The Promoter Structure Differentiation of a MYB Transcription Factor <i>RLC1</i> Causes Red Leaf Coloration in Empire Red Leaf Cotton under Light

<div><p>The red leaf coloration of Empire Red Leaf Cotton (ERLC) (<i>Gossypium hirsutum</i> L.), resulted from anthocyanin accumulation in light, is a well known dominant agricultural trait. However, the underpin molecular mechanism remains elusive. To explore this, we compared the molecular biological basis of anthocyanin accumulation in both ERLC and the green leaf cotton variety CCRI 24 (<i>Gossypium hirsutum</i> L.). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077891#s1" target="_blank">Introduction</a> of R2R3-MYB transcription factor <i>Rosea1</i>, the master regulator anthocyanin biosynthesis in <i>Antirrhinum majus</i>, into CCRI 24 induced anthocyanin accumulation, indicating structural genes for anthocyanin biosynthesis are not defected and the leaf coloration might be caused by variation of regulatory genes expression. Expression analysis found that a transcription factor <i>RLC1</i> (Red Leaf Cotton 1) which encodes the ortholog of <i>PAP1/Rosea1</i> was highly expressed in leaves of ERLC but barely expressed in CCRI 24 in light. Ectopic expression of <i>RLC1</i> from ERLC and CCRI 24 in hairy roots of <i>Antirrhinum majus</i> and CCRI 24 significantly enhanced anthocyanin accumulation. Comparison of <i>RLC1</i> promoter sequences between ERLC and CCRI 24 revealed two 228-bp tandem repeats presented in ERLC with only one repeat in CCRI 24. Transient assays in cotton leave tissue evidenced that the tandem repeats in ERLC is responsible for light-induced <i>RLC1</i> expression and therefore anthocyanin accumulation. Taken together, our results in this article strongly support an important step toward understanding the role of R2R3-MYB transcription factors in the regulatory menchanisms of anthocyanin accumulation in red leaf cotton under light.</p></div

Analysis of <i>RLC1</i> and gene expression levels in cotton by RT-PCR. UBI 7 was used as a positive control.

<p><b>A</b>, <i>RLC1</i> 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>B</b>, Comparison of the expression levels of <i>RLC1</i> in mature leaves of ERLC grown in shade, light, and combined conditions. <b>C</b>, Comparison of color accumulation in transformed hairy roots of CCRI 24. <b>a</b>, Hairy root transformed with pBI121; <b>b</b>, Hairy root transformed with pBI35S::<i>RLC1.</i> Scale bar indicates 1 cm. <b>D</b>, Expression analysis of structural genes in hairy roots of transformed CCRI 24 with the negative controls pBI121 and pBI35S::<i>RLC1</i>, respectively.</p

Analysis of <i>RLC1</i> promoter activity by infiltration.

<p><b>A</b>, Diagrams of constructs for the analysis of <i>RLC1</i> promoter activity. R_−pro: 2300-bp promoter region of <i>RLC1</i> of ERLC; G_−pro: 2080-bp promoter region of <i>RLC1</i> of CCRI 24 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077891#pone-0077891-g006" target="_blank">Fig. 6</a>). <b>B</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. <b>a</b>, A leaf infiltrated with 35::<i>RLC1</i> and cultured for three days in light; <b>b</b>, A leaf infiltrated with R−pro::<i>RLC1</i> and cultured for three days in light; <b>c</b>, A leaf infiltrated with G₋pro::<i>RLC1</i> and cultured for three days in light; <b>d</b>, A GUS-stained leaf infiltrated with pBI121 and cultured for three days in light; <b>e</b>, A GUS stained leaf infiltrated with R−pro::<i>GUS</i> and cultured for three days in light; <b>f</b>, A leaf infiltrated with G₋pro::<i>GUS</i> and cultured for three days in light. Scale bar is 0.1 cm.</p

Comparison of leaf colors and analysis of total anthocyanin concentrations of leaves from ERLC and CCRI 24 cultivars grown in light and shade conditions.

<p>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 <i>Agrobacterium</i> strain GV3101/pBI35S::<i>ROSEA1</i> (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.</p

Comparison of the <i>RLC1</i> alleles between ERLC and CCRI 24.

<p>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 <i>A. majus</i> by <i>A. rhizhogenes-</i>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 <i>A. majus</i> 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.</p

Phylogenetic analysis, subcellular localization, and expression pattern of <i>GhSnRK2</i> in cotton plant.

<p>(A) A phylogenetic tree of <i>GhSnRK2</i> and other <i>SnRK2</i> proteins from different plants was constructed using the neighbor-joining method with MEGA 5. The sequences used for analysis are listed by accession number: <i>Litchi chinensis</i> (<i>LcSnRK2</i>), AFX72761.1; <i>Arabidopsis thaliana</i> (<i>AtSNRK2.2</i>), CP002686.1; <i>Arabidopsis thaliana</i> (<i>AtSnRK2.3</i>), AED98274.1; <i>AtSnRK2.1</i>, AED91326.1; <i>AtSnRK2.9</i>, AEC07398.1; <i>AtSnRK2.10</i>, AEE33751.1; <i>AtSnRK2.4</i>, AEE28666.1; <i>AtSnRK2.5</i>, AED97781.1; <i>AtSnRK2.7</i>, AEE87152.1; <i>Populus tremula</i> (<i>PtreSnRK2.6a</i>), AGW51610.1; <i>Zea mays</i> (<i>ZmSnRK2.2</i>), NM_001137717.1; <i>Solanum tuberosum</i> (<i>StSnRK2.8</i>), AFR68945.1; <i>Oryza sativa</i> (<i>RK1</i>), ABB89146.1; <i>Oryza sativa</i> (<i>SAPK4)</i>, BAD18000.1; <i>Sorghum bicolor</i> (<i>SAPK4</i>), AGM39623.1; and <i>Zea mays</i> (<i>SAPK5</i>), ACG42286.1. The bootstrap values are shown on the tree branches. (B) Subcellular localization of the <i>GhSnRK2-GFP</i> protein. (2 and 5) GFP alone; (4 and 6) <i>GhSnRK2-GFP</i> in onion epidermal cells; (1 and 3) corresponding bright-field images. (C) The expression pattern of the <i>GhSnRK2</i> gene in cotton plants subjected to 10% PEG stress. The gene expression data were normalized to that of the cotton histone 3 gene. The values are presented as the means of three experimental replicates. The vertical axis represents the relative expression level. The values from1 to 6 indicate the time (h) of PEG treatment. Asterisk denotes a significant difference (P<0.05) compared with the control (0 h). (D) Relative expression levels of the <i>GhSnRK2</i> gene in various cotton plant tissues. Samples from root (RT) stem (ST), cauline leaves (CL), rosettes leaves (RL) and flowers (FL) were analyzed. The vertical axis represents the relative expression level. The letters denote significant differences (P<0.05) based on Duncan's multiple range tests. The cotton histone 3 gene was used as an internal control for normalization of the gene expression data.</p

Phenotypic comparison between Empire red leaf cotton (ERLC) (upper panel) and green leaf cotton CCRI 24 (bottom panel) cultivars grown in light.

<p>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.</p

Comparison of the deduced amino acid sequence of RLC1 with verified MYB genes of other plant species.

<p>A, Phylogenetic tree of <i>RLC1</i> 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.</p

Plant transformation vector and the expression pattern of the <i>GhSnRK2</i> gene in the transgenic lines.

<p>(A) Schematic representation of the T-DNA region of the binary vector <i>pCAMBIA2301-GhSnRK2</i>. (B) Expression pattern of the <i>GhSnRK2</i> gene in the transgenic plants. Various upregulated expression patterns of the <i>GhSnRK2</i> gene in transgenic lines were detected, as indicated by the vertical axis. The values are presented as the means of three experimental replicates; the error bars indicate the standard deviations. The <i>AtACT2</i> gene was used as an internal control for normalization of gene expression.</p

Cloning of <i>Gossypium hirsutum</i> Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (<i>GhSnRK2</i>) and Its Overexpression in Transgenic <i>Arabidopsis</i> Escalates Drought and Low Temperature Tolerance

<div><p>The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the <i>Gossypium hirsutum</i> sucrose non-fermenting 1-related protein kinase 2 (<i>GhSnRK2</i>) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that <i>GhSnRK2</i> participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the <i>GhSnRK2</i>-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing <i>GhSnRK2</i> exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that <i>GhSnRK2</i> acts as a positive regulator in response to cold and drought stresses. Plants overexpressing <i>GhSnRK2</i> displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of <i>GhSnRK2</i> expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as <i>AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1</i>, and <i>AtABI5</i>, were increased in the <i>GhSnRK2</i>-overexpressing plants compared to the wild-type plants. <i>GhSnRK2</i> gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the <i>GhSnRK2</i> gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.</p></div