Interactome analysis of the six cotton 14-3-3s that are preferentially expressed in fibres and involved in cell elongation

Abstract

Proteins of the 14-3-3 family regulate a divergent set of signalling pathways in all eukaryotic organisms. In this study, several cDNAs encoding 14-3-3 proteins were isolated from a cotton fibre cDNA library. The Gh14-3-3 genes share high sequence homology at the nucleotide level in the coding region and at the amino acid level. Real-time quantitative RT-PCR analysis indicated that the expression of these Gh14-3-3 genes is developmentally regulated in fibres, and reached their peak at the stage of rapid cell elongation of fibre development. Furthermore, overexpression of Gh14-3-3a, Gh14-3-3e, and Gh14-3-3L in fission yeast promoted atypical longitudinal growth of the host cells. Yeast two-hybrid analysis revealed that the interaction between cotton 14-3-3 proteins is isoform selective. Through yeast two-hybrid screening, 38 novel interaction partners of the six 14-3-3 proteins (Gh14-3-3a, Gh14-3-3e, Gh14-3-3f, Gh14-3-3g, Gh14-3-3h, and Gh14-3-3L), which are involved in plant development, metabolism, signalling transduction, and other cellular processes, were identified in cotton fibres. Taking these data together, it is proposed that the Gh14-3-3 proteins may participate in regulation of fibre cell elongation. Thus, the results of this study provide novel insights into the 14-3-3 signalling related to fibre development of cotton.

Fig. 1.

Sequence alignment among the six Gh14-3-3 proteins. Sequences of Gh14-3-3s were aligned. The amino acid residues identical among the sequences are indicated in black, while similar residues are shown in grey, and the regions of the nine conserved antiparallel α-helices (α1–α9) are underlined.

Fig. 2.

Phylogenetic relationship of cotton 14-3-3 proteins to Arabidopsis 14-3-3 proteins. The minimum evolution tree was constructed in MEGA3.1 from 1000 bootstrap replicates. The accession numbers of cotton and Arabidopsis 14-3-3 proteins in GenBank are: Gh14-3-3a (EU189220), Gh14-3-3b (EU189221), Gh14-3-3c (EU189222), Gh14-3-3d (EU189223), Gh14-3-3e (EU189224), Gh14-3-3f (GU451708), Gh14-3-3g (GU451709), Gh14-3-3h (GU451710), Gh14-3-3L (DQ402076), AtGF14κ (AAD51783), AtGF14λ (AAD51781), AtGF14χ (AAA96254), AtGF14ϕ (AAB62224), AtGF14ω (AAA96253), AtGF14ν (AAD51782), AtGF14υ (AAB62225), AtGF14ψ (AAA96252), AtGF14μ (AAD51784), AtGF14ι (AAK11271), AtGF14° (AAG47840), AtGF14ε (AAD51785), and AtGF14π (NP-565174).

Fig. 3.

The structures of four Gh14-3-3 genes. Exons are denoted by black boxes. Introns, 5′-flanking regions, and 3′-UTRs are denoted by lines. The length of the intron in base pairs is indicated. The position of substitution is denoted by a diagonal line.

Fig. 4.

Real-time quantitative RT-PCR analysis of expression of Gh14-3-3 genes in cotton tissues. Relative values of Gh14-3-3 gene expression in cotton tissues are shown as a percentage of GhUBI1 expression activity. R, roots; H, hypocotyls; C, cotyledons; L, leaves; A, anthers; P, petals; F, 10 DPA (days post-anthesis) fibres; O, 10 DPA ovules.

Fig. 5.

Expression of Gh14-3-3 genes during fibre development. Relative values of Gh14-3-3 gene expression in developing fibres of cotton are shown as a percentage of GhUBI1 expression activity. 3d, 5d, 6d, 9d, 10d, 12d, 15d, 18d, and 20d refer to 3, 5, 6, 9, 10, 12, 15, 18, and 20 DPA fibres.

Fig. 6.

Overexpression of Gh14-3-3 genes stimulated the longitudinal growth of fission yeast cells. Micrographs were taken using dark-field microscopy. Yeast cells harbouring the constructed vectors pREP5N-Gh14-3-3L (A and E), pREP5N-Gh14-3-3a (B and F), and pREP5N-Gh14-3-3e (C and G), and empty plasmid (pREP5N; D and H) were cultured in non-induction medium (A–D) or induction medium (E–H). Bar=100 μm.

Fig. 7.

Statistical analysis of the length of various fission yeast cell lines. C, controls (yeast cells with empty vectors); L1–L10, transformed cell lines (yeast cells harbouring Gh14-3-3 genes). A single asterisk represents a significant difference (P-value <0.05), and double asterisks represent very significant differences (P-value <0.01) between the transformed cell lines and the control by t-test. The experiments were repeated three times; means represent average values of cell length (n=100 cells each line), and bars show standard errors.

Fig. 8.

Isoform-specific interactions among the Gh14-3-3 proteins. The coding sequences of the Gh14-3-3 genes were cloned into the yeast two-hybrid vectors pGADT7 and pGBKT7, and introduced into yeast cells (see Materials and methods). Interactions among the Gh14-3-3 proteins were analysed by yeast mating. Transformants were assayed for growth on QDO nutritional selection medium.

Fig. 9.

Venn diagram of classification of the isolated Gh14-3-3 interactors. The proteins identified in cotton fibres by yeast two-hybrid screening were classified into seven classes.

Fig. 10.

Isoform-specific interactions between Gh14-3-3s and target proteins. Seven interactors were selected to analyse the interaction with each Gh14-3-3 protein by yeast mating. Transformants were assayed for growth on QDO nutritional selection medium.