Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

The functional role of expansin-like A and B family members remains unclear in plants. In this study, we investigate the functional role of Brassica rapa expansin-like B1 (BrEXLB1) by overexpressing BrEXLB1 sense, antisense and BrEXLB1 specific promoter: GUS in Arabidopsis. The overexpressors of sense BrEXLB1, antisense BrEXLB1 showed variable and unstable pleiotropic effects on leaf growth. Interestingly, overexpressors of sense BrEXLB1 enhances plant height than antisense overexpressors and wild types. GUS reporter-aided analysis of BrEXLB1 promoter showed their activity prominently in seeds, roots and carpels. This is further confirmed by relative quantification of BrEXLB1 among various tissue samples of B. rapa by semi-quantitative Reverse transcription-polymerase chain reaction assay (RT-PCR). We found that BrEXLB1 promoter has several cis-acting elements including light-responsive, phytohormone-responsive, environmental/stress-responsive and tissue-specific elements in promoter sequences. Hence, we analysed the promoter activity of BrEXLB1 by GUS reporter-aided approach, in which stress, phytohormones and other factors regulated changes in GUS transcripts were measured using quantitative RT-PCR. This study suggests that promoter activity is inducible to exogenous application of phytohormones such as indole-3-acetic acid, jasmonic acid, and other factors such as white light and drought stress. These results suggest that BrEXLB1 under its specific promoters may participate in regulation of leaf, plant growth and responds to hormone availability, light quality, dark periods, developmental stages, and drought conditions.

Plant uridine 5′-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina–millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.