Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species

Abstract

Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.

Keywords: PHD finger; ZmMs7; dominant male-sterility system; protein-protein interaction.

Fig. 1.

Phenotypic and cytological comparison of WT, ms7-6007 mutant, and ZmMs7 knockout line generated via the CRISPR-Cas9 method. (A) Comparison of tassels (A1), anthers (A2), and pollen grains stained with I₂-KI (A3) among WT, ms7-6007 mutant, and the Cas9-ZmMs7-01 line. (Scale bars, 1 mm in A2 and 200 μm in A3.) (B) Transverse section analysis of anthers in WT (B1 to B3) and ms7-6007 mutant (B4 to B6) from stages 9 to 11 during maize anther development. (Scale bar, 50 μm.) (C) SEM analysis of pollen grain development in WT (C1 to C3) and ms7-6007 mutant (C4 to C6). (Scale bar, 20 μm.) (D) TEM analysis of Ubisch body in WT (D1 to D3) and ms7-6007 mutant (D4 to D6). (Scale bar, 0.5 μm.) (E) TEM analysis of pollen exine in WT (E1 to E3) and ms7-6007 mutant (E4 to E6) (Scale bar, 0.5 μm.) Ba, bacula; CMsp, collapsed microspore; E, epidermis; En, endothecium; F, foot layer; ML, middle layer; Msp, microspore; Ta, tapetum; Te, tectum; and Ub, Ubisch body.

Fig. 2.

Gene expression, transcriptional activity, and transcriptome and lipidome analyses of ZmMs7. (A) RT-qPCR analysis of ZmMs7 expression in different organs of maize. Data are means ± SD, n = 3. (B) Subcellular localization of ZmMs7 in maize protoplasts. DAPI staining was used as a nuclear marker. (Scale bar, 10 μm.) (C) Transcriptional activation assay of ZmMs7 using a dual-luciferase system in maize protoplasts. GAL4 DNA binding domain (BD) and transcriptional activator VP16 were used as negative and positive controls, respectively. Data are means ± SD, n = 4. Asterisks indicate significant difference compared to BD (**P < 0.01, Student’s t test). (D) Male sterile phenotype of pZmMs7:ZmMS7-SRDX transgenic maize plants. Comparison of tassels, anthers, and pollen grains stained with I₂-KI between WT and transgenic plants. (Scale bar, 100 μm.) (E) The numbers of DEGs between WT and ms7-6007 mutant anthers at stages 8, 9, and 10, respectively. (F) Total amount of anther cutin and wax per unit surface area and (G) total amount of anther internal lipid per dry weight (DW) in WT and ms7-6007 mutant. Data are means ± SD, n = 5. Asterisks indicate significant difference in comparison (***P < 0.001, Student’s t test).

Fig. 3.

Interaction of ZmMs7 with ZmNF-Y subunits. (A) Y2H and Co-IP assays of ZmMs7 interaction with NF-YA6 and NF-YC9/12/15. For Y2H assay, LAM and P53 were used as negative and positive controls, respectively. DDO, double dropout medium (SD-Trp-Leu); QDO, quadruple dropout medium (SD-Trp-Leu-His-Ade). Co-IP assays were performed using a transient expression system in maize protoplasts; the nYFP-FLAG was used as a negative control. The asterisk indicates a nonspecific band. (B) Y2H and Co-IP assays of NF-YA6 interaction with NF-YC9/12/15. IG1-nYFP-FLAG was used as a negative control. Others are as in A. (C) Y2H and Co-IP assays of NF-YB2 interaction with ZmMs7N, NF-YA6, and NF-YC9/12/15. Others are as in A. (D) BiFC analysis of in vivo interaction between ZmMs7, NF-YA6, NF-YB2, and NY-YC9/12/15 in maize protoplasts. Protein name-n indicates nYFP fusions. DAPI staining was used as a nuclear marker. The fluorescence was detected using confocal microscopy. (Scale bar, 10 μm.)

Fig. 4.

