Analysis of gene expression in cotton fiber initials

<p>Abstract</p> <p>Background</p> <p>Cotton (<it>Gossypium hirsutum </it>L.) fibers are trichomes that initiate from the ovule epidermis. Little is known about the developmental pathway causing fiber to differentiate from ovular epidermal cells even though limits on the number of cells that differentiate into fiber will limit yield.</p> <p>Results</p> <p>A method was developed to isolate RNA from fiber initials 1 day post anthesis (dpa). Complementary DNA libraries representing 1 dpa fibers and other cotton tissues were sequenced and analyzed. Assembly of <it>G. hirsutum </it>Expressed Sequenced Tags (ESTs) identified over 11,000 sequences not previously represented in GenBank. New genes identified among these ESTs were represented on microarrays. The microarrays were used to identify genes enriched in fiber initials (1 dpa fibers) and elongating fibers. Analyses of Gene Ontologies (GO) of differentially expressed genes determined that terms associated with the "membranes" were statistically over represented among genes increased in expression in fiber initials and 10 dpa fibers. Staining ovules with a fluorescent dye confirmed an increase in Endoplasmic Reticulum (ER) occurred in fiber initials on the day of anthesis, persisted through 3 dpa and was absent in a <it>fiberless </it>mutant. Two genes similar to the CAPRICE/TRIPTYCHON (CPC) gene that inhibits differentiation of leaf trichomes in <it>Arabidopsis </it>were also characterized. Genes associated with novel regulation of brassinosterols, GTP mediated signal transduction and cell cycle control and components of a Ca⁺²mediated signaling pathway were identified. Staining of cellular Ca⁺²indicated that fiber initials had more Ca⁺²than other ovule cells supporting a role for Ca⁺²in fiber development.</p> <p>Conclusion</p> <p>Analysis of genes expressed in fiber initials identified a unique stage in fiber development characterized by an increase in ER and Ca⁺²levels that occurred between 0 and 1 dpa. The gene similar to CPC has a MYB domain but appears to lack a transcription activating domain similar to the <it>Arabisopsis </it>gene. The method used to stain the ER also can be used to count fiber initials and showed fiber cells develop from adjacent cells unlike leaf trichomes.</p

Expression of genes associated with carbohydrate metabolism in cotton stems and roots

<p>Abstract</p> <p>Background</p> <p>Cotton (<it>Gossypium hirsutum </it>L) is an important crop worldwide that provides fiber for the textile industry. Cotton is a perennial plant that stores starch in stems and roots to provide carbohydrates for growth in subsequent seasons. Domesticated cotton makes these reserves available to developing seeds which impacts seed yield. The goals of these analyses were to identify genes and physiological pathways that establish cotton stems and roots as physiological sinks and investigate the role these pathways play in cotton development during seed set.</p> <p>Results</p> <p>Analysis of field-grown cotton plants indicated that starch levels peaked about the time of first anthesis and then declined similar to reports in greenhouse-grown cotton plants. Starch accumulated along the length of the stem and the shape and size of the starch grains from stems were easily distinguished from transient starch. Microarray analyses compared gene expression in tissues containing low levels of starch with tissues rapidly accumulating starch. Statistical analysis of differentially expressed genes indicated increased expression among genes associated with starch synthesis, starch degradation, hexose metabolism, raffinose synthesis and trehalose synthesis. The anticipated changes in these sugars were largely confirmed by measuring soluble sugars in selected tissues.</p> <p>Conclusion</p> <p>In domesticated cotton starch stored prior to flowering was available to support seed production. Starch accumulation observed in young field-grown plants was not observed in greenhouse grown plants. A suite of genes associated with starch biosynthesis was identified. The pathway for starch utilization after flowering was associated with an increase in expression of a glucan water dikinase gene as has been implicated in utilization of transient starch. Changes in raffinose levels and levels of expression of genes controlling trehalose and raffinose biosynthesis were also observed in vegetative cotton tissues as plants age.</p

A VISUAL AID FOR STATISTICIANS AND MOLECULAR BIOLOGISTS WORKING WITH MICROARRAY EXPERIMENTS

The use of microarrays to measure the expression of large numbers of genes simultaneously is increasing in agriculture research. Statisticians are expected to help biologists analyze these large data sets to identify biologically important genes that are differentially regulated in the samples under investigation. However, molecular biologists are often unfamiliar with the statistical methods used to analyze microarrays. Presented here are methods developed to graphically represent microarray data and various types of errors commonly associated with microarrays to help visualize sources of error. Two case studies were used. In case study one, genes differentially regulated when two corn lines, one resistant and one sensitive, were treated with Aspergillus flavus isolate NRRL 3357 or left untreated were investigated. Analyses and images showing 3 types of variation are shown. Genes were ranked according to fold change and re-ranked after adjusting for potential sources of error. In case two, cotton genes differentially regulated in 1-day-old fiber compared to whole ovules or older fibers were investigated. Data and sources of error were imaged as described for case one and genes with significant changes in gene expression were identified

