Ectopic Expression of MADS-Box Transcription Factor VvAGL12 from Grape Promotes Early Flowering, Plant Growth, and Production by Regulating Cell-Wall Architecture in Arabidopsis

Abstract

The MADS-box family, a substantial group of plant transcription factors, crucially regulates plant growth and development. Although the functions of AGL12-like subgroups have been elucidated in Arabidopsis, rice, and walnut, their roles in grapes remain unexplored. In this study, we isolated VvAGL12, a member of the grape MADS-box group, and investigated its impact on plant growth and biomass production. VvAGL12 was found to localize in the nucleus and exhibit expression in both vegetative and reproductive organs. We introduced VvAGL12 into Arabidopsis thaliana ecotype Columbia-0 and an agl12 mutant. The resulting phenotypes in the agl12 mutant, complementary line, and overexpressed line underscored VvAGL12's ability to promote early flowering, augment plant growth, and enhance production. This was evident from the improved fresh weight, root length, plant height, and seed production, as well as the reduced flowering time. Subsequent transcriptome analysis revealed significant alterations in the expression of genes associated with cell-wall modification and flowering in the transgenic plants. In summary, the findings highlight VvAGL12's pivotal role in the regulation of flowering timing, overall plant growth, and development. This study offers valuable insights, serving as a reference for understanding the influence of the VvAGL12 gene in other plant species and addressing yield-related challenges.

Keywords: VvAGL12; cell wall; grape; plant growth.

Figure 1

Multiple sequence alignment and evolutionary tree analysis of VvAGL12 and other plant AGL12 proteins. (A) Sequence alignment of VvAGL12 and homologous AGL12 proteins from various plants. The protein accession numbers were as follows: VvAGL12 (GenBank: XP_002278239.1), OsMADS26 (GenBank: NP_001390263.1), AtAGL12 (GenBank: NP_565022), SlAGL12 (GenBank: NP_001233764.2), NtAGL12 (GenBank: NP_001312055.11), AcAGL12 (GenBank: PSS00173.1), PtAGL12 (GenBank: XP_006376144.2), and CsAGL12 (GenBank: XP_028060316.1). Conserved domains were highlighted in red and green lines. (B) Phylogenetic tree of VvAGL12 with homologous AGL12 proteins from other plants.

Figure 2

Subcellular localization and tissue-specific expression analysis of VvAGL12 protein. (A) Subcellular localization of VvAGL12 protein. (B) Tissue-specific expression analysis of VvAGL12 in various tissues of Pinot Noir plants. YL: young leaves; OL: old leaves; S: stem; T: tendrils; I: inflorescence; F: small fruit; R: root.

Figure 3

VvAGL12 promoted elongation of the main roots and the growth of Arabidopsis seedlings. (A) Phenotypes of wild-type, mutant, and transgenic Arabidopsis seedlings grown vertically on MS plates for 9 d. Scale bar = 1 cm. (B) Fresh weight and (C) primary root length calculated for four lines: Col-0 (wild-type Arabidopsis), agl12 (mutant of AGL12), Vv12:cs (VvAGL12 complementary Arabidopsis mutant agl12), and Vv12:col (overexpressed VvAGL12 in wild-type). Asterisks indicate significant differences between the wild-type (Col-0) and other three lines (* p < 0.05; ** p < 0.01).

Figure 4

Overexpression of VvAGL12 promoted flowering in Arabidopsis thaliana. (A) Representative images of 20-day-old Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines. Compared to wild-type Col-0, Vv12:col had a larger shoot size, whereas agl12 had a smaller shoot size. Scale bar = 1.5 cm. (B) Overexpression of VvAGL12 led to early bolting in Arabidopsis. Representative images of 25-day-old Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines were captured. Vv12:col and Vv12:cs exhibited early bolting, whereas Col-0 and agl12 did not. Scale bar = 1.5 cm. (C) Overexpression of VvAGL12 showed early flowering in Arabidopsis. The 30-day-old Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines were photographed. Scale bar = 5 cm. (D) Days to bolting of Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines. (E) Days to flowering in Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines. (F) Number of rosette leaves in Col-0 (wild-type), agl12 (mutant), and VvAGL12 transgenic Arabidopsis lines. Col-0 was a wild-type Arabidopsis, agl12 was a mutant of AGL12, Vv12:cs was the VvAGL12 complementary Arabidopsis mutant agl12, and Vv12:col was overexpressed VvAGL12 in the wild-type. Asterisks indicate significant differences between the wild-type (Col-0) and other three lines (* p < 0.05; ** p < 0.01).

Figure 5

Overexpression of VvAGL12 increased plant height and fresh weight in transgenic Arabidopsis during the pod stage. Plants were observed for 28 days after culturing in the soil. The phenotypes of the four lines were photographed (A,B). (A) Scale bar = 9 cm. (B) Scale bar = 6 cm. A total of 10 Arabidopsis plants of each variety were selected for analysis of (C) plant height and (D) fresh weight. Col-0 was a wild-type Arabidopsis, agl12 was a mutant of AGL12, Vv12:cs was the VvAGL12 complementary Arabidopsis mutant agl12, and Vv12:col was overexpressed VvAGL12 in the wild-type. Asterisks indicate significant differences between the wild-type (Col-0) and the other three lines (* p < 0.05; ** p < 0.01).

Figure 6

Overexpression of VvAGL12 enhanced Arabidopsis seed yield. (A) Silique phenotypes of the four lines (Col-0, agl12, Vv12:cs, and Vv12:col). Overexpression of VvAGL12 enhanced the (B) seed size, (C) silique number, (D) silique length, (E) seed number per silique, and (F) seed weight in Arabidopsis. (A) Scale bar = 4 mm. (B) Scale bar = 1 mm. Col-0 was a wild-type Arabidopsis, agl12 was a mutant of AGL12, Vv12:cs was the VvAGL12 complementary Arabidopsis mutant agl12, and Vv12:col was overexpressed VvAGL12 in the wild-type. Asterisks indicate significant differences between the wild-type and the other three lines (* p < 0.05; ** p < 0.01).

Figure 7

Transcriptome analysis of Arabidopsis wild-type, mutant, and overexpressed plants (Vv12:col). For each line, 10-day-old seedlings were collected for sequencing. Three comparisons (mutant vs WT, VvAGL12 overexpressing plants (vv12) vs WT, and VvAGL12 overexpressing plants (vv12) vs mutant) were conducted to filter the different genes. (A) Number of differentially expressed genes in the three comparisons. The red box indicates the up-regulated genes, and the blue box indicates the down-regulated genes. The numbers were listed in the respective boxes. (B) Venn diagram of vv12 vs WT and vv12 vs mutant comparisons. A total of 81 common genes were identified in both the comparisons. (C) GO enrichment of the 81 common genes triggered by VvAGL12. The number of genes is listed on the right-hand side of the panel. (D) Fold changes in cell wall-related genes in vv12 vs WT and vv12 vs mutant.