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Cellulose synthase catalytic subunits (CesAs) are the catalytic sites within a multisubunit complex for cellulose biosynthesis in plants. CesAs have been extensively studied in diploid plants, but are not well characterized in polyploid plants. Gossypium hirsutum is an allotetraploid cotton species producing over 90% of the world's cotton fibers. Although G. hirsutum CesAs (GhCesAs) are responsible for cellulose production in cotton fiber, very limited numbers of GhCesA genes have been identified. Here, we report isolating and characterizing a pair of homeologous CesA2 genes and their full-length cDNAs from allotetraploid cotton. The GhCesA2-A(T) gene from the A-subgenome and GhCesA2-D(T) gene from the D-subgenome were screened from a G. hirsutum BAC library. These genes shared 92% sequence similarity throughout the entire sequence. The coding sequences were nearly identical, and the deduced amino acid sequences from GhCesA2-A(T) (1,039 amino acids) and GhCesA2-D(T) (1,040 amino acids) were identical except four amino acids, whereas the noncoding sequences showed divergence. Sequence analyses showed that all exons of GhCesA2-A(T) contained consensus splice donor dinucleotides, but one exon in GhCesA2-D(T) contained nonconsensus splice donor dinucleotides. Although the nonconsensus splice donor dinucleotides were previously suggested to be involved in alternative splice or pseudogenization, our results showed that a majority of GhCesA2-A(T) and GhCesA2-D(T) transcripts consisted of functional and full-length transcripts with little evidence for alternative mRNA isoforms in developing cotton fibers. Expression analyses showed that GhCesA2-A(T) and GhCesA2-D(T) shared common temporal and spatial expression patterns, and they were highly and preferentially expressed during the cellulose biosynthesis stage in developing cotton fibers. The observations of higher expression levels of both GhCesA2-A(T) and GhCesA2-D(T) in developing fibers of one near-isogenic line (NIL) with higher fiber bundle strength over the other NIL with lower fiber bundle strength suggested that the differential expression of genes associated with secondary cell wall cellulose biosynthesis in developing fiber might affect cotton fiber properties.

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.

Agrobacterium rhizogenes-induced cotton (Gossypium hirsutum L.) hairy roots were evaluated as a model system for studying molecular cotton-nematode interactions. Hairy root cultures were developed from the root-knot nematode (RKN) (Meloidogyne incognita [Kofoid and White] Chitwood, race 3)-resistant breeding line M315 and from the reniform nematode (RN) (Rotylenchulus reniformis Linford & Oliveira)-resistant accession GB713 (G. barbadense L.) and compared to a nematode-susceptible culture derived from the obsolete cultivar DPL90. M315, GB713, and DPL90 hairy roots differed significantly in their appearance and growth potential; however, these differences were not correlated with transcript levels of the A. rhizogenes T-DNA genes rolB and aux2 which help regulate hairy root initiation and proliferation. DPL90 hairy roots were found to support both RKN and RN reproduction in tissue culture, whereas M315 and GB713 hairy roots were resistant to RKN and RN, respectively. M315 hairy roots showed constitutive up-regulation of the defense gene MIC3 (Meloidogyne Induced Cotton3) compared to M315 whole-plant roots and DPL90 hairy roots. Our data show the potential use of cotton hairy roots in maintaining monoxenic RKN and RN cultures and suggest hairy roots may be useful in evaluating the effect of manipulated host gene expression on nematode resistance in cotton.

