Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).

Eui-Jung Kim,Yu-Jin Kim,Woo-Jong Hong,Ki-Hong Jung

doi:10.3390/ijms23010239

Abstract

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

Highlights

Plants use pollen grains to deliver sperm cells to the ovary
In the case of Oryza sativa, OsMADS62, OsMADS63, and OsMADS68 have high expression in the pollen grains at the tricellular stage; OsMADS62 and OsMADS63 belong to the MIKC* S-clade, whereas OsMADS68 belongs to the MIKC* P-clade [30]
We generated homozygous triple loss-of-function mutants of three OsMADS genes belonging to the MIKC*-type (OsMADS62, OsMADS63, and OsMADS68) that are preferentially expressed in mature pollen grains

Summary

Introduction

Plants use pollen grains (the male gamete) to deliver sperm cells to the ovary. When the pollen maturing inside the anther reaches the tricellular stage, anthesis and dehiscence occur, and pollen grains are released [1]. MADS-box protein is a transcription factor (TF) with a highly conserved MADS domain and essential functions in floral organ development, seed development, floral organ identity determination, and regulation of flowering time [17,18,19,20] It regulates the expression of downstream genes by directly binding to the CArG motif of the promoter. In the case of Oryza sativa, OsMADS62, OsMADS63, and OsMADS68 have high expression in the pollen grains at the tricellular stage; OsMADS62 and OsMADS63 belong to the MIKC* S-clade, whereas OsMADS68 belongs to the MIKC* P-clade [30] These three proteins can form homodimers and heterodimers through diverse combinations, and the interactions of OsMADS62/68 and OsMADS63/68 are the strongest [30]. We generated homozygous triple loss-of-function mutants of three OsMADS (box) genes belonging to the MIKC*-type (OsMADS62, OsMADS63, and OsMADS68) that are preferentially expressed in mature pollen grains. Because OsMADS62, OsMADS63, and OsMADS68 function in the late stage of pollen maturation, germination, and tube elongation, transcriptome analysis using triple mutations should provide an important molecular basis for future research

Results

OsMADStri Is Defective in the Late Maturation Stage of Pollen

Nucleic Acids Extraction and RT-qPCR

Vector Construction and Plant Transformation

Phenotypic Analysis

RNA Sequencing and Bioinformatic Analysis