Gary N. Drews

Class III Homeodomain-Leucine Zipper Gene Family Members Have Overlapping, Antagonistic, and Distinct Roles in Arabidopsis Development

The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.

Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors

Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON (LEC) genes LEC1, LEC1-LIKE, LEC2, and FUS3. The rest represent different classes of TFs with unknown roles in seed development. Promoter-β-glucuronidase (GUS) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.

Female gametophyte development.

Early in their evolution, plants acquired a life cycle that alternates between a multicellular haploid organism, the gametophyte, and a multicellular diploid organism, the sporophyte. Angiosperms have both female and male gametophytes. The female gametophyte is critical to many steps of the

Megagametogenesis in Arabidopsis wild-type and the Gf mutant

Abstract The female gametophyte is an essential structure for angiosperm reproduction that mediates a host of reproductive functions and, following fertilization, gives rise to most of the seed. Here, we describe a rapid method to analyze Arabidopsis female gametophyte structure using confocal laser scanning microscopy (CLSM). We present a comprehensive description of megagametogenesis in wild-type Arabidopsis. Based on our observations, we divided Arabidopsis megagametogenesis into eight morphologically distinct stages. We show that synergid cell degeneration is triggered by pollination, that dramatic nuclear migrations take place during the four-nucleate stage, and that megagametogenesis within a pistil is fairly synchronous. Finally, we present a phenotypic analysis of the previously reported Gf mutant (Redei 1965) and show that it affects an early step of megagametogenesis.

MYB98 Is Required for Pollen Tube Guidance and Synergid Cell Differentiation in Arabidopsis

The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process, including guidance of pollen tube growth to the female gametophyte. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. MYB98 is a member of the R2R3-MYB gene family, the members of which likely encode transcription factors. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed

Significance Seeds are complex structures that are comprised of the embryo, endosperm, and seed coat. Despite their importance for food, fiber, and fuel, the cellular processes that characterize different regions of the seed are not known. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets provide unique insights into the cellular processes that occur in understudied seed regions, revealing unexpected overlaps in the functional identities of seed regions and enabling predictions of gene regulatory networks. This dataset is an essential resource for studies of seed biology. Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution, providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these datasets as an essential resource for the study of seed biology.

Genetic Analysis of Female Gametophyte Development and Function

The female gametophyte is an absolutely essential structure for angiosperm reproduction. It produces the egg cell and central cell (which give rise to the embryo and endosperm, respectively) and mediates several reproductive processes including pollen tube guidance, fertilization, the induction of seed development, and perhaps also maternal control of embryo development. Although much has been learned about these processes at the cytological level, specific molecules mediating and controlling megagametogenesis and female gametophyte function have not been identified. A genetic approach to the identification of such molecules has been initiated in Arabidopsis and maize. Although genetic analyses are still in their infancy, mutations affecting female gametophyte function and specific steps of megagametogenesis have already been identified. Large-scale genetic screens aimed at identifying mutants affecting every step of megagametogenesis and female gametophyte function are in progress; the characterization of genes identified in these screens should go a long way toward defining the molecules that are required for female gametophyte development and function.

AGL80 Is Required for Central Cell and Endosperm Development in Arabidopsis

During plant reproduction, the central cell of the female gametophyte becomes fertilized to produce the endosperm, a storage tissue that nourishes the developing embryo within the seed. The molecular mechanisms controlling the specification and differentiation of the central cell are poorly understood. We identified a female gametophyte mutant in Arabidopsis thaliana, fem111, that is affected in central cell development. In fem111 female gametophytes, the central cells nucleolus and vacuole fail to mature properly. In addition, endosperm development is not initiated after fertilization of fem111 female gametophytes. fem111 contains a T-DNA insertion in AGAMOUS-LIKE80 (AGL80). FEM111/AGL80 is a member of the MADS box family of genes that likely encode transcription factors. An AGL80–green fluorescent protein fusion protein is localized to the nucleus. Within the ovule and seed, FEM111/AGL80 is expressed exclusively in the central cell and uncellularized endosperm. FEM111/AGL80 expression is also detected in roots, leaves, floral stems, anthers, and young flowers by real-time RT-PCR. FEM111/AGL80 is required for the expression of two central cell–expressed genes, DEMETER and DD46, but not for a third central cell–expressed gene, FERTILIZATION-INDEPENDENT SEED2. Together, these data suggest that FEM111/AGL80 functions as a transcription factor within the central cell gene regulatory network and controls the expression of downstream genes required for central cell development and function.

The AGL62 MADS Domain Protein Regulates Cellularization during Endosperm Development in Arabidopsis

Endosperm, a storage tissue in the angiosperm seed, provides nutrients to the embryo during seed development and/or to the developing seedling during germination. A major event in endosperm development is the transition between the syncytial phase, during which the endosperm nuclei undergo many rounds of mitosis without cytokinesis, and the cellularized phase, during which cell walls form around the endosperm nuclei. The molecular processes controlling this phase transition are not understood. In agl62 seeds, the endosperm cellularizes prematurely, indicating that AGL62 is required for suppression of cellularization during the syncytial phase. AGL62 encodes a Type I MADS domain protein that likely functions as a transcription factor. During seed development, AGL62 is expressed exclusively in the endosperm. During wild-type endosperm development, AGL62 expression is strong during the syncytial phase and then declines abruptly just before cellularization. By contrast, in mutant seeds containing defects in some FERTILIZATION-INDEPENDENT SEED (FIS) class Polycomb group genes, the endosperm fails to cellularize and AGL62 expression fails to decline. Together, these data suggest that AGL62 suppresses cellularization during the syncytial phase of endosperm development and that endosperm cellularization is triggered via direct or indirect AGL62 inactivation by the FIS polycomb complex.

Mitochondrial GFA2 Is Required for Synergid Cell Death in Arabidopsis

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain–containing protein. Of the J-domain–containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.