De Ye

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.

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.

Comparison of MADS Box Gene Expression in Developing Male and Female Flowers of the Dioecious Plant White Campion

The MADS box motif is common to genes that regulate the pattern of flower development. To determine whether MADS box genes also play a role in differentiation of the sexes in dioecious plants, we isolated cDNAs (SLM1 to SLM5, for Silene latifolia MADS) with MADS box homology from transcripts of male flower buds of the model dioecious species white campion and compared their expression in developing female and male flowers. SLM1 had extensive sequence similarity to the snapdragon MADS box gene PLENA, SLM2 to GLOBOSA, SLM3 to DEFICIENS, and both SLM4 and SLM5 were similar to SQUAMOSA. Each of the white campion MADS box genes was expressed in the same floral whorls as their respective most homologous snapdragon genes. The sex of the plant affected the pattern of SLM2 and SLM3 expression in the petal and stamen whorls, resulting in a smaller fourth whorl in male flowers than in female flowers. This was correlated with repressed gynoecium development in male flowers. The expression of SLM4 and SLM5 in both sexes differed from that of SQUAMOSA in one important aspect. Unlike SQUAMOSA, they were expressed in inflorescence meristems. This may reflect differences in growth pattern between white campion and snapdragon.

SLOW WALKER1, Essential for Gametogenesis in Arabidopsis, Encodes a WD40 Protein Involved in 18S Ribosomal RNA Biogenesis

The progression of mitotic division cycles and synchronous development between and within the male and female reproductive organs are essential for plant sexual reproduction. Little is known about the genetic control of the progression of mitotic cycles of the haploid genome during gametogenesis in higher plants. Here, we report the phenotypic and molecular characterization of an Arabidopsis thaliana mutant, slow walker1 (swa1), in which the progression of the mitotic division cycles of the female gametophyte was disrupted. Confocal microscopy revealed that megagametophyte development was asynchronous in swa1, causing embryo sacs to arrest at two-, four-, or eight-nucleate stages within the same pistil. A delayed pollination experiment showed that a small fraction of the swa1 embryo sacs were able to develop into functional female gametophytes. The swa1 mutation also showed a slight reduction in penetrance through the male gametophyte, although the pollen grains were morphologically normal. Molecular analysis indicates that SWA1 encodes a protein with six WD40 repeats that is localized in the nucleolus in interphase cells. The SWA1 gene is expressed in cells undergoing active cell divisions, including functional megaspores and the female gametophytic cells. RNA interference results indicated that knockout of SWA1 inhibited root growth significantly and led to the accumulation of unprocessed 18S pre-rRNA. These data suggest that SWA1 most likely plays a role in rRNA biogenesis that is essential for the progression of the mitotic division cycles during gametogenesis in plants.

Overexpression of TAPETUM DETERMINANT1 Alters the Cell Fates in the Arabidopsis Carpel and Tapetum via Genetic Interaction with EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS

Previously, we reported that the TAPETUM DETERMINANT1 (TPD1) gene is required for specialization of tapetal cells in the Arabidopsis (Arabidopsis thaliana) anther. The tpd1 mutant is phenotypically identical to the excess microsporocytes1 (ems1)/extra sporogenous cells (exs) mutant. The TPD1 and EMS1/EXS genes may function in the same developmental pathway in the Arabidopsis anther. Here, we further report that overexpression of TPD1 alters the cell fates in the Arabidopsis carpel and tapetum. When TPD1 was expressed ectopically in the wild-type Arabidopsis carpel, the number of cells in the carpel increased significantly, showing that the ectopic expression of TPD1 protein could activate the cell division in the carpel. Furthermore, the genetic analysis showed that the activation of cell division in the transgenic carpel by TPD1 was dependent on EMS1/EXS, as it did not happen in the ems1/exs mutant. This result further suggests that TPD1 regulates cell fates in coordination with EMS1/EXS. Moreover, overexpression of TPD1 in tapetal cells also delayed the degeneration of tapetum. The TPD1 may function not only in the specialization of tapetal cells but also in the maintenance of tapetal cell fate.

A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis.

In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, Thermosensitive Male Sterile 1 (TMS1), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37 degrees C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30 degrees C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.

WBC27, an Adenosine Tri‐phosphate‐binding Cassette Protein, Controls Pollen Wall Formation and Patterning in Arabidopsis

In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores.

POD1 Regulates Pollen Tube Guidance in Response to Micropylar Female Signaling and Acts in Early Embryo Patterning in Arabidopsis

This work examines the pollen defective in guidance1 (pod1) mutant and finds that it is defective in pollen tube guidance to the micropyle. Homozygous pod1 embryos also show lethality. POD1 is an endoplasmic reticulum (ER) luminal protein involved in ER protein retention. The pollen tube germinates from pollen and, during its migration, it perceives and responds to guidance cues from maternal tissue and from the female gametophyte. The putative female cues have recently been identified, but how the pollen tube responds to these signals remains to be unveiled. In a genetic screen for male determinants of the pollen tube response, we identified the pollen defective in guidance1 (pod1) mutant, in which the pollen tubes fail to target the female gametophyte. POD1 encodes a conserved protein of unknown function and is essential for positioning and orienting the cell division plane during early embryo development. Here, we demonstrate that POD1 is an endoplasmic reticulum (ER) luminal protein involved in ER protein retention. Further analysis shows that POD1 interacts with the Ca2+ binding ER chaperone CALRETICULIN3 (CRT3), a protein in charge of folding of membrane receptors. We propose that POD1 modulates the activity of CRT3 or other ER resident factors to control the folding of proteins, such as membrane proteins in the ER. By this mechanism, POD1 may regulate the pollen tube response to signals from the female tissues during pollen tube guidance and early embryo patterning in Arabidopsis thaliana.