The flavour of grape colour: anthocyanin content tunes aroma precursor composition by altering the berry microenvironment

Anthocyaninless (white) instead of black/red (coloured) fruits develop in grapevine cultivars without functional VviMYBA1 and VviMYBA2 genes, and this conditions the colour of wines that can be produced. To evaluate whether this genetic variation has additional consequences on fruit ripening and composition, we performed comparisons of microenvironment, transcriptomics, and metabolomics of developing grapes between near-isogenic white- and black-berried somatic variants of Garnacha and Tempranillo cultivars. Berry temperature was as much as 3.5 ºC lower in white- compared to black-berried Tempranillo. An RNA-seq study combined with targeted and untargeted metabolomics revealed that ripening fruits of white-berried variants were characterized by the up-regulation of photosynthesis-related and other light-responsive genes and by their higher accumulation of specific terpene aroma precursors, fatty acid-derived aldehyde volatiles, and phenylpropanoid precursor amino acids. MYBA1-MYBA2 function proved essential for flavonol trihydroxylation in black-berried somatic variants, which were also characterized by enhanced expression of pathogen defence genes in the berry skin and increased accumulation of C6-derived alcohol and ester volatiles and γ-aminobutyric acid. Collectively, our results indicate that anthocyanin depletion has side-effects on grape composition by altering the internal microenvironment of the berry and the partitioning of the phenylpropanoid pathway. Our findings show how fruit colour can condition other fruit features, such as flavour potential and stress homeostasis

Structural comparison of CpMan5B with <i>Fervidobacterium</i><i>nodosum</i> Rt17-B1 endoglucanase (FnCel5A).

<p>A, The FnCel5A enzyme (PDB ID: 3RJY) bound to three glucose molecules (yellow and red heteroatoms) and phosphate (orange and red heteroatoms) is superimposed onto CpMan5B (tan). B, Close-up view of the proposed catalytic triad. The figure is drawn in the same orientation as in A. Sugar subsite is indicated in black font.</p

Crystal structure of <i>C</i>.

<div><p><b><i>polysaccharolyticus</i> Man5B</b>. </p> <p>A, The structure is shown as a ribbon representation with a color spectrum from blue (N-terminus) to red (C-terminus). B, Orthogonal views of the molecular surfaces of CpMan5B colored according to the electrostatic potential. C, Close-up view of the Tris molecule bound to the active site of CpMan5B. The Tris molecule and the contacting residues are highlighted by a stick representation.</p></div

Specific activities of wild-type and mutant CpMan5B enzymes and the CbMan5D enzyme.

<p>Rates of product formation from either mannohexaose (Panels A, C) or cellohexaose (Panels B, D) were determined after 10 minutes and 8 hours, respectively, when the rates of product formation by the wild-type enzymes were linear with time. The amino acid changes in the x-axes labels indicate the site-specific mutants of the CpMan5B enzyme. Parenthetical values above the bars show the percentage of wild-type CpMan5B activity, and asterisks indicate that the raw data are significantly different (P<0.05, Student's paired t test) from those of the wild-type CpMan5B enzyme in the same experiment. Abbreviations: M1, mannose; M2, mannobiose; M3, mannotriose; G1, glucose; G2, cellobiose; G3, cellotriose; n.d., end product(s) was not detected under these assay conditions; IU, international units (μmol min^-1 mg^-1); mIU, milli-international units (nmol min^-1 mg^-1). Lower limits for detection were <2 IU mg^-1 for mannosaccharides and <2 mIU mg^-1 for cellosaccharides.</p

Structural comparison of CpMan5B with <i>Clostridium</i><i>thermocellum</i> cellulase (CtCel5C).

<p>A, The CtCel5C-cellobiose complex (green) is superimposed onto CpMan5B (tan). B, Close-up view of the active site. The figure is drawn in the same orientation as in A. The cellobiose molecule bound to CtCel5C is shown in a stick model colored yellow and red for carbon and oxygen atoms, respectively. Contacting residues of CtCel5C (green), and the corresponding residues of CpMan5B (tan) are shown in panel B. Sugar subsites are indicated in black font.</p

Structural comparison of CpMan5B with <i>Thermotoga</i><i>maritima</i> cellulase (TmCel5A).

<p>A, The TmCel5A-mannotriose complex (green, PDB ID: 3AZS) is superimposed onto CpMan5B (tan). B, Close-up view of the active site. The mannotriose molecule bound to TmCel5A is shown by a stick model colored yellow and red for carbon and oxygen atoms, respectively. Contacting residues of TmCel5A (green), and the corresponding residues of CpMan5B (tan) are shown in panel B. Sugar subsites are indicated in black font. C. The active site has been rotated 180° around the y-axis relative to the view in panel B.</p

Amino acid sequence alignment of CpMan5B, CbMan5D, and TmCel5A.

<p>The three polypeptides were aligned using ClustalW with the Blosum62 similarity matrix (<a href="http://www.genome.jp/tools/clustalw/" target="_blank">http://www.genome.jp/tools/clustalw/</a>). Boxes indicate similar residues and dark shading indicates identical residues. The GenBank accession numbers for CpMan5B, CbMan5D, and TmCel5A are ADK22147, ACM59384, and AAD36816, respectively.</p

Mutational and Structural Analyses of <i>Caldanaerobius polysaccharolyticus</i> Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

<div><p>CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from <i>Caldicellulosiruptor bescii</i> as a mannanase with minor endo-glucanase activity.</p> </div