Ectopic Expression of a Pak-choi YABBY Gene, BcYAB3, Causes Leaf Curvature and Flowering Stage Delay in Arabidopsis thaliana

Abstract

The YABBY family are a group of seed plant-specific transcription factors, which are involved in the specification of abaxial polarity in lateral organs. In Arabidopsis thaliana, YABBY3 (YAB3) plays a critical role in regulating abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, the BcYAB3 gene was isolated from Pak-choi (Brassica rapa subsp. chinensis). The tissue-specific expression analysis indicated that the BcYAB3 gene has significantly high transcript levels in stem, leaf, and flower. We investigated the subcellular localization of BcYAB3 and found the protein to be expressed in the nucleus. In the transgenic Arabidopsisthaliana plants expressing the BcYAB3 gene, the leaves were curling downward with the plant growth, and the bolting and flowering stages were delayed. These results not only validate the function of BcYAB3 in the leaf and flower development in Arabidopsis, but also contribute to unravel the molecular regulatory mechanism of YAB3 gene in the establishment of adaxial-abaxial polarity of the lateral organs in Pak-choi.

Keywords: Arabidopsis; BcYAB3; Pak-choi; adaxial–abaxial polarity; leaf development.

Figure 1

Phylogenetic tree analysis of BcYAB3. Phylogenetic relationships of YABBY genes involving with Arabidopsis thaliana, Oryza sativa, Zea mays, Solanum lycopersicum, Vitis vinifera, Brassica oleracea, and Pak-choi. The phylogenetic tree was constructed using the neighbor-joining method, and the bootstrap value was set at 1000.

Figure 2

Multiple sequence alignment of BcYAB3. The BcYAB3 gene was aligned with YABBY genes and have been reported to be in Antirrhinum majus, Triticum aestivum, Vitis pseudoreticulata, and Arabidopsis thaliana. The conserved C2C2 zinc finger domain and YABBY domain are presented using an orange box and green box, respectively.

Figure 3

Expression patterns of BcYAB3 in different tissues. The expression levels of BcYAB3 with qRT-PCR amplification in leaf, stem, root, petiole, hypocotyl, flower bud, flower, and pod. The data represent the means of three replicates, and error bars represent the standard deviations of means.

Figure 4

Subcellular localization of BcYAB3. (A) The construct of 35S:GFP and 35S:BcYAB3-GFP. GFP: green fluorescent protein; NOS: nopaline synthase gene. (B) Transient expression of 35S:BcYAB3-GFP fusion protein in tobacco. 35S:GFP was used as a control. Fluorescence images of mCherry (a nuclear marker), GFP, and merged were captured with confocal laser scanning microscopy and are displayed in red, green, and yellow, respectively. Scale bars = 50 um.

Figure 5

Type I phenotype of 35S:BcYAB3–GUS transgenic plants in Arabidopsis thaliana. (A) Fourteen days of BcYAB3 transgenic plants grown on Murashige and Skoog (MS) medium with hygromycin. Scale bar = 2 cm. (B) The leaves of 22-day-old seedlings. Scale bar = 5 cm. (C) Phenotypes of the whole plant and leaves during bolting stage. Scale bars indicate 5 cm and 1 cm, respectively. (D) Leaf length and width statistics of the rosette leaves of 25-day-old plants. Error bars represent standard deviation of the mean number of 25 plants for each line. ** means significant differences compared to wild type (P < 0.01). (E) Phenotype of BcYAB3 and wild type plants during flowering and fruiting stage. WT: wild-type Arabidopsis thaliana; OE3/9/12: transgenic lines of BcYAB3. Scale bar = 5 cm.

Figure 6

Type II phenotype of transgenic Arabidopsis with BcYAB3 gene. (A) Phenotype of 10-old-day seedlings. Scale bar = 2 cm. (B) Phenotype of 18-old-day seedlings and characteristic of abaxial side of leaves in wild type and transgenic plants. Scale bar = 1 cm. (C) Phenotype of transgenic plants and wild type during flowering and fruiting stages. The phenotype was observed in a front view and top view, respectively. Scale bars = 5 cm. Comparison of the adaxial and abaxial sides of the leaves of wild type and transgenic plants. Scale bar = 1 cm. WT: wild-type Arabidopsis thaliana; OE1/6/7/10: transgenic lines of BcYAB3.