ABSTRACT Flavonoids are important specialized metabolites that contribute to plant pigmentation, stress adaptation, and nutritional value. In banana, a major global staple crop, their accumulation is highly tissue-specific, with very low levels in the edible pulp, and the mechanisms underlying this spatial distribution remain unclear. Here, integrative transcriptomic and metabolomic analyses across vegetative and reproductive tissues reveal that light-responsive regulatory networks control tissue-specific flavonoid biosynthesis. We identified a B-box transcription factor MaBBX21 as a key positive regulator of flavonoid biosynthesis. Its overexpression enhances flavonoid accumulation, whereas knockdown leads to a reduction in flavonoid levels in banana. Mechanistically, MaBBX21 interacts with MaHY5 and directly activates anthocyanin biosynthesis genes ( MaDFR2 and MaANS ). It also regulates metabolic flux by binding to the MaWRKY23 promoter, promoting flavonol biosynthesis through activation of MaFLS1 while partially repressing the anthocyanin branch. In addition, MaBBX21 introduces an epigenetic layer by activating the histone acetyltransferase MaGCN5, increasing H3K9 acetylation at target promoters, including MaDFR2, MaANS, and MaBBX21 itself, thereby forming a positive feedback loop. Functionally, MaBBX21 overexpression enhances flavonoid accumulation, ROS scavenging, and tolerance to heat and UV-B stress, whereas knockdown lines show reduced metabolite levels and increased stress sensitivity. Collectively, these results define a MaBBX21–MaWRKY23/MaGCN5 regulatory axis that integrates transcriptional regulation, and chromatin modification to control flavonoid biosynthesis, providing a foundation for improving nutritional quality and stress resilience in banana.

Comparative metabolic profiling of new genetic multiple flavonoid r2r3-myb mutants show that different types of R2R3–MYBs activate the early flavonoid biosynthesis genes. A revised model for flavonoid biosynthesis in Arabidopsis thaliana is proposed that integrates the regulatory roles of these R2R3–MYBs across early and late biosynthetic steps. Flavonoids are a large group of specialized plant metabolites. Their biosynthesis is mainly transcriptionally regulated by a sophisticated network of different transcription factors from various families, with R2R3–MYB factors being the main determinant of specific flavonoid class formation. The early biosynthetic steps, leading to the formation of non-visible flavonoids, have been proposed to be regulated by three R2R3–MYBs, PRODUCTION OF FLAVONOL GYLCOSIDE1-3 (PFG1-3), while the later biosynthetic steps leading to the formation of visible anthocyanin and proanthocyanidin pigments are reported to be regulated by four R2R3–MYBs, PRODUCTION OF ANTHOCYANIN PIGMENT1-4 (PAP1-4) and TRANSPARENT TESTA2 (TT2), respectively. Several studies have indicated that this model for the transcriptional regulation of flavonoid biosynthesis may be incomplete. To address this issue, especially regarding the regulation of the early biosynthesis genes by PAP1-4 and TT2, we generated several multiple r2r3-myb mutant lines. We characterized the pfg1-3, pfg1-3 tt2 and pfg1-3 pap1-4 mutants and did comparative metabolite profiling. This revealed that only the pfg1-3 tt2 mutant was deficient in proanthocyanidins and only the pfg1-3 pap1-4 mutant was deficient in anthocyanins. We demonstrate that PAP and TT2 R2R3–MYBs are also capable of activating the early biosynthesis genes required for dihydroflavonol formation. Our results provide evidence that the traditional view of distinct branch-specific R2R3–MYB regulators in flavonoid biosynthesis is overly simplistic. We, therefore, propose a revised model for the transcriptional regulation of flavonoid biosynthesis.

Plant flavonols act primarily as ultraviolet radiation absorbers, reactive oxygen species scavengers, and phytoalexins, and they contribute to biotic and abiotic stress tolerance in plants. Banana (Musa acuminata), an herbaceous monocot and important fruit crop, accumulates flavonol derivatives in different organs, including the edible fruit pulp. Although flavonol content varies greatly in different organs, the molecular mechanisms involving transcriptional regulation of flavonol synthesis in banana are not known. Here, we characterized three SG7-R2R3 MYB transcription factors (MaMYBFA1, MaMYBFA2, and MaMYBFA3) and heat shock transcription factor (MaHSF11), to elucidate the molecular mechanism involved in transcriptional regulation of flavonol biosynthesis in banana. MaMYBFA positively regulates flavonol synthase 2 (MaFLS2) and downregulates MaFLS1. We show these transcription factors to be weak regulators of flavonol synthesis. Overexpression of MaHSF11 enhances flavonol contents, particularly that of myricetin, and promotes flavonol B-ring hydroxylation, which contributes to the diversity of flavonol derivatives. MaHSF11 directly interacts with the MaFLS1 and flavonoid 3',5'-hydroxylase1 (MaF3'5'H1) promoters, both in vitro and in vivo. MaHSF11 activates the expression of MaDREB1 directly, which is known to promote cold and chilling tolerance in banana fruit. Overall, our study elucidates a regulatory mechanism for flavonol synthesis in banana and suggests possible targets for genetic optimization to enhance nutritional value and stress responses in this globally important fruit crop.

