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. 2022 Aug 7;23(15):8767.
doi: 10.3390/ijms23158767.

miR3633a- GA3ox2 Module Conducts Grape Seed-Embryo Abortion in Response to Gibberellin

Yunhe Bai  1 , Xiaowen Zhang  1 , Xuxian Xuan  1 , Ehsan Sadeghnezhad  1   2 , Fei Liu  1 , Tianyu Dong  1 , Dan Pei  1 , Jinggui Fang  1 , Chen Wang  1
Affiliations

miR3633a- GA3ox2 Module Conducts Grape Seed-Embryo Abortion in Response to Gibberellin

Yunhe Bai et al. Int J Mol Sci. .

Abstract

Seedlessness is one of the important quality and economic traits favored by grapevine consumers, which are mainly affected by phytohormones, especially gibberellin (GA). GA is widely utilized in seedless berry production and could effectively induce grape seed embryo abortion. However, the molecular mechanism underlying this process, like the role of RNA silencing in the biosynthesis pathway of GA remains elusive. Here, Gibberellin 3-β dioxygenase2 (GA3ox2) as the last key enzyme in GA biosynthesis was predicated as a potential target gene for miR3633a, and two of them were identified as a GA response in grape berries. We also analyzed the promoter regions of genes encoding GA biosynthesis and found the hormone-responsive elements to regulate grape growth and development. The cleavage interaction between VvmiR3633a and VvGA3ox2 was validated by RLM-RACE and the transient co-transformation technique in tobacco in vivo. Interestingly, during GA-induced grape seed embryo abortion, exogenous GA promoted the expression of VvmiR3633a, thereby mainly repressing the level of VvGA3ox2 in seed embryos. We also observed a negative correlation between down-regulated VvGA20ox2/VvGA3ox2 and up-regulated VvGA2ox3/VvGA2ox1, of which GA inactivation was greater than GA synthesis, inhibited active GA content, accompanied by the reduction of VvSOD and VvCAT expression levels and enzymatic activities. These series of changes might be the main causes of grape seed embryo abortion. In conclusion, we have preliminarily drawn a schematic mode of GA-mediated VvmiR3633a and related genes regulatory network during grape seed abortion induced by exogenous GA. Our findings provide novel insights into the GA-responsive roles of the VvmiR3633a-VvGA3ox2 module in the modulation of grape seed-embryo abortion, which has implications for the molecular breeding of high-quality seedless grape berries.

Keywords: GA3ox2; embryo abortion; gibberellin; grapevine; miR3633a.

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Conflict of interest statement

The authors declare no conflict of interest in the content of this manuscript.

Figures

Figure 1
Figure 1
Morphological changes of grape berries and embryos after GA treatment. The appearance of grape and ovule changed after exogenous GA treatment. The appearance of the berry and ovule (A), the changes in the transverse and longitudinal diameter of berries (B), changes in transverse and longitudinal diameters of ovules (C), and changes in berry and ovule weight per grain (D) in response to GA exogenous. d in horizontal axes represents days after grape flowering.
Figure 2
Figure 2
Identification of microRNA3633a and its target genes. Chromosomal location of VvmiR3633a (A); The stem-loop structure of pre-VvmiR3633a and its mature sequence and star sequences (B); Cloning of VvMIR3633a gene (C); The interaction pattern and mismatch rate of VvmiR3633a and its VvGA3ox2 target genes (D).
Figure 3
Figure 3
Sequence analysis and subcellular localization of target genes. Cloning of VvGA3ox2 (A); VvGA3ox2 amino acid sequence deduction (B); Subcellular localization (C); Alignment of GA3ox2 amino acid sequence homology and conserved domains in different species (D,E); GA3ox2 evolutionary analysis (F); GA3ox2 gene structure analysis (G); Analysis of Conserved Motifs of GA3ox Protein Sequences in Different Species (H).
Figure 4
Figure 4
Classification of cis-acting elements in promoter regions (A) and promoter activity of VvMIR3633a and VvGA3ox2 in response to GA (B,C).
Figure 5
Figure 5
Validation of target genes cleavage by VvmiR3633a. The cleavage of VvGA3ox2 by VvmiR3633a was verified by the RLM-RACE technique (A) and tobacco transient expression technique (B,C); GUS activity (D).
Figure 6
Figure 6
Expression analysis and correlation of miR3633a-GA3ox2 module in response to GA in berries (A,C) and ovules (B,D).
Figure 7
Figure 7
Expression modes of gibberellin catabolic enzymes according to time course experiment during grape and seed development.
Figure 8
Figure 8
The activity of SOD and CAT enzymes (A) and the expression profile of members belonged to SOD and CAT family (B) during seed development in response to GA.
Figure 9
Figure 9
Regulation network of grape embryo abortion induced by exogenous GA.
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