Hairy root cultures were induced by inoculating cotyledonary leaves and hypocotyl segments from two cotton species, Gossypium hirsutum and Gossypium barbadense, with Rhizobium rhizogenes 15834. For both species, more hairy roots formed on inoculation sites on cotyledonary leaves than on hypocotyls. The addition of sucrose to basal Murashige–Skoog media increased the frequency of hairy root formation, whereas the addition of naphthalene acetic acid (0.54 μM) did not. After transfer to a liquid culture, hairy root growth was very rapid. After 3 wk in liquid culture, both cotton species produced gossypol, a di-sesquiterpene secondary metabolite with known anticancer activity, and two related methylated derivatives. Most (60–95%) gossypol produced by cultures was retained within the hairy root tissues, but some was found in the media. The average gossypol level observed among 96 different cultures was 15 mg/g of dry culture mass; however, some cultures produced >40 mg/g of dry culture mass. Variation in gossypol levels was greater for cultures arising from different transformation events than for multiple subclones of a single transformant. The high level of gossypol production attained by most of these cultures suggests that they will be valuable for studying the biochemical and molecular aspects of gossypol biosynthesis, capable of producing large amounts of gossypol and related compounds, and useful for generating modified forms of gossypol (e.g., radio-labeled gossypol) for understanding bioactivity mechanisms.

Hydrogen peroxide and other reactive oxygen species are important signaling molecules in diverse physiological processes. Previously, we discovered superoxide dismutase (SOD) activity in extracellular protein preparations from fiber-bearing cotton (Gossypium hirsutum L.) seeds. We show here, based on immunoreactivity, that the enzyme is a Cu/Zn-SOD (CSD). Immunogold localization shows that CSD localizes to secondary cell walls of developing cotton fibers. Five cotton CSD cDNAs were cloned from cotton fiber and classified into three subfamilies (Group 1: GhCSD1; Group 2: GhCSD2a and GhCSD2b; Group 3: GhCSD3 and GhCSD3s). Members of Group 1 and 2 are expressed throughout fiber development, but predominant during the elongation stage. Group 3 CSDs are also expressed throughout fiber development, but transiently increase in abundance at the transition period between cell elongation and secondary cell wall synthesis. Each of the three GhCSDs also has distinct patterns of expression in tissues other than fiber. Overexpression of cotton CSDs fused to green fluorescent protein in transgenic Arabidopsis demonstrated that GhCSD1 localizes to the cytosol, GhCSD2a localizes to plastids, and GhCSD3 is translocated to the cell wall. Subcellular fractionation of proteins from transgenic Arabidopsis seedlings confirmed that only c-myc epitope-tagged GhCSD3 co-purifies with cell wall proteins. Extracellular CSDs have been suggested to be involved in lignin formation in secondary cell walls of other plants. Since cotton fibers are not lignified, we suggest that extracellular CSDs may be involved in other plant cell wall growth and development processes.

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

Gene expression during the early stages of fiber cell development and in allopolyploid crops is poorly understood. Here we report computational and expression analyses of 32 789 high-quality ESTs derived from Gossypium hirsutum L. Texas Marker-1 (TM-1) immature ovules (GH_TMO). The ESTs were assembled into 8540 unique sequences including 4036 tentative consensus sequences (TCs) and 4504 singletons, representing approximately 15% of the unique sequences in the cotton EST collection. Compared with approximately 178 000 existing ESTs derived from elongating fibers and non-fiber tissues, GH_TMO ESTs showed a significant increase in the percentage of genes encoding putative transcription factors such as MYB and WRKY and genes encoding predicted proteins involved in auxin, brassinosteroid (BR), gibberellic acid (GA), abscisic acid (ABA) and ethylene signaling pathways. Cotton homologs related to MIXTA, MYB5, GL2 and eight genes in the auxin, BR, GA and ethylene pathways were induced during fiber cell initiation but repressed in the naked seed mutant (N1N1) that is impaired in fiber formation. The data agree with the known roles of MYB and WRKY transcription factors in Arabidopsis leaf trichome development and the well-documented phytohormonal effects on fiber cell development in immature cotton ovules cultured in vitro. Moreover, the phytohormonal pathway-related genes were induced prior to the activation of MYB-like genes, suggesting an important role of phytohormones in cell fate determination. Significantly, AA sub-genome ESTs of all functional classifications including cell-cycle control and transcription factor activity were selectively enriched in G. hirsutum L., an allotetraploid derived from polyploidization between AA and DD genome species, a result consistent with the production of long lint fibers in AA genome species. These results suggest general roles for genome-specific, phytohormonal and transcriptional gene regulation during the early stages of fiber cell development in cotton allopolyploids.

