Anhthu Q. Bui

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

Abstract We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsisthaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsisthaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis.

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.

LEAFY COTYLEDON1-LIKE Defines a Class of Regulators Essential for Embryo Development

Arabidopsis LEAFY COTYLEDON1 (LEC1) is a critical regulator required for normal development during the early and late phases of embryogenesis that is sufficient to induce embryonic development in vegetative cells. LEC1 encodes a HAP3 subunit of the CCAAT binding transcription factor. We show that the 10 Arabidopsis HAP3 (AHAP3) subunits can be divided into two classes based on sequence identity in their central, conserved B domain. LEC1 and its most closely related subunit, LEC1-LIKE (L1L), constitute LEC1-type AHAP3 subunits, whereas the remaining AHAP3 subunits are designated non-LEC1-type. Similar to LEC1, L1L is expressed primarily during seed development. However, suppression of L1L gene expression induced defects in embryo development that differed from those of lec1 mutants, suggesting that LEC1 and L1L play unique roles in embryogenesis. We show that L1L expressed under the control of DNA sequences flanking the LEC1 gene suppressed genetically the lec1 mutation, suggesting that the LEC1-type B domains of L1L and LEC1 are critical for their function in embryogenesis. Our results also suggest that LEC1-type HAP3 subunits arose from a common origin uniquely in plants. Thus, L1L, an essential regulator of embryo development, defines a unique class of plant HAP3 subunits.

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.

Interorgan regulation of ethylene biosynthetic genes by pollination.

Pollination initiates a syndrome of developmental events that contribute to successful reproduction, including perianth senescence, changes in pigmentation, and ovule differentiation in preparation for impending fertilization. In orchid flowers, initiation of each of these processes in distinct floral organs is strictly and coordinately controlled by pollination, thus providing a unique opportunity to study the signals that coordinate interorgan postpollination development. Because ethylene has been implicated in contributing to regulation of severa1 aspects of postpollination development, we focused on determining the expression of its biosynthetic genes and their possible role in regulation. The abundance of mRNA encoding both 1-aminocyclopropane-l-carboxylic acid (ACC) synthase and ACC oxidase in the stigma, ovary, and labellum was found to be coordinately regulated by emasculation, auxin, and ethylene. Although petals contribute up to 26% of total flower ethylene and accumulate high levels of ACC oxidase mRNA and activity following pollination, no ACC synthase mRNA or activity was detected in this tissue. Together, these results support a model of interorgan regulation of postpollination development that depends on pollination-stimulated accumulation of mRNA encoding ethylene biosynthetic enzymes in a developmentally regulated and tissue-specific manner. This model relies on the translocation of a soluble hormone precursor, ACC, rather than on the translocation of the hormone itself. In this way, ACC serves to actuate the response already initiated by ethylene perceived by other parts of the flower. Thus, ACC may function as a secondary transmissible signal that coordinates postpollination development in diverse floral organs.

Using Genomics to Study Legume Seed Development

Seeds are essential for flowering plant reproduction because they protect, nourish, and contain the developing embryo that represents the next sporophytic generation. In addition, seeds contain energy resources that sustain the young sporophyte during germination before photosynthesis begins. In

Regional and cell-specific gene expression patterns during petal development.

We investigated gene expression patterns that occur during tobacco petal development. Two petal mRNA classes were identified that are present at elevated levels relative to other organs. One class is represented equally in the unpigmented tube and pigmented limb regions of the corolla. The other class accumulates preferentially within the limb region. Limb-specific mRNAs accumulate at different times during corolla development, peak in prevalence prior to flower opening, and are localized in either the epidermal cell layers or the mesophyll. The epidermal- and mesophyll-specific mRNAs change abruptly in concentration within a narrow zone of the limb/tube border. Preferential accumulation of at least one limb-specific mRNA occurs within the corolla upper region early in development prior to limb maturation and pigment accumulation. Limb-specific mRNAs also accumulate preferentially within the unpigmented corolla limb region of Nicotiana sylvestris, a diploid progenitor of tobacco. Runoff transcription studies and experiments with chimeric beta-glucuronidase genes showed that petal gene organ, cell, and region specificities are controlled primarily at the transcriptional level. We conclude that during corolla development transcriptional processes act coordinately on limb-specific genes to regulate their regional expression patterns, but act individually on these genes to define their cell specificities.

