vascular cells, and they also show a reduction in vascular cell elongation. Neither cvp1 nor cvp2 mutants displayed altered auxin perception, biosynthesis, or transport, suggesting that auxin metabolism is not generally affected in these mutants.     

The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.     

Modification of cell proliferation patterns alters leaf vein architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Formation of leaf vascular pattern requires regulation of a number of cellular processes, including cell proliferation. To assess the role of cell proliferation during vein order formation, leaf development in genetic lines exhibiting aberrant cell proliferation patterns due to altered expression patterns of ANT and ICK1 genes was analyzed. Modification of cell proliferation patterns alters the number of higher order veins and the number of minor tertiary veins remodeled as intersecondary veins in Arabidopsis rosette leaves. Minor vein complexity, as indicated by branch point and freely ending veinlet number, is highly correlated with a decrease or increase in cell proliferation. Observations of procambial strand formation in modified cell proliferation pattern lines showed that vein pattern is specified early in leaf development and that formation of freely ending veinlets is temporally correlated with the expansion of ground meristem when cell proliferation is terminated prematurely. Taken together, our observations indicate that: (1) genes that modulate cell proliferation play a key role in regulating the meristematic competence of ground meristem cells to form procambium and vein pattern during leaf development, and (2) ANT is a crucial part of this regulation.     

IDENTIFICATION OF PROCAMBIUM IN THE PRIMARY ROOT OF TRIFOLIUM PRATENSE (FABACEAE)

Histochemical and morphometric analyses were used to identify and define an early stage of procambial differentiation in 1.5–2.0-cm-long (48 hr after germination) primary roots of Trifolium pratense L. Esterase activity was used as a histochemical marker for early differentiation of procambium. Morphometric analysis of cell length and width as a function of distance from the root cap junction was performed on the same tissue using brightfield and Nomarski DIC optics. This combination of techniques allowed the identification of esterase activity in both the cell wall and cytoplasm and permitted the determination of the exact location, size, and shape of the histochemically stained cells within the apex. Esterase activity identified the proendodermis and procambial cylinder (six to seven cells in diameter) two to three cells proximal to the root cap junction. In this system, esterase activity proved to be an earlier marker for procambial differentiation than morphometric or cytological changes. It is suggested that these techniques will be useful in characterizing procambial pattern development in more complex shoot systems.     

cdc2a expression in Arabidopsis is linked with competence for cell division.

A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34(cdc2), is encoded by the cdc2 gene. We studied the control of the expression of the Arabidopsis cdc2a gene in cell suspensions and during plant development. In cell cultures, arrest of the cell cycle did not significantly affect cdc2a mRNA levels, but nutrient conditions were important for cdc2a expression. During plant development, the pattern of cdc2a expression was strongly correlated with the cell proliferation potential. The effects of external signals on cdc2a expression were analyzed. Wounding induced expression in leaves. Lack of light altered temporal regulation of cdc2a in the apical but not root meristem of seedlings. Differential cdc2a responses were obtained after different hormone treatments. Signals present only in intact plants were necessary to mediate these responses. Although other control levels have yet to be analyzed, these results suggest that the regulation of cdc2a expression may contribute greatly to spatial and temporal regulation of cell division in plants. Our results also show that cdc2a expression is not always coupled with cell proliferation but always precedes it. We propose that cdc2a expression may reflect a state of competence to divide, and that the release of other controls is necessary for cell division to occur.