Co-regulation of exine wall patterning, pollen fertility and anther dehiscence by Arabidopsis apyrases 6 and 7.

An NCBI nucleotide blast keyed to apyrase (ATP-diphosphohydrolases, EC 3.6.1.5) conserved regions revealed five apyrases, AtAPYs (3-7), in addition to the previously identified AtAPY1 and 2. Here we report the functional analyses of two of the newly defined apyrases, AtAPY6 and AtAPY7. We analyzed tissue specificity of AtAPY6 and 7 expression by qRT-PCR and promoter:GUS fusion assays. We characterized the phenotypes of single and double knockout mutants for AtAPY6 and 7 in anther and pollen by light microscopy and electron microscopy. The transcripts of both AtAPY6 and 7 are expressed in mature pollen grains. Single knockout mutants of AtAPY6 and 7 displayed a minor change in pollen exine pattern under scanning electron microscopy without obvious change in fertility. Double knockout mutants of AtAPY6 and 7 (apy6apy7) displayed severe defects in pollen exine pattern, deformed pollen shape and reduced male fertility. An analysis of pollen from heterozygous apy6apy7 plants suggests that the defects in pollen exine wall are determined by the diploid genome. Our findings demonstrate that AtAPY6 and AtAPY7 are enzymes that play an important role in exine development of pollen grains, possibly through regulating the production of key polysaccharides needed for proper assembly of the exine layer.

Ectopic expression of an annexin from Brassica juncea confers tolerance to abiotic and biotic stress treatments in transgenic tobacco.

Plant annexins belong to a multigene family and are suggested to play a role in stress responses. A full-length cDNA for a gene encoding an annexin protein was isolated and characterized from Brassica juncea (AnnBj1). AnnBj1 message levels were regulated by abscisic acid, ethephon, salicylic acid, and methyl jasmonate as well as chemicals that induce osmotic stress (NaCl, Mannitol or PEG), heavy metal stress (CdCl(2)) and oxidative stress (methyl viologen or H(2)O(2)). In order to determine if AnnBj1 functions in protection against stress, we generated transgenic tobacco plants ectopically expressing AnnBj1 under the control of constitutive CaMV 35S promoter. The transgenic tobacco plants showed significant tolerance to dehydration (mannitol), salt (NaCl), heavy metal (CdCl(2)) and oxidative stress (H(2)O(2)) at the seedling stage and retained higher chlorophyll levels in response to the above stresses as determined in detached leaf senescence assays. The transgenic plants also showed decreased accumulation of thiobarbituric acid-reactive substances (TBARS) compared to wild-type plants in response to mannitol treatments in leaf disc assays. AnnBj1 recombinant protein exhibited low levels of peroxidase activity in vitro and transgenic plants showed increased total peroxidase activity. Additionally, the transgenic plants showed enhanced resistance to the oomycete pathogen, Phytophthora parasitica var. nicotianae, and increased message levels for several pathogenesis-related proteins. Our results demonstrate that ectopic expression of AnnBj1 in tobacco provides tolerance to a variety of abiotic and biotic stresses.

Profile and analysis of gene expression changes during early development in germinating spores of Ceratopteris richardii.

Analysis of an expressed sequence tag library with more than 5,000 sequences from spores of the fern Ceratopteris richardii reveals that more than 3,900 of them represent distinct genes, and almost 70% of these have significant similarity to Arabidopsis (Arabidopsis thaliana) genes. Eight genes are common between three very different dormant plant systems, Ceratopteris spores, Arabidopsis seeds, and Arabidopsis pollen. We evaluated the pattern of mRNA abundance over the first 48 h of spore development using a microarray of cDNAs representing 3,207 distinct genes of C. richardii and determined the relative levels of RNA abundance for 3,143 of these genes using a Bayesian method of statistical analysis. More than 900 of them (29%) show a significant change between any of the five time points analyzed, and these have been annotated based on their sequence similarity with the Arabidopsis proteome. Novel data arising from these analyses identify genes likely to be critical for the germination and subsequent early development of diverse cells and tissues emerging from dormancy.

Disruption of apyrases inhibits pollen germination in Arabidopsis.

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive beta-glucuronidase staining in the pollen of apyrase promoter:beta-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.