MOLECULAR BIOLOGY AND PHYSIOLOGY Fiber Initiation in 18 Cultivars and Experimental Lines of Three Gossypium Species

ABSTRACT A new technique was developed to study the cotton fiber initiation process and fiber initial densities. The goal was to provide an additional tool to cotton breeders and geneticists interested in fiber improvement. The objectives were to assess whether fiber initiation patterns reported for some Gossypium hirsutum L. and G. barbadense L. cultivars extend to a more diverse range of cultivars/lines; and to test if there is a relationship between fiber initials density, lint percentage, and fiber characteristics. This study was performed with 17 cultivars and experimental lines of G. hirsutum (upland cotton) and G. barbadense (Pima cotton) and one G. arboreum L. accession. The ovules were extracted from flowers at the day of anthesis and 1 and 2 d thereafter, stained with a fluorescent dye, DiOC 6 (3) (3,3′-dihexyloxacarbocyanine iodide), and observed under a light microscope. Staining cotton fiber initials with DiOC 6 (3) enabled the evaluation of a greater number of samples than the more time-consuming scanning electron microscopy protocols to assess the fiber initiation process. There were significant differences in fiber initial densities among cultivars and lines but these did not cluster into &quot;upland&quot; and &quot;Pima&quot; types as had been expected. Upland and Pima fiber initiation occurred at anthesis, whereas Sea Island Pima initiation was delayed a day. A delay in fiber initiation was also confirmed in fuzzless mutant lines. Fiber initial densities were not found to be useful predictors of lint percentage

THE CHROMATIN STRUCTURE OF SOME CROWN GALL TUMOR GENES (NUCLEOSOME, TRANSFER-DNA)

I have investigated the nucleosome organization and the relative deoxyribonuclease I (DNA\u27se I) sensitivity of the T-DNA of an octopine-type and a nopaline-type crown gall tumor. The T-DNA of the octopine-type tumor was found to be in an altered nucleosomal array, to be preferentially DNA\u27se I sensitive over the bulk of the chromatin, and to contain no detectable DNA\u27se I hypersensitive sites. The T-DNA of the nopaline-type tumor was found in the typical nucleosome array, was preferentially DNA\u27se I sensitive over the bulk of the chromatin, and was probably lacking any detectable DNA\u27se I hypersensitive sites. The preferential DNA\u27se I sensitivity of the T-DNA in both crown gall tumor lines suggests that the majority of the T-DNA in these cell lines is transcriptionally competent. The altered nucleosome array of the octopine-type T-DNA may indicate that the majority of the T-DNA in this line is rapidly transcribed. The plant portion of a plant/T-DNA junction was isolated and characterized. The sequences homologous to the plant portion of the plant/T-DNA junction were found in the typical nucleosome array in the octopine-type tumor. The sequences homologous to the plant portion of the junction fragment did not share the altered nucleosome structure of the majority of the T-DNA in the octopine-type tumor investigated. I have also investigated the nucleosome structure of the rRNA in a variety of Nicotiana cell lines. Of seven tumorous and nontumorous cell lines investigated only the two octopine type crown gall tumors had their rDNA in an altered nucleosomal conformation. A correlation was established between the altered nucleosome structure of the rDNA in a cell line and a substantial reduction of the rDNA copy number in the same cell lines. It is likely that the altered nucleosome structure of the rDNA is a result of rapid transcription of the remaining rDNA necessitated by the reduction in their copy number

MOLECULAR BIOLOGY AND PHYSIOLOGY Nitrogen Metabolism in Cotton Stems and Roots During Reproductive Development

ABSTRACT Cotton is a perennial plant grown as an annual row crop in much of the world. Cotton stems and roots store starch prior to reproduction that is subsequently available to support reproduction. Aspects of nitrogen metabolism in cotton stems and roots were investigated to determine whether these tissues also provided nitrogen to support reproduction. Measurements of total nitrogen, soluble proteins, and individual amino acids indicated that nitrogen metabolism was altered after flowering began. Analysis of gene expression from previous microarray experiments also showed patterns consistent with a role of altered nitrogen metabolism during seed set. Changes in transcript levels of genes associated with amino acid biosynthesis, biosynthesis of nitrogenous compounds, and protein turnover also indicated that nitrogen metabolism changed in roots during seed set. We confirm that stem and roots provide nitrogen to support reproduction, and propose that the transport of nitrogenous compounds from these tissues to reproductive tissue could affect seed set by communicating the nitrogen status of the plant

Alignment of CPC and other MYBs: GhCPC1(contig16590), GhCPC2(Contig17149), AtCPC(NP 182164), AtGL1(NP 189430), GhMYB2(translation of MYB2)

<p><b>Copyright information:</b></p><p>Taken from "Analysis of gene expression in cotton fiber initials"</p><p>http://www.biomedcentral.com/1471-2229/7/22</p><p>BMC Plant Biology 2007;7():22-22.</p><p>Published online 16 May 2007</p><p>PMCID:PMC1906782.</p><p></p> Regions of identity shared by all clones lightly shaded. Regions shared by majority of clones darkly shaded. MYB motifs underlined