Flavonoids are specialized metabolites that play important roles in plants, including interactions with the environment. The high structural diversity of this metabolite group is largely due to enzyme-mediated modifications of flavonoid core skeletons. In particular, glycosylation with different sugars is very common. In this study, the functions of the Arabidopsis thaliana glycoside hydrolase family 1-type glycosyltransferase proteins BGLU1, BGLU3, and BGLU4 were investigated, using a reverse genetics approach and untargeted metabolic fingerprinting. We screened for metabolic differences between A. thaliana wild type, loss-of-function mutants, and overexpression lines and partially identified differentially accumulating metabolites, which are putative products and/or substrates of the BGLU enzymes. Our study revealed that the investigated BGLU proteins are glycosyltransferases involved in the glycosylation of already glycosylated flavonoids using different substrates. While BGLU1 appears to be involved in the rhamnosylation of a kaempferol diglycoside in leaves, BGLU3 and BGLU4 are likely involved in the glycosylation of quercetin diglycosides in A. thaliana seeds. In addition, we present evidence that BGLU3 is a multifunctional enzyme that catalyzes other metabolic reactions with more complex substrates. This study deepens our understanding of the metabolic pathways and enzymes that contribute to the high structural diversity of flavonoids.

Transcription factors control gene expression during development and in response to a broad range of internal and external stimuli. They regulate promoter activity by directly binding cis-regulatory elements in DNA. The angiosperm Arabidopsis (Arabidopsis thaliana) contains more than 1,500 annotated transcription factors, each containing a DNA-binding domain that is used to define transcription factor families. Analyzing the binding motifs of 686 and the binding sites of 335 Arabidopsis transcription factors, as well as motifs of 92 transcription factors from other plants, we identified a constrained vocabulary of 74 conserved motifs spanning 50 families in plants. Among 21 transcription factor families, we found 1 core motif for all analyzed members and between 2% and 72% overlapping binding sites. Five families show conservation of the motif along phylogenetic clades. Five families, including the C2H2 zinc finger family, show high diversity among motifs in plants, suggesting potential for the neofunctionalization of duplicated transcription factors based on the motif recognized. We tested whether conserved motifs remained conserved since at least 450 million years ago by determining the binding motifs of 17 transcription factors from 11 families in Marchantia (Marchantia polymorpha) using amplified DNA affinity purification sequencing. We detected nearly identical binding motifs as predicted from the angiosperm data. Our findings show a large repertoire of overlapping binding sites within a transcription factor family and species and a high degree of binding motif conservation for at least 450 million years, indicating more potential for evolution in cis- rather than trans-regulatory elements.

In land plants, one of the processes vital to cope with environmental changes is the accumulation of photoprotective flavonoids such as flavonols and anthocyanins. The inactivation of SUCROSE NON-FERMENTING1 RELATED PROTEIN KINASE1 (SnRK1), which acts in a chloroplast-derived sugar signalling pathway, permits the activation of flavonoid biosynthesis in high light. The present study provides genetic evidence that SnRK1 acts upstream of PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), encoding a crucial transcription factor that activates the anthocyanin branch of flavonoid biosynthesis during high light acclimation. A time-resolved expression analysis indicates a two-phasic suppression of MYB-LIKE2 (MYBL2), a repressor of anthocyanin production, involving SnRK1 inactivation for anthocyanin accumulation during prolonged high light exposure. Furthermore, overexpression of PAP1 resulted in the marked suppression of MYB11, MYB12, MYB111, and FLAVONOL SYNTHASE1, initiating the flavonol branch of the pathway. Analysis of a flavonoid-deficient CHALCONE SYNTHASE mutant overexpressing PAP1 suggests that regulation of flavonoid biosynthesis is PAP1 dependent but independent of flavonoid intermediates and end-products. It is proposed that PAP1 suppresses the flavonol branch by an unknown mechanism, thereby promoting the consumption of carbon building blocks for anthocyanin production to permit the fine-tuning of the pathway.

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.

Background Flavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxygenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each other’s reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana , stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. Results To address this issue, we generated a f3h / fls1 / ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h / fls1 / ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h / fls1 and f3h / ans , was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta . Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3’-hydroxylase (F3’H) activity. Conclusions We describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h / fls1 / ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for the creation of novel food supplements.

