Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Genetic origin and expression patterns of acetohydroxyacid synthase multigene family in Brassica juncea and B. carinata and their progenitors.

Acetohydroxyacid synthase (AHAS), the key enzyme in the branched-chain amino acids leucine, isoleucine, and valine biosynthesis pathway, has gained intensive investigation because it is the target of five different AHAS herbicides widely used to control weeds in farmland. In the present study, the AHAS gene family in Brassica juncea and B. carinata and their progenitor species was characterized in combination with bioinformatics, gene-specific PCR and qRT-PCR analyses. The results indicated that B. juncea contains four AHAS genes, of them, BjuAHAS3 and BjuAHAS4 originated from the A genome donor of B. rapa, whereas BjuAHAS6 and BjuAHAS7 from the B genome donor of B. nigra. BjuAHAS3 and BjuAHAS6 are predicted to be functional and constitutively expressed in all vegetative and reproductive tissues in the tested B. juncea accessions. B. carinata contains five AHAS genes, of them, BcaAHAS1, BcaAHAS2, and BcaAHAS5 originated from the C genome donor of B. oleracea, whereas BcaAHAS6 and BcaAHAS7 came from the B genome donor of B. nigra. BcaAHAS1, BcaAHAS2, and BcaAHAS6 are predicted to be functional. BcaAHAS1 and BcaAHAS6 are constitutively expressed in all vegetative and reproductive tissues in the tested B. carinata accessions, however, BcaAHAS2 is mainly expressed in siliques. In addition, translocation events for the AHAS1, AHAS2, and AHAS7 genes occurred when the three amphidiploids species B. napus, B. juncea, and B. carinata were formed by hybridization of their respective diploid species. The findings in this study will provide important basic information for the breeding of herbicide-resistant varieties in B. juncea and B. carinata.

BnERF114.A1, a Rapeseed Gene Encoding APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture through Auxin Accumulation in the Apex in Arabidopsis.

Plant architecture is crucial for rapeseed breeding. Here, we demonstrate the involvement of BnERF114.A1, a transcription factor for ETHYLENE RESPONSE FACTOR (ERF), in the regulation of plant architecture in Brassica napus. BnERF114.A1 is a member of the ERF family group X-a, encoding a putative 252-amino acid (aa) protein, which harbours the AP2/ERF domain and the conserved CMX-1 motif. BnERF114.A1 is localised to the nucleus and presents transcriptional activity, with the functional region located at 142-252 aa of the C-terminus. GUS staining revealed high BnERF114.A1 expression in leaf primordia, shoot apical meristem, leaf marginal meristem, and reproductive organs. Ectopic BnERF114.A1 expression in Arabidopsis reduced plant height, increased branch and silique number per plant, and improved seed yield per plant. Furthermore, in Arabidopsis, BnERF114.A1 overexpression inhibited indole-3-acetic acid (IAA) efflux, thus promoting auxin accumulation in the apex and arresting apical dominance. Therefore, BnERF114.A1 probably plays an important role in auxin-dependent plant architecture regulation.

Melatonin Represses Oil and Anthocyanin Accumulation in Seeds.

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of melatonin in seed oil and anthocyanin accumulation, and corresponding underlying mechanisms, remain unclear. Here, we found that serotonin N-acetyltransferase1 (SNAT1) and caffeic acid O-methyltransferase (COMT) genes were ubiquitously and highly expressed and essential for melatonin biosynthesis in Arabidopsis developing seeds. We demonstrated that blocking endogenous melatonin biosynthesis by knocking out SNAT1 and/or COMT significantly increased oil and anthocyanin content of mature seeds. In contrast, enhancement of melatonin signaling by exogenous application of melatonin led to a significant decrease in levels of seed oil and anthocyanins. Further gene expression analysis through RNA sequencing and reverse-transcription quantitative PCR demonstrated that the expression of a series of important genes involved in fatty acid and anthocyanin accumulation was significantly altered in snat1-1 comt-1 developing seeds during seed maturation. We also discovered that SNAT1 and COMT significantly regulated the accumulation of both mucilage and proanthocyanidins in mature seeds. These results not only help us understand the function of melatonin and provide valuable insights into the complicated regulatory network controlling oil and anthocyanin accumulation in seeds, but also divulge promising gene targets for improvement of both oil and flavonoids in seeds of oil-producing crops and plants.

