Abstract
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 VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera 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 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE 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 VvβVPE 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.
Highlights
Seedless grapes are classified into three types based on their pollination and fruit setting characteristics: spontaneous parthenocarpy, pseudoparthenocarpy and irritating parthenocarpy
The kDa ppVPE is transformed into a kDa precursor Vacuolar processing enzymes (VPEs) in the endoplasmic reticulum through signal peptide removal and glycosylation, transported into the dissolved vacuole, where it is transformed into a 43 kDa intermediate VPE via autocatalysis, and into the 40 kDa mature VPE [6]
The grapevine genome contains 3 VPE family members, which we designated VvβVPE, VvγVPE and VvδVPE according to phylogenetic comparison with Arabidopsis thaliana VPEs (Fig 1) The full-length complementary DNAs (cDNAs) of 6 identified VPEs were isolated from ‘V. vinifera cv
Summary
Seedless grapes are classified into three types based on their pollination and fruit setting characteristics: spontaneous parthenocarpy, pseudoparthenocarpy and irritating parthenocarpy. Vacuolar processing enzymes (VPEs), which show caspase-1-like activity and regulate the programmed cell death (PCD), are cysteine peptidases in charge of the maturation of multifarious vacuolar proteins in higher plants. VPEs process proteins in the vacuole and show caspase-1-like activity to regulate PCD [7,8]. Many researchers found that γVPE deficiency enhanced the susceptibility of plants after pathogen invasion and demonstrated that γVPE shows caspaselike activity [21,22,23] These results illustrated that γVPE participate in PCD regulation of plants. The expression of δVPE is highly specific and is observed only transiently during early development It is considered an essential factor in the apoptosis of these two cell layers, which is significant during the forming process of seed coat [27]
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