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. 2022 Oct 1;23(19):11661.
doi: 10.3390/ijms231911661.

Characterization of the ABC Transporter G Subfamily in Pomegranate and Function Analysis of PgrABCG14

Qing Yu  1   2 , Jiyu Li  2 , Gaihua Qin  2 , Chunyan Liu  2 , Zhen Cao  2 , Botao Jia  2 , Yiliu Xu  2 , Guixiang Li  1   2 , Yuan Yang  2 , Ying Su  1 , Huping Zhang  1
Affiliations

Characterization of the ABC Transporter G Subfamily in Pomegranate and Function Analysis of PgrABCG14

Qing Yu et al. Int J Mol Sci. .

Abstract

ATP-binding cassette subfamily G (ABCG) proteins play important roles in plant growth and development by transporting metabolites across cell membranes. To date, the genetic characteristics and potential functions of pomegranate ABCG proteins (PgrABCGs) have remained largely unknown. In this study, we found that 47 PgrABCGs were divided into five groups according to a phylogenetic analysis; groups I, II, III, and IV members are half-size proteins, and group V members are full-size proteins. PgrABCG14, PgrABCG21, and PgrABCG47 were highly expressed in the inner seed coat but had very low expression levels in the outer seed coat, and the expression levels of these three PgrABCG genes in the inner seed coats of hard-seeded pomegranate 'Dabenzi' were higher than those of soft-seeded pomegranate 'Tunisia'. In addition, the expression of these three PgrABCG genes was highly correlated with the expression of genes involved in lignin biosynthesis and hormone signaling pathways. The evolution of PgrABCG14 presents a highly similar trend to the origin and evolution of lignin biosynthesis during land plant evolution. Ectopic expression of PgrABCG14 in Arabidopsis promoted plant growth and lignin accumulation compared to wild type plants; meanwhile, the expression levels of lignin biosynthesis-related genes (CAD5, C4H, and Prx71) and cytokinin response marker genes (ARR5 and ARR15) were significantly upregulated in transgenic plants, which suggests the potential role of PgrABCG14 in promoting plant growth and lignin accumulation. Taken together, these findings not only provide insight into the characteristics and evolution of PgrABCGs, but also shed a light on the potential functions of PgrABCGs in seed hardness development.

Keywords: ABCG transporter; evolution; inner seed coat; lignin biosynthesis; pomegranate; seed hardness.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic analysis of ABCG proteins from pomegranate, grape, Arabidopsis, and eucalyptus. The tree was generated with MEGA X using the neighbor-joining method based on 1000 bootstrap replicates.
Figure 2
Figure 2
Expression profile of PgrABCG genes in different tissues of pomegranate based on RNA-Seq data. The transcript data were calculated with log2 normalization based on FPKM values. “DAP” = days after pollination, “ISC” = Inner Seed Coat, “OSC” = Outer Seed Coat.
Figure 3
Figure 3
Relative expression levels of the candidate PgrABCG genes in the inner seed coats of hard-seeded pomegranate ‘Dabenzi’ and soft-seeded pomegranate ‘Tunisia’ at five developmental stages. “DBZ” = ‘Dabenzi;’ “TNS” = ‘Tunisia;’ “DAP” = days after pollination. Error bars indicate standard deviation (n = 3). Different letters indicate a significant difference at p < 0.05 using Tukey’s test.
Figure 4
Figure 4
Co-expression network and evolution of the candidate PgrABCG genes. (A) The red dots represent PgrABCG genes, the blue dots represent genes involved in phenylpropanoid biosynthesis pathways, and the orange dots represent genes involved in hormone signaling pathways. (B) Rooted evolutionary trees of the candidate PgrABCG genes and the homologous genes in typical plant species. Different colors represent different plant species.
Figure 5
Figure 5
Subcellular localization of PgrABCG14-GFP fusion protein. (A) Subcellular localization of PgrABCG14-GFP in tobacco epidermal cells. Scale bar, 50 μm. (B) Subcellular localization of PgrABCG14-GFP in transgenic Arabidopsis plants. Scale bar, 100 μm.
Figure 6
Figure 6
GUS activity in transgenic Arabidopsis plants expressing the GUS reporter gene under the control of PgrABCG14 promoter. (A) 10-day rosette leaves, (B) root, (C) 45-day-old leaf, (D) inflorescences, (E) silique.
Figure 7
Figure 7
Phenotypes of PgrABCG14 OE Arabidopsis plants under normal growth conditions. (A) Phenotypes of the wild type and PgrABCG14 OE lines grown in soil for 4 weeks. (B) Biomass production of plants grown as in (A). (C) Lignin content in the shoots of plants grown as in (A). (D) Lignin staining of the stems and siliques of wild-type and PgrABCG14 OE lines. (E) Expression levels of lignin biosynthesis-related genes and cytokinin response marker genes in the shoots of wild-type and PgrABCG14 OE plants. Data are means ± SD (n = 3). Asterisks indicate values significantly different from those of the wild type (* p < 0.05, ** p < 0.01, Student’s t test). Bars: (D) 200 mm.
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