Rice ß-Glucosidase 4 (Os1ßGlu4) Regulates the Hull Pigmentation via Accumulation of Salicylic Acid.

Salicylic acid (SA) is a stress hormone synthesized in phenylalanine ammonia-lyase (PAL) and the branching acid pathway. SA has two interconvertible forms in plants: SAG (SA O-ß-glucoside) and SA (free form). The molecular mechanism of conversion of SA to SAG had been reported previously. However, which genes regulate SAG to SA remained unknown. Here, we report a cytoplasmic ß-glucosidase (ß-Glu) which participates in the SA pathway and is involved in the brown hull pigmentation in rice grain. In the current study, an EMS-generated mutant brown hull 1 (bh1) displayed decreased contents of SA in hulls, a lower photosynthesis rate, and high-temperature sensitivity compared to the wild type (WT). A plaque-like phenotype (brown pigmentation) was present on the hulls of bh1, which causes a significant decrease in the seed setting rate. Genetic analysis revealed a mutation in LOC_Os01g67220, which encodes a cytoplasmic Os1ßGlu4. The knock-out lines displayed the phenotype of brown pigmentation on hulls and decreased seed setting rate comparable with bh1. Overexpression and complementation lines of Os1ßGlu4 restored the phenotype of hulls and normal seed setting rate comparable with WT. Subcellular localization revealed that the protein of Os1ßGlu4 was localized in the cytoplasm. In contrast to WT, bh1 could not hydrolyze SAG into SA in vivo. Together, our results revealed the novel role of Os1ßGlu4 in the accumulation of flavonoids in hulls by regulating the level of free SA in the cellular pool.

A homologous gene of OsREL2/ASP1, ASP-LSL regulates pleiotropic phenotype including long sterile lemma in rice.

BACKGROUND: Panicle is a harvesting organ of rice, and its morphology and development are closely associated with grain yield. The current study was carried on a mutant screened through an EMS (ethyl-methane sulphonate) mutagenized population of a Japonica cultivar Kitaake (WT). RESULTS: A mutant, named as asp-lsl (aberrant spikelet-long sterile lemma), showed a significant decrease in plant height, number of tillers, thousand-grains weight, seed setting rate, spikelet length, kernel length and effective number of grains per panicle as compared to WT. Asp-lsl showed a pleiotropic phenotype coupled with the obvious presence of a long sterile lemma. Cross-sections of lemma showed an increase in the cell volume rather than the number of cells. Genetic segregation analysis revealed its phenotypic trait is controlled by a single recessive nuclear gene. Primary and fine mapping indicated that candidate gene controlling the phenotype of asp-lsl was located in an interval of 212 kb on the short arm of chromosome 8 between RM22445 and RM22453. Further sequencing and indels markers analysis revealed LOC_Os08g06480 harbors a single base substitution (G→A), resulting in a change of 521st amino acid(Gly→Glu. The homology comparison and phylogenetic tree analysis revealed mutation was occurred in a highly conserved domain and had a high degree of similarity in Arabidopsis, corn, and sorghum. The CRISPR/Cas9 mutant line of ASP-LSL produced a similar phenotype as that of asp-lsl. Subcellular localization of ASP-LSL revealed that its protein is localized in the nucleus. Relative expression analysis revealed ASP-LSL was preferentially expressed in panicle, stem, and leaves. The endogenous contents of GA, CTK, and IAA were found significantly decreased in asp-lsl as compared to WT. CONCLUSIONS: Current study presents the novel phenotype of asp-lsl and also validate the previously reported function of OsREL2 (ROMOSA ENHANCER LOCI2), / ASP1(ABERRANT SPIKELET AND PANICLE 1).