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Cytological and comparative proteomic analyses on male sterility in Brassica napus L. induced by the chemical hybridization agent monosulphuron ester sodium.

ABSTRACT: Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.

SUBMITTER: Cheng Y

PROVIDER: S-EPMC3828188 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:Chemical hybridization agent (CHA)-induced male sterility is becoming one of the most useful tools for the crop heterosis in seed production. We had previously discovered monosulfuron ester sodium (Mes), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, could induce rapeseed (Brassica napus L.) male sterility. To investigate the mechanism of Mes inducing male sterility, a cytological analysis and comparative transcriptome analysis were performed between Mes-treated plants leaves and different developmental stage anthers and those of the Mock-treated plants. Cytological analysis exhibited that Mes destroyed plastids ultrastructure and depressed the materials accumulation during anther development and maturation process. Comparative transcriptome analysis identified a total of 1501 transcripts differentially expressed in the leaves and different developmental stage anthers. Subcellular localization analysis, functional analysis and pathway analysis were carried out to obtain the clues that plastid was the seriously damaged organelle, and many genes involved in carbon and lipid metabolism and cellular transport differentially expressed. Detailed expression pattern analysis of genes related with these functions were conducted and verified by RT-PCR. In addition, several transcription factors, protein kinases and hormone related genes were identified. Carbohydrate content analysis confirmed carbon metabolism were influenced by Mes. Taken together, we proposed a putative action mode that Mes inhibited activity of acetolactate synthase, which localized in plastid in plants, and then disturbed the normal supply of carbon and lipid metabolite for anther development, finally displayed male sterility. This results have important significance for uncovering the metabolic gene regulation during anther development and may provide more potential targets for developing new male sterility inducing CHAs in rapeseed breeding. Rapeseed (Brassica napus L.) plants were foliar sprayed with 0.1Î¼g mL-1 Monosulfuron ester sodium (Mes) solution containing 50ppm DMF and 5ppm Tween 80 for about 15 ml per plant. Young leaves (C2Y1, C2Y2, C2Y3; T2Y1, T2Y2, T2Y3) of the main inflorescences and developing anthers (including small buds (C2S1, C2S2, C2S3; T2S1, T2S2, T2S3), middle anthers (C2M1, C2M2, C2M3; T2M1, T2M2, T2M3), and large anthers (C2L1, C2L2, C2L3; T2L1, T2L2, T2L3)) from Mes-treated and Mock-treated plants were collected for each experiment. Each tissue has three independent biological replicates.

Project description:BackgroundChemical hybridization agents (CHAs) are often used to induce male sterility for the production of hybrid seeds. We previously discovered that monosulfuron ester sodium (MES), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, can induce rapeseed (Brassica napus L.) male sterility at approximately 1% concentration required for its herbicidal activity. To find some clues to the mechanism of MES inducing male sterility, the ultrastructural cytology observations, comparative transcriptome analysis, and physiological analysis on carbohydrate content were carried out in leaves and anthers at different developmental stages between the MES-treated and mock-treated rapeseed plants.ResultsCytological analysis revealed that the plastid ultrastructure was abnormal in pollen mother cells and tapetal cells in male sterility anthers induced by MES treatment, with less material accumulation in it. However, starch granules were observed in chloroplastids of the epidermis cells in male sterility anthers. Comparative transcriptome analysis identified 1501 differentially expressed transcripts (DETs) in leaves and anthers at different developmental stages, most of these DETs being localized in plastid and mitochondrion. Transcripts involved in metabolism, especially in carbohydrate and lipid metabolism, and cellular transport were differentially expressed. Pathway visualization showed that the tightly regulated gene network for metabolism was reprogrammed to respond to MES treatment. The results of cytological observation and transcriptome analysis in the MES-treated rapeseed plants were mirrored by carbohydrate content analysis. MES treatment led to decrease in soluble sugars content in leaves and early stage buds, but increase in soluble sugars content and decrease in starch content in middle stage buds.ConclusionsOur integrative results suggested that carbohydrate and lipid metabolism were influenced by CHA-MES treatment during rapeseed anther development, which might responsible for low concentration MES specifically inducing male sterility. A simple action model of CHA-MES inducing male sterility in B. napus was proposed. These results will help us to understand the mechanism of MES inducing male sterility at low concentration, and might provide some potential targets for developing new male sterility inducing CHAs and for genetic manipulation in rapeseed breeding.

Project description:Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Project description:BACKGROUND:Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS:Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS:Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.

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Cytological and comparative proteomic analyses on male sterility in Brassica napus L. induced by the chemical hybridization agent monosulphuron ester sodium.

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