Proteomic and phosphoproteomic analyses reveal extensive phosphorylation of regulatory proteins in developing rice anthers

Abstract

SUMMARYAnther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms modulating protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4984 proteins and 3203 phosphoproteins with 8973 unique phosphorylation sites (p-sites). Among those detected here, 1544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P 3 DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that 'DNA repair','transcription regulation' and 'signaling' related proteins were overrepresented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support 46 kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification affecting protein activity.

Proteomic analysis of lysine acetylation provides strong evidence for involvement of acetylated proteins in plant meiosis and tapetum function

“…Consistently, most of those proteins are also involved in distinct metabolic pathways related to tapetum function, for instance callose and pollen wall development. A comparison of our previously reported RAM phosphoproteome (Ye et al ., ) and the lysine acetylome here revealed that proteins encoding transcription factors are preferentially modified by phosphorylation, while those functioning as enzymes are more likely to be acetylated (Figure ). In particular, among known functional proteins involved in tapetum and pollen development, we found that eight were acetylated.…”

“…To obtain clues about the subcellular localization of the KAC proteins in RAMs we performed enrichment of Gene Ontology (GO) term annotations for these proteins. The results showed that the largest proportion was assigned to cytoplasm, followed by membrane and nucleus (Figure a), similar to the pattern in the RAM phosphoproteome in our previous report (Ye et al ., ). In particular, 12.3%, 10.2%, 2.7% and 1.5% of the KAC proteins are located in the chloroplast, mitochondrion, endoplasmic reticulum (ER) and chromosome, respectively (Figure a).…”

“…Interestingly, we found that OsUGP1 has nine lysine‐acetylated sites, providing direct evidence for multiple lysine modification for proteins in callose metabolism. In addition, we previously found that OsUGP1 is also modified by phosphorylation (Ye et al ., ), suggesting that the function of OsUGP1 might be precisely modulated by distinct post‐translational modifications. To learn whether other detected KAC sites are in potential tapetum‐related proteins in rice, we searched for rice homologs of known tapetum and pollen development genes in Arabidopsis.…”

“…Plant growth conditions were described previously (Ye et al ., ). Rice meiotic stage anther collection, total protein extraction and trypsin digestion were conducted as described previously (Ye et al ., ).…”

bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice

“…Apart from this, the other possible reason behind biphasic protein accumulation of bHLH142 may lie in the enhancement of its protein stability. In the recent report, proteomic and phospho-proteomic analysis of rice meiotic anthers showed presence of bHLH142 protein in phospho-proteome43. Detection of bHLH142 protein in anther phospho-proteome suggested its post-translational level regulation that may change its stability.…”