Cytoskeleton assembly plays an important role in determining cotton fiber cell length and morphology and is developmentally regulated. As in other plant cells, it is not clear how cytoskeletal assembly in fibers is regulated. Recently, several Rac/Rop GTPases in Arabidopsis were shown to regulate isotropic and polar cell growth of root hairs and pollen tubes by controlling assembly of the cytoskeleton. GhRac1, isolated from cottonseeds, is a member of the Rac/Rop GTPase family and is abundantly expressed in rapidly growing cotton tissues. GhRac1 shows the greatest sequence similarity to the group IV subfamily of Arabidopsis Rac/Rop genes. Overexpression of GhRac1 in E. coli led to the production of a functional GTPase as shown by in vitro enzyme activity assay. In contrast to other Rac/Rop GTPases found in cotton fiber, GhRac1 is highly expressed during the elongation stage of fiber development with expression decreasing dramatically when the rate of fiber elongation declines. The association of highest GhRac1 expression during stages of maximal cotton fiber elongation suggests that GhRac1 GTPase may be a potential regulator of fiber elongation by controlling cytoskeletal assembly.

There are only a few cells in the plant kingdom that are as exaggerated in their size or composition as cotton fibers. It is precisely their highly elongated structure and exceptional chemical make-up that establishes cotton fiber as an ideal model for studies of plant cell elongation and cell wall

Nearly 30 years ago the conditions for culturing immature cotton ovules were established to serve as a working research tool for investigating the physiology and biochemistry of fiber development. Not only has this tissue culture method been employed to characterize the biochemistry of plant cell expansion and secondary cell wall synthesis, but ovule cultures have contributed to numerous other aspects of plant cell physiology and development as well. In addition to basic studies on fiber development, cotton ovule cultures have been used to examine plant-fungal interactions, to model low temperature stress responses, to elucidate the pathways responsible for pigment formation in naturally pigmented fiber and to probe how cytoskeletal elements regulate cell wall organization. Success in rescuing Gossypium interspecific hybrids was dependent on ovule culture media formulations that could support early embryo development in ovulo. As tissues produced in culture are analyzed by increasingly more sophisticated techniques, there appear to be some differences between ovule growth in planta and ovule growth in vitro. Discerning how ovule culture fiber development is different from fiber development in field-grown plants can contribute valuable information for crop improvement. Cotton ovule cultures are an especially attractive model system for studying the effects of gravity on cell elongation, cellulose biosynthesis and embryo development and are excellent targets for examining transient expression of introduced gene constructs. With only minor modification, the procedure originally described by C. A. Beasley and I. P. Ting for growing cotton ovules in vitro will continue to be useful research tool for the foreseeable future.

Cytoplasts from cotton ( Gossypium hirsutum L.) fiber cells retain microtubule and microfilament cytoskeletons through extraction with non-ionic detergent and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid. Tubulin and actin are the most abundant proteins in extracted cytoplasts; however, many other less abundant proteins are also present. To determine if minor proteins were associated with the cytoskeleton, microtubules and microfilaments were selectively removed from extracted cytoplasts by detergent extraction in an alkaline Ca 2+ solution. Under these extraction conditions, microtubules and microfilaments were fragmented and depolymerized unless previously stabilized by taxol and phalloidin. Associated proteins were identified by their loss in conjunction with either microtubules or microfilaments. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one protein, of roughly 115 kDa, appeared to be associated with microfilaments since it was present in Ca 2+ -extracted preparations only when microfilaments were stabilized with phalloidin. The failure of most minor proteins to associate with microtubules and microfilaments suggests that caution must be used when interpreting co-isolation as evidence for an association of low abundance proteins with cytoskeletons.