Differentiation and degeneration of cells that play a major role in tobacco anther dehiscence

Dehiscence is the terminal step in anther development that releases pollen grains from the wall of each theca at a specific site between the two locules. In tobacco, two groups of cells—the circular cell cluster and the stomium—are required for anther dehiscence and define the position at which pollen is released. The processes responsible for the differentiation of the circular cell cluster and the stomium from cells in specific anther regions are unknown. Nor is it understood what initiates the programmed degeneration of these cell types that ultimately is responsible for pollen release from the anther. We characterized stomium and circular cell cluster differentiation and degeneration using both light and transmission electron microscopy throughout anther development, from the emergence of stamen primordia to anther dehiscence at flower opening. We observed that histological changes within primordium L1 and L2 cells destined to become the stomium and circular cell cluster occur at the same time after the differentiation of surrounding locule regions. Sub-epidermal cells that differentiate into the circular cell cluster divide, enlarge, and generate vacuoles with calcium oxalate crystals prior to any detectable changes in pre-stomium epidermal cells. Differentiation and division of cells that generate the stomium occur after cell degeneration initiates in the circular cell cluster. Prior to dehiscence, the stomium consists of a small set of cytoplasmically dense cells that are easily distinguished from their larger, highly vacuolate epidermal neighbors. Plasmodesmata connections within and between cells of the stomium and circular cell cluster were observed at different developmental stages, suggesting that these cells communicate with each other. Circular cell cluster and stomium cell death is programmed developmentally and occurs at different times. Degeneration of the circular cell cluster occurs first, contributes to the formation of a bilocular anther, and generates the site of anther wall breakage. The stomium cell death process is complete at flower opening and provides an opening for pollen release from each theca. We used laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reactions to demonstrate that stomium cells can be isolated from developing anthers and studied for the presence of specific mRNAs. Our data suggest that a cascade of unique gene expression events throughout anther development is required for the dehiscence program, and that the differentiation of the stomium and circular cell cluster in the interlocular region of the anther probably involves cell signaling processes.

Abundance of an mRNA encoding a high mobility group DNA-binding protein is regulated by light and an endogenous rhythm.

A cDNA clone encoding an HMG1 protein from Pharbitis nil was characterized with regard to its sequence, genomic organization and regulation in response to photoperiodic treatments that control floral induction. The HMG1 cDNA contains an open reading frame of 432 nucleotides encoding a 144 amino acid protein of approximately 16 kDa. The predicted polypeptide has the characteristic conserved motifs of the HMG1 and HMG2 class of proteins including an N-terminal basic region, one of two HMG-box domains, and a polyacidic carboxy terminus. Within the HMG-box region, Pharbitis HMG1 deduced amino acid sequence shares 47%, 67% and 69% identity with its animal, maize, and soybean counterparts, respectively. Southern blot hybridization analysis suggests that HMG1 is a member of a multigene family. Analysis of mRNA abundance indicates that the HMG1 gene is expressed to higher levels in dark-grown tissue, such as roots, and at lower levels in light-grown tissue, such as cotyledons and stems. Following the transition to darkness, the levels of HMG1 mRNA in cotyledons were initially stable, however, after a lag time of 8 h or more, HMG1 mRNA increased in abundance to a peak level at 20 h. A second peak in mRNA levels was observed about 24 h later, indicating that the expression of the HMG1 gene is regulated by an endogenous circadian rhythm. Abundance of the HMG1 mRNA during a dark period was dramatically affected by brief light exposure (night break), a treatment which inhibits floral induction. These data indicate that the expression of HMG1 is regulated by both an endogenous rhythm and the light/dark cycle and are consistent with a role for HMG1 in maintaining patterns of circadian-regulated gene expression activated upon the transition from light to darkness.

Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development

An RNA interference screen of 53 transcription factor mRNAs that accumulate specifically during soybean seed development identified a homolog of an epidermal factor required to initiate stomatal cell lineages during embryo development. We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and INDUCER OF C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT-BINDING FACTOR EXPRESSION1/SCREAM2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development.