ZmMs7-NF-YA/YB/YC complexes directly activate target gene expression. (A) Transient dual-luciferase assay of ZmMT2C promoter activity activated by ZmMs7-NF-Y complexes in maize protoplasts. Data are means ± SD, n = 3. Different letters above each column indicate significant difference (P < 0.01, Student’s t test). (B) EMSA assay of ZmNF-YA6 binding to the CCAAT box in ZmMT2C promoter region. (C) The enrichments of ZmMT2C promoter analyzed by ChIP-qPCR with the primer sets (P1, P2, and P3), using the anther samples of ZmMs7-3x Myc transgenic maize plants. Data are means ± SD, n = 3. +Ab, presence of anti-c-Myc antibody; −Ab, absence of the antibody. The asterisks indicate significant difference between +Ab and −Ab (P < 0.01, Student’s t test). (D) Detection of DNA fragmentation by TUNEL assay in WT and ms7-6007 anthers. (Scale bar, 50 μm.) (E) TEM of tapetum degeneration in WT and ms7-6007 anthers. Ta, tapetum. (Scale bar, 10 μm.) (F) Model of ZmMs7-NF-YA/YB/YC complexes controlling maize male fertility through directly activating ZmMT2C expression and indirectly regulating cutin and sporopollenin biosynthesis-related genes such as ZmMs6021, ZmLAP5, ZmDRL1/2, and ZmACOS5.

Fig. 5.

Phenotypic and cytological comparison of WT and the p5126-ZmMs7M-01 dominant male-sterility line. (A) Comparison of tassels (A1), anthers (A2), and pollen grains stained with I₂-KI (A3) between WT and the p5126-ZmMs7M-01 line. (Scale bars, 1 mm in A2 and 200 μm in A3.) (B) Comparison of ear phenotypes between WT and the p5126-ZmMs7M-01 line under bright light (B1, B4, and B5) and green excitation light with red fluorescence filter I (GREEN.L, China) (B2 and B6) and red fluorescence filter II (NIGHTSEA, United States) (B3 and B7). (C) Transverse section analysis of anthers in WT (C1 to C3) and the p5126-ZmMs7M-01 line (C4 to C6) from stage 8b to 10 during maize anther development. (Scale bar, 50 μm.) (D) SEM analysis of microspores in WT (D1 to D3) and the p5126-ZmMs7M-01 line (D4 to D6). (Scale bar, 20 μm.) (E) TEM analysis of Ubisch body in WT (E1 to E3) and the p5126-ZmMs7M-01 line (E4 to E6). (Scale bar, 0.5 μm.) (F) TEM analysis of pollen exine in WT (F1 to F3) and the p5126-ZmMs7M01 line (F4 to F6). (Scale bar, 0.5 μm.) Ba, bacula; CMsp, collapsed microspore; E, epidermis; En, endothecium; F, foot layer; LD, lipid droplet; ML, middle layer; Msp, microspore; Ta, tapetum; Tds, tetrads; Te, tectum; and Ub, Ubisch body.

Fig. 6.

p5126 promoter drives premature expression of ZmMs7-activated genes and results in the dominant male-sterility phenotype of the p5126-ZmMs7M-01 line. (A) The 126 shared DEGs between 144 down-regulated DEGs in the ms7-6007 mutant and 244 up-regulated DEGs in the p5126-ZmMs7M-01 line (A1). The expression patterns of the 50 representative genes in anther transcriptomes of ms7-6007 mutant at stages 8 to 9 (A2) and in the p5126-ZmMs7M-01 line at stages 6 to 10 (A3) are shown, respectively. (B) Gene ontology enrichment analysis of the 126 shared genes. (C) RT-qPCR analysis of ZmMs7 and five putative activated genes in WT, ms7-6007, and the p5126-ZmMs7M-01 line anthers at stages 6 to 10.

Fig. 7.

Dominant male-sterility phenotype of p5126-ZmMs7 transgenic rice and Arabidopsis plants. (A) Four rice transgenic plants expressing p5126-ZmMs7-mCherry show complete male-sterility phenotypes. Comparison of whole plants after heading, panicles at anthesis, anthers (Scale bars, 1 mm.), pollen grains with I₂-KI staining (Scale bars, 100 μm.), and seeds under bright field and a red fluorescence filter (Scale bars, 0.5 cm.) among WT and four p5126-ZmMs7 transgenic lines. (B) Three Arabidopsis plants expressing p5126-ZmMs7-myc show complete male sterility of pollen grains. Comparison of siliques, the surface of stigmas and anthers, pollen grains stained with I₂-KI and pollen germination among WT and three transgenic lines. (Scale bars, 100 μm.) The numbers in A and B indicate different independent transgenic events.