We have analyzed the maize leaf transcriptome using Illumina sequencing. We mapped more than 120 million reads to define gene structure and alternative splicing events and to quantify transcript abundance along a leaf developmental gradient and in mature bundle sheath and mesophyll cells. We detected differential mRNA processing events for most maize genes. We found that 64% and 21% of genes were differentially expressed along the developmental gradient and between bundle sheath and mesophyll cells, respectively. We implemented Gbrowse, an electronic fluorescent pictograph browser, and created a two-cell biochemical pathway viewer to visualize datasets. Cluster analysis of the data revealed a dynamic transcriptome, with transcripts for primary cell wall and basic cellular metabolism at the leaf base transitioning to transcripts for secondary cell wall biosynthesis and C(4) photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.

INTRODUCTIONThe pattern and ontogeny of leaf venation appear to guide or limit many aspects of leaf cell differentiation and function. Photosynthetic, supportive, stomatal, and other specialized cell types differentiate in positions showing a spatial relationship to the vascular system. These spatial relationships are of obvious importance to leaf function, which relies on venation for the servicing of cells engaged in photosynthesis, gas exchange, and other leaf processes.Although the need for coordinated organization of cell types around the vascular system is clear, the means by which this is achieved during development is not well understood. In the few systems in which it has been possible to follow the ontogeny of the venation along with the differentiation and function of surrounding cell types (e.g., in C4 grasses), observations suggest that the developing vascular system may have a role in providing positional landmarks that guide the differentiation of other cell types. Another possible explanation is that an underlying pattern guides the differentiation of both venation and surrounding cells.Whether the process of vascularization creates or reveals a pattern, studies to date are largely descriptive, and little is understood of the underlying mechanisms. These mechanisms must be highly regulated, as evidenced by the successful use of species-specific leaf vascular pattern as a taxonomic characteristic (e.g., Klucking, 1992) and by the predictable effect of certain mutations. In this review, we summarize the vascular patterns and their ontogenies in dicots and monocots, referring extensively but not exclusively to Arabidopsis and maize as examples. We also discuss a variety of models that seek to explain vascular pattern formation, and we provide a summary of molecular and genetic investigations of the process. VASCULAR PAlTERN IN MATURE LEAVES DicotsThe relatively small simple leaves of Arabidopsis illustrate many of the characteristic features of leaf venation found in

The regulation of members of the knotted1-like homeobox (knox) gene family is required for the normal initiation and development of lateral organs. The maize rough sheath2 (rs2) gene, which encodes a Myb-domain protein, is expressed in lateral organ primordia and their initials. Mutations in the rs2 gene permit ectopic expression of knox genes in leaf and floral primordia, causing a variety of developmental defects. Ectopic KNOX protein accumulation in rs2 mutants occurs in a subset of the normal rs2-expressing cells. This variegated accumulation of KNOX proteins in rs2 mutants suggests that rs2 represses knox expression through epigenetic means.

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Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 ( cvp1 ) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization. INTRODUCTIONThe plant vascular system is formed progressively during embryogenesis and postembryonic development. In organ primordia, provascular cells appear among ground cells in hierarchical patterns that give rise to the patterns of veins (Nelson and Dengler, 1997). Provascular cells proliferate and elongate in axialized files that appear to depend on the intercellular polar transport of auxin through the developing tissue. Screens for pattern-defective mutants have identified several genes that regulate or influence this process. The monopteros mutation causes a discontinuous venation pattern associated with improper vascular differentiation (Przemeck et al., 1996) and corresponds to an auxin response transcription factor (Hardtke and Berleth, 1998).In gnom/emb30 mutants, which correspond to a brefeldin A-sensitive ADP-ribosylation factor guanine-nucleotide exchange factor (ARF-GEF), veins are unaxialized and patterned irregularly (Mayer et al., 1993;Shevell et al., 1994;Steinmann et al., 1999;Grebe et al., 2000;Koizumi et al., 2000). Additional venation pattern mutants have been described, such as the van mutants, lop1 / tornado1 , and scarface , but their gene products have not been identified (Carland and McHale, 1996;Cnops et al., 2000;Deyholos et al., 2000;Koizumi et al., 2000).We recently described a number of additional mutants with various effects on cotyledon vascular pattern (Carland et al., 1999). One of these, cotyledon vascular pattern1 ( cvp1 ), resembles the monopteros phenotype in that a discontinuous, poorly axialized venation pattern is formed. Here, we show that cvp1 corresponds to a defect in sterol biosynthesis.Derivatives of the sterol pathway in plants and animals include both membrane sterols and signaling steroids. Recently, the membrane sterol cholesterol was shown to influence signaling and vectorial processes through its key role in the organization of lipid rafts (Simons and Ikonen, 1997). Lipid rafts appear to provide scaffolding for protein localization and distribution, membrane trafficking, and membrane si...

The functions of the plant body rely on interactions among distinct and nonequivalent cell types. The comparison of transcriptomes from different cell types should expose the transcriptional networks that underlie cellular attributes and contributions. Using laser microdissection and microarray profiling, we have produced a cell type transcriptome atlas that includes 40 cell types from rice (Oryza sativa) shoot, root and germinating seed at several developmental stages, providing patterns of cell specificity for individual genes and gene classes. Cell type comparisons uncovered previously unrecognized properties, including cell-specific promoter motifs and coexpressed cognate binding factor candidates, interaction partner candidates and hormone response centers. We inferred developmental regulatory hierarchies of gene expression in specific cell types by comparison of several stages within root, shoot and embryo.

Plant sterols are structural components of cell membranes that provide rigidity, permeability, and regional identity to membranes. Sterols are also the precursors to the brassinosteroid signaling molecules. Evidence is accumulating that specific sterols have roles in pattern formation during development. COTYLEDON VASCULAR PATTERNING1 (CVP1) encodes C-24 STEROL METHYLTRANSFERASE2 (SMT2), one of three SMTs in Arabidopsis (Arabidopsis thaliana). SMT2 and SMT3, which also encodes a C-24 SMT, catalyze the reaction that distinguishes the synthesis of structural sterols from signaling brassinosteroid derivatives and are highly regulated. The deficiency of SMT2 in the cvp1 mutant results in moderate developmental defects, including aberrant cotyledon vein patterning, serrated floral organs, and reduced stature, but plants are viable, suggesting that SMT3 activity can substitute for the loss of SMT2. To test the distinct developmental roles of SMT2 and SMT3, we identified a transcript null smt3 mutant. Although smt3 single mutants appear wild type, cvp1 smt3 double mutants show enhanced defects relative to cvp1 mutants, such as discontinuous cotyledon vein pattern, and produce novel phenotypes, including defective root growth, loss of apical dominance, sterility, and homeotic floral transformations. These phenotypes are correlated with major alterations in the profiles of specific sterols but without significant alterations to brassinosteroid profiles. The alterations to sterol profiles in cvp1 mutants affect auxin response, demonstrated by weak auxin insensitivity, enhanced axr1 auxin resistance, ectopically expressed DR5:b-glucuronidase in developing embryos, and defective response to auxin-inhibited PIN2-green fluorescent protein endocytosis. We discuss the developmental roles of sterols implied by these results.