Background: Acetolactate synthase (ALS)-inhibiting herbicides amidosulfuron (Hoestar) is an efficient gametocide that can induce male sterility in rapeseed (Brassica napus L.). We conducted an integrated study of cytological, transcriptomic, and physiological analysis to decipher the gametocidal effect of amidosulfuron.Results: In the first several days after exposure to amidosulfuron at a gametocidal dose of ca. 1 μg per plant, the plants showed the earliest symptoms including short retard of raceme elongation, slight chlorosis on leaf, and decrease of photosynthesis rate. Chloroplasts in leaf and anther epidermis, and tapetal plastids were deformed. Both tapetal cell and uni-nucleate microspore showed autophagic vacuoles and degenerated quickly. The amidosulfuron treatment caused reduction of photosynthetic rate and the contents of leaf chlorophyll, soluble sugar and pyruvate, as well as content alteration of several free amino acids in the treated plants. A comparison of transcriptomic profiling data of the young flower buds of the treated plants with the control identified 142 up-regulated and 201 down-regulated differential expression transcripts with functional annotations. Down-regulation of several interesting genes encoding PAIR1, SDS, PPD2, HFM1, CSTF77, A6, ALA6, UGE1, FLA20, A9, bHLH91, and putative cell wall protein LOC106368794, and up-regulation of autophagy-related protein ATG8A indicated functional abnormalities about cell cycle, cell wall formation, chloroplast structure, and tissue autophagy. Ethylene-responsive transcription factor RAP2-11-like was up-regulated in the flower buds and ethylene release rate was also elevated. The transcriptional regulation in the amidosulfuron-treated plants was in line with the cytological and physiological changes.Conclusions: The results suggested that metabolic decrease related to photosynthesis and energy supply are associated with male sterility induced by amidosulfuron. The results provide insights into the molecular mechanisms of gametocide-induced male sterility and expand the knowledge on the transcriptomic complexity of the plants exposure to sulfonylurea herbicide.

The thermo-sensitive genic male sterility (TGMS) line SP2S is a spontaneous rapeseed mutation with several traits that are favorable for the production of two-line hybrids. To uncover the key cellular events and genetic regulation associated with TGMS expression, a combined study using cytological observation, transcriptome profiling, and gene expression analysis was conducted for SP2S and its near-isogenic line SP2F grown under warm conditions. Asynchronous microsporocyte meiosis and abnormal tapetal plastids and elaioplasts were demonstrated in the anther of SP2S. The tetrad microspore did not undergo mitosis before the cytoplasm degenerated. Delayed degradation of the tetrad wall, which led to tetrad microspore aggregation, resulted in postponement of sexine (outer layer of pollen exine) formation and sexine fusion in the tetrad. The nexine (foot layer of exine) was also absent. The delay of tetrad wall degradation and abnormality of the exine structure suggested that the defective tapetum lost important functions. Based on transcriptomic comparisons between young flower buds of SP2S and SP2F plants, a total of 465 differentially expressed transcripts (DETs) were identified, including 303 up-regulated DETs and 162 down-regulated DETs in SP2S. Several genes encoding small RNA degrading nuclease 2, small RNA 2′-O-methyltransferase, thioredoxin reductase 2, regulatory subunit A alpha isoform of serine/threonine-protein phosphatase 2A, glycine rich protein 1A, transcription factor bHLH25, leucine-rich repeat receptor kinase At3g14840 like, and fasciclin-like arabinogalactan proteins FLA19 and FLA20 were greatly depressed in SP2S. Interestingly, a POLLENLESS3-LIKE 2 gene encoding the Arabidopsis MS5 homologous protein, which is necessary for microsporocyte meiosis, was down-regulated in SP2S. Other genes that were up-regulated in SP2S encoded glucanase A6, ethylene-responsive transcription factor 1A-like, pollen-specific SF3, stress-associated endoplasmic reticulum protein 2, WRKY transcription factors and pentatricopeptide repeat (PPR) protein At1g07590. The tapetum-development-related genes, including BnEMS1, BnDYT1, and BnAMS, were slightly up-regulated in 3-mm-long flower buds or their anthers, and their downstream genes, BnMS1 and BnMYB80, which affect callose dissolution and exine formation, were greatly up-regulated in SP2S. This aberrant genetic regulation corresponded well with the cytological abnormalities. The results suggested that expression of TGMS associates with complex transcriptional regulation.

Premise of the study:SP2S is a spontaneous thermo-sensitive genic male sterility (TGMS) mutation that facilitates two-line hybrid breeding in Brassica napus (Brassicaceae). De novo assembly of the floral bud transcriptome of SP2S can provide a foundation for deciphering the transcriptional regulation of SP2S in response to temperature change.Methods:mRNAs of the young floral buds of SP2S and its near-isogenic line SP2F grown under cool (16°C)/warm (22°C) conditions were sequenced on an Illumina Solexa platform, producing 239.7 million short reads with a total length of 19.95 Gbp.Results:The reads were assembled de novo using the Trinity program, resulting in 135,702 transcripts with an average length of 784 bp, an N50 value of 1221 bp, and a total length of 107 Mbp. We identified 24,157 cDNA-derived simple sequence repeats in the assembly. We found 137 and 195 single-nucleotide polymorphisms and 49 and 51 differentially regulated KEGG orthology groups when comparing sample SP2S at 22°C vs. SP2S at 16°C and sample SP2S at 22°C vs. SP2F at 22°C, respectively.Discussion:The numerous differentially expressed genes and the derived single-nucleotide polymorphisms show abnormal transcriptional regulation in the TGMS system. These results outline an intricate transcriptional regulation that occurred in the rapeseed TGMS SP2S when the temperature changed.

We conducted a proteomic comparison between thermo-sensitive genic male sterility (TGMS) SP2S and its near-isogenic line SP2F grown at 22°C. The proteomes at microsporocyte meiosis and uninucleate microspore stages were profiled using 2-dimensional gel electrophoresis. Twenty-five well-reproducible spots (10 spots at microsporocyte meiosis stage and 15 spots at uninucleate microspore stage) containing 28 proteins were successfully identified by MALDI-TOF/TOF mass spectrometry. An elongation factor EF-2 at microsporocyte stage and 4 proteins (aconitate hydratase, triosephosphate isomerase, serine/arginine-rich mRNA splicing factor and glutathione S-transferase) at uninucleate microspore stage accumulated in SP2S, but more proteins were lost or reduced, including those associated with amino acid metabolism, photosynthesis, synthesis and degradation of protein, lipid metabolism, cytoskeleton, RNA modification, oxidoreductase and defence response. The dramatic decrease of tubulin, actin and Translationally controlled tumour protein (TCTP) crucial for microtubule and cell division and three enzymes for amino acid metabolite at early stage indicated a serious defect on the cytokinesis. They were important clues for us to search the TGMS genes and its interacting genes.