A putative SUBTILISIN-LIKE SERINE PROTEASE 1 (SUBSrP1) regulates anther cuticle biosynthesis and panicle development in rice

Abstract

Introduction: Panicle abortion is a severe physiological defect and causes a reduction in grain yield.

Objectives: In this study, we aim to provide the characterization and functional analysis of a mutant apa1331 (apical panicle abortion1331).

Methods: The isolated mutant from an EMS-mutagenized population was subjected to SSR analysis and Mutmap assay for candidate gene mapping. We performed phenotypic analysis, anthers cross-sections morphology, wax and cutin profiling, biochemical assays and phylogenetic analysis for characterization and evaluation of apa1331. We used CRISPR/Cas9 disruption for functional validation of its candidate gene. Furthermore, comparative RNA-seq and relative expression analysis were performed to get further insights into mechanistic role of the candidate gene.

Results: The anthers from the apical spikelets of apa1331 were degenerated, pollen-less and showed defects in cuticle formation. Transverse sections of apa1331 anthers showed defects in post-meiotic microspore development at stage 8-9. Gas Chromatography showed a significant reduction of wax and cutin in anthers of apa1331 compared to Wildtype (WT). Quantification of H₂O₂ and MDA has indicated the excessive ROS (reactive oxygen species) in apa1331. Trypan blue staining and TUNEL assay revealed cell death and excessive DNA fragmentation in apa1331. Map-based cloning and Mutmap analysis revealed that LOC_Os04g40720, encoding a putative SUBTILISIN-LIKE SERINE PROTEASE (OsSUBSrP1), harbored an SNP (A > G) in apa1331. Phenotypic defects were only seen in apical spikelets due to highest expression of OsSUBSrP1 in upper panicle portion. CRISPR-mediated knock-out lines of OsSUBSrP1 displayed spikelet abortion comparable to apa1331. Global gene expression analysis revealed a significant downregulation of wax and cutin biosynthesis genes.

Conclusions: Our study reports the novel role of SUBSrP1 in anther cuticle biosynthesis by ROS-mediated programmed cell death in rice.

Keywords: Anther cuticle; Cell death; Spikelet development; Subtilisin like protease; Wax and cutin.