- Results: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. - Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - Here, we aimed to bridge some of the gaps currently existing in berry fruit RNA-seq resources by generating and analyzing the fruit transcriptomes of 12 species as well as the leaf transcrip- tome of an additional species as part of the the BacH- Berry (BACterial Hosts for production of Bioactive phenolics from bERRY fruits) collaborative project [26].. - Plant-based products like fruits and berries are essen- tial parts of the human diet and are considered healthy and nutritious foods (reviewed in [27. - Many berries and fruits are valued for their high content of bioactive compounds, including specialised metabolites of the phenylpropanoid pathway such as flavonoids (flavonols, flavones, isoflavones, anthocyanins and proanthocyani- dins). - Members of sev- eral protein superfamilies mediate the transcriptional regulation of the flavonoid biosynthetic pathway, namely the MYB transcription factors (TFs), basic helix-loop- helix (bHLH) TFs and conserved WD40 repeat (WDR) proteins [53].. - MYB TFs are often specific for the genes and pathway/pathway branches they target, such as the flavonol-specific activators of the R2R3 MYB subgroup (SG) 7 (e.g., AtMYB12 [56]) whereas others are confined to regulating anthocyanin (MYB SG6, A. - multiple regulatory targets [61] and can control tran- scription of several branches of the flavonoid pathway as shown, for instance, by AtTT8 from A. - The third component of the MBW complex, partici- pating in flavonoid/anthocyanin biosynthesis, is the WDR protein. - Fruits and leaves utilised for tran- scriptome analysis were collected by members of the BacHBerry Consortium [26] in Chile, China, Portugal, Russia and the UK (Additional file 1: Table S1). - whereas the distribution of the other species is mostly restricted to their native habitats, for example, A. - uliginosum grows in cool temperate re- gions of the Northern Hemisphere and C. - The majority of the berry fruit species that were used for RNA sequencing and analysis (Table 1) lacked an available reference genome sequence, therefore, de novo assembly of the Illumina reads was carried out for each species using Trinity software. - and its BLAST portal [68] were developed to allow mining of the transcriptomic data of the 13 wild and cultivated berry fruit species.. - We analysed the phylogenetic relationship of the twelve berry fruit transcriptomes and one leaf transcriptome together with the genome sequences of seven reference species. - The single-copy gene ortholo- gues of the 20 species underwent homology searches to produce a super alignment matrix for the assembly of a phylogenetic tree (Fig. - The branching order displayed in the tree reflected the expected phylogenetic group classification for the clades, orders and families of the Angiosperms with members of the Rosids clade (A. - vinifera) and the clade of the Asterids (C. - trichopoda represents a basal group of the. - Published sequences from a total of 68 regulatory pro- teins (45 MYB TFs, 18 bHLH TFs and five WDRs) and 120 modifying and decorating enzymes (18 acyltrans- ferases, 31 glucosyltransferases, 29 methyltransferases, 26 hydroxylases, nine reductases, two aurone synthases, two dehydrogenases, two dehydratases and one dirigent protein) from a range of plant species were also used in BLAST searches against the transcriptome sequences of the 13 species. - In total, 1248 sequences homologous to regulatory genes and 5150 sequences homologous to enzymes of the general phenylpropanoid pathway and its decoration and modification were identified from the different. - Approximately a third of the hydroxylases and glucosyltransferases were detected with high levels of aa similarity.. - In addition to the gene mining of the phenylpropanoid pathway, protein-coding sequences were predicted and functionally annotated in the transcriptomes of all the 13 species. - Regulatory genes of the anthocyanin biosynthetic pathway isolated from R. - Prestige (abbreviated to Ri), several candidate regulatory genes of the anthocyanin biosynthetic pathway were identified in both species, cloned and characterised. - domestica MdMYB10 as a representative member of the R2R3-type MYB gene subgroup 6 (SG6) family, re- sponsible for the regulation of anthocyanin and PA bio- synthesis [54, 71] which led to the isolation of RgMyb10 and RiMyb10. - thaliana AtMYB12 as a member of the R2R3-type MYB TFs of SG7 that control the activa- tion of flavonol and flavone synthesis [54] which resulted in the isolation of RgMyb12 and RiMyb12. - The MYB domain consisting of the imperfect repeats R2 and R3 with regularly spaced tryptophan residues (R2 [−W-(x 19 )-W-(x 19 )-W. - [54]) was highly conserved in the N-terminus of the four Rubus MYB TFs. - Several regulators of the anthocyanin and PA pathways have been shown to contain an add- itional aa signature motif for bHLH interaction ([D/. - R]x 2 [F/Y] which lies downstream of the MYB domain as well as the small conserved ‘box A’ motif ([A/S/G]NDV) in the R3 repeat of the DNA binding domain [72]. - Table 4 Cloning and functional analysis of regulatory genes of the phenylpropanoid pathway in R. - Phylo- genetic analysis of the Rubus and several other R2R3- type MYB TFs showed clear separation of the flavonoid MYB regulators into two distinct clades (equivalent to SG6 and SG7 in A. - Of