Characterization of tribenuron-methyl-induced male sterility in Brassica juncea L.

Significant heterosis has been documented in Brassica juncea L. that are grown as agriculturally important oilseeds, vegetables and condiments crops. Male sterility induced by chemical hybridizing agents is an important pollination control system in hybrid crop breeding. Herein, we show that tribenuron-methyl (TBM), a sulfonylurea herbicide, is an effective male gametocide in B. juncea when used at a very low dosage. In the present study, foliar application of various rates of TBM induced a significant increase in pollen sterility in B. juncea (90.57-100%). TBM-treated plants exhibited reductions in size of floral organ and yield components; however, lower dose of TBM (0.075 g a.i. ha-1) did not cause a significant reduction in seed yield per plant. Tapetum cells of TBM-treated plants were hypertrophied and degenerated earlier, and abnormal meiosis was observed at the meiotic stage. A significant decrease of acetohydroxyacid synthase (AHAS) activities was detected in buds of plants treated with 0.10 g a.i. ha-1 TBM, and RT-qPCR analysis showed that TBM exposure perturbed AHAS expression in small buds, which support that TBM induces male sterility in B. juncea by targeting AHAS expression. Our results suggest that TBM could be used as an efficient chemical hybridization agent in B. juncea, which has practical implications for the application of hybrid breeding in B. juncea.

Cytological observation of anther structure and genetic investigation of a thermo-sensitive genic male sterile line 373S in Brassica napus L.

BACKGROUND: Photoperiod and/or thermo-sensitive male sterility is an effective pollination control system in crop two-line hybrid breeding. We previously discovered the spontaneous mutation of a partially male sterile plant and developed a thermo-sensitive genic male sterile (TGMS) line 373S in Brassica napus L. The present study characterized this TGMS line through cytological observation, photoperiod/ temperature treatments, and genetic investigation. RESULTS: Microscopic observation revealed that the condensed cytoplasm and irregular exine of microspores and the abnormal degradation of tapetum are related to pollen abortion. Different temperature and photoperiod treatments in field and growth cabinet conditions indicated that the fertility alteration of 373S was mainly caused by temperature changes. The effects of photoperiod and interaction between temperature and photoperiod were insignificant. The critical temperature leading to fertility alteration ranged from 10 °C (15 °C/5 °C) to 12 °C (17 °C/7 °C), and the temperature-responding stage was coincident with anther development from pollen mother cell formation to meiosis stages. Genetic analysis indicated that the TGMS trait in 373S was controlled by one pair of genes, with male sterility as the recessive. Multiplex PCR analysis revealed that the cytoplasm of 373S is pol type. CONCLUSIONS: Our study suggested that the 373S line in B. napus has a novel thermo-sensitive gene Bnmst1 in Pol CMS cytoplasm background, and its fertility alteration is mainly caused by temperature changes. Our results will broaden the TGMS resources and lay the foundation for two-line hybrid breeding in B. napus.

AGC protein kinase AGC1-4 mediates seed size in Arabidopsis.

KEY MESSAGE: AGC1-4 kinase plays a crucial role in the regulation of seeds by mediating cell proliferation and embryo development in Arabidopsis. Seed size is a crucial factor to influence final seed yield in plants. However, the molecular mechanisms that set final seed size still need to be investigated. Here, we identified a novel AGC protein kinase AGC1-4, which encodes a serine-threonine kinase, belongs to the AGC VIIIa subfamily. The seeds of agc1-4 mutant were significantly larger than that in the wild type. Overexpression of the AGC1-4 gene reduced seed size. Regulation of AGC1-4 seed size is dependent on embryonic cell number. To further determine AGC1-4 functions in seed size, we analyzed AGC1-4 phosphoproteins using label-free quantitative phosphoproteomics coupled to the transcriptome of agc1-4 using RNA sequencing (RNA-seq). The RNA-seq analysis showed 1611 differentially expressed genes (DEGs), which cover a wide range of functions, such as cell cycle and embryo development. The 262 unique phosphoproteins were detected by phosphoproteomics analysis. The differentially phosphorylated proteins were involved in cell cycle and post-embryo development. Overlay of the RNA-seq and phosphoproteomics results demonstrated AGC1-4 as an important factor that influences seed size by mediating cell proliferation and embryo development. The results in this study provide novel data on the serine-threonine kinase AGC1-4 mediating seed size in Arabidopsis.

