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Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

ABSTRACT: Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

SUBMITTER: Schilbert HM

PROVIDER: S-EPMC8548573 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

Schilbert Hanna Marie HM Schöne Maximilian M Baier Thomas T Busche Mareike M Viehöver Prisca P Weisshaar Bernd B Holtgräwe Daniela D

Frontiers in plant science 20211013

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed fla ...[more]

PMID: 34721462

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Project description:Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.

Project description:BackgroundSBP-box genes belong to one of the largest families of transcription factors. Though members of this family have been characterized to be important regulators of diverse biological processes, information of SBP-box genes in the third most important oilseed crop Brassica napus is largely undefined.ResultsIn the present study, by whole genome bioinformatics analysis and transcriptional profiling, 58 putative members of SBP-box gene family in oilseed rape (Brassica napus L.) were identified and their expression pattern in different tissues as well as possible interaction with miRNAs were analyzed. In addition, B. napus lines with contrasting branch angle were used for investigating the involvement of SBP-box genes in plant architecture regulation. Detailed gene information, including genomic organization, structural feature, conserved domain and phylogenetic relationship of the genes were systematically characterized. By phylogenetic analysis, BnaSBP proteins were classified into eight distinct groups representing the clear orthologous relationships to their family members in Arabidopsis and rice. Expression analysis in twelve tissues including vegetative and reproductive organs showed different expression patterns among the SBP-box genes and a number of the genes exhibit tissue specific expression, indicating their diverse functions involved in the developmental process. Forty-four SBP-box genes were ascertained to contain the putative miR156 binding site, with 30 and 14 of the genes targeted by miR156 at the coding and 3'UTR region, respectively. Relative expression level of miR156 is varied across tissues. Different expression pattern of some BnaSBP genes and the negative correlation of transcription levels between miR156 and its target BnaSBP gene were observed in lines with different branch angle.ConclusionsTaken together, this study represents the first systematic analysis of the SBP-box gene family in Brassica napus. The data presented here provides base foundation for understanding the crucial roles of BnaSBP genes in plant development and other biological processes.

Project description:Auxin response factor (ARF) is a member of the plant-specific B3 DNA binding superfamily. Here, we report the results of a comprehensive analysis of ARF genes in allotetraploid Brassica napus (2n = 38, AACC). Sixty-seven ARF genes were identified in B. napus (BnARFs) and divided into four subfamilies (I-IV). Sixty-one BnARFs were distributed on all chromosomes except C02; the remaining were on Ann and Cnn. The full length of the BnARF proteins was highly conserved especially within each subfamily with all members sharing the N-terminal DNA binding domain (DBD) and the middle region (MR), and most contained the C-terminal dimerization domain (PBI). Twenty-one members had a glutamine-rich MR that may be an activator and the remaining were repressors. Accordingly, the intron patterns are highly conserved in each subfamily or clade, especially in DBD and PBI domains. Several members in subfamily III are potential targets for miR167. Many putative cis-elements involved in phytohormones, light signaling responses, and biotic and abiotic stress were identified in BnARF promoters, implying their possible roles. Most ARF proteins are likely to interact with auxin/indole-3-acetic acid (Aux/IAA) -related proteins, and members from different subfamilies generally shared many common interaction proteins. Whole genome-wide duplication (WGD) by hybridization between Brassica rapa and Brassica oleracea and segmental duplication led to gene expansion. Gene loss following WGD is biased with the An-subgenome retaining more ancestral genes than the Cn-subgenome. BnARFs have wide expression profiles across vegetative and reproductive organs during different developmental stages. No obvious expression bias was observed between An- and Cn-subgenomes. Most synteny-pair genes had similar expression patterns, indicating their functional redundancy. BnARFs were sensitive to exogenous IAA and 6-BA treatments especially subfamily III. The present study provides insights into the distribution, phylogeny, and evolution of ARF gene family.

Dataset Information

Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

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Characterization of the <i>Brassica napus</i> Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

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