the seven bHLH homologues cloned (Table 4), three encoded isoforms of RgAn1 (termed RgAn1-1, RgAn1-2, RgAn1-3 with 99% aa identity among the iso- forms), RiAn1, RgDel and two isoforms of RiDel (named RiDel-1 and RiDel-2 that shared 99% identity at the aa level) had the general structure of flavonoid bHLH TFs (being about 600 aa in length, reviewed by [53]) (Add- itional file 10: Figure S3). - The characteristic H-E-R aa motif (−H-(x 3 )-E-(x 3 )-R- [63]) within the basic part of the bHLH domain is preserved in all cloned bHLH TFs of the two Rubus species. - Phylogenetic analysis showed clustering of the different Rubus bHLH TFs together with other plant bHLH homologues in two conserved clades of bHLH regulatory proteins (SGIIIf-1: PhAN1/AtTT8 clade and SGIIIf-2: AmDEL/PhJAF13 clade. - Functional characterisation of regulatory genes of the anthocyanin biosynthetic pathway isolated from R.. - However, when combined with most of the cloned Ru- bus bHLH TFs, inoculation of the Rubus Myb10 genes induced a strong red-purple colouration in infiltrated leaf patches to a level easily detectable by the naked eye (Fig. - Those SG6 TFs that require an added bHLH for anthocyanin induction likely require specific interact- ing bHLH partners for pigment formation, either in a hierarchical regulatory cascade or directly in the MBW complex that activates the expression of the genes en- coding the enzymes of anthocyanin biosynthesis. - 20:1, v/v/v) from leaves of the N. - benthami- ana leaves were carried out with the putative compo- nents of the R. - Anthocyanin accumulation increased approximately 4.3-fold between 4 and 7 dpi in the pres- ence of a WDR component compared to an approxi- mately 1.8-fold increase without a WDR co-factor and therefore, the addition of the WDR co-factor RiTTG1 almost doubled the anthocyanin content in N.. - At 1 dpi, anthocyanin formation was not visible by the naked eye nor detectable in methanol extracts of infiltrated leaves (eight leaf discs per treatment) and equalled to the one of the mock infil- trated leaves of avgA 530nm of 0.06. - At 2 dpi, the effect of the addition of a WDR protein was already evident, as anthocyanin formation could be observed by the naked eye in RiMyb10, RiAn1 and RiTTG1-co-infil- trated leaves and pigment formation was estimated in methanol extracts of leaf discs as avgA 530nm of 0.20. - Use of the NT accession for infiltrations confirmed all our observations using the JIC-LAB strain of N. - benthamiana leaf and stem explants with RiMyb10 or RgMyb10 under the con- trol of the constitutive CaMV 35S promoter led to anthocyanin induction with and without bHLH and/or WDR co-factors from the same species. - Our data indicated that transient assays do not always reflect the metabolic changes observed in stable transformations, the latter being more sensitive indicators (at specific stages during regeneration) of the ability of regulatory genes to ectopi- cally induce anthocyanin production. - BacHBerryEXP also con- tains a BLAST tool [95] to identify candidate transcript homologues for differential expression analysis of the two Rubus species.. - For antho- cyanin regulators such as the Rubus R2R3-type MYB, bHLH and WDR homologues cloned in this study, the expression levels of the cloned transcripts were assessed (Fig. - 3b) showed strong induction of transcript levels during ripening in line with the function of UFGT in stabilizing anthocyanidins by glucosylating them on the hydroxyl group of carbon 3, prior to transport to the vacuoles of the cells in coloured ripe fruits. - Expression of genes encoding regulators of flavonoid biosynthesis during fruit development in Rubus species Expression of the regulatory genes such as RiMyb10 and RgMyb10 was strongly upregulated during fruit ripening, especially from unripe to intermediate ripe fruits, whereas RiMyb12, RgMyb12, RiAn1 and RiDel were not differentially expressed at different stages of fruit. - This suggests that the highly abundant PhAN1-type bHLH TFs (RgAN1/RiAN1) are the dominant players and partners of the MYB10 TFs regulating anthocyanin production and. - 3 Transcriptome profiling of candidate genes encoding enzymes of the flavonoid core pathway, anthocyanin transporters, P-ATPases and flavonoid regulatory proteins during fruit maturation in R. - b Candidate genes encoding enzymes of the phenylpropanoid core pathway and modifying proteins: Top panel - R. - 3d), the closest homologue of the activators of flavonol biosynthesis in G. - This was confirmed by the identification of the PAs catechin and epicatechin in intermediate to ripe fruits (approximately 1/10 of the anthocyanin content) [51]. - functional genomic studies in berry fruit species as well as contributing to the understanding of the synthesis of polyphenols, the molecular mechanisms underlying phe- nylpropanoid, and particularly flavonoid, synthesis and the regulatory processes controlling phenylpropanoid metabolism during fruit ripening. - The usefulness of these transcriptomic resources has been demonstrated by the cloning and characterisation of regulators of the anthocyanin pathway from these berry fruit species, namely R2R3-type MYBs, bHLH and WDR homologues, which regulate anthocyanin and PA biosynthesis in two Rubus species. - However, the DEL homologue from wild blackberry was unable to induce pigment formation with Ri/RgMYB10 perhaps as a result of the few aa differ- ences found between RgDEL