OsVDE, a xanthophyll cycle key enzyme, mediates abscisic acid biosynthesis and negatively regulates salinity tolerance in rice.

MAIN CONCLUSION: OsVDE, a lipocalin-like protein in chloroplasts, negatively regulated the ABA biosynthesis and stomatal closure under salt stress in rice seedlings. Violaxanthin de-epoxidase (VDE) is a key enzyme of xanthophyll cycle. It plays a critical role in abscisic acid (ABA) biosynthesis, growth and stress responses in plants. Although functions of several VDE genes have been characterized, it is largely unknown whether OsVDE regulates the ABA biosynthesis and salt stress tolerance in rice. In this study, we generated the OsVDE overexpressing and CRISPR-Cas9-mediated gene-editing transgenic lines, and identified that the gene-editing mutant lines showed the dwarfism, shorter panicle and lower seed-setting rate than the wild type whereas the overexpression lines did not exhibit the difference from the wild type. In addition, the gene-editing transgenic lines were hypersensitive to exogenous ABA during germination. Under salt stress, the gene-editing transgenic seedlings had a higher ABA level, higher stomatal closure percentage and higher survival rate than the wild type. The qRT-PCR analysis confirmed that OsVDE negatively regulated the OsNECD2/4/5 expressions, ABA biosynthesis and salt stress tolerance in rice seedlings. These results provide new evidence that VDE plays an essential role in ABA biosynthesis and salt stress tolerance in plants.

OsCML16 interacts with a novel CC-NBS-LRR protein OsPi304 in the Ca2+/Mg2+ dependent and independent manner in rice.

In plants, many target proteins of calmodulins (CaMs) have been identified in cellular metabolism and responses. However, calmodulin-like proteins (CMLs) and their target proteins have not been discovered in stress responses in rice. In this study, a novel CC-NBS-LRR protein was obtained in screening a cold stress rice seedlings yeast cDNA library with OsCML16 as bait. Furthermore, yeast two-hybrid and BiFC assays demonstrated that the full length, CC region in the N-terminus and LRR in the C-terminus of Pi304 protein could interact with OsCML16. More interestingly, OsCML16 bound to the 1-10 motif rather than 1-14 motif in the Ca2+ or Mg2+ dependent manner in vitro. In addition, transcript levels of OsCML16 and OsPi304 were induced more markedly in Nipponbare than in 9311 under cold stress. Taken together, these data indicates that they are involved in the cold stress signaling and response in rice.

Overexpressing OsFBN1 enhances plastoglobule formation, reduces grain-filling percent and jasmonate levels under heat stress in rice.

In higher plants, Fibrillins (FBNs) constitute a conserved plastid-lipid-associated (PAPs) protein family and modulate the metabolite transport and lipid metabolism in plastids of dicot species. However, FBNs have not functionally characterized in monocot species. In this study, the function of rice fibrillin 1 (OsFBN1) was investigated. The subcellular localization assay showed that the N-terminal chloroplast transport peptide (CTP) could facilitate the import of OsFBN1 into chloroplast. OsFBN1 specifically bound C18- and C20- fatty acids in vitro. Overexpressing OsFBN1 increased the tiller number but decreased the panicle length, grain-filling percent and JA levels compared to the wild type and RNAi silencing lines under heat stress. In addition, the overexpressing lines had more plastoglobules (PGs) than the wild type and RNAi silencing lines under both normal and heat stress conditions. Moreover, overexpressing OsFBN1 affected the transcription levels of OsAOS2 in JA synthesis, OsTHF1, OsABC1K7 and OsPsaE in thylakoid stability and photosynthesis, OsABC1-4 and OsSPS2 in ubiquinone-metabolism, OsHDR, OsDXR, and OsFPPS in isoprenoid metabolism. Collectively, these findings suggest the essential role of rice OsFBN1 in PG formation and lipid metabolism in chloroplasts, which coordinately regulate the growth and grain filling of the overexpressing lines under heat stress.