Multiple isoforms of α‐ and β‐tubulin accumulate in higher plant cells, including cotton (Gossypium hirsutum L.) fiber. Isotypes may originate either by transcription of distinct genes or by posttranslational modification of gene products. The existence of two types of posttranslational modification in cotton fiber α‐tubulin has been examined by immunoblotting cotton fiber proteins from two developmental stages and by probing with specific monoclonal antibodies to acetylated (6‐11B‐1) or tyrosinated (YL 1/2 ) α‐tubulin. Control experiments were conducted with an antibody (YOL 1/34) that recognizes a conserved region in plant and animal α‐tubulins. No acetylated forms of α‐tubulin were found in either of two varieties of cotton fiber at 10 or 20 d postanthesis. One isotype of α‐tubulin, isotype 6, failed to cross‐react with YL 1/2 at 10 d postanthesis, which indicated that the protein was detyrosinated, and isotype 8 appeared to be detyrosinated at 20 d postanthesis. Since the carboxyl terminus of higher p...

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate α-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of α-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that α-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total α-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the α-tubulins was specific to fiber cells.

Young cotton (Gossypium hirsutum) ovules will produce fiber in vitro when floated on a defined culture medium. Our laboratory is interested in examining the effects of altered gravity environments on fiber development as a model for the effects of gravity on cell expansion and cellulose biosynthesis. Since liquid culture media are unsuitable for altered gravity experiments, addition of gelling agents to cotton ovule culture media is necessary. In this study we have systematically examined the effects of four gelling agents at several concentrations on fiber production in culture. A rapid screening method using toluidine blue O staining indicated that after 3 wk in culture, fiber growth on 0.15% (wt/vol) Phytagel™ medium was similar to fiber growth on liquid medium. More detailed analysis of fiber development revealed that fiber length was not influenced by the addition of Phytagel™. Accumulation of cellulose, however, was reduced 50–60% compared with fibers produced in liquid media after 3 wk in culture. The fiber cellulose content rose with additional time in culture for both solid and liquid media treatments. By 4 wk in culture, the difference in cellulose content of fiber cell walls grown on solid versus liquid media was less than 20%. This variance in growth response on gelled media could be due to differences in media matric potential, to the immobility of ions trapped within the gel, or to toxicity of contaminants copurifying with Phytagel™. By identifying why ovule growth and fiber cellulose biosynthesis are reduced in cultures grown on gelled media, it will be possible to reveal new information about these processes in system that is less complicated than physiological systems at the whole plant level.

Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca2+-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.

When cotton (Gossypium hirsutum, DPL 90ne) ovules are removed from the carpel on the day of anthesis and placed on an appropriate medium, fiber cells will differentiatein vitro from the ovule epidermis. Alpha-amanitin, an inhibitor of poly (A)+ RNA synthesis was added to ovule cultures at selected times after culture initiation to determine the timing of events in fiber development. Replicate cultures were initiated from day of anthesis, cotton ovules with α-amanitin being added to media at final concentrations ranging from 0.05μg/ml to 5.0 μg/ml. After 21 d in culture, ovules were evaluated for fiber length, percentage of ovules producing fiber, fiber dry weight, and fiber cellulose content. Cultures treated with α-amanitin on the day of anthesis to 2 d postanthesis were prevented from forming normal numbers of fiber cells. Addition of α-amanitin to cultures between 2 and 10 d postanthesis reduced the length that fiber cells attained in culture after 21 d. Addition of α-amanitin to cultures at 4 d postanthesis had as much an effect on the accumulation of cellulose in the fiber cell wall as the later addition of the inhibitor at 8 to 10 d postanthesis. The inhibitory effect of α-amanitin on fiber growth was reversible when ovules were transferred to amanitin-free medium within 2 d after culture initiation. Ovules placed on media containing α-amanitin for 5 d or longer could not overcome the inhibitory effects when transferred to media lacking the inhibitor. These results indicate critical time periods when RNA transcripts important for specific phases of development are being synthesized.