Inheritance and Molecular Characterization of a Novel Mutated AHAS Gene Responsible for the Resistance of AHAS-Inhibiting Herbicides in Rapeseed (Brassica napus L.).

The use of herbicides is an effective and economic way to control weeds, but their availability for rapeseed is limited due to the shortage of herbicide-resistant cultivars in China. The single-point mutation in the acetohydroxyacid synthase (AHAS) gene can lead to AHAS-inhibiting herbicide resistance. In this study, the inheritance and molecular characterization of the tribenuron-methyl (TBM)-resistant rapeseed (Brassica napus L.) mutant, K5, are performed. Results indicated that TBM-resistance of K5 was controlled by one dominant allele at a single nuclear gene locus. The novel substitution of cytosine with thymine at position 544 in BnAHAS1 was identified in K5, leading to the alteration of proline with serine at position 182 in BnAHAS1. The TBM-resistance of K5 was approximately 100 times that of its wild-type ZS9, and K5 also showed cross-resistance to bensufuron-methyl and monosulfuron-ester sodium. The BnAHAS1544T transgenic Arabidopsis exhibited higher TBM-resistance than that of its wild-type, which confirmed that BnAHAS1544T was responsible for the herbicide resistance of K5. Simultaneously, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS1544T. In addition, a method for the fast screening of TBM-resistant plants at the cotyledon stage was developed. Our research identified and molecularly characterized one novel mutative AHAS allele in B. napus and laid a foundation for developing herbicide-resistant rapeseed cultivars.

Exposure to trace amounts of sulfonylurea herbicide tribenuron-methyl causes male sterility in 17 species or subspecies of cruciferous plants.

BACKGROUND: For most cruciferous plants, which are known as important crops and a number of weeds, hybrid breeding is hampered by the unavailability of a pollination control system. Male sterility induced by a gametocide can be useful for the utilization of plant heterosis. RESULTS: The gametocidal effect of sulfonylurea herbicide tribenuron-methyl was tested across seventeen cruciferous species or subspecies including Brassica juncea, B. carinata, B. oleracea ssp. capitata, B. oleracea ssp. acephala, B. rapa ssp. pekinensis, B. rapa ssp. chinensis, B. rapa ssp. parachinensis, B. nigra, Orychophragmus violaceus, Matthiola incana, Raphanus sativa, Sisymbrium altissimum, Eruca sativa, Sinapis alba, Sinapis arvensis, Capsella bursa-pastoris and Camelina sativa. The plants of 23 cultivars in these species or subspecies were foliar sprayed with 10 ml of 0.2 or 0.4 mg/L of tribenuron-methyl before the vacuolated microspore formed in the largest flower buds; the application was repeated ten to twelve days afterwards. Tribenuron-methyl exposure significantly changed the flowering phenology and reproductive function. The treated plants demonstrated a one to four day delay in flowering time and a shortened duration of flowering, as well as other slight phytotoxic effects including a reduction in plant height and floral organ size. Approximately 80% to 100% male sterility, which was estimated by both pollen staining and selfing seed-set rate, was induced in the plants. As a result, plants were rendered functionally able to out-cross, with an average 87% and 54% manually pollinated seed-set rate compared to the corresponding controls at the 0.2 mg/L and 0.4 mg/L doses, respectively. CONCLUSIONS: The results suggested that male reproductive function was much more sensitive to tribenuron-methyl exposure than female function. This sulfonylurea herbicide has a promising use as the gametocide for hybrid production in cruciferous plants.

TaCYP78A5 regulates seed size in wheat (Triticum aestivum).

Seed size is an important agronomic trait and a major component of seed yield in wheat. However, little is known about the genes and mechanisms that determine the final seed size in wheat. Here, we isolated TaCYP78A5, the orthologous gene of Arabidopsis CYP78A5/KLUH in wheat, from wheat cv. Shaan 512 and demonstrated that the expression of TaCYP78A5 affects seed size. TaCYP78A5 encodes the cytochrome P450 (CYP) 78A5 protein in wheat and rescued the phenotype of the Arabidopsis deletion mutant cyp78a5. By affecting the extent of integument cell proliferation in the developing ovule and seed, TaCYP78A5 influenced the growth of the seed coat, which appears to limit seed growth. TaCYP78A5 silencing caused a 10% reduction in cell numbers in the seed coat, resulting in a 10% reduction in seed size in wheat cv. Shaan 512. By contrast, the overexpression of TaCYP78A5 increased the number of cells in the seed coat, resulting in seed enlargement of ~11-35% in Arabidopsis. TaCYP78A5 activity was positively correlated with the final seed size. However, TaCYP78A5 overexpression significantly reduced seed set in Arabidopsis, possibly due to an ovule development defect. TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation during seed development. Accordingly, a working model of the influence of TaCYP7A5 on seed size was proposed. This study provides direct evidence that TaCYP78A5 affects seed size and is a potential target for crop improvement.

Comparative transcriptome analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male sterility induced by the chemical hybridization agent monosulfuron ester sodium.

BACKGROUND: Chemical 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. RESULTS: Cytological 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. CONCLUSIONS: Our 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.

Identification of nuclear pore complexes (NPCs) and revealed outer-ring component BnHOS1 related to cold tolerance in B. napus.

Nuclear pore complexes (NPCs) consist of ~30 different nucleoporins (Nups), are the unique channels that govern development, hormonal response, and roles in both biotic and abiotic responses, as well as the transport and information exchange of biomacromolecules between nucleoplasms. Here, we report the comprehensive identification of 77 BnNups throughout the zhongshuang11 (ZS11) genome, which were classified into 29 distinct categories based on their evolutionary connections. We compared and contrasted different BnNups by analyzing at their gene structures, protein domains, putative three-dimensional (3D) models and expression patterns. Additional examples of genome-wide duplication events and cross-species synteny are provided to demonstrate the proliferation and evolutionary conservation of BnNups. When BnHOS1 was modified using CRISPR/Cas9 technology, the resulting L10 and L28 lines exhibited substantial freezing resistance. This not only demonstrated the negative regulatory impact of BnHOS1 on cold stress, but also offered a promising candidate gene for cold tolerance breeding and augmented the available B. napus material. These findings not only help us learn more about the composition and function of BnNPCs in B. napus, but they also provide light on how NPCs in other eukaryotic organism functions.

Genome-Wide Survey of Leucine-Rich Repeat Receptor-Like Protein Kinase Genes and CRISPR/Cas9-Targeted Mutagenesis BnBRI1 in Brassica napus.

The leucine-rich repeat receptor-like protein kinase (LRR-RLK) family represents the largest group of RLKs in plants and plays vital roles in plant growth, development and the responses to environmental stress. Although LRR-RLK families have been identified in many species, they have not yet been reported in B. napus. In this study, a total of 444 BnLRR-RLK genes were identified in the genome of Brassica napus cultivar "Zhongshuang 11" (ZS11), and classified into 22 subfamilies based on phylogenetic relationships and genome-wide analyses. Conserved motifs and gene structures were shared within but not between subfamilies. The 444 BnLRR-RLK genes were asymmetrically distributed on 19 chromosomes and exhibited specific expression profiles in different tissues and in response to stress. We identified six BnBRI1 homologs and obtained partial knockouts via CRISPR/Cas9 technology, generating semi-dwarf lines without decreased yield compared with controls. This study provides comprehensive insight of the LRR-RLK family in B. napus. Additionally, the semi-dwarf lines expand the "ideotype" germplasm resources and accelerate the breeding process for B. napus.

Systematic analysis of MADS-box gene family in the U's triangle species and targeted mutagenesis of BnaAG homologs to explore its role in floral organ identity in Brassica napus.

