Clone and sequence analysis of SiPKSA
Sesame polyketide synthase, the orthologue of Arabidopsis PKSA, designated SiPKSA (Sesamum indicum Polyketide Synthase A), was cloned using the cDNA of sesame anthers at the tetrad stage as the template. The open reading frame (ORF) of SiPKSA was 1191 bp, encoding 396 amino acids, with an isoelectric point of 6.41, a fat index of 95.33, and a molecular weight of 43.6 kDa.
Multiple sequence alignment of SiPKSA with other PKSA proteins reported in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and rape (Brassica napus) showed that SiPKSA contained typical chalcone/stilbene synthase N-terminal domain (25–242 bp), C-terminal domain (252–396 bp) and various conserved active sites, such as polypeptide binding site, malonyl-CoA binding site, and product binding site (Fig. 1A). As well, it contained catalytic triad Cys-His-Asn, which is common and important in the PKSs family (Fig. 1A). Phylogenetic analysis further confirmed that SiPKSA clustered together with PKSA proteins in other species (Fig. 1B), confirming that it encoded a type III polyketide synthase A protein.
In addition, the promoter region of the SiPKSA was analyzed using the PLACE and PLANTCARE databases. It was found that a large number of formal elements were annotated as MYB recognition sites, light-responsive elements, and auxin-responsive elements. Notably, multiple copies of AGAAA, which was considered as an anther- and/or pollen-specific cis-regulatory motif, were predicted in the promoter region of SiPKSA. Other motifs, such as Q-element and GTGA-motif, related to pollen development were also predicted in the promoter region (Table S2). These results indicated that SiPKSA was a type III polyketide synthase A protein and might be involved in pollen development in sesame.
SiPKSA is predominantly expressed in reproductive tissue
The expression profile of SiPKSA in different tissues was analyzed. It was shown that SiPKSA was especially expressed in flower buds (Fig. 2A and 2B), indicating its function in sesame might be related to plant reproduction. Further, to determine the potential role of SiPKSA in sesame male sterility, we compared the expression pattern of SiPKSA in sterile and fertile sesame anthers at different developmental stages. We observed that the expression of SiPKSA was significantly higher in sterile sesame anthers at the tetrad stage and microspore development stage compared with the fertile anthers (Fig. 2C and 2D), suggesting that SiPKSA might be related to sesame male fertility.
Overexpressing SiPKSA in Arabidopsis caused transgenic plants male sterile
To further assess the in vivo function of SiPKSA in the anther development, SiPKSA was overexpressed in Arabidopsis. Several T3 generation transgenic plants were harvested, and two homozygous lines, in which SiPKSA expression was highly overexpressed, were selected for phenotypic and functional analysis (Fig. 3A). The expression level of the Arabidopsis homologous gene AtPKSA was also detected. The results showed that there were no significant differences among SiPKSA-overexpressing plants, wild type and plants transformed with empty vector in terms of the expression level of AtPKSA both in seedlings and anthers at different developmental stages (Fig. S1A-C), suggesting that the phenotype of transgenic plants in the present study was caused by SiPKSA.
As shown in Fig. 3B, the siliques of SiPKSA-overexpressing plants were flat and short compared with those of the wild type (WT) and plants transformed with empty vector (EV) (Fig. 3C-E). The acetocarmine staining showed that the pollen grains of WT and EV were plump and darkly stained (Fig. 3F and 3H). While, the pollen grains of SiPKSA-overexpressing plants were uncolored and smaller compared with the controls, indicating the pollen grains of SiPKSA-overexpressing plants were poor vigor (Fig. 3G). Scanning electron microscope (SEM) further confirmed that the WT pollen grains were normal and those of the SiPKSA-overexpressing plants were abnormal. The WT pollen grains were full with a typical sculpture surface (Fig. 3I and 3J). In contrast, the pollen grains of the SiPKSA-overexpressing plants were shrunken and collapsed (Fig. 3K and 3L). These results indicated that SiPKSA-overexpressing plants were male sterile.
Abnormal pollen and pollen wall development in SiPKSA-overexpressing Arabidopsis plants
Transmission electron microscopy (TEM) observation was further performed to investigate the ultrastructure of pollen development. According to the cytological events, Arabidopsis anther development was characterized into 14 stages, and the microspores were differentiated into mature pollen during stages 9–12 [8]. Our results showed that, during stages 9–10, the tapetum of WT contained small vacuoles and abundant plastids filled with plastoglobuli (Fig. 4A). The microspores of WT were rich in inclusions and embedded with a regular primary structure of the exine (Fig. 4B). However, in the tapetal cells of SiPKSA-overexpressing plants, large cavities and less substance were observed (Fig. 4C), the microspores of SiPKSA-overexpressing plants were obviously abnormal, with few cytoplasmic components, and the sporopollenin particles were abundant accumulated on the microspore surface (Fig. 4D) During stage 11–12, the pollen of WT exhibited a typical normal pollen wall with obvious exine and intine layers (Fig. 4E). In contrast, the pollen grains of SiPKSA-overexpressing plants were seriously collapsed, with irregular exine and intine (Fig. 4F). Further measurement of the thickness of the pollen exine during the stages 9–10 and 11–12 showed that, compared with the WT, the exine layer of SiPKSA-overexpressing plants was significantly thicker than WT (Fig. 5A-E).
Overexpression of SiPKSA disrupted the expression of sporopollenin biosynthesis-related genes
To further explore whether sporopollenin biosynthesis was affected in anthers of SiPKSA-overexpressing plants, the expression of several sporopollenin synthesis-related genes was analyzed by qRT-PCR, including AtMS2, AtCYP704B1, AtTKPR1, and AtTKPR2. As shown in Fig. 6, comparing to WT, the expression of AtMS2, AtCYP704B1, AtTKPR1 and AtTKPR2 were up-regulated in anthers of SiPKSA-overexpressing plants during stage 6–8. During stages 9–10, there were no significant differences among SiPKSA-overexpressing plants, WT, and EV in terms of the expression of AtMS2, AtCYP704B1, AtTKPR1, except for AtTKPR2, which expression was also up-regulated. These results indicated that the expressions of sporopollenin biosynthesis-related genes were influenced by the high expression of SiPKSA in transgenic Arabidopsis.
Disrupted fatty acids metabolism occurred in anthers of SiPKSA-overexpressing plants
Pollen exine is mainly composed of sporopollenin, which was a complex biopolymer constituted of fatty acids, phenols, and their derivatives [38]. Therefore, the contents of fatty acids and their derivates were detected by GC-MS analysis. The results showed that, in comparison with the WT, the content of several long-chain fatty acids such as C15:1, C16:0, ethyl-C16, C18:1, C22:1, and C22:2 changed greatly in anthers of SiPKSA-overexpressing plants during the anther development compared with the WT (Fig. 7A). In addition, three phenolic compounds (C14-OH, C15-OH, and C25-2OH) and one fatty alcohol (Phytol) were increased significantly at stages 6–8 in SiPKSA-overexpressing plants (Fig. 7B). The results indicated that over-expression of SiPKSA affected fatty acid metabolism in anthers of transgenic Arabidopsis.