Table_1_An Easy and Rapid Transformation Protocol for Transient Expression in Cotton Fiber.xlsx

Cotton fiber is the most important natural textile material in the world. Identification and functional characterization of genes regulating fiber development are fundamental for improving fiber quality and yield. However, stable cotton transformation is time-consuming, low in efficiency, and technically complex. Moreover, heterologous systems, such as Arabidopsis and tobacco, did not always work to elucidate the function of cotton fiber specifically expressed genes or their promoters. For these reasons, constructing a rapid transformation system using cotton fibers is necessary to study fiber’s specifically expressed genes. In this study, we developed an easy and rapid Agrobacterium-mediated method for the transient transformation of genes and promoters in cotton fibers. First, we found that exogenous genes could be expressed in cotton fibers via using β-glucuronidase (GUS) and green fluorescence protein (GFP) as reporters. Second, parameters affecting transformation efficiency, including LBA4404 Agrobacterium strain, 3 h infection time, and 2-day incubation time, were determined. Third, four different cotton genes that are specifically expressed in fibers were transiently transformed in cotton fibers, and the transcripts of these genes were detected ten to thousand times increase over the control. Fourth, GUS staining and activity analysis demonstrated that the activity profiles of GhMYB212 and GhFSN1 promoters in transformed fibers are similar to their native activity in developmental fibers. Furthermore, the transient transformation method was confirmed to be suitable for subcellular localization studies. In summary, the presented Agrobacterium-mediated transient transformation method is a fast, simple, and effective system for promoter characterization and protein expression in cotton fibers.</p

Heat map of the <i>COBLs</i> expression during the fiber developmental stages of <i>G</i>. <i>hirsutum</i> acc. TM-1.

<p>The fiber developmental stages involved in initiation (from -3 DPA to 0 DPA), elongation (from 0 DPA to 10 DPA) and secondary wall biosynthesis (from 20 DPA to 25 DPA) were sampled for detecting the expression levels of <i>COBL</i> genes during the fiber development. The relative expression levels were shown as Log₂ (FPKM). A_t and D_t were derived from the A-genome and D-genome progenitor in the tetraploid cotton.</p

Phylogenetic relationship, gene structure and motif compositions of the <i>COBLs</i> in <i>G</i>. <i>raimondii</i> and <i>AtCOBRA</i>, <i>AtCOBL7</i> in <i>A</i>. <i>thaliana</i>.

<p>A. The phylogenetic tree was conducted using MEGA 5.0 software with the maximum likelihood method. B. Exon/intron organization of <i>GrCOBL</i> members. Black boxes and black lines represented the exons and introns. The length of nucleotides was shown below. C. Schematic representation of the conserved motifs of <i>GrCOBL</i> proteins elucidated by MEME and SMART. Each motif was marked by the different colors and the ω-sites were showed in red font. The length of amino acids was shown below.</p

Chromosomal distribution of the <i>COBL</i> family genes in <i>G</i>. <i>raimondii</i>.

<p>The chromosome numbers were consistent with the interspecific genetic map (D1 to D13) in allotetraploid cultivated cotton species [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145725#pone.0145725.ref034" target="_blank">34</a>] and the scaffolds (Chr.1 to Chr.13) in the genomic data of <i>G</i>. <i>raimondii</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145725#pone.0145725.ref030" target="_blank">30</a>]. The nomenclature of <i>COBLs</i> was based on the order of the chromosomes in <i>G</i>. <i>raimondii</i>. Lines were drawn to connect the duplicated genes.</p

Heat map of the <i>COBLs</i> expression in different tissues of <i>G</i>. <i>hirsutum</i> acc. TM-1.

<p>The eight different tissues of <i>G</i>. <i>hirsutum</i> acc. TM-1 were involved here: 1: root; 2: stem; 3: leaf; 4: petal; 5: anther; 6: 0 DPA ovule; 7: 10 DPA fiber; 8: 20 DPA fiber. Log₂ (FPKM) indicated the different transcriptome profiling (FPKM: fragments per kb per million mapped reads). A_t and D_t were derived from the A-genome and D-genome progenitor in the tetraploid cotton.</p

Physical integration of <i>GhCOBL9</i> and <i>GhCOBL13</i> with QTLs associated with fiber quality traits.

<p>Fiber quality traits included fiber length (FL), fiber strength (FS), fiber micronaire (FM), fiber elongation (FE) and fiber uniformity (FU). Different symbols represented the QTLs with the references followed.</p

Phylogenetic analyses of <i>COBL</i> family genes in eudicot (<i>A</i>. <i>thaliana</i>, <i>G</i>. <i>raimondii</i>) and monocot (<i>O</i>. <i>sativa</i>, <i>Z</i>. <i>mays</i>).

<p>Amino acid sequences were aligned using ClustalW and the phylogenetic tree was conducted using MEGA 5.0 software with the maximum likelihood method. Four different color fonts were represented for the four species.</p

Expression patterns of <i>GhCOBL9</i> and <i>GhCOBL13</i> with the <i>GhCESA</i> family genes in <i>G</i>. <i>hirsutum</i> acc. TM-1.

<p>The X axis indicated the different fiber development stages and the Y axis indicated the expression levels.</p

qRT-PCR analysis of <i>COBL9</i> (A) and <i>COBL13</i> (B) during the fiber development stages in TM-1 and Hai7124.

<p>The A_t and D_t were derived from A-subgenome and D-subgenome specific in tetraploid cotton species respectively. The Y axis indicated relative expression levels and the X axis indicated the different fiber development stages. “*” and “**” denoted the differences at P < 0.05 and P < 0.01 between TM-1 and Hai7124 respectively.</p