Yariv reagent [1,3,5-tris(4-β-d-glucopyranosyloxyphenylazo)-2,4,6-trihydroxybenzene] has been used in the purification/quantification of arabinogalactan-proteins and as a histochemical reagent to localize arabinogalactan-proteins in plant tissues. When developing cotton fiber cells were stained with β-glucosyl Yariv reagent, interference from other cell wall polymers, especially cellulose, was suspected. In order to examine this potential interference, we have developed two dye-binding assays to measure the binding of Yariv reagent in the presence of various carbohydrate polymers. Reaction conditions such as pH, ionic strength, and exposure time in the standard binding assays were investigated. The results show that β-glucosyl and α-galactosyl Yariv reagents bind to cellulose and several other glucans. Treatments with excess glucose in the assay to inhibit binding were only partially effective in preventing β-glucosyl Yariv reagent from binding to cellulose. Keywords: Arabinogalactan protein; cellulose; g...

Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H2O2 had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils.

Background Cotton, one of the world’s leading crops, is important to the world’s textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. Results We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome, even though G. raimondii contains a D genome (D5). Conclusions The library represents the first BIBAC library in cotton and related species, thus providing tools useful for integrative physical mapping, large-scale genome sequencing and large-scale functional analysis of the Upland cotton genome. Comparative sequence analysis provides insights into the Upland cotton genome, and a possible mechanism underlying the divergence and evolution of polyploid Upland cotton from its diploid putative progenitor species, G. raimondii.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the protein profiles of cotton (Gossypium hirsutum L.) fiber cells at two developmental stages. In the first stage, the cells ...

Secondary wall thickness is a function of maturity in cotton fibers. Fibers begin to elongate at anthesis, and elongation continues for approximately 20 days. As this elongation period ends, deposition of the secondary wall begins and continues for 25- 30 days until the boll "matures" and opens. The exact period of elongation and sec ondary thickening depends on factors such as variety, growing temperature, and light level. This microscopical study illustrates rates of secondary wall development in three cotton varieties, and shows effects of drastic changes in light and temperature, as well as differences in fiber development within a single boll.

Our objective was to develop a rapid, specific, and inexpensive method for measuring glucose concentrations in bolls of cotton (Gossypium hirsutum L.). A new procedure was developed, based on a reagent strip for measuring blood glucose in diabetic patients, and compared with three other glucose methods by assaying glucose standard solutions. The strip results were significantly correlated (r = 0.948) with known concentrations of glucose and comparable to other methods in both precision and accuracy. For aqueous extracts of locules (lint and seed) from 15 days-post-anthesis (DPA) cotton bolls, glucose levels were ∼ 10 mg g −1 fresh weight. The raegent strip strongly correlated to glucose hexokinae, glucose oxidase, and anthrone methods with correlation coefficients (r) of 0.983, 0.994, and 0.974, respectively. Thus, the strip technique should be useful for routine glucose determinations in cotton bolls considering the time (∼ 2 min/sample), cost (∼ $0.70/sample), and portability

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five-to sixfold ploidy increase approximately 60million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A t D t (in which t' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups. © 2012 Macmillan Publishers Limited. All rights reserved.

The expression of [alpha]- and [beta]-tubulin proteins in developing fibers and several other tissues of cotton (Gossypium hirsutum, cv Texas Marker 1) have been analyzed by immunoblots of one- and two-dimensional gels utilizing anti-tubulin antibodies as probes. As a percentage of total protein, fibers had greater amounts of tubulin than did hypocotyls, roots, leaves, or cotyledons. Both [alpha]- and [beta]-tubulin, having apparent molecular masses of approximately 50 kD and isoelectric points between pH 5 and pH 6, were resolved on a single two-dimensional gel. Under the conditions used, [alpha]-tubulin was less acidic in the isoelectric focusing dimension and migrated slightly faster in the sodium dodecyl sulfate dimension than did [beta]-tubulin. Nine [alpha]-tubulin isotypes that formed two distinct groups were identified on immunoblots of two-dimensional gels. The three most abundant [alpha]-tubulin isotypes were common to all tissues examined. Seven distinct [beta]-tubulin isotypes were also identified. Although their level of accumulation differed, four of the [beta]-tubulin isotypes were common to all tissues. Preferential accumulation of isotypes was more apparent in fibers than in the other tissues examined. Two [alpha]-tubulin isotypes and two [beta]-tubulin isotