Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers

In animals, the small GTP-binding proteins, Rac and Rho, of theras superfamily participate in the signal rransduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

Developmental and gene expression analyses of a cotton naked seed mutant

Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Quantitative trait loci affecting cotton fiber are linked to the t1 locus in upland cotton

Pilose (T ₁), a dominant marker in upland cotton, has been associated with coarse, short fibers. Pilose was, thereby, considered to be pleiotropic on fiber fineness and length. However, a pilose-expressing line with a fiber of average fineness was recently identified. This finding does not support pleiotropy between T ₁ and fiber traits, but is indicative of linkage between pilose and loci influencing fiber characteristics. To understand the relationship between T ₁ and fiber traits, a pilose line with short, coarse fiber was crossed to two t ₁ lines with standard fiber characteristics. One hundred and forty-nine F₂-derived F₃ lines were developed from one cross, and 60 F₂-derived F₃ lines from the other. Seven fiber traits (elongation, maturity, micronaire reading, perimeter, 2.5% span length, strength, and wall thickness) were measured. Segregation was normal, as indicated by allelic frequencies of 0.5 for T ₁ and t ₁, and segregation ratios of 121 for marker genotypes. The association of homozygous T ₁ lines with fibers of average fineness was again observed. Linkage between T ₁ and loci affecting micronaire, perimeter, 2.5% span length, strength, and wall thickness was found in both populations. Significant additive and non-additive gene effects for each of these traits at the marker locus were found as well. The pilose marker accounted for 10–75% of the phenotypic variation associated with each trait. In conclusion, the t ₁ locus is linked to numerous loci that influence fiber traits, and this linkage has previously been misinterpreted as pleiotropy.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton

Background

The cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical post-translational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0 days post-anthesis (DPA).

Results

A total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P³DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation.

Conclusions

Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-466) contains supplementary material, which is available to authorized users.     

Characterization and expression analysis of two cotton genes encoding putative UDP-Glycosyltransferases

UDP-Glycosyltransferases (UGT) are a large family of enzymes, which catalyze the transfer of a sugar from an activated sugar donor to an acceptor molecule. Both in plants and in mammals, they are important in the maintenance of cellular homeostasis. In this study, two genes (designated GhUGT1 and GhUGT2, respectively) encoding putative UGT were isolated from the cotton fiber cDNA library. The deduced proteins contain the signature sequences of plant UGTs in the C-terminal region. The GhUGT1 gene encodes a polypeptide of 457 amino acids, and displays homology at amino acid levels with the known glycosyltransferase genes. Sequence analysis revealed that the GhUGT2 merely encodes a small protein, as there is a nucleotide substitution that results in formation of a stop codon in its open reading frame. Real-time RT-PCR analysis revealed that the expression of GhUGT1 is higher in the fast growth tissues, such as in fibers and roots. GhUGT2 has also higher expression in roots, but with lower expression levels in fibers and other tissues. The results also showed that the expression of GhUGT1 is higher than GhUGT2. Further study showed that GhUGT1 and GhUGT2 expressions are regulated under osmotic stress, suggesting they may be involved in plants responding to osmotic stress. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 1, pp. 50–58. The text was submitted by the authors in English.     

In vitro selection and regeneration of cotton resistant to high temperature stress

Summary Cell suspension cultures of cotton (Gossypium hitirsutum L. cv. Coker 312) were exposed to various temperature:time treatments in order to select cell lines resistant to high temperature stress. Cells were exposed to 45°C for 3 h each day until the total accumulated hours of stress were: 0 h, 10 h, 75 h, 100 h, or 105 h (81 h pulsed then 24 h continuous). After the stress treatments, the cells were plated onto embryo development medium and plants were recovered. The embryogenic calli that were recovered were subcultured monthly for 6 months and tested for increased resistance to the temperature:time treatments previously determined to be lethal and to water stress as imposed by PEG. All of the selected cell lines were more resistant to both types of stress than the control cell lines. Leaf tissue of stress selected (Ro) formed and maintained callus growth when incubated at 38°C; whereas, tissue excised from nonselected controls rarely formed callus and calli which did form quickly became necrotic. These cells and plants will provide a tool for determining the mechanisms involved in resistance to high temperature stress.     

Storage protein accumulation patterns in somatic embryos of cotton (Gossypium hirsutum L.)

Summary The storage protein content of somatic embryos of Gossypium hirsutum L. cv. Coker 201 was determined using extinction level, antigen/antibody association detection methods. Mature storage protein was first detected in early globular-stage somatic embryos at a total concentration of 0.36% of the embryo protein mass. Tulip-stage and mature somatic embryos were comprised of 3.0% and 1.3% mature storage protein, respectively. Maximum storage protein synthesis was found to occur during early globular- and early heart-stages. During this period of development, significant levels of protein precursors were found also to accumulate. The pattern of storage protein synthesis, processing and accumulation paralleled the pattern that has been reported for the zygotic system, although somatic embryos accumulate storage protein at much earlier stages and to a lesser degree. The possibility of using complex biochemical pathways to monitor embryogenic systems in vitro is discussed.     

Somatic embryogenesis in cotton (Gossypium) I. Effects of source of explant and hormone regime

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

Studies of pollen maturation in cotton: the storage reserve accumulation phase

Summary This study follows the maturation of the pollen grain of cotton (Gossypium hirsutum L.), particularly the development of the vegetative cytoplasm and the various storage products formed. CTEM, HVEM, stereoscopy, and cyto-histochemistry were used to examine the events occurring during the 9 days before anthesis. Starch began to accumulate in plastids at anthesis minus 9 days and reached a peak concentration shortly before anthesis; lipid deposition followed a similar pattern, but started at 6 days before anthesis. Lipid bodies were always seen closely oppressed to the endoplasmic reticulum (ER). Dictyosomes appear active during the entire 9 days; first producing vesicles involved in the formation of the intine and, later, producing vesicles stored in the pollen grain. The dictyosome vesicles appear to contain polysaccharides and concentrate in layers around the lipid bodies. Ribosomes increase in number from 6 days before anthesis and are particularly numerous in the mature pollen. From anthesis minus 6 days until anthesis, the ER cisternae become increasingly inflated and, in the hours immediately before pollen release, form pockets filled with lipid bodies and dictysosome vesicles. The mature pollen has a core region filled with ER pockets and a peripheral cytoplasm in which such pockets are generally lacking.This research was supported in part by NSF Grant BMS575-22-23 and Grant N.RR-00592 from the Division of Research Resources, National Institutes of Health     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

The rate of cellulose increase is highly related to cotton fibre strength and is significantly determined by its genetic background and boll period temperature

This experiment was conducted to study the relationship between the increase in cellulose content in developing cotton bolls and their final cotton fibre strength. The rate of cellulose increase over time was estimated using logistical regression, and the logistic equation parameters were then used to compare different cotton cultivars in different temperature environments. The increase in cellulose content followed a typical “S” curve, with the boll period time divided into slow-fast-slow stages. In different cultivars, the final fibre strength was closely related to the characters of the fast cellulose content increasing stage, negatively related to the maximal cellulose increasing rate (P < 0.05), and positively related to the duration of the fast cellulose content increasing stage (P < 0.01). In the same cultivar, low temperature reduced the maximal cellulose increasing rate and prolonged the duration of the fast cellulose increasing stage. The results indicate that, in diverse genetic background, long-lasting and tempered cellulose growth during the rapid cellulose increasing stage is of significant benefit to high strength fibre development. For closely related cotton cultivars, decreasing the maximal cellulose increasing rate and the termination of rapid cellulose increasing stage reduced fibre strength that often occurs when temperatures are low.