The R3-MYB gene GhCPC negatively regulates cotton fiber elongation

Abstract

Cotton (Gossypium spp.) fibers are single-cell trichomes that arise from the outer epidermal layer of seed coat. Here, we isolated a R3-MYB gene GhCPC, identified by cDNA microarray analysis. The only conserved R3 motif and different expression between TM-1 and fuzzless-lintless mutants suggested that it might be a negative regulator in fiber development. Transgenic evidence showed that GhCPC overexpression not only delayed fiber initiation but also led to significant decreases in fiber length. Interestingly, Yeast two-hybrid analysis revealed an interaction complex, in which GhCPC and GhTTG1/4 separately interacted with GhMYC1. In transgenic plants, Q-PCR analysis showed that GhHOX3 (GL2) and GhRDL1 were significantly down regulated in -1-5 DPA ovules and fibers. In addition, Yeast one-hybrid analysis demonstrated that GhMYC1 could bind to the E-box cis-elements and the promoter of GhHOX3. These results suggested that GhHOX3 (GL2) might be downstream gene of the regulatory complex. Also, overexpression of GhCPC in tobacco led to differential loss of pigmentation. Taken together, the results suggested that GhCPC might negatively regulate cotton fiber initiation and early elongation by a potential CPC-MYC1-TTG1/4 complex. Although the fibers were shorter in transgenic cotton lines than in the wild type, no significant difference was detected in stem or leaf trichomes, even in cotton mutants (five naked seed or fuzzless), suggesting that fiber and trichome development might be regulated by two sets of genes sharing a similar model.

Figure 1. Protein comparison of GhCPC (R3 MYB) and AtCPC (R3 MYB).

A. Sequence alignment of GhCPC and AtCPC R3 proteins. Shaded letters indicate identical residues. Green lines shows the positions of the three helices (h1, h2, h3) forming R3 MYB. B. Helical diagrams of h1, h2 and h3 in GhCPC R3 and AtCPC R3 with non polar residues in blue, polar residues in yellow, acidic residues in red and basic residues in green.

Figure 2. Phylogenetic tree and amino acid sequence alignment among the R2R3 MYB and R3 MYB regions.

A. Neighbor joining phylogenetic tree of the amino acid sequence of the R2R3 MYB regions (AtWER, AtGL1, AtPAP1, GhMYB2, GhMYB25, GhMYB25-like, GhMYB36, GhMYB109) and R3 MYB regions (AtETC1, AtTRY, AtCPC, GhTRY, GhCPC). B. Sequence alignment of R2R3 MYB and R3 MYB members using ClustalX software. R2 and R3 domains are marked with black bars under the corresponding residues. Three helices of both the R2 and R3 domains are indicated with red and green boxes, respectively. The conserved MYB-bHLH interaction motif on the first and second helices of the R3 domain is underlined with a blue bar.

Figure 3. Q-PCR analysis of GhCPC expression in different cotton species.

A. Spatial and temporal expression patterns in roots, stems, leaves, −3–0 DPA ovules and 3–9 DPA fibers in TM-1. B. Differential expression pattern in ovules of wild type (TM-1), three fiberless mutants (XZ142 FLM, MD17 and SL1-7-1) and two naked-seed mutants (N1N1 and n2n2).

Figure 4. Q-PCR analysis of GhCPC expression in two RIL populations.

A, B showed different expression pattern in 0–1 DPA ovules of pure lines of (MD17×TM-1) and (SL1-7-1×TM-1) RIL F₅ population, respectively. W, L and FL on abscissa represent linted-fuzzy, linted-fuzzless and lintless-fuzzless lines, respectively. Wa, La and FLa on abscissa represent the average expression levels in the linted-fuzzy, linted-fuzzless and lintless-fuzzless lines, respectively.

Figure 5. Compared to the wild type, overexpression of GhCPC leads to delayed fiber differention and decreased fiber length in the T3 generation Wild type was separated from the T0 generation.

A, B and C respectively show significantly different expression of GhCPC, GhHOX3 and GhRDL1 in CPC sense transgenic lines S21-2 and wild type. D, Different initial development of fiber cells of 0 DPA between CPC-overexpression T3 S21-2 and wild type . a, b, c Wild type ovules exhibited normal differentiation and rapid emergence of fiber cells from the surface (a-c). However, CPC-overexpression ovules exhibited the opposite morphology in which the surfaces of ovules from the transgenic plants were smooth with no appearance of fiber initiation. The fiber cells were observed at 50×, 300× and 1,000× magnification. E, Mature fibers in the wild type (g) and S21-2 (h), respectively, corresponding to D-a and D-f. F, Mature fiber comparison among wild type and CPC overexpression lines. The white line represents 1 mm. G, Measurement of fiber length showed that the fiber length in transgenetic lines was shorter than that in the wild type. Bars represent SD of three measurements and ** represent p-value ≤ 0.01 (t-test).

Figure 6. Interactions of different proteins.

A. Yeast two-hybrid assays examining the interactions between CPC, MYC1 and ribosomal proteins. The vectors pGADT7/pGBKT-53 and pGADT7/pGBKT-Lam were separately used as positive and negitave controls. B. Mapping of trucated domains of MYC1 to bind to CPC. As shown, both the MYB/MYC domain and the bHLH domain are required for the interaction with CPC. C. Of the four WD40 proteins, only TTG1 and TTG4 had weak interactions with CPC. D. Interactions between different function genes. E. Different concentrations (cell/ml) were plated onto SD/-Ade-Leu-Trp-His medium to examine the intensity of the interaction in the positve control by the yeast two-hybrid assay.

Figure 7. Transcriptional activation of ProHOX3, 4×E-box-WT and 4×E-box-Mutant by GhMYC1.

(1) Yeast Y1H integrating ProHOX3, (2) 4×E-box-WT, (3) 4×E-box-Mutant or (4) p53-pAbAi were respectively transformed with GhMYC1.

Figure 8. The potential CPC-MYC1-TTG1 regulatory complex in cotton.

In this network, CPC-MYC1-TTG4 complex can regulate the expression levels of GhHOX3 and GhRDL1 by binding to their promoters. GhCPC and GaMYB2 may have opposite effects on fiber development by completely binding to GhMYC1.