MADS-box transcription factors play an important role in regulating floral organ development and participate in environmental responses. To date, the MADS-box gene family has been widely identified in Brassica rapa (B. rapa), Brassica oleracea (B. oleracea), and Brassica napus (B. napus); however, there are no analogous reports in Brassica nigra (B. nigra), Brassica juncea (B. juncea), and Brassica carinata (B. carinata). In this study, a whole-genome survey of the MADS-box gene family was performed for the first time in the triangle of U species, and a total of 1430 MADS-box genes were identified. Based on the phylogenetic relationship and classification of MADS-box genes in Arabidopsis thaliana (A. thaliana), 1430 MADS-box genes were categorized as M-type subfamily (627 genes), further divided into Mα, Mß, Mγ, and Mδ subclades, and MIKC-type subfamily (803 genes), further classified into 35 subclades. Gene structure and conserved protein motifs of MIKC-type MADS-box exhibit diversity and specificity among different subclades. Comparative analysis of gene duplication events and syngenic gene pairs among different species indicated that polyploidy is beneficial for MIKC-type gene expansion. Analysis of transcriptome data within diverse tissues and stresses in B. napus showed tissue-specific expression of MIKC-type genes and a broad response to various abiotic stresses, particularly dehydration stress. In addition, four representative floral organ mutants (wtl, feml, aglf-2, and aglf-1) in the T0 generation were generated by editing four AGAMOUS (BnaAG) homoeologs in B. napus that enriched the floral organ variant phenotype. In brief, this study provides useful information for investigating the function of MADS-box genes and contributes to revealing the regulatory mechanisms of floral organ development in the genetic improvement of new varieties.

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

BACKGROUND: Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products. RESULTS: Three sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus. CONCLUSION: We used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.

Alternatively spliced BobCAL transcripts alter curd morphotypes in a collection of Chinese cauliflower accessions.

The curd of cauliflower (Brassica oleracea L. var. botrytis) is a modified inflorescence that is consumed as a vegetable. Curd formation is proposed to be due to a mutation in the BobCAULIFLOWER (BobCAL) gene, but the genetic relationship between BobCAL variation and curd morphotypes remains obscure. To address this question, we collected and classified a collection of 78 cauliflower accessions into four subpopulations according to curd surface features: smooth, coarse, granular, and hairy curd morphotypes. Through the cDNA sequencing of BobCAL alleles, we showed that smooth and coarse accessions characterized by inflorescence meristem arrest presented a strong association with the 451T SNP (BobCAL_T), whereas granular and hairy accessions marked with floral organ arrest presented an association with 451G (BobCAL_G). Interestingly, all BobCAL alleles were alternatively spliced, resulting in a total of four alternative splice (AS) variants due to the retention of the fourth and/or seventh introns. Among accessions with BobCAL_G alleles, the total expression of all these AS variants in granular plants was almost equal to that in hairy plants; however, the expression of the individual AS variants encoding intact proteins relative to those encoding truncated proteins differed. Hairy accessions showed relatively high expression of the individual variants encoding intact proteins, whereas granular accessions displayed relatively low expression. In smooth cauliflower, the overexpression of the BobCAL_Ga variant caused an alteration in the curd morphotype from smooth to hairy, concurrent with an increase in the expression levels of downstream floral identity genes. These results reveal that alternative splicing of BobCAL transcripts is involved in the determination of cauliflower curd morphotypes.

Cytological and genetic characterisation of dominant GMS line Shaan-GMS in Brassica napus L.

Genic male sterility (GMS) is an effective pollination control system applied in the hybrid breeding of Brassica napus L. Shaan-GMS is a spontaneous mutant of dominant GMS in B. napus. In this research, anther abortion in the homozygous two-type line 9A15AB derived from Shaan-GMS was characterised with the combined use of light microscopy and transmission electron microscopy. Results indicated that the most striking differences between the fertile and sterile plants occurred in the tapetum in the early microsporocyte stage. In sterile plants, the tapetal cells were irregularly arranged, multi-layered and occupied the growing space of microsporocytes. When entering into meiosis, the tapetum cells degraded and the cytoplasm fused. Some oval monolayer or bilayer membrane organelles existed in the tapetal cells in sterile anthers. Mitochondria in the tapetal cells were abnormal, and middle layer cells degraded early. Pollen mother cells of Shaan-GMS degenerated at the start of meiosis and ceased at the anaphase I stage, with no dyads or tetrads formed. The combined effects of the abnormal development of the tapetum, the middle layer cells and meiosis lead to male sterility in Shaan-GMS. Inheritance of male sterility of Shaan-GMS is controlled by a monogenically multiallelic locus with three different alleles (Ms, ms and Mf), with a relationship expressed as Mf > Ms and Ms > ms. The findings help lay the foundation for illustrating the mechanism of male sterility and the utilisation of Shaan-GMS in rapeseed.

Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L.

Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1544T ), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be(-KX)) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.

Acetolactate Synthase-Inhibiting Gametocide Amidosulfuron Causes Chloroplast Destruction, Tissue Autophagy, and Elevation of Ethylene Release in Rapeseed.

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.