An ATP-binding cassette transporter GhWBC1 from elongating cotton fibers

Abstract

We have isolated a cDNA (GhWBC1) from cotton (Gossypium hirsutum) that encodes an ATP-binding cassette transporter of the WBC (white/brown complex) subfamily. Members of this subfamily are half-sized transporters and are reported to mediate lipid and drug excretion in human (Homo sapiens). GhWBC1 is highly expressed in developing fiber cells, but transcripts were also detectable in other tissues except roots. The transcript level peaked in rapidly expanding fibers from 5 to 9 DPA and then decreased. The GhWBC1 expression was weak in fiber cells of an li (ligon-lintless) mutant, which is defective in fiber cell elongation. These data indicate that GhWBC1 gene expression correlates with cotton fiber elongation. Transient expression of enhanced green fluorescence protein-GhWBC1 fusion protein in onion (Allium cepa) epidermal cells revealed plasma membrane localization. The GhWBC1 cDNA driven by a constitutive 35S promoter was introduced into Arabidopsis. About 13% of the transformants produced short siliques (SSs), whereas others had normal siliques (long siliques [LSs]). In siliques of SS lines, most embryos were severely shriveled, and only several seeds per silique could be found at maturity. The transgene expression level was higher in SS lines than in LS lines. Expression of AtWBC11, the closest homolog of GhWBC1 in Arabidopsis, was not altered in either SS or LS transgenic plants examined. These data suggest that GhWBC1 interferes with substance translocation that is required for Arabidopsis seed and silique development. Characterization of Arabidopsis WBC members, particularly AtWBC11, will help to dissect the role of GhWBC1 in cotton fiber development and elongation.

Figure 1.

RT-PCR analysis of GhWBC1 (Zhu93) gene expression in developing ovules of cotton cv Xu-142 and fl mutant, collected at 5 DPA. The cotton Histone-3 (GhHIS3) was amplified as a control; in negative control (–), no template DNA was added. The PCR was performed with primers GH1 and GH2.

Figure 2.

Alignment of amino acid sequences of GhWBC1, Arabidopsis AtWBC11 (At1g17840), a rice putative ABC transporter (AAM92819), human ABCG5 (AAG40003), and fruitfly White protein (P10090). All the proteins aligned here are WBC members of the ABC transporter superfamily. The sequences were aligned with ClustalW (Thompson et al., 1994) using default parameters through EMBnet (http://www.ch.embnet.org/software/ClustalW.html), and the box shade was created by BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html).

Figure 3.

Northern-blot analysis of spatial and temporal pattern of GhWBC1 expression. A, Expression in seedlings (10 d old) and developing ovules (9 DPA); total RNA was isolated from roots (1), hypocotyls (2), cotyledons (3), young true leaves (4), fiber cells (5), wild-type intact ovules (6), and fl ovules (7); 20 μg of total RNA was loaded each lane. B, Expression of GhWBC1 in developing fibers of 5, 9, 15, 20, and 25 DPA, respectively; 1.25 μg of total RNA was loaded each lane. C and D, expression of GhWBC1, E6, and Expansin genes in li mutant and wild-type cotton cv Xu-142 ovules. C, GhWBC1 expression in 1- and 2-DPA ovules; D, GhWBC1, E6, and Expansin expression in 5- and 9-DPA ovules. Ten micrograms of total RNA was loaded each lane.

Figure 4.

RT-PCR analysis of GhWBC1 expression in different aerial organs of cotton cv Xu-142 and fl mutant. Total RNAs were isolated from hypocotyls (H), cotyledons (C), and young true leaves (L) of the 10-d-old seedlings and from 9-DPA ovules (O) or fibers (F). The PCR was conducted by 27 cycles of PCR amplification with primers GH3 and GH4.

Figure 5.

Subcellular localization of EGFP-GhWBC1 fusion protein in onion epidermal cells. A, Cell structure under light microscopy. B, Green fluorescence signal of EGFP-GhWBC1 after plasmolysis with 20% (w/v) Suc for 5 min. Plasma membrane and GFP signal shrank together from cell wall.

Figure 6.

Phenotype of transgenic Arabidopsis plants overexpressing GhWBC1 under a 35S promoter. A, Inflorescence of mature plants of WT (left) and SS-29 transgenic line (right); B, siliques of WT (left) and SS-29 line (right); C, seeds in the 3-d-old siliques of WT (left) and of SS-29 line (right).

Figure 7.

Silique length and seed number per silique of WT and GhWBC1-transgenic lines of Arabidopsis. A, Length of siliques; B, seed number per silique. LS and SS represent the long (normal) silique lines and the SS lines of transgenic Arabidopsis, respectively. For each line, around 10 siliques were measured.

Figure 8.

A, RT-PCR analysis of transcripts of GhWBC1 and AtWBC11 in siliques of GhWBC1 transgenic lines of Arabidopsis; higher level of GhWBC1 gene expression was detected in siliques of SS lines. B, RT-PCR analysis of AtWBC11 expression in WT Arabidopsis plants. 1, Roots; 2, leaves; 3, stem; 4, inflorescence; –, negative control without template DNA. The PCR was performed by 30 cycles of amplification. Arabidopsis ACTIN2 (AtACT) was amplified as a control.