Timothy Nelson

The developmental dynamics of the maize leaf transcriptome

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 C4 photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

Leaf Vascular Pattern Formation.

The 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.

Laser Capture Microdissection of Cells from Plant Tissues

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.

A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies

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.

The Identification of CVP1 Reveals a Role for Sterols in Vascular Patterning

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.

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.

Sex Determination in Monoecious and Dioecious Plants.

Angiosperm species that produce unisexual flowers pres? ent the opportunity for separate analysis of the male and female programs for floral differentiation and gametogenesis. A vast literature describes the genetic and physio? logical basis of sex determination in these species. (For reviews, see Westergaard, 1958; Grant, 1975; Frankel and Galun, 1977; Durand and Durand, 1984.) Recently, it has become feasible to pursue the molecular genetic basis of the male and female differentiation programs in certain plant species, as has already been profitably undertaken in several animal species (Hodgkin, 1987, 1989). This article reviews the unisexual flowering schemes found in angiosperms and summarizes available data on the control of floral polymorphism in the monoecious species maize (Zea mays) and in the dioecious species mercury (Mercurialis annua).

Arabidopsis thickvein Mutation Affects Vein Thickness and Organ Vascularization, and Resides in a Provascular Cell-Specific Spermine Synthase Involved in Vein Definition and in Polar Auxin Transport

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.

COTYLEDON VASCULAR PATTERN2–Mediated Inositol (1,4,5) Triphosphate Signal Transduction Is Essential for Closed Venation Patterns of Arabidopsis Foliar Organs

Vein patterns in leaves and cotyledons form in a spatially regulated manner through the progressive recruitment of ground cells into vascular cell fate. To gain insight into venation patterning mechanisms, we have characterized the cotyledon vascular pattern2 (cvp2) mutants, which exhibit an increase in free vein endings and a resulting open vein network. We cloned CVP2 by a map-based cloning strategy and found that it encodes an inositol polyphosphate 5′ phosphatase (5PTase). 5PTases regulate inositol (1,4,5) triphosphate (IP3) signal transduction by hydrolyzing IP3 and thus terminate IP3 signaling. CVP2 gene expression is initially broad and then gradually restricted to incipient vascular cells in several developing organs. Consistent with the inferred enzymatic activity of CVP2, IP3 levels are elevated in cvp2 mutants. In addition, cvp2 mutants exhibit hypersensitivity to the plant hormone abscisic acid. We propose that elevated IP3 levels in cvp2 mutants reduce ground cell recruitment into vascular cell fate, resulting in premature vein termination and, thus, in an open reticulum.

Patterns of leaf development in C4 plants.

The differentiation of cell types in plants depends on the continuous interpretation of positional information. Plant organs are established by patterns of cell division that are often highly variable, yet the final arrangement of cell types within each organ remains the same. A current challenge is to understand the means by which cells of varying clonal history arrive at the same differentiated fate in the development of a plant organ. The ideal system for the study of this developmental control would be an organ with few cell types, with defined patterns of cell origin, and with molec? ular markers for the differentiation of the individual cell