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Item Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana(Oxford University Press, 2024) Naik, Jogindra; Tyagi, Shivi; Rajput, Ruchika; Kumar, Pawan; Pucker, Boas; Bisht, Naveen C.; Misra, Prashant; Stracke, Ralf; Pandey, AshutoshFlavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. Despite the ubiquitous nature and multifunctionality of flavonols in land plants, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis (Arabidopsis thaliana) loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). Our RNA-seq results indicated that flavanols modulate various biological and metabolic pathways, with significant alteration in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor–mediated upregulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly upregulated aliphatic glucosinolate biosynthesis genes compared to kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.Item The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat(Springer Nature Publishing AG, 2022) Rajput, Ruchika; Tyagi, Shivi; Naik, Jogindra; Pucker, Boas; Stracke, Ralf; Pandey, AshutoshChickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein–protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.Item Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata)(John Wiley & Sons, 2022) Rajput, Ruchika; Naik, Jogindra; Stracke, Ralf; Pandey, AshutoshProanthocyanidins are oligomeric flavonoids that promote plant disease resistance and benefit human health. Banana is one of the world's most extensively farmed crops and its fruit pulp contain proanthocyanidins. However, the transcriptional regulatory network that fine-tunes proanthocyanidin biosynthesis in banana remains poorly understood. We characterized two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana. Heterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs to transactivate anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase genes in vitro and form functional MBW complexes with MaTTG1 to transactivate these promoters in vivo. Overexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and transcription of proanthocyanidin biosynthesis-related genes. MaMYBPR repressors are also shown to interact with MaMYCs forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis via the formation of competitive MBW complex. Our results reveal regulatory modules of R2R3 MYB- that fine-tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation for nutritional improvement of banana.