OBJECTIVE:Bananas are one of the most popular fruits in the world, providing food security and employment opportunities in several developing countries. Increasing the anthocyanin content of banana fruit could improve the health-promoting properties. Anthocyanin biosynthesis is largely regulated at the transcriptional level. However, relatively little is known about the transcriptional activation of anthocyanin biosynthesis in banana.RESULTS:We analysed the regulatory activity of three Musa acuminata MYBs that were predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana. MaMYBA1, MaMYBA2 and MaMYBPA2 did not complement the anthocyanin-deficient phenotype of the Arabidopsis thaliana pap1/pap2 mutant. However, co-transfection experiments in A. thaliana protoplasts showed that MaMYBA1, MaMYBA2 and MaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, the so called MBW complex, resulting in the activation of the A. thaliana ANTHOCYANIDIN SYNTHASE and DIHYDROFLAVONOL 4-REDUCTASE promoters. The activation potential of MaMYBA1, MaMYBA2 and MaMYBPA2 was increased when combined with the monocot Zea mays bHLH ZmR instead of the dicot AtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. It will also facilitate research towards increased anthocyanin content in banana and other monocot crops.

Predicting gene expression from promoter sequence requires understanding of the different signal integration points within a promoter. Sequence-specific transcription factors (TFs) binding to their cognate TF binding motifs control gene expression in eukaryotes by activating and repressing transcription. Their interplay generates complex expression patterns in reaction to environmental conditions and developmental cues. We hypothesized that signals are not only integrated by different TFs binding various positions in a promoter, but also by single TF binding motifs onto which multiple TFs can bind. Analyzing 2,190 binding motifs, we identified only 76 core TF binding motifs in plants. Twenty-one TF protein families act highly specific and bind a single conserved motif. Four TF families are classified as semi-conserved as they bind up to four motifs within a family, with divisions along phylogenetic groups. Five TF families bind diverse motifs. Expression analyses revealed high competition within TF families for the same binding motif. The results show that singular binding motifs act as signal integrators in plants where a combination of binding affinity and TF abundance likely determine the output.

Plant flavonols act primarily as ultraviolet radiation absorbers, reactive oxygen species scavengers, and phytoalexins, and they contribute to biotic and abiotic stress tolerance in plants. Banana ( Musa acuminata ), an herbaceous monocot and important fruit crop, accumulates flavonol derivatives in different organs, including the edible fruit pulp. Although flavonol content varies greatly in different organs, the molecular mechanisms involving transcriptional regulation of flavonol synthesis in banana are not known. Here, we characterized three SG7-R2R3 MYB transcription factors MaMYBFA1, MaMYBFA2, and MaMYBFA3) and their upstream regulators, heat shock transcription factor (MaHSF11) and dehydration responsive element binding factor (MaDREB1), to elucidate the molecular mechanism involved in transcriptional regulation of flavonol biosynthesis in banana. MaMYBFA positively regulate flavonol synthase 2 ( MaFLS2) and downregulates MaFLS1 . We show these transcription factors to be weak regulators of flavonol synthesis. Overexpression of MaHSF11 enhances flavonol contents, particularly that of myricetin, and promotes flavonol B-ring hydroxylation, which contributes to the diversity of flavonol derivatives. MaHSF11 directly interacts with the MaFLS1 and flavonoid 3′, 5′-hydroxylase 1 ( MaF3′5′H1) promoters, both in vitro and in vivo . MaHSF11 activates the expression of MaDREB1 directly, which in turn regulates the expression of MaMYBFA3 . Overall, our study elucidates a key regulatory mechanism for flavonol synthesis in banana and suggests possible targets for genetic optimization to enhance nutritional value and stress responses in this globally important fruit crop.

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.

Flavonols are structurally and functionally diverse molecules playing roles in plant biotic and abiotic stress tolerance, auxin transport inhibition, pollen development, etc. Despite their ubiquitous occurrence in land plants and multifunctionality, the effect of perturbation of flavonol biosynthesis over global gene expression and pathways other than flavonoid biosynthesis has not been studied in detail. To understand the signaling role of different flavonol metabolites, herein, we used the flavonol deficient Arabidopsis thaliana loss-of-function mutant flavonol synthase1 (fls1-3) as object of study. Comparative transcriptome and metabolic profiling were used to study the effects of genetic flavonol deficiency and exogenous supplementation with flavonol derivatives (kaempferol, quercetin and rutin) on different cellular processes in the seedling. Various flavonol biosynthesis-related regulatory and structural genes were found to be up-regulated in the fls1-3 mutant which could be reversed by exogenous flavonol feeding. Our manifold comparative studies indicated the modulation of various biological processes and metabolic pathways by flavonols. Camalexin biosynthesis was found to be negatively regulated by flavonols. Interestingly, flavonols appeared to promote the accumulation of aliphatic glucosinolate through transcription factor-mediated up-regulation of biosynthesis genes. Overall, this study provides new insights into molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets and adds new knowledge to the expanding plethora of biological activity of flavonols in plants. Significance Comparative transcriptome and metabolomic profiling of genetic flavonol deficiency and exogenous flavonol supplementation in A. thaliana seedlings, for the first-time revealed the inverse regulation of interconnected specialized metabolite pathways by flavonol aglycones, and -glycosides. Flavonols negatively regulate camalexin biosynthesis, while promoting the accumulation of aliphatic glucosinolates. Our study adds new insights into the expanding plethora of biological activity of flavonols in plants and will help to uncover the molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets.

