Venkatesan Sundaresan

Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis

The plant life cycle involves an alternation of generations between sporophyte and gametophyte. Currently, the genes and pathways involved in gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify 130 mutants of Arabidopsis thaliana with defects in female gametophyte development and function. A wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. Unexpectedly, nearly half of the mutants isolated in this study were found to be primarily defective in post-fertilization processes dependent on the maternal allele, suggesting that genes expressed from the female gametophyte or the maternal genome play a major role in the early development of plant embryos. Sequence identification of the genes disrupted in the mutants revealed genes involved in protein degradation, cell death, signal transduction and transcriptional regulation required for embryo sac development, fertilization and early embryogenesis. These results provide a first comprehensive overview of the genes and gene products involved in female gametophyte development and function within a flowering plant.

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis.

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5′ end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell.org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

VANGUARD1 Encodes a Pectin Methylesterase That Enhances Pollen Tube Growth in the Arabidopsis Style and Transmitting Tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.

The indeterminate Gene Encodes a Zinc Finger Protein and Regulates a Leaf-Generated Signal Required for the Transition to Flowering in Maize

Flowering in plants is a consequence of the transition of the shoot apex from vegetative to reproductive growth in response to environmental and internal signals. The indeterminate1 gene (id1) controls the transition to flowering in maize. We show by cloning the id1 gene that it encodes a protein with zinc finger motifs, suggesting that the id1 gene product functions as a transcriptional regulator of the floral transition. id1 mRNA expression studies and analyses of transposon-induced chimeric plants indicate that id1 acts non-cell-autonomously to regulate the production of a transmissible signal in the leaf that elicits the transformation of the shoot apex to reproductive development. These results provide molecular and genetic data consistent with the florigen hypothesis derived from classical plant physiology studies.

TAPETUM DETERMINANT1 Is Required for Cell Specialization in the Arabidopsis Anther

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel TAPETUM DETERMINANT1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.

Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

Rice mutant resources for gene discovery

With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines required for functional analyses, extensive international collaboration is needed for this next goal. The aims of this review are to summarize the current status of rice mutant resources, key tools for functional analysis of genes, and our perspectives on how to accelerate rice gene discovery through collaboration.

The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence

BACKGROUND Several processes of plant development, such as abscission, pollen release, fruit dehiscence, and seed dispersal, require organs or tissues to physically disassociate or split open. Due to the immobility of plant cells, these processes occur through coordinated mechanisms of cell separation that are not found in animals. Arabidopsis produces dry dehiscent fruits (siliques) making it a convenient system for the genetic study of cell separation associated with dehiscence. RESULTS We describe here a novel mutation in Arabidopsis called alcatraz (alc), which prevents dehiscence of fruit by specifically blocking the separation of the valve cells from the replum. The ALC gene is shown to encode a protein related to the myc/bHLH family of transcription factors and is expressed in the valve margins of the silique, which is the site of cell separation during dehiscence. Detailed studies using TEM indicates that ALC enables cell separation in Arabidopsis fruit dehiscence by promoting the differentiation of a strip of labile nonlignified cells sandwiched between layers of lignified cells. Transgenic plants expressing antisense or dominant-negative ALC are defective in silique dehiscence. CONCLUSIONS Cell separation in fruit dehiscence requires a specialized cell layer which is nonlignified and capable of autolysis, specified by a myc/bHLH protein encoded by ALC. These findings may have relevance to other processes requiring cell separation, as well as for the practical design of crops with reduced seed losses.

Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project.

The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes.

Auxin-dependent patterning and gamete specification in the Arabidopsis female gametophyte

Flower Functionalization The development of the specialized cells that make up the female reproductive unit in flowering plants, the gametophyte, requires the hormone auxin. However, auxins function and movement to and within these cells are unclear. Pagnussat et al. (p. 1684, published online 4 June; see the Perspective Friedman) provide evidence that auxin is synthesized at specific positions within the female gametophyte and exerts a positional effect and that a gradient of auxin controls patterning of these specialized cells. An auxin gradient is involved in cell fate specification of the female sex cells in flowering plants. The female reproductive unit of flowering plants, the haploid female gametophyte, is highly reduced relative to other land plants. We show that patterning of the Arabidopsis female gametophyte depends on an asymmetric distribution of the hormone auxin during its syncitial development. Furthermore, this auxin gradient is correlated with location-specific auxin biosynthesis, rather than auxin efflux that directs patterning in the diploid sporophytic tissues comprising the rest of the plant. Manipulation of auxin responses or synthesis induces switching of gametic and nongametic cell identities and specialized nonreproductive cells to exhibit attributes presumptively lost during angiosperm evolution. These findings may account for the unique egg cell specification characteristic of angiosperms and the formation of seeds with single diploid embryos while containing endosperm that can have variable numbers of parental haploid genomes.