Developing Fiber Specific Promoter-Reporter Transgenic Lines to Study the Effect of Abiotic Stresses on Fiber Development in Cotton

<div><p>Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (<i>Expansin</i>, <i>E6</i>, <i>Rac13</i>, <i>CelA1</i>, <i>LTP</i>, <i>and Fb late</i>), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via <i>Agrobacterium</i> mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.</p></div

Photomicrographs of cotton pollen following a high temperature challenge.

<p>Representative photomicrographs showing pollen germination at 28°C according to the procedure described by Burke et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref017" target="_blank">17</a>] from plants grown under control temperatures (31°C/27°C day/night temperatures) and elevated temperatures (43°C/28°C day/night temperatures). (A) Plant 52 Control, (B) Plant 40 Control, (C) Plant 52 from a greenhouse with elevated day/night temperatures, (D) Plant 40 from a greenhouse with elevated day/night temperatures.</p

GUS activities measured by fluorometric quantification at different fiber developmental stages in transgenic plants.

<p>(A) C22- <i>Expansin</i>::<i>GUS</i>, (B) C21-<i>CelA1</i>::<i>GUS</i>, (C) C28-<i>E6</i>::<i>GUS</i> and (D) <i>Rac13</i>::<i>GU</i>S grown under normal or stressed conditions. The GUS activities were determined by measuring fluorescence on Tecan 200 fluorometer using 360nm and 465nm as excitation and emission wavelengths, respectively and expressed as pmol of 4-MU per min per μg total protein.</p

Photographs of anthers from high temperature grown cotton plants.

<p>(A) Line 39 [AtHSP101 minus], (B) Line 40 [AtHSP101 plus], (C) Line 52 [AtHSP101 minus], (D) Line 24 [AtHSP101 plus] flowers from plants grown under high temperatures (43°C/28°C day/night) in a greenhouse. Pollen dehiscence is apparent in all lines evaluated.</p

Photographs of RT-PCR and Western Blot analysis showing the expression of <i>AtHSP101</i> and presence of HSP101 proteins in transgenic cotton lines.

<p>RT-PCR products from pollen (A) and leaf (B) tissues of transgenic line 40 plants grown in 31°C/27°C greenhouse; Western blot analysis of HSP101 for: mature pollen of transgenic line 40 (C) and null line 52 (F, AtHSP101 minus) plants grown in a greenhouse set to 31°C/27°C day/night; leaf tissues of transgenic line 40 (D) and null line 52 (G) plants grown in a greenhouse set to 31C°/27°C day/night; leaf tissues of transgenic line 40 (E) and null line 52 (H) plants grown in a hot greenhouse set to 43°C/28°C day/night temperatures.</p

Photomicrographs of cotton pollen following a high temperature challenge.

<p>(A) pollen from plant 39 (AtHSP101 minus) germination at 28°C after treating flowers for five hours at 23°C, (B) pollen from plant 39 (AtHSP101 minus) germination at 28°C after treating flowers for five hours at 37°C, (C) pollen from plant 40 (AtHSP101 plus) germination at 28°C after treating flowers for five hours at 23°C, and (D) pollen from plant 40 (AtHSP101 plus) germination at 28°C after treating flowers for five hours at 37°C.</p

Diagram of pE1801-ocs/mas ‘superpromoter’-HSP101 plasmid.

<p><b>PAg7</b> = Transcription termination and poly-Adenylation signal sequence from Octopine Ti-Plasmid T-DNA (gene for transcript #7) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref057" target="_blank">57</a>]; <b>NptII</b> = Neomycin phosphotransferase II coding region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref058" target="_blank">58</a>]; <b>Pnos</b> = Nopaline synthase promoter from Nopaline Ti-Plasmid T-DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref059" target="_blank">59</a>]; <b>Aocs X 3</b> = Octopine synthase enhancer element (3 copies) from Octopine T-Plasmid T-DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref060" target="_blank">60</a>]; <b>AmasPmas</b> = Manopine synthase promoter from Octopine Ti-Plasmid T-DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref061" target="_blank">61</a>]; <b>HSP101</b> = Heat shock protein (101 kdalton molecular weight) from <i>A</i>. <i>thaliana</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref032" target="_blank">32</a>]; <b>Ags-ter</b> = Transcription termination and poly-Adenylation signal sequence from Octopine Ti-Plasmid T-DNA (Agropine synthase gene) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122933#pone.0122933.ref062" target="_blank">62</a>].</p

Segregation of T₂ seedlings of selected C30 and C31 cotton transgenic lines for rooting on hygromycin-selection medium and for the presence/absence of hygromycin DNA fragment in PCR products.

<p>^†: Transgenic line contains 2wo or more copies of CFSP-reporter transgene;</p><p>^‡: This transgenic line was discarded due to weak expression of the reporter gene;</p><p>^§: Not applied. No T2 seeds were harvested from this line.</p><p>Segregation of T₂ seedlings of selected C30 and C31 cotton transgenic lines for rooting on hygromycin-selection medium and for the presence/absence of hygromycin DNA fragment in PCR products.</p

Boll accumulation on control and heat-treated cotton plants.

<p>Photograph of cotton bolls harvested from individual plants of plant line 24 (AtHSP101 plus) and plant line 39 (AtHSP101 minus) that were grown in a greenhouse with a 43°C/28°C day/night temperature regime. Paired plants (line 24 and 39) were randomly distributed throughout the greenhouse because of a measured uneven temperature distribution (set point ± 3°C) during the winter months.</p

Photomicrographs of tobacco pollen following a high temperature challenge.

<p>SR1 (A), SR1-GS2 (B), and SR1-GS3 (C) tobacco pollen germination when incubated at 30°C for 3 hours; and SR1 (D), SR1-GS2 (E), SR1-GS3 (F) pollen germination when incubated at 46°C for 3 hours.</p