Characterization of VvmiR166s-Target Modules and Their Interaction Pathways in Modulation of Gibberellic-Acid-Induced Grape Seedless Berries

Abstract

Exogenous GA is widely used to efficiently induce grape seedless berry development for significantly improving berry quality. Recently, we found that VvmiR166s are important regulators of response to GA in grapes, but its roles in GA-induced seedless grape berry development remain elusive. Here, the precise sequences of VvmiR166s and their targets VvREV, VvHB15 and VvHOX32 were determined in grape cv. 'Rosario Bianco', and the cleavage interactions of VvmiR166s-VvHB15/VvHOX32/VvREV modules and the variations in their cleavage roles were confirmed in grape berries. Exogenous GA treatment significantly induced a change in their expression correlations from positive to negative between VvmiR166s and their target genes at the seeds during the stone-hardening stages (32 DAF-46 DAF) in grape berries, indicating exogenous GA change action modes of VvmiR166s on their targets in this process, in which exogenous GA mainly enhanced the negative regulatory roles of VvmiR166s on VvHB15 among all three VvmiR166s-target pairs. The transient OE-VvmiR166a-h/OE-VvHB15 in tobacco confirmed that out of the VvmiR166 family, VvmiR166h/a/b might be the main factors in modulating lignin synthesis through inhibiting VvHB15, of which VvmiR166h-VvHB15-NtPAL4/NtCCR1/NtCCR2/NtCCoAMT5/NtCOMT1 and VvmiR166a/b-VvHB15-NtCAD1 are the potential key regulatory modules in lignin synthesis. Together with the GA-induced expression modes of VvmiR166s-VvHB15 and genes related to lignin synthesis in grape berries, we revealed that GA might repress lignin synthesis mainly by repressing VvCAD1/VvCCR2/VvPAL2/VvPAL3/Vv4CL/VvLac7 levels via mediating VvmiR166s-VvHB15 modules in GA-induced grape seedless berries. Our findings present a novel insight into the roles of VvmiR66s that are responsive to GA in repressing the lignin synthesis of grape seedless berries, with different lignin-synthesis-enzyme-dependent action pathways in diverse plants, which have important implications for the molecular breeding of high-quality seedless grape berries.

Keywords: HD-Zip III; VvmiR166s; gibberellin; lignin; seedless grape.

Figure 1

Morphological changes in berries and seeds in response to GA application. The changes in grape berries and seeds at 32 d, 46 d, 60 d and 86 d after GA treatment; the red arrow points to the berry brush (A). Single fruit weight changes after GA treatment (B). Changes in fruit shape index after GA treatment (C). Fruit seedless rate after 86 DAF of GA treatment (D).

Figure 2

Comparison of VvmiR166s and their precursor sequences with the alignment. Comparison of VvmiR166s sequence in miRbase 21.0 and cloned VvmiR166s sequence, where red regions are different sequences and the yellow regions are the same sequences (A). The genetic relationship of miR166s in different species (B). miR166s precursor sequence alignment diagram (C). Stem-loop structure of miR166s precursor sequence (D).

Figure 3

MiR166 and its target gene alignment and cutting validation. Comparison of VvmiR166s sequence in miRbase 2.1.0 and their target gene, and the mismatch bases of VvmiR166s with its target genes, where ‘X’ represents completely mismatch and ‘O’ represents the 0.5 mismatch.

Figure 4

Phylogenetic tree of HB15, HOX32 and REV across various plant species. VvHB15, VvHOX32 and VvREV CDS sequences and their translated proteins; yellow indicates the Homeodomain (HD), red indicates the Leucine-Zipper domain (Zip), blue indicates the Steroidogenic Acute Regulatory Protein-Related Lipid Transfer domain (START), green is the MEKHLA domain (A). Comparison of VvHB15, VvHOX32 and VvREV amino acid sequences in different species (1: HD; 2: Zip; 3: START; 4: MEKHLA domain) (B). Evolutionary tree analysis of VvHB15, VvHOX32 and VvREV in different species (C). Analysis of VvHB15, VvHOX32 and VvREV gene structure in different species (D).

