Expression of <i>VvβVPE</i> gene in different development stages of ovule in seed and seedless grapes.

Relative expression level of VvβVPE gene in different development stages (15, 20, 25, 30, 35, 40, and 45 DAF) of ovule in ‘V. vinifera cv. Thompson Seedless ‘, ‘V. vinifera cv. Youngle’, ‘V. vinifera cv. Pinot Noir’ and ‘V. vinifera cv.Flame Seedless’. DAF (days after full-bloom) (error bars indicate ±SD).</p

Gene Cloning, Expression and Enzyme Activity of <i>Vitis vinifera</i> Vacuolar Processing Enzymes (<i>VvVPEs</i>)

<div><p>Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the <i>VPE</i> gene family, we identified 3 <i>Vitis vinifera VPE</i> genes (<i>VvβVPE</i>, <i>VvγVPE</i>, and <i>VvδVPE</i>) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘<i>Vitis vinifera</i> cv. Pinot Noir’ and ‘<i>Vitis vinifera</i> cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 <i>Arabidopsis thaliana</i> and 6 <i>Vitis vinifera</i> VPEs revealed that the 10 <i>VPE</i>s form 3 major branches. Furthermore, the 6 grapevine <i>VPE</i>s share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine <i>VPEs</i> are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the <i>Pichia Pastoris</i> expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that <i>VvδVPE</i> is only expressed in flowers, buds and ovules, that <i>VvγVPE</i> is expressed in various tissues, and that <i>VvβVPE</i> was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that <i>VvβVPE</i> in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that <i>VvβVPE</i> is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.</p></div

Gene analysis of <i>VPEs</i> in <i>Vitis vinifera</i>.

<p>Gene analysis of <i>VPEs</i> in <i>Vitis vinifera</i>.</p

Enzymatic activity of grapevine VPEs.

<p>CK-1 (ddH₂O); CK-2 (ddH₂O and fluorescent VPE-specific substrate); GS115 (ddH₂O, fluorescent VPE-specific substrate and GS115 thallus); pPICZαA (ddH₂O, fluorescent VPE-specific substrate and GS115 thallus tansformed with pPICZαA plasmid); βVPE, γVPE and δVPE (ddH₂O, fluorescent VPE-specific substrate and GS115 thallus transformed with recombinant plasmid). Enzymatic activity of recombinant grapevine βVPE, γVPE and δVPE were testified by using Ac-ESEN-MCA substrates at pH 5.5. Y-axes are scales of relative fluorescence values (error bars indicate ±SD).</p

Multi-sequence alignment of <i>Vitis vinifera</i> and <i>Arabidopsis thaliana</i> VPE proteins.

<p>Amino acid sequences from 6 grapevine and 4 <i>Arabidopsis thaliana VPE</i> genes share similar catalytic dyad His (177), Cys (219) and the substrate binding pocket consisting of Arg (112), Arg (389), and Ser (395), with the exception of Ser (395) in VvγVPE, which was replaced with Ala (395).</p

Expression of <i>VvδVPE</i> gene in different development stages of ovule in seed and seedless grapes.

<p>Relative expression level of <i>VvδVPE</i> gene in different development stages (15, 20, 25, 30, 35, 40, and 45 DAF) of ovule in ‘<i>V</i>. <i>vinifera</i> cv. Thompson Seedless ‘, ‘<i>V</i>. <i>vinifera</i> cv. Youngle’, ‘<i>V</i>. <i>vinifera</i> cv. Pinot Noir’ and ‘<i>V</i>. <i>vinifera</i> cv.Flame Seedless’. DAF (days after full-bloom) (error bars indicate ±SD).</p

Expression of <i>VPE</i> genes in different grapevine tissues.

<p>Expression of <i>VPE</i> genes analyzed by sqRT-PCR in root, stem, leaf, tendril, alabastrum, flowers, pericarp, pulp, and ovule tissues of <i>Vitis vinifera</i> cv. Pinot Noir.</p

Intron and exon organization of <i>Vitis vinifera VPE</i> genes.

<p>Intron and exon organization of <i>Vitis vinifera VPE</i> genes.</p

Phylogenetic analysis of grape and <i>Arabidopsis thaliana</i> VPE family proteins.

<p>Phylogenetic analysis of grape and <i>Arabidopsis thaliana</i> VPE family proteins.</p

Additional file 3 of “The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola”

Additional file 3: Figure S1: Predict the conservative motif of PLCPs by online MEME website