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. 2021 Sep;19(9):1824-1838.
doi: 10.1111/pbi.13596. Epub 2021 May 7.

VvHDZ28 positively regulate salicylic acid biosynthesis during seed abortion in Thompson Seedless

Zhiqian Li  1   2   3 , Yuntong Jiao  1   2   3 , Chen Zhang  1   2   3 , Mengru Dou  1   2   3 , Kai Weng  2   4 , Yuejin Wang  1   2   3 , Yan Xu  1   2   3
Affiliations

VvHDZ28 positively regulate salicylic acid biosynthesis during seed abortion in Thompson Seedless

Zhiqian Li et al. Plant Biotechnol J. 2021 Sep.

Abstract

Seedlessness in grapes is one of the features most appreciated by consumers. However, the mechanisms underlying seedlessness in grapes remain obscure. Here, we observe small globular embryos and globular embryos in Pinot Noir and Thompson Seedless from 20 to 30 days after flowering (DAF). From 40 to 50 DAF, we observe torpedo embryos and cotyledon embryos in Pinot Noir but aborted embryos and endosperm in Thompson Seedless. Thus, RNA-Seq analyses of seeds at these stages from Thompson Seedless and Pinot Noir were performed. A total of 6442 differentially expressed genes were identified. Among these, genes involved in SA biosynthesis, VvEDS1 and VvSARD1, were more highly expressed in Thompson Seedless than in Pinot Noir. Moreover, the content of endogenous SA is at least five times higher in Thompson Seedless than in Pinot Noir. Increased trimethylation of H3K27 of VvEDS1 and VvSARD1 may be correlated with lower SA content in Pinot Noir. We also demonstrate that VvHDZ28 positively regulates the expression of VvEDS1. Moreover, over-expression of VvHDZ28 results in seedless fruit and increased SA contents in Solanum lycopersicum. Our results reveal the potential role of SA and feedback regulation of VvHDZ28 in seedless grapes.