and RiDEL.. - There is a growing interest in the exploitation of wild fruits and berries as part of the rising demand for novel health promoting foods. - Plant tissues from 13 berry fruit species were collected by partners of the BacHBerry Consortium [26] in Chile, China, United Kingdom, Portugal and Russia (Table 1 and Additional file 1: Table S1). - molinae was isolated from 200 mg of frozen fruit tissue based on a protocol for plant tissues rich in polyphenols and polysaccharides [100] and included an additional DNase I treatment (RQ1 RNase-Free DNase, Promega) before phenol: chloroform extraction within step III of the protocol. - The library preparation involved several quality control analysis steps, including the use of the Quant-iT™ RNA Assay Kit (Life Technologies) for RNA quantification, the Quant- iT™ dsDNA Assay Kit (Life Technologies) for double- stranded DNA quantification as well as the LabChip GX Automated Electrophoresis System (PerkinElmer) and High Sensitivity DNA kit (Agilent) for RNA/dsDNA quantification and verification of the cDNA library insert size. - Illumina data from total RNA samples of the 13 berry fruit species were assembled by the Earlham Institute (Norwich, UK) into individual de novo transcriptomes using Trinity [101] and these assemblies were then used as a reference for mapping, quantification of expression and functional annotation. - Qual- ity control of the raw data was performed using FastQC [103] and the contamination screening and filtering tool Kontaminant [104]. - The BacHBerryGEN database [67] was created to de- posit the transcriptomic data of the 13 berry fruit spe- cies. - The phylogenetic analysis of the transcriptomes of the 13 berry fruit species together with the genome sequences of seven reference species (A. - A multiple alignment for each of the 214 single-copy gene families was produced using MUSCLE v . - using the TBLASTN programme of the BacHBerryGEN BLAST server [68] and a protein sequence query (Additional file 7: Table S6).. - To facilitate the cloning of the various TF RT-PCR products, the CaMV 35S promoter (pro) and soybean poly(A) (SPA) terminator (ter) sequences [125] were cloned into three basic vectors (i) pGreenII0029 (containing a NOS-pro::nptII::NOS-ter plant selectable marker gene) [126], (ii) pGreenII00179 (harbouring a CaMV35S-pro::hpt::CaMV35S-ter plant selectable marker gene) [126] and (iii) pGreenII00229 (possessing a NOS- pro::bar::NOS-ter plant selectable marker gene) [126]. - The RT-PCR fragments of the Rubus regulatory genes were inserted between the CaMV 35S-pro and SPA-ter se- quences as blunt-end fragments or BamHI/XbaI-PstI/. - The presence of the different regulatory genes in the Agrobacterium strains was confirmed by PCR amplification of the full-length genes from Agrobacterium plasmid preps and sequencing of the PCR products. - predicted to be of the same origin as the LAB isolate of [80. - A so-called empty vector strain without a gene of interest (pGreenII00179 + pBOOST-S in AGL1) was used as a negative control or as a component of the co-infiltration mixes to complement for co-factors (e.g., WDR). - Schematic representation of the phylogenetic relationship among the 13 berry fruit species studied.. - BLAST search output summaries for transcript candidates involved in the phenylpropanoid pathway for 13 berry fruit species: Enzymes of the core pathway and its decorating, modifying and regulatory proteins.. - Identification and cloning of regulatory genes of the phenylpropanoid pathway from R. - Primers used for the cloning of regulatory genes of the phenylpropanoid pathway from R. - Homologues of the regulatory genes cloned in this study in the collection of the 13 berry fruit species.. - Prestige during three fruit ripening stages: Examples of candidate enzymes of the general phenylpropanoid pathway, flavonoid regulatory enzymes, transporters, decorating and modifying enzymes.. - Additional file 16: Step-by-step guide of the phylogenetic analysis of the transcriptomes of the 13 berry fruit species together with the reference genomes of A. - We would like to thank the coordinators of the BacHBerry project (BACterial Hosts for production of Bioactive phenolics from bERRY fruits, http://www.. - RR-G performed the phylogenetic analysis of the transcriptome sequences. - The funding bodies had no role in the design of the study, collection, analysis and interpretation of data nor in writing the manuscript.. - Metabolomics datasets of the 13 berry fruit species studied in this. - A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. - De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidants. - De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.. - The plasticity of the grapevine berry transcriptome. - Anthocyanin composition and content of the Vaccinium uliginosum berry. - Identification and microbial production of the raspberry phenol salidroside that is active against Huntington's disease. - Current understanding of the pathways of flavonoid biosynthesis in model and crop plants. - Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. - Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. - An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. - Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. - Genome sequence of the palaeopolyploid soybean. - Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data
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