Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation

Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes.     

Exogenous Jasmonic Acid Inhibits Cotton Fiber Elongation

Jasmonic acid (JA) is a well-characterized phytohormone that acts in various ways to influence plant development. Its role in cotton fiber development, however, has not yet been thoroughly explored. In this study, JA was proven to be an inhibitor of ovule and fiber development in vitro. Continuous exogenous JA application inhibited fiber elongation. This effect was dependent on development stage and dosage. Fibers and ovules at three different stages of development and different JA dosages were compared. The most serious suppression was detected when ovules 1?day before anthesis (–1?DPA) were cultured in medium with 2.5?μM JA. Genes related to trichome and fiber development responded differently to JA treatment between –1?DPA and 1?day post anthesis (1 DPA). JAs (JA and JA-Ile) quantification showed that JAs content was sharply decreased from –1?DPA to 5?DPA ovules, which indicated that JA was negatively associated with fiber elongation in vivo. In addition, gene expression analysis showed the same trend. Our results demonstrate that there was a negative relationship of JA with fiber elongation in vitro and in vivo. These results are meaningful for uncovering the mechanism of fiber elongation in cotton.     

A beta-tubulin-like cDNA expressed specifically in elongating cotton fibers induces longitudinal growth of fission yeast

Using cDNA Representational Difference Analysis (RDA) techniques, we isolated a cDNA that was expressed specifically in cotton fibers but not in the ovules of a fuzzless-lintless mutant (fl). We designated it as Gh-BTubL for it shares high sequence identity with known plant and yeast beta-tubulins. RT-PCR and robotic cDNA dot blot analyses indicated that the expression of Gh-BTubL was correlated with the elongation pattern of cotton fibers. In situ hybridization results verified that there was no Gh-BTubL mRNA in fl ovules while it was easily detected in the elongating wild type cotton fiber cells. Overexpression of Gh-BTubL in fission yeast induced longitudinal growth of the host cells by 1.74-fold, with no apparent effect on other aspects of the host cells. We suggest that Gh-BTubL plays an important role in cotton fiber elongation and we believe that elucidation of the control mechanisms for expression of tubulin-like proteins may help improve fiber quality and productivity.     

The role of phytohormones in cotton fiber development

Since 1974, when Beasley and Ting discovered that fertilized ovules of cotton can be cultured in media supplemented with GA along with auxin, the effect of all types of phytohormones on fiber development has been widely studied. Many phytohormones, including GA, IAA, brassinosteroid (Br), ABA, ethylene (Et), and cytokinins (Ck), all have been demonstrated to play important roles during cotton fiber development. In recent years, the rapid development of genomic analysis and the accumulation of high-quality cotton ESTs allowed us to probe phytohormonal gene expression during fiber development. Many phytohormonal genes, including GA-, IAA-, ABA-, Br-, Et-, and Ck-related genes, participating in phytohormone biosynthesis pathways and signal transduction pathway accumulated in the process of cotton fiber development.     

Levels of Cytokinins in the Ovules of Cotton Mutants with Altered Fiber Development

Endogenous levels of cytokinin and abscisic acid (ABA) were determined in ovules of normal cotton (TM-1) and four fiber differentiation mutants (n2, Ligon lintless, H10, and Xu142) before and after flowering by enzyme-linked immunosorbent assays. The fluctuation patterns of ABA levels in ovules of normal cotton and mutants were similar. At the fiber elongation stage, ABA content was low, and from 1 day after flowering, the ABA content decreased steadily. On the other hand, the peaks of isopentenyladenine and isopentenyladenosine in ovules of TM-1 were observed 1 day before flowering. The level of cytokinins decreased after flowering in TM-1, whereas in the mutants it increased steadily. These results indicate that endogenous ABA is probably not the main inhibitor for fiber elongation and that endogenous cytokinins likely play a dual role in fiber development. Before flowering, cytokinins function as one of the stimuli for the initiation of fibers, but after flowering, cytokinins inhibit fiber growth. Received February 18, 1997; accepted June 11, 1997     

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H⁺)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Characterization of the Brassinosteroid Insensitive 1 Genes of Cotton

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.