Keywords: VvHDZ28; VvEDS1 and VvSARD1; embryo abortion; salicylic acid; seedless grapes; trimethylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Seeds and morphological analyses of samples taken 20, 30, 40 and 50 days after flowering (DAF) from Pinot Noir and Thompson Seedless. (A) Seeds and berries of 20, 30, 40 and 50 DAF from Pinot Noir and Thompson Seedless. (B) Longitudinal diameters, transverse diameters and fresh weights of seeds, 30 berries were used for data analysis. (C) Morphological analyses of seeds. a‐d, small globular embryo, globular embryo, torpedo embryo and cotyledon embryo for seeds at 20, 30, 40 and 50 DAF of Pinot Noir, e‐g, small globular embryos, globular embryos, embryo has disappeared and endosperm begins to disintegrate, endosperm disintegrated and seed coat left for seeds at 20, 30, 40 and 50 DAF from Thompson Seedless. Embryo location is denote by red arrow. Bar: a, b = 5 μm, c, d = 100 μm,g, h = 1 mm.
Figure 2
Figure 2
DEG distribution in Pinot Noir and Thompson Seedless. (A) Number of differential genes expressed in Pinot Noir and Thompson Seedless. Red, up‐regulated genes; blue, down‐regulated genes. Upper number, differentially expressed genes; lower number in brackets represent differentially expressed transcription factors. (B) Venn diagram of differentially expressed genes in Pinot Noir and Thompson Seedless. (C) Distribution of different expressed transcription factor among Pinot Noir and Thompson Seedless, the number of genes in each family is shown. (D) Hierarchical clustering of expression profiles of the differentially expressed genes and transcription factors. Representative functions and genes are exhibited in the middle frame. Colors represent expression values of the corresponding alternatively spliced transcripts.
Figure 3
Figure 3
Hormonal content of seed development in PN and TS and hormonal co‐expressed gene analysis. (A) SA, salicylic acid, GA, gibberellin, ABA: abscisic acid, BR: Brassinosteroids, IAA: auxin, CTK: cytokinin, JA: jasmonic acid, ACC: aminocyclopropane −1‐carboxylic acid, precursor of ethylene. Capital letter represents comparison of PN and TS at each stage (P < 0.05); lowercase letters represent comparison among different stages of PN and TS (P < 0.05). (B) Hormonal correlation analysis. (C) Genes co‐expressed with endogenous hormone in PN and TS. (D) Heat map of 463 genes co‐expressed with at least two hormones.
Figure 4
Figure 4
Expressions of downstream genes of SA and VvHDZ28 treatment by SA. Expression of genes (qRT‐PCR) related to SA biosynthesis, SARD1 and EDS1 (a), and gene response to SA (b). (c, d) pHDZ28 injected into abaxial surfaces of tobacco leaves, treated with 1 mm salicylic acid, GUS staining and activity was determined three days later. ** indicates statistical significance of difference from controls by a one‐sided paired t‐test with a confidence level of P < 0.01.
Figure 5
Figure 5
Increased H3 acetylation correlates with altered expression of EDS1 and SARD1 in Pinot Noir. (a, c) Regions of EDS1 and SARD1 used for ChIP ‐qRT‐PCR assays. (b, d) ChIP‐qRT‐PCR analyses of promoter fragments (region 1) and exon fragments (region 2) of SARD1 and EDS1 in Pinot Noir and Thompson Seedless using anti‐H3K27me3 and anti‐H3AC antibodies. ChIP values are normalized to their respective DNA inputs. PN and TS are indicated by black and grey columns, respectively. The results are representative of three biological repetitions. White bars represent input controls for each sample. Error bars indicate SEs from three biological. ** indicates significant difference assessed by a one‐sided paired t‐test with confidence level P < 0.01, and asterisks are comparison between region 1 and region 2.
Figure 6
Figure 6
Effect of OE‐HDZ28 on flower development and endogenous hormone salicylic acid (SA). (a, f) flower, (b, c and g, h) anatomical observation of stamen and ovary, (e, j) transverse sections of fruit of WT and OE‐HDZ28. Bar = 100 μm. Wild‐type pistils and OE‐HDZ28 were pollinated with wild‐type pollen and subsequently stained with aniline blue 24 h after pollination. Ovary after pollination 24 h of WT (k) and OE‐HDZ28 (n), pollen germinated after pollination 24 h on stigma of WT (l) and OE‐HDZ28 (o), pollen tube in ovary of WT (m) and OE‐HDZ28 (p), Bars = 100 μm. (k) and (n) were created using multiple microscopic images. Q˜T, fruits develop after pollination with WT pollen, WT fruit with seeds (q), OE‐HDZ28 fruits without seeds (s), with seeds (r) and shrivelled flower (t). (s) Number of seeds in WT and OE‐HDZ28. Data represent means and SEs from three fruits. (u) Endogenous SA content in OE‐HDZ28, (v, w) Expression of SARD and EDS in OE‐HDZ28. Flower buds about 5 mm long; green flower ˜ green flower before anthesis about 8 mm long, flowers at anthesis. Data represent means and SEs from three biological replicates. * and ** indicate statistical significance of difference assessed by a one‐sided paired t‐test with confidence levels of P < 0.05 or P < 0.01, respectively.
Figure 7
Figure 7
A proposed model of genetic and molecular interactions in the regulatory network during embryogenesis and embryo abortion in grape. In TS, VvHDZ28 up‐regulate expression of VvEDS1 by binding to its promoter while up‐regulate VvSARD1 in an indirect way; genes involved in SA biosynthesis were expressed at higher levels in TS. Consequently, higher levels of SA accumulated in TS, which we propose plays an important role in the PCD in seed abortion. In PN, SA has lower content possibly due to altered histone modifications (such as increased H3K27me3) in VvSARD1 and VvEDS1.
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