Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana.

Flavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. However, 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 loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). RNA-seq results indicated that flavonols modulate various biological and metabolic pathways, with significant alterations in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor-mediated up-regulation 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 up-regulated aliphatic glucosinolate biosynthesis genes compared with 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-SG7 transcription factor CaMYB39 orchestrates surface phenylpropanoid metabolism and pathogen resistance in chickpea.

Flavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced the accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced reactive oxygen species levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight (AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density, and defense against necrotrophic fungal infection in chickpea.

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.

MAIN CONCLUSION: We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum. 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.

Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata).

Proanthocyanidins 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 characterised 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 MYB-bHLH-WD Repeat (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 through the formation of competitive MBW complexes. 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.

Molecular components associated with the regulation of flavonoid biosynthesis.

Flavonoids exhibit amazing structural diversity and play different roles in plants. Besides, these compounds have been associated with several health benefits in humans. Several exogenous and endogenous cues, for example, light, temperature, nutrient status, and phytohormones have been reported as modulators of biosynthesis and accumulation of flavonoids. Thus, multiple hormones and stress-related signaling pathways are involved in the regulation of gene expression associated with this pathway. The transcriptional regulators belonging to the MYB and bHLH family transcription factors are well documented as the direct regulators of the structural genes associated with flavonoid biosynthesis. Recent studies also suggest that some of these factors are regulated by molecular components involved in stress and hormone signaling pathways. Adapter proteins for transcriptional activation or repression via recruitment of co-activators and co-repressors, respectively, E2 ubiquitin ligases, miRNA processing complex, and DNA methylation/demethylation factors have been recently discovered in various plants to play key roles in fine-tuning flavonoids synthesis. In the present review, we aim to provide comprehensive information about the role of different factors in the regulation of flavonoid biosynthesis. Besides, we describe the potential upstream regulators involved in the regulation of flavonoid biosynthesis within the context of available information. To sum up, the present review furnishes an updated account of signal transduction pathways modulating the biosynthesis of flavonoids.

The R2R3-MYB transcription factor MtMYB134 orchestrates flavonol biosynthesis in Medicago truncatula.

KEY MESSAGE: Our results provide insights into the flavonol biosynthesis regulation of M. truncatula. The R2R3-MYB transcription factor MtMYB134 emerged as tool to improve the flavonol biosynthesis. 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.