Figure 5

Subcellular localization of VvHB15, VvHOX32 and VvREV in agroinfiltrated tobacco leaves. VvHB15, VvHOX32 and VvREV subcellular localization prediction (A). (B) Validation of subcellular localization of VvHB15, VvHOX32 and VvREV in tobacco leaves.

Figure 6

Motif analysis of promoters of VvMIR166s and their potential targets. Cis-acting element type and ratio column contained in the VvmiR166s promoter (A). Type and proportion of cis-acting elements contained in the promoter of VvmiR166s target gene (B). Type and proportion of hormone acting elements contained in VvmiR166s promoter (C). Type and proportion of hormone acting elements contained in the promoter of VvmiR166s target gene (D).

Figure 7

Expression profiles of VvMIR166s in the various tissues of diverse-stage grape berries. Expression patterns of VvMIR166s in seeds, flesh and peel (A). Expression patterns of VvREVOLUTA, VvHB15 and VvHOX32 in seeds, flesh and peel (B). 32DAF: 32nd day after flowering, 46DAF: 46th day after flowering, 60DAF: 60th day after flowering, 86DAF: 86nd day after flowering. Each reaction was repeated three times and the standard error is indicated with bars in the diagram. Each PCR assay was carried out using three biological replicates, and each replicate corresponded to three technological repeats of separate experiments.

Figure 8

Expression modes of VvMIR166s responsive to GA in various tissues of diverse-stage grape berries. The column chart shows the relative expression of the untreated tissue, and the red dot shows the relative expression of the GA treatment tissue. Each PCR assay was carried out using three biological replicates, and each replicate corresponded to three technological repeats of separate experiments. Asterisks indicate statistically significant differences at (* p < 0.05; ** p < 0.01) as determined by Student’s t-test.

Figure 9

Expression modes of VvHB15, VvREV and VvHox32 responsive to GA in various tissues of diverse-stage grape berries. The column chart shows the relative expression of the untreated tissue, and the red dot shows the relative expression of the GA treatment tissue. Each PCR assay was carried out using three biological replicates, and each replicate corresponded to three technological repeats of separate experiments. Asterisks indicate statistically significant differences at (* p < 0.05; ** p < 0.01) as determined by Student’s t-test.

Figure 10

Comparison of Pearson’s correlation of VvmiR166s and VvHB15 expression between controls and GA treatments. This chart mainly focused on the comparison of expression correlation of VvmiR166s and VvHB15 from control and GA treatments at the stone-hardening stage (from 32DAF to 46DAF).

Figure 11

Transient expression verification of VvmiR166 family and its target gene VvHB15 in tobacco leaves. VvmiR166a-h and VvHB15 were integrated into the plant expression vector pCAMBIA1302 through double digestion (A). The obtained vector was independently transformed into tobacco plants using an Agrobacterium-mediated method. The expression vector pCAMBIA1302 was used as the control. After 3 days of dark culture, DNA was extracted, and specific primers were used for PCR detection. After gel electrophoresis, sequencing was performed, which was identical to the sequence of interest (B). Agrobacterium-mediated injection of tobacco leaves (C). After overexpressing VvmiR166a-h and VvHB15, the expression of NtHB15 and NtHB15-like changes. Each PCR assay was carried out using three biological replicates, and each replicate corresponded to three technological repeats of separate experiments (D). After overexpressing VvmiR166a-h and VvHB15, the expression changes of genes involved in the tobacco lignin synthesis pathway were detected using real-time quantitative PCR (E). ‘a, b, c, d, e, f, g, h’ and ‘hb’ indicate the overexpression of VvmiR166a-h and VvHB15, respectively. ‘ck’ is the control.

Figure 12

Expression of genes related to lignin synthesis in grape seeds and its correlation with VvMIR166s and VvHB15 expression. Expression of genes related to lignin synthesis in grape seeds (A). Correlation of lignin-synthesis-related genes with VvMIR166s and VvHB15 expression (B).

Figure 13

GA-VvmiR166h/b/a-VvHB15-genes in lignin synthesis. (A,B) Pathways of lignin synthesis regulated by miR166-HB15 modules in tobacco and grape, respectively.