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Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica. [ABSTRACT FROM AUTHOR]

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum, Alternaria brassicae, pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot.

The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum , Alternaria brassicae , pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2 n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot. [ABSTRACT FROM AUTHOR]

Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1,185 nt and an N50 of 1,672 nt. Among these unigenes, 46,535 (97%) unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Clusters of Orthologous Groups of proteins (COGs). The tissue expression pattern profiles revealed that 3,489, 1,361 and 8,482 unigenes were predominantly expressed in the leaves, stems and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs) were identified, and 12,830 pairs of primers were developed for 2,522 SSR-containing unigenes. Additionally, the glucosinolate (GSL) and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1 and CYP79F1 expressions. Glutathione was extensively converted into phytochelatin in roots, but it was actively converted to the oxidized form in leaves, indicating the different mechanisms in the two tissues. This transcriptome will not only benefit basic research and molecular breeding of S. alba but also be useful for the molecular-assisted transfer of beneficial traits to other crops.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleotide binding proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalogue of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins. [ABSTRACT FROM AUTHOR]