Brassinosteroids control male fertility by regulating the expression of key genes involved in Arabidopsis anther and pollen development

Abstract

The development of anther and pollen is important for male reproduction, and this process is coordinately regulated by many external and internal cues. In this study, we systematically examined the male reproductive phenotypes of a series of brassinosteroid biosynthetic and signaling mutants and found that, besides the expected cell-expansion defects, these mutants also showed reduced pollen number, viability, and release efficiency. These defects were related with abnormal tapetum and microspore development. Using both real-time quantitative RT-PCR and microarray experiments, we found that the expression of many key genes required for anther and pollen development was suppressed in these mutants. ChIP analysis demonstrated that BES1, an important transcription factor for brassinosteroid signaling, could directly bind to the promoter regions of genes encoding transcription factors essential for anther and pollen development, SPL/NZZ, TDF1, AMS, MS1, and MS2. Taken together, these data lead us to propose that brassinosteroids control male fertility at least in part via directly regulating key genes for anther and pollen development in Arabidopsis. Our work provides a unique mechanism to explain how a phytohormone regulates an essential genetic program for plant development.

Fig. 1.

Comparison of developing anthers and microspores among bri1-116, cpd, and the wild type. MMC, microspore mother cell; Msp, microspore; T, tapetum; Tds, tetrads. Micrographs of 6-μm thick anther transverse sections at different anther stages in bri1-116, cpd, and the wild type (Col-0). (A–C) bri1-116; (D–F) cpd; (G–I) Col-0. (A, D, and G) stage 5; (B, E, and H) stage 7; (C, F, and I) stage 9. (Scale bars, 50 μm.) (J) Microspore number per locule at different stages of anther development in bri1-116, cpd, and the wild type. Relative number per locule was calculated through multiplying anther diameter by pollen number per locule counted with longitudinal sections of anther stages 5, 6, 8, and 9; at stage 12 the pollen number was calculated with transverse sections (Experimental Procedures). MMC was counted at stage 5, meiocytes were counted at stage 6, microspores were counted at stages 8 and 9, and pollen grains were counted at stage 12.

Fig. 2.

bri1-116 and cpd are defective in exine pattern formation. (A–C) Scanning electron micrographs of dehiscent anthers of bri1-116 (A), cpd (B) and Col-0 (C). (Scale bars, 50 μm.) (D–F) After pollination, more pollen grains are retained on the inner wall of the anthers of bri1-116 (D) and cpd (E) than Col-0 (F). (Scale bars, 50 μm.) (G–I) Scanning electron micrographs of mature pollen grains: wild type (I), bri1-116 (G), and cpd (H). (Scale bars, 10 μm.) (J–L) Transmission electron micrographs of transverse section of microspores at stage 8: bri1-116 (J), cpd (K), and wild type (L). Ba, bacula; Msp, microspore; Te, tectum. (Scale bars, 1 μm.)

Fig. 3.

The expression of MS1 and its target genes, and several essential genes in anther and pollen development, is regulated by BRs. Real-time qRT-PCR analysis of AMS, AtMYB103, MS2, MS1, and target genes of MS1 in bri1-116, cpd, and Col-0. The floral buds approximately containing anthers at stages 8 to 10 were collected and used for this analysis. The expression level of each gene in Col-0 was defined as “1.” Bars indicate the SE.

Fig. 4.

A model to illustrate the roles of BRs in regulating male fertility in Arabidopsis. Besides the roles in regulating filament elongation and pollen tube growth via promoting cell elongation, the BR signaling also controls the tapetum and microspore development in Arabidopsis by directly regulating the expression of SPL/NZZ, TDF1, MYB103, MS2, MS1, and MS1-target genes. Solid arrows indicate a direct regulation on gene expression. Open arrows indicate the positive effect on the final developmental process. Genes highlighted with green indicate a down-regulation in BR biosynthetic or signaling mutants, and genes highlighted with red indicate an up-regulation. The first and second numbers in the parenthesis indicate fold-changes of gene expression in bri1-116 and cpd, respectively, compared to wild type.

Fig. 5.

BES1 directly binds to the promoter regions of key regulatory genes in anther and pollen development. (A) Anti-BES1 antibodies or an unrelated antibody as negative control were used to precipitate chromatin prepared from 5-week-old Col-0 adult plants. TA3 was used as an internal control. (B) Real-time qPCR analysis of ChIP products. The fold-changes were calculated based on the relative change in anti-BES1 compared with an unrelated antibody, which was used as a control and defined as “1.” (C) BES1 binds to MS2 promoter in vitro. Without probe (Lane 1), with labeled probe (Lane 2), labeled probe with 50× or 250× unlabeled wild-type (Lanes 3 and 4) or the mutant sequences (Lanes 5 and 6). Anti-BES1 antibody was added (Lane 7).