Figure 1.
Phenotypic comparison between Empire red leaf cotton (ERLC) (upper panel) and green leaf cotton CCRI 24 (bottom panel) cultivars grown in light.
A, A 7-day-old young seedling of ERLC;B, A young leaf from a 4-week-old ERLC plant; C, stems of ERLC; D, A mature flower of ERLC; E, A boll of ERLC; F, A 7-day-old seedling of CCRI 24; G, A young leaf from a 4-week-old plant of CCRI 24; H, stems of CCRI 24; I, A mature flower of CCRI 24; J, A boll of CCRI 24. Scale bar indicates 1 cm.
Figure 2.
Comparison of leaf colors and analysis of total anthocyanin concentrations of leaves from ERLC and CCRI 24 cultivars grown in light and shade conditions.
A, Comparison of leaf colors. The cultivars are indicated on the left and conditions are indicated above. The bar indicates 1; b,A mature leaf of ERLC grown in shade; c, A mature leaf of CCRI 24 grown in light; d, A mature leaf of CCRI 24 grown in shade. B, Total anthocyanin extracted from three fully opened young leaves of each cultivar, respectively, measured using a UV spectrometer. Means of three replicates with error bars indicating standard error (± SD). C, Transient analysis was performed on the leaves of CCRI 24, the Agrobacterium strain GV3101/pBI35S::ROSEA1 (left), and the negative control GV3101/pBI121 (right). The treated cotton leaves were cultured at 25°C in, 16 h light for three days, and observed for color accumulation by microscopy. Scale bar is 0.4 cm.
Figure 3.
Comparison of the deduced amino acid sequence of RLC1 with verified MYB genes of other plant species.
A, Phylogenetic tree of RLC1 and selected R2R3-MYBs from other plant species. The multiple sequence alignment was performed with the R2R3 domain of MYB proteins. The tree was constructed using the neighbor-Joining method using MEGA software. Numbers along the branches indicate bootstrap support determined from 1,000 trials, and the bar indicates an evolutionary distance of 0.05%. B, Alignment of deduced amino acid sequences of RLC1 with MYB transcriptional regulators. The R2 and R3 repeat domains are indicated by lines above, and the conserved region of the bHLH interacting motif ([DE]Lx2[RK]x3Lx6Lx3R) and the conserved KPRPR[S/T]F motif are underlined.
Figure 4.
Phenotypic comparison of the hairy roots of A. majus transformed with pBI121, pBI35S::ROSEA1, and pBI35S::RLC1, and expression analysis of genes involved in the anthocyanin pathway in A. majus by RT-PCR.
Hairy roots transformed with A, pBI121; B, pBI35S::RLC1; C, pBI35S::ROSEA1. D, A shoot regenerated from hairy roots transformed with pBI35S::RLC1. Anthocyanin pigmentation is apparent in the leaves and stems. E, RT-PCR analysis of gene expression in the hairy roots of A. majus transformed with pBI121 (negative control), pBI35S::ROSEA 1 (positive control), and pBI35S::RLC1, respectively.
Figure 5.
Analysis of RLC1 and gene expression levels in cotton by RT-PCR. UBI 7 was used as a positive control.
A, RLC1 expression analysis was performed in roots, seedlings, leaves, and mature petals of ERLC and CCRI 24 cultivars grown in light by semi-quantitative RT-PCR. B, Comparison of the expression levels of RLC1 in mature leaves of ERLC grown in shade, light, and combined conditions. C, Comparison of color accumulation in transformed hairy roots of CCRI 24. a, Hairy root transformed with pBI121; b, Hairy root transformed with pBI35S::RLC1. Scale bar indicates 1 cm. D, Expression analysis of structural genes in hairy roots of transformed CCRI 24 with the negative controls pBI121 and pBI35S::RLC1, respectively.
Figure 6.
Comparison of the RLC1 alleles between ERLC and CCRI 24.
A, The coding sequence is shown in gray boxes and the non-coding sequence is shown as a black line. Location of the putative TATA box is shown by an empty square. Repeat fragments (R) are indicated by black squares. The exons, introns, and promoter region are labeled. The location of a nucleotide change site is also indicated in exon 2 of CCRI 24. Numbers refer to the position relative to the first nucleotide of the start codon. B, DNA sequence of the 228-bp fragment, with the location of putative I-box and G-box (underlined). C, Functional analysis of the RLC1a region of CCRI 24 (Fig. 6A, bottom) in the hairy roots of A. majus by A. rhizhogenes-mediated transformation. The pBI35S::RLC1a expression vector contained the RLC1a region on pBI121 driven by the cauliflower mosaic virus 35S promoter. a, a red pigmented mass developed on the end of hypocotyl segment of A. majus two weeks after infection; b, red pigmented hairy root developed from the ends of hypocotyl segments four weeks after infection. The pigmented mass (a) and hairy roots (b) are indicated by small arrows.
Figure 7.
Analysis of RLC1 promoter activity by infiltration.
A, Diagrams of constructs for the analysis of RLC1 promoter activity. R−pro: 2300-bp promoter region of RLC1 of ERLC; G−pro: 2080-bp promoter region of RLC1 of CCRI 24 (Fig. 6). B, promoter activity tests were performed using mature young leaves of CCRI 24 using the combination of expression vectors described above. Treated cotton leaves were cultured at 25°C with 16-h light periods for three days, and the leaves were used for color observation or GUS staining. a, A leaf infiltrated with 35::RLC1 and cultured for three days in light; b, A leaf infiltrated with R−pro::RLC1 and cultured for three days in light; c, A leaf infiltrated with G−pro::RLC1 and cultured for three days in light; d, A GUS-stained leaf infiltrated with pBI121 and cultured for three days in light; e, A GUS stained leaf infiltrated with R−pro::GUS and cultured for three days in light; f, A leaf infiltrated with G−pro::GUS and cultured for three days in light. Scale bar is 0.1 cm.