OsGRF4AA compromises heat tolerance of developing pollen grains in rice

Abstract

Extreme high temperature at the meiosis stage causes a severe decrease in spikelet fertility and grain yield in rice. The rice variety grain size on chromosome 2 (GS2) contains sequence variations of OsGRF4 (Oryza sativa growth-regulating factor 4; OsGRF4^AA ), escaping the microRNA miR396-mediated degradation of this gene at the mRNA level. Accumulation of OsGRF4 enhances nitrogen usage and metabolism, and increases grain size and grain yield. In this study, we found that pollen viability and seed-setting rate under heat stress (HS) decreased more seriously in GS2 than in its comparator, Zhonghua 11 (ZH11). Transcriptomic analysis revealed that, following HS, genes related to carbohydrate metabolic processes were expressed and regulated differentially in the anthers of GS2 and ZH11. Moreover, the expression of genes involved in chloroplast development and photosynthesis, lipid metabolism, and key transcription factors, including eight male sterile genes, were inhibited by HS to a greater extent in GS2 than in ZH11. Interestingly, pre-mRNAs of OsGRF4, and a group of essential genes involved in development and fertilization, were differentially spliced in the anthers of GS2 and ZH11. Taken together, our results suggest that variation in OsGRF4 affects proper transcriptional and splicing regulation of genes under HS, and that this can be mediated by, and also feed back to, carbohydrate and nitrogen metabolism, resulting in a reduction in the heat tolerance of rice anthers.

Keywords: OsGRF4; carbohydrate metabolism; gene transcription; heat tolerance; pre-mRNA alternative splicing; rice.

Figure 1

Phenotypes of Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2) under different temperature conditions at the pollen mother cell meiosis stage. (A–D) Photographs of whole plants, seed setting, and pollen grains in ZH11 with and without HS treatment. (A, C) Without heat stress (HS) treatment. (B, D) After HS treatment. (E–H) Photographs of whole plants, seed setting, and pollen grains in GS2 with and without HS treatment. (E, G) Without HS treatment. (F, H) After HS treatment. Scale bars in (C, D, G, H) represent 100 µm. (I) Ratios of dead pollen grains. (J) Ratios of fertilized seeds. Significance level for t-test: **p < 0.01; ***p < 0.001.

Figure 2

Gene expression patterns in Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2) under different temperature conditions. (A) Mean–variance trend of raw read counts (before filtering). (B) Mean–variance trend of filtered read counts (poorly expressed reads removed). (C) Principal component analysis (PCA) plots of 12 libraries. (D) Differentially expressed gene (DEG) numbers of four separate comparisons. The green box shows up-regulated genes and the blue box shows down-regulated genes. (E) Specific and overlapping DEG numbers across four different comparisons.

Figure 3

Heat-responsive differentially expressed genes (DEGs) in anthers from different genotypes. (A) Gene ontology analysis of genes up-regulated by heat stress (HS) in both Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2). (B) Gene ontology analysis of genes down-regulated by HS in both ZH11 and GS2. (C) Numbers of specific and overlapping DEGs in our experiment and in two other studies. (D) Expression of 87 genes (overlapping DEGs in three studies) in ZH11 and GS2 under normal and HS conditions.

Figure 4

Expression patterns of differentially expressed genes (DEGs) between Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2) after heat stress (HS). (A) A heatmap showing expression patterns of DEGs in ZH11 and GS2 anthers after HS treatment. (B) Numbers of overlapping and specific DEGs between four comparisons in our study, namely ZH11HS < GS2HS (DEGs with lower expression levels in ZH11 than in GS2 after HS), ZH11&GS2_Up by HS (DEGs up-regulated by HS treatment in both ZH11 and GS2), ZH11&GS2_Down by HS (DEGs down-regulated by HS treatment in both ZH11 and GS2), and ZH11HS > GS2HS (DEGs with higher expression levels in ZH11 than in GS2 after HS). (C) Reverse transcription-quantitative PCR (qRT-PCR) validation of RNA sequencing (RNA Seq) data with some of these genes: BBX19, DREB1A, Pox1, HSFA2a, ABA8ox2, and OsMS2. Significance level for t-test: *p < 0.05; **p < 0.01. ns, non-significant.

Figure 5

Differentially expressed genes (DEGs) essential for male fertility in anthers from Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2) after heat stress (HS) treatment. (A) Gene ontology (GO) analysis of DEGs with higher levels of expression in ZH11. (B) GO analysis of DEGs with lower levels of expression in ZH11. (C) Expression patterns of 60 stress-responsive DEGs with higher levels of expression levels ZH11. (D) Expression patterns of those DEGs involved in chloroplast development and photosynthesis. (E) The overlap of male sterile genes (MSGs) and those expressed at a higher level in ZH11 than in GS2. (F) Gene identifiers (IDs) and relative expression patterns of the eight MSGs in E.

Figure 6

Expressions of growth regulation factors (GRFs) under different temperature conditions. (A) Expression patterns of GRFs in RNA sequencing (RNA-Seq) data. (B) Expression of OsGRF4 (Oryza sativa growth-regulating factor 4) measured by reverse transcription-quantitative PCR (qRT-PCR). (C) Visualization of coverage of sequencing reads annotated to OsGRF4 gene using the IGV program. (D) Reverse transcription-PCR (RT-PCR) validation of the intron retention (IR) event detected in OsGRF4. (E) RT-PCR amplification of OsGRF4 by using leaf cDNA as a template. (F) Amino acid residues of the conventional OsGRF4 protein and that of the potential protein variant translated from the intron-retained mRNA isoform. Only amino acid residues from position 380 on are shown. * stands for the terminal point of the amino acid sequence.

Figure 7

Detection of differential alternative splicing (DAS) genes in Zhonghua 11 (ZH11) and grain size on chromosome 2 (GS2). (A) Number of DAS events in four different comparisons. (B) Overlap of differentially expressed genes (DEGs) and DAS genes in the comparison of the ZH11 HS group and the GS2 HS group. (C) Gene ontology (GO) analysis of DAS genes in the comparison of the ZH11 HS group and the GS2 HS group. (D–J) Alternative splicing (AS) events on seven genes as determined through reverse transcription-PCR (RT-PCR) (upper) and the IGV program (bottom).