Summary 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.

Key message Decreased absorptance and increased singlet oxygen production may cause photoinhibition of both PSII and PSI in birch leaves during autumn senescence; however, photosynthetic electron transfer stays functional until late senescence. Abstract During autumn senescence, deciduous trees degrade chlorophyll and may synthesize flavonols. We measured photosynthetic parameters, epidermal flavonols, singlet oxygen production in vivo and photoinhibition of the photosystems (PSII and PSI) from green and senescing silver birch ( Betula pendula ) leaves. Chlorophyll a fluorescence and P 700 absorbance measurements showed that the amounts of both photosystems decreased throughout autumn senescence, but the remaining PSII units stayed functional until ~ 90% of leaf chlorophyll was degraded. An increase in the chlorophyll a to b ratio, a decrease in > 700 nm absorbance and a blue shift of the PSI fluorescence peak at 77 K suggest that light-harvesting complex I was first degraded during senescence, followed by light-harvesting complex II and finally the photosystems. Senescing leaves produced more singlet oxygen than green leaves, possibly because low light absorption by senescing leaves allows high flux of incident light per photosystem. Senescing leaves also induced less non-photochemical quenching, which may contribute to increased singlet oxygen production. Faster photoinhibition of both photosystems in senescing than in green leaves, under high light, was most probably caused by low absorption of light and rapid singlet oxygen production. However, senescing leaves maintained the capacity to recover from photoinhibition of PSII. Amounts of epidermal flavonols and singlet oxygen correlated neither in green nor in senescing leaves of silver birch. Moreover, Arabidopsis thaliana mutants, incapable of synthesizing flavonols, were not more susceptible to photoinhibition of PSII or PSI than wild type plants; screening of chlorophyll absorption by flavonols was, however, small in A. thaliana . These results suggest that flavonols do not protect against photoinhibition or singlet oxygen production in chloroplasts.

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.

Transcription initiation of the genes coding for small nuclear RNA (snRNA) has been extensively analyzed in humans and fruit fly, but only a single ortholog of a snRNA-activating protein complex (SNAPc) subunit has so far been characterized in plants. The genome of the model plant Arabidopsis thaliana encodes orthologs of all three core SNAPc subunits, including A. thaliana SNAP complex 4 (AtSNAPc4)-a 4R-MYB-type protein with four-and-a-half adjacent MYB repeat units. We report the conserved role of AtSNAPc4 as subunit of a protein complex involved in snRNA gene transcription and present genetic evidence that AtSNAPc4 is an essential gene in gametophyte and zygote development. We present experimental evidence that the three A. thaliana SNAPc subunits assemble into a SNAP complex and demonstrate the binding of AtSNAPc4 to snRNA promoters. In addition, co-localization studies show a link between AtSNAPc4 accumulation and Cajal bodies, known to aggregate at snRNA gene loci in humans. Moreover, we show the strong evolutionary conservation of single-copy 4R-MYB/SNAPc4 genes in a broad range of eukaryotes and present additional shared protein features besides the MYB domain, suggesting a conservation of the snRNA transcription initiation machinery along the course of the eukaryotic evolution.

Bananas ( Musa ) are non-grass, monocotyledonous, perennial plants that are well-known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta . Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

Different Musa species, subspecies, and cultivars are currently investigated to reveal their genomic diversity. Here, we compare the genome sequence of one of the commercially most important cultivars, Musa acuminata Dwarf Cavendish, against the Pahang reference genome assembly. Numerous small sequence variants were detected and the ploidy of the cultivar presented here was determined as triploid based on sequence variant frequencies. Illumina sequence data also revealed a duplication of a large segment on the long arm of chromosome 2 in the Dwarf Cavendish genome. Comparison against previously sequenced cultivars provided evidence that this duplication is unique to Dwarf Cavendish. Although no functional relevance of this duplication was identified, this example shows the potential of plants to tolerate such aneuploidies.

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.