Project description:Tandem mass tags (TMT)-based quantitative analysis was used to investigate the profiles of proteins related to lipid and carbohydrate metabolism in 4-, 6- and 8.5-mm O. longistaminata spikelets before flowering. O. longistaminata plants were sampled at three important stages of pollen development: 4-mm spikelet (Ls-4mm, primary sporogenous cells, Stage 4), 6-mm spikelet (Ls-6mm, microspore mother cells, Stage 6) and 8.5-mm spikelet (Ls-8.5mm, second mitosis cells, Stage 12) .
Project description:Transcriptional profiling of mitotic archesporial cells, early pollen mother cells and pollen mother cells comparing control tapetal cells, pollen, parenchyma cells and seedling. Goal was explain the mitosis/meiosis transition at the molecular level.
Project description:The profiling was conducted with the Rice 3'-Tiling 135k Microarray designed from 31,439 genes deposited at IRGSP, RAP2 database (http://rapdb.lab.nig.ac.jp). We have identified and characterized a T-DNA insert rice mutant (Osfuct) with loss of α1,3-fucosyltransferase function. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycan revealed the lack of α1,3-fucose in the N-glycan structure of rice Osfuct mutant. The mutant displayed the pleiotropic developmental defects such as diminished growth, shorter plant height, less number of tillers, shorter panicle lengths and internode, impaired anther and pollen development. In addition, the anther was curved, pollen grains shapes were shriveled, pollen viability and pollen number per anther was dramatically decreased in Osfuct mutant. The complementation test of Osfuct mutant clearly exhibited that the phenotype is caused by the loss of α1,3-fucosyltransferase function bescause complementation line is rescued. Transcriptome profiling data revealed that several genes essential in plant developmental processes were significantly altered in Osfuct mutant including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis and hypothetical proteins. Moreover, Osfuct mutant exhibited the enhanced salt insensitivity. Taken together, these findings demonstrated that Osfuct plays a critical role in growth, anther, pollen development and salt stress response.
Project description:- Pollen tube growth is important process for successful double fertilization, which is critical for grain yield in crop plants. Despite much progress in identification of rapid alkalization factors (RALFs) which serve as ligand for signaling transduction during fertilization in Arabidopsis, there is no functional study of RALF in mono-cotyledon plant. - We functionally characterized two pollen specific RALF in rice (Oryza sativa) using multiple CRISPR/Cas9 induced loss-of-function mutants, peptide treatment, expression analyses, tag reporter lines. - OsRALF17 is specifically expressed in pollen and pollen tube as the strongest level among 41 RALF members in rice. Exogenously applied OsRALF17 inhibits pollen tube germination and elongation at high concentration, but enhances tube elongation at low concentration, indicating the regulation of growth balance. Double mutant of OsRALF17 with OsRALF19 exhibit almost male sterile, with defect on pollen germination and tube elongation. - Our study revealed that functionally-redundant OsRALF17 and 19 peptides binds to the OsMTD2, CrRLK1L family member, and transmits ROS signal for pollen tube germination and integrity maintenance in rice. We provide new insights into the role of RALF and expanding our understanding of the biological role of RALF in regulating rice fertilization.
Project description:Pollen tube growth is essential for successful fertilization and stable crop yields. We constructed loss-of-function/knock-out mutants that simultaneously target two rice genes using the CRISPR/Cas9 mutagenesis system. The selected OsRALF17 and OsRALF19 genes are strongly expressed in rice bicellular/tricellular pollen and have essential functions in the pollen tube growth. For the corresponding transcriptomic analysis, we sampled mature pollen anthers from a control group and an OsRALF17/19 knock-out mutant.
Project description:We isolated tricellular pollen (TCP) and pollen mother cells (PMC) of rice using laser microdissection, and did microarray analysis with Agilent 44k rice array.
Project description:In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In this study, proteins from egg and sperm cells obtained from Oryza sativa flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. Database was searched using Mascot software (ver.2.2.1, Matrix Science, MA, USA) with the following parameters. The fixed modification was propionasmide (Cys) and variable modification parameters were pyro-Glu, acetylation (protein N-terminus), and oxidation (Met). The maximum missed cleavage was set at 3 with a peptide mass tolerance of +/– 15 ppm. Peptide charges from +2 to +4 states and MS/MS tolerances of +/– 0.8 Da were allowed.
Project description:We created a triple loss-of-function/knockout mutant targeting three rice genes simultaneously. The three selected genes are as follows: OsADF1 (LOC_Os02g44470), OsADF6 (LOC_Os04g46910), and OsADF9 (LOC_Os07g30090). These three ADFs are strongly transcriptional expressed in the rice mature anthers (stages 13) and bi-/tricelluler pollen. The triple mutant of these OsADFs does not produce self-fertilizing seeds due to the short length of the pollen tube (male-sterile). This data is about mature anther transcriptome data about the triple mutant of OsADFs (ADFmT). We sampled mature anther for the analysis.
Project description:Oryza longistaminata is an African wild rice species that possesses special traits for breeding applications. Self-incompatibility is the main cause of sterility in O. longistaminata, but here we demonstrated that its pollen fertility and vitality are normal. Lipid and carbohydrate metabolism were active throughout pollen development. In this study, transcriptomics quantitative analysis was used to investigate the profiles of genes related to lipid and carbohydrate metabolism in 4-, 6- and 8.5-mm O. longistaminata spikelets before flowering. We documented cytological changes throughout important stages of anther development, including changes in reproductive cells as they formed mature pollen grains through meiosis and mitosis. RNA-seq and proteome association analysis indicated that fatty acids were converted to sucrose after the 6-mm spikelet stage, based on the abundance of most key enzymes of the glyoxylate cycle and gluconeogenesis. In conclusion, our study provides novel insights into the pollen viability of O. longistaminata at the transcriptome level, which can be used to improve the efficiency of male parent pollination in hybrid rice breeding applications.