Inositolphosphorylceramide synthases, OsIPCSs, regulate plant height in rice.

Inositolphosphorylceramide synthase (IPCS) catalyses ceramides and phosphatidylinositol (PI) into inositolphosphorylceramide (IPC), which is involved in the regulation of plant growth and development. A total of three OsIPCS family genes have been identified in rice. However, most of their functions remain unknown. Here, the functions of OsIPCSs were analyzed by CRISPR/Cas9 technology, lipidomics analysis, and transcriptomics analysis. Single-gene mutation of OsIPCSs resulted in dwarf phenotype. Among them, the phenotype of osipcs3 mutant was more severe. Multi-gene mutation of OsIPCS genes led to more severe phenotypes, indicating the additive effects of OsIPCSs. We further determined that a significant decrease in epidermal cell elongation of internode in the mutants. There was a significant decrease in the content of IPC detected in the osipcs2/3 and osipcs1/2/3 mutants. The contents of glycosyl inositol phosphoryl ceramide (GIPC) were also decreased by 20% and 10% in osipcs2/3 and osipcs1/2/3, respectively. The results of RNA-seq showed that numerous DEGs found to be associated with cellular component organization, anatomical structure morphogenesis, and cell growth in the osipcs2, osipcs2/3, and osipcs1/2/3. Taken together, OsIPCSs may be involved in the regulation of plant height through affecting cell growth and sphingolipid metabolism in rice.

New Fructokinase, OsFRK3, Regulates Starch Accumulation and Grain Filling in Rice.

Plant fructokinase (FRK) guarantees the growth and development of higher plants by participating in carbohydrate metabolism. In this study, a new fructokinase, OsFRK3, was identified using bioinformatics analysis, enzyme assay, bacterial growth assay, and yeast complementation test. Then, we created OsFRK3 knockout transgenic lines (osfrk3-1 and osfrk3-2) by the CRISPR/Cas9 technology. We found that the 1000-grain weight decreased notably (approximately -3.6% and -6.1%, respectively) in osfrk3-1 and osfrk3-2. Evidently decreased grain width, grain thickness, and endosperm filling rate were detected in the osfrk3 mutants (osfrk3-1 and osfrk3-2) compared with those of the WT. In addition, the content of seed total starch was significantly decreased by 3.42 and 4.80% in osfrk3 lines, compared with that in the WT. The level of maltose was significantly reduced in the mutants, while that of sucrose and fructose was obviously increased in the mutants. The transcript levels of OsGBSS1, OsBEIIb, OsPFP1ß, and OsAGPL1 were significantly decreased in the osfrk3 mutants. These results suggest that OsFRK3 may positively regulate the accumulation of starch through influencing the sugar metabolism.

Glycine- and Proline-Rich Protein OsGPRP3 Regulates Grain Size and Quality in Rice.

The grain size and shape of rice are limited by the growth of the spikelet hulls and are important selective target during domestication and breeding. In this study, we identified a glycine- and proline-rich protein (OsGPRP3), which belongs to a conserved family rarely studied. We found that OsGPRP3 was highly expressed in the seed at 10 days after pollination (DAP) using qRT-PCR, pOsGPRP3::GUS and in situ hybridization. Knockout and knockdown of OsGPRP3 led to significant decrease of 1000-grain weight, grain width, and grain thickness. We further found that the content of storage protein and total lipid were decreased in osgprp3 lines. In particular, the contents of C14:0 (myristic acid), C16:0 (palmitic acid), C18:1 (oleic acid), and C18:2 (linoleic acid) were reduced in osgprp3 lines. Cytological experiments revealed that the cell width of spikelet hull in osgprp3 lines was significantly reduced than that in WT. Taken together, our results reveal that OsGPRP3 regulates the grain size and shape of rice by influencing the cell width of spikelet hulls and the accumulation of storage protein and lipids.