Abstract
Galactomannan is a polymer of high economic importance and is extracted from the seed endosperm of clusterbean (C. tetragonoloba). In the present study, we worked to reveal the stage-specific galactomannan biosynthesis and its regulation in clusterbean. Combined electron microscopy and biochemical analysis revealed high protein and gum content in RGC-936, while high oil bodies and low gum content in M-83. A comparative transcriptome study was performed between RGC-936 (high gum) and M-83 (low gum) varieties at three developmental stages viz. 25, 39, and 50 days after flowering (DAF). Total 209,525, 375,595 and 255,401 unigenes were found at 25, 39 and 50 DAF respectively. Differentially expressed genes (DEGs) analysis indicated a total of 5147 shared unigenes between the two genotypes. Overall expression levels of transcripts at 39DAF were higher than 50DAF and 25DAF. Besides, 691 (RGC-936) and 188 (M-83) candidate unigenes that encode for enzymes involved in the biosynthesis of galactomannan were identified and analyzed, and 15 key enzyme genes were experimentally validated by quantitative Real-Time PCR. Transcription factor (TF) WRKY was observed to be co-expressed with key genes of galactomannan biosynthesis at 39DAF. We conclude that WRKY might be a potential biotechnological target (subject to functional validation) for developing high gum content varieties.
Highlights
GO classification and KEGG enrichment analysis of Differentially expressed genes (DEGs)
RNA extracts from two genotypes (RGC-936 and M-83) during different pod development stages were subjected to Illumina Hiseq X Ten sequencing, which generated a total of 286.85 Mb raw sequencing reads from 12 RNA libraries
At 39DAF, higher expression was observed for genes involved in metabolic pathways, glutathione biosynthesis, secondary metabolite biosynthesis and cysteine methionine metabolism; while late developmental stage i.e., 50DAF showed higher expression of genes involved in metabolic pathways, secondary metabolite biosynthesis, antibiotic biosynthesis, carbon metabolism, oxidative phosphorylation, amino acid biosynthesis, and carboxy acid metabolism
Summary
GO classification and KEGG enrichment analysis of DEGs. GO annotation of the DEGs from the 15 pairwise comparisons (mentioned above) were used to classify genes into three main GO categories (present in the gene ontology annotations) based on total gene count and their role in different functional processes: information storage and processing category, metabolic and cellular function. KEGG enrichment analysis allowed mapping of DEGs to top 20 pathways. At 25DAF (early development), highly expressed genes belonged to metabolic pathways, ribosome, oxidative phosphorylation, photosynthesis and secondary metabolite biosynthesis. At 39DAF (mid development), higher expression was observed for genes involved in metabolic pathways, glutathione biosynthesis, secondary metabolite biosynthesis and cysteine methionine metabolism; while late developmental stage i.e., 50DAF showed higher expression of genes involved in metabolic pathways, secondary metabolite biosynthesis, antibiotic biosynthesis, carbon metabolism, oxidative phosphorylation, amino acid biosynthesis, and carboxy acid metabolism. Genes involved in metabolic pathways and secondary metabolite biosynthesis were common to all the three seed developmental stages, while oxidative phosphorylation genes were prominent at 25 and 50 DAF
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