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

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

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

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

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

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

Additional file 9: of Analysis of transcriptome in hickory (Carya cathayensis), and uncover the dynamics in the hormonal signaling pathway during graft process

The expression level of cytokinin signaling pathway related genes. (XLSX 11 kb

DataSheet_1_Rice DST transcription factor negatively regulates heat tolerance through ROS-mediated stomatal movement and heat-responsive gene expression.pdf

Plants are frequently subjected to a broad spectrum of abiotic stresses including drought, salinity and extreme temperatures and have evolved both common and stress-specific responses to promote fitness and survival. Understanding the components and mechanisms that underlie both common and stress-specific responses can enable development of crop plants tolerant to different stresses. Here, we report a rice heat stress-tolerant 1 (hst1) mutant with increased heat tolerance. HST1 encodes the DST transcription factor, which also regulates drought and salinity tolerance. Increased heat tolerance of hst1 was associated with suppressed expression of reactive oxygen species (ROS)-scavenging peroxidases and increased ROS levels, which reduced water loss by decreasing stomatal aperture under heat stress. In addition, increased ROS levels enhanced expression of genes encoding heat shock protein (HSPs) including HSP80, HSP74, HSP58 and small HSPs. HSPs promote stabilization of proteins and protein refolding under heat stress and accordingly mutation of HST1 also improved reproductive traits including pollen viability and seed setting under high temperature. These results broaden the negative roles of DST in abiotic stress tolerance and provide important new insights into DST-regulated tolerance to diverse abiotic stresses through both shared and stress-specific mechanisms.</p

Additional file 5: of Analysis of transcriptome in hickory (Carya cathayensis), and uncover the dynamics in the hormonal signaling pathway during graft process

The information of the classification of the KEGGs. (XLSX 9 kb