Browsing by Author "Pucker, Boas"

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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, Ashutosh
Flavonols 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.
The R2R3-MYB gene family in banana (Musa acuminata): Genome-wide identification, classification and expression patterns
(PLOS, 2020) Pucker, Boas; Pandey, Ashutosh; Weisshaar, Bernd; Stracke, Ralf
The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, defense responses and metabolite accumulation. To date MYB family genes have not yet been comprehensively identified in the major staple fruit crop banana. In this study, we present a comprehensive, genome-wide analysis of the MYB genes from Musa acuminata DH-Pahang (A genome). A total of 285 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Organ- and development-specific expression patterns were determined from RNA-Seq data. For 280 M. acuminata MYB genes for which expression was found in at least one of the analysed samples, a variety of expression patterns were detected. The M. acuminata R2R3-MYB genes were functionally categorised, leading to the identification of seven clades containing only M. acuminata R2R3-MYBs. The encoded proteins may have specialised functions that were acquired or expanded in Musa during genome evolution. This functional classification and expression analysis of the MYB gene family in banana establishes a solid foundation for future comprehensive functional analysis of MaMYBs and can be utilized in banana improvement programmes.
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, Ashutosh
Chickpea (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.
The R2R3‑MYB transcription factor MtMYB134 orchestrates favonol biosynthesis in Medicago truncatula
(Springer Nature Publishing AG, 2021) Naik, Jogindra; Rajput, Ruchika; Pucker, Boas; Stracke, Ralf; Pandey, Ashutosh
Flavonols are plant specialized metabolites with vital roles in plant development and defense and are known as diet compound beneficial to human health. In leguminous plants, the regulatory proteins involved in flavonol biosynthesis are not well characterized. Using a homology-based approach, three R2R3-MYB transcription factor encoding genes have been identified in the Medicago truncatula reference genome sequence. The gene encoding a protein with highest similarity to known flavonol regulators, MtMYB134, was chosen for further experiments and was characterized as a functional flavonol regulator from M. truncatula. MtMYB134 expression levels are correlated with the expression of MtFLS2, encoding a key enzyme of flavonol biosynthesis, and with flavonol metabolite content. MtMYB134 was shown to activate the promoters of the A. thaliana flavonol biosynthesis genes AtCHS and AtFLS1 in Arabidopsis protoplasts in a transactivation assay and to interact with the Medicago promoters of MtCHS2 and MtFLS2 in yeast 1-hybrid assays. To ascertain the functional aspect of the identified transcription factor, we developed a sextuple mutant, which is defective in anthocyanin and flavonol biosynthesis. Ectopic expression of MtMYB134 in a multiple myb A. thaliana mutant restored flavonol biosynthesis. Furthermore, overexpression of MtMYB134 in hairy roots of M. truncatula enhanced the biosynthesis of various flavonol derivatives. Taken together, our results provide insight into the understanding of flavonol biosynthesis regulation in M. truncatula and provides MtMYB134 as tool for genetic manipulation to improve flavonol synthesis.