Dissection of sexual organ ontogenesis: a genetic analysis of ovule development in Arabidopsis thaliana.

Understanding organogenesis remains a major challenge in biology. Specification, initiation, pattern formation and cellular morphogenesis, have to be integrated to generate the final three-dimensional architecture of a multicellular organ. To tackle this problem we have chosen the ovules of the flowering plant Arabidopsis thaliana as a model system. In a first step towards a functional analysis of ovule development, we performed a large-scale genetic screen and isolated a number of sterile mutants with aberrant ovule development, We provide indirect genetic evidence for the existence of proximal-distal pattern formation in the Arabidopsis ovule primordium. The analysis of the mutants has identified genes that act at an intermediate regulatory level and control initiation of morphogenesis in response to proximal-distal patterning. A second group of genes functions at a subordinate control level and regulates general cellular processes of morphogenesis. A large group of male and female sterile mutants shows defects restricted to early or late gametogenesis. In addition, we propose that the mature ovule obtains its overall curved shape by at least three different processes that act in only one domain of the ovule.

Identification of genes required for pollen-stigma recognition in Arabidopsis thaliana.

In higher plants, cell-cell recognition reactions taking place following pollination allow the selective restriction of self-pollination and/or interspecific pollination. Many of these systems function by regulating the process of water transfer from the cells found at the stigmatic surface to the individual pollen grain. Interspecific pollination studies on the cruciferous weed Arabidopsis thaliana revealed only a broad specificity of pollen recognition such that pollen from all tested members of the crucifer family were recognized, whereas pollen from almost all other species failed to hydrate. Genetic analysis of A. thaliana has identified three genes that are essential for this recognition process. Recessive mutations in any of these genes result in male sterility due to the production of pollen grains that fail to hydrate when placed on the stigma, but that are capable of hydrating and growing a pollen tube in vitro. Results from mixed pollination experiments suggest that the mutant pollen grains specifically lack a functional pollen-stigma recognition system. All three mutations described also result in a defect in the wax layer normally found on stems and leaves, similar to previously described eceriferum (cer) mutations. Genetic complementation and mapping experiments demonstrated that the newly identified mutants are allelic to the previously identified genes cer1, cer3 and cer6. TEM analysis of the ultrastructure of the pollen coating revealed that all of the mutant pollen grains bear coatings of normal thickness and that tryphine lipid droplets are missing in cer1-147, are reduced in size in cer6-2654 and appear normal in cer3-2186.(ABSTRACT TRUNCATED AT 250 WORDS)

Semi-constitutive expression of an Arabidopsis thaliana alpha-tubulin gene.

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple alpha-tubulin and beta-tubulin genes. Previous evidence suggested that the TUA2 alpha-tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5'-flanking DNA fused to the beta-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.

Molecular genetics of cell interactions in Arabidopsis.

Many events in plant development are regulated by the interactions of neighboring cells. We are interested in determining what sorts of molecules act as signals and/or receptors in these interactions and how these mechanisms relate to those used in animals and fungi. We are presently working on two different types of systems to try to address this question. In one case we are starting at the molecular level and characterizing a family of receptor protein kinase genes which seem natural candidates for mediating cellular interactions. By analyzing the expression patterns of these genes as well as the phenotypes of transgenic plants bearing altered genes we hope to determine what roles these proteins play in plant development. In the second case we are starting from the organismic level and using genetics to identify genes essential to a whole range of cellular interactions which are required for proper male gametophyte development during reproduction. These interactions involve both recognition of the pollen grain to verify that it is from the correct species and also a transfer of positional information from the female to the male which first allows the pollen tube to determine the polarity of the stigmatic cell on which it has germinated and later provides 'guidance' for the elongating tube to find the ovule.

Alpha-tubulin gene family of maize (Zea mays L.). Evidence for two ancient alpha-tubulin genes in plants.

Among 81 alpha-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different alpha-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different alpha-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93%. The results of hybridizations of genomic DNAs to alpha-tubulin coding region probes and to 3' non-coding region probes constructed from six different alpha-tubulin cDNA clones indicated that the maize alpha-tubulin gene family contains at least eight members. Comparison of deduced alpha-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that alpha-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient alpha-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily II genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.

The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes.

The small genome of Arabidopsis contains at least nine expressed beta-tubulin (TUB) genes, in contrast to the large genomes of vertebrate animals, which contain a maximum of seven expressed beta-tubulin genes. In this study, we report the structures of seven new TUB genes (TUB2, TUB3, TUB5, TUB6, TUB7, TUB8, and TUB9) of Arabidopsis. The sequences of TUB1 and TUB4 had been reported previously. Sequence similarities and unique structural features suggest that the nine TUB genes evolved by way of three branches in the plant beta-tubulin gene evolutionary tree. Two genes (TUB2 and TUB3) encode the same beta-tubulin isoform; thus, the nine genes predict eight different beta-tubulins. In contrast to the alpha-tubulin (TUA) genes with their divergent intron patterns, all nine TUB genes contain 2 introns at conserved positions. Noncoding 3' gene-specific hybridization probes have been constructed for all nine TUB genes and used in RNA gel blot analyses to demonstrate that all nine genes are transcribed. Two-dimensional protein immunoblot analyses have resolved at least seven different beta-tubulin isoforms in Arabidopsis, indicating that most, if not all, of the TUB transcripts are translated.

The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes.

The goal of our investigations is to define the genetic control of microtubule-based processes in a higher plant. The available evidence suggests that we have achieved our first objective: the characterization of the complete alpha-tubulin and beta-tubulin gene families of Arabidopsis. Four additional alpha-tubulin genes (TUA2, TUA4, TUA5, and TUA6) of Arabidopsis have been cloned and sequenced to complete the analysis of the gene structure for all six alpha-tubulin genes detectable on DNA gel blots of Arabidopsis genomic DNA hybridized with alpha-tubulin coding sequences. TUA1 and TUA3 were characterized earlier in our laboratory. Noncoding gene-specific hybridization probes have been constructed for all six alpha-tubulin genes and used in RNA gel blot analyses to demonstrate that all six genes are transcribed. The six genes encode four different alpha-tubulin isoforms; TUA2 and TUA4 encode a single isoform, as do TUA3 and TUA5. Two-dimensional protein gel immunoblot analyses have resolved at least four alpha-tubulin isoforms from plant tissues, suggesting that all of the predicted TUA gene products are synthesized in vivo.