CRISPR/Cas9‐based discovery of maize transcription factors regulating male sterility and their functional conservation in plants

Abstract

Summary Identifying genic male‐sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9‐mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9‐1/‐2/‐3, ZmTGA10, ZmMYB84, ZmMYB33‐1/‐2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9‐1/‐2/‐3 triple‐gene mutants and ZmMYB33‐1/‐2 double‐gene mutants displayed complete male sterility, but their double‐ or single‐gene mutants showed male fertility. Similarly, ZmLBD10/27 double‐gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co‐segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14‐type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male‐sterility lines in maize.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

“…DNA sequencing of three target sites revealed that up to 84% of T 0 plants contained mutations in one site at least, while five plants generated from one event were homozygous triple mutants. We identified a homozygous loss-of-function triple mutant of ZmTGA9-1/-2/-3 with frameshift and truncation mutations from the five plants mentioned above (Figure S2A), including 1-bp deletion in ZmTGA9-1, and 1-bp insertion in ZmTGA9-2 and ZmTGA9-3, respectively (Figure 1(A2),B), which is a new allelic mutant differing from ZmTGA9 triple mutants obtained previously [10]. Since maize T 0 transgenic plants could not usually produce seeds by self-pollination in the greenhouse, we pollinated the T 0 plants with pollen grains from maize inbred line Zheng58, and then harvested F 1 seeds that could be divided into Cas9-positive (transgene) and Cas9-negative (non-transgene) segregation.…”

“…However, only ZmTGA9-1, ZmTGA9-2, and ZmTGA9-3 are expressed during maize anther development based on RNA-seq data of W23 anther (Figure S1(A2)) [32], indicating the potentially critical roles of the three genes in controlling maize male reproduction. Based on co-introduced of two vectors containing Cas9 and sgRNA expression cassettes into maize, our previous research revealed that ZmTGA9 triple mutant displayed complete male sterility, while single and double mutants of ZmTGA9-1/-2/-3 showed normal fertility [10]. However, the co-transformation method influences transformation efficiency and especially increases the complexity of molecular identification.…”

“…Thus far, more than 100 GMS genes have been identified and characterized in plants, most of which are reported to encode transcription factors (TFs) or lipid biosynthesis and transport proteins, including 49 in Arabidopsis, 38 in rice, and 24 in maize [2,[8][9][10]. GMS genes isolated in maize, compared with Arabidopsis and rice, are relatively few.…”

Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat

“…Studying genes by map-based cloning is time-consuming, and sometimes cloning of homologous genes is fast and effective. By analyzing the putative orthologs of GMS genes in Arabidopsis and rice, a putative regulatory network for anther and pollen development in maize was constructed (Wan et al, 2019;Jiang et al, 2021). In our study, DEGs/DEPs are involved in fatty acid biosynthesis process, lipid transport, L-phenylalanine metabolic process and polysaccharide metabolism in AL18A's anthers development (Figure 7).…”

“…Anther development is one of the most critical events in the reproductive phase of plant development, involving hundreds of genes and proteins. Transcriptional factors (TFs) are the key connection points in regulatory networks associated with tapetal function and pollen development (Jiang et al, 2021). By comparing AL18A and AL18B, we analyzed the expression pattern of TFs in DEGs during the anther development (Figure 7B; Supplementary Table S20).…”