Additional file 2: of Characterization and functional analysis of GhWRKY42, a group IId WRKY gene, in upland cotton (Gossypium hirsutum L.)

Table S2. Primers used in this study. (DOCX 32 kb

Over-expression of <i>GhWRKY17</i> can promote leaf senescence in <i>Arabidopsis thaliana</i>.

<p>(A) Flowering phenotypes of WT and OE1, OE2 and OE3 transgenic lines. (B) Senescent phenotypes of WT and OE1, OE2 and OE3 transgenic lines. (C) Confirmation of transgenic lines by qRT-PCR. (D-F) Relative expression of the senescence-associated genes <i>AtSAG12</i>, <i>AtSAG13</i> and <i>AtWRKY53</i>. OE1, OE2 and OE3 represent three transgenic lines. When qRT-PCR was performed, <i>AtUBQ10</i> was used as a reference gene. The data are presented as the means±standard error. Values significantly different from WT at the 0.01 confidence level.</p

Expression patterns of group IIa <i>GhWRKY</i> genes in various tissues and at different leaf senescence stages.

<p>(A) Expression patterns of group IIa <i>GhWRKY</i> genes in root, stem, leaf, petal, pistil, stamen, torus, calycle, fiber 5, fiber 10, fiber 20 and fiber 25 tissues. Fiber 5, fiber 10, fiber 20 and fiber 25 indicate fiber development stages at 5, 10, 20, and 25 days after anthesis. (B) Expression patterns of group IIa <i>GhWRKY</i> genes in leaf senescence. New-1 and old-1 indicate leaves from the three-leaf stage. New-2 and old-2 indicate leaves from the maturation/senescence stages.</p

Identification of the group IIa WRKY subfamily and the functional analysis of <i>GhWRKY17</i> in upland cotton (<i>Gossypium hirsutum</i> L.)

<div><p>WRKY transcription factors play important roles in plant defense, stress response, leaf senescence, and plant growth and development. Previous studies have revealed the important roles of the group IIa <i>GhWRKY</i> genes in cotton. To comprehensively analyze the group IIa <i>GhWRKY</i> genes in upland cotton, we identified 15 candidate group IIa <i>GhWRKY</i> genes in the <i>Gossypium hirsutum</i> genome. The phylogenetic tree, intron-exon structure, motif prediction and Ka/Ks analyses indicated that most group IIa <i>GhWRKY</i> genes shared high similarity and conservation and underwent purifying selection during evolution. In addition, we detected the expression patterns of several group IIa <i>GhWRKY</i> genes in individual tissues as well as during leaf senescence using public RNA sequencing data and real-time quantitative PCR. To better understand the functions of group IIa <i>GhWRKYs</i> in cotton, <i>GhWRKY17</i> (KF669857) was isolated from upland cotton, and its sequence alignment, promoter <i>cis</i>-acting elements and subcellular localization were characterized. Moreover, the over-expression of <i>GhWRKY17</i> in <i>Arabidopsis</i> up-regulated the senescence-associated genes <i>AtWRKY53</i>, <i>AtSAG12</i> and <i>AtSAG13</i>, enhancing the plant’s susceptibility to leaf senescence. These findings lay the foundation for further analysis and study of the functions of WRKY genes in cotton.</p></div

Nuclear localization of <i>GhWRKY17</i> in onion epidermal cells.

<p>(A) Plasmid sketch of the <i>35S-GFP</i> empty vector and <i>35S-GhWRKY17</i>::<i>GFP</i> fusion construct. <i>GhWRKY17</i> was fused to the N-terminus of GFP driven by the <i>CaMV 35S</i> promoter. (B) Transient expression of both <i>35S-GFP</i> and <i>35S-GhWRKY17</i>::<i>GFP</i> fusion proteins in onion epidermal cells in bright field (Bright), dim field (GFP), and overlapped field (Merge).</p

Motif composition in group IIa <i>GhWRKY</i> subfamily proteins.

<p>Each colored box indicates a different putative motif. The scale plate indicates the protein length. The combined E-value was calculated by the MEME online software.</p

Tissue-specific and senescence expression patterns of <i>GhWRKY17</i>, <i>GhWRKY39</i> and <i>GhWRKY140</i>.

<p>(A) Relative expression of <i>GhWRKY17/39/140</i> in root, stem, leaf, petal, pistil, stamen, fiber and ovule tissues. The root and stem were sampled from two-week-old seedlings. The leaf, petal, pistil and stamen were sampled at the full flower period. The fiber and ovule were sampled 10 days after anthesis. (B) Five different senescence degrees of true leaves in CCRI74. Stage 1, an expanded new leaf; Stage 2, a mature but non-senescent leaf; Stage 3, a leaf with 25% senescence area; Stage 4, a leaf with 50% senescence area; and Stage 5, a leaf with at least 75% senescence area. (C) Relative expression of <i>GhNAP</i> and <i>GhWRKY17/39/140</i> in cotton. When qRT-PCR was performed, <i>GhActin</i> was used as a reference gene. The data are presented as the means±standard error.</p

Motif composition in group IIa <i>GhWRKY</i> subfamily proteins.

<p>Each colored box indicates a different putative motif. The scale plate indicates the protein length. The combined E-value was calculated by the MEME online software.</p

The predicted <i>cis</i>-acting elements in the promoter of <i>GhWRKY17</i>.

<p>The predicted <i>cis</i>-acting elements in the promoter of <i>GhWRKY17</i>.</p

The Ka/Ks ratios for duplicate group IIa <i>GhWRKY</i> genes.

<p>The Ka/Ks ratios for duplicate group IIa <i>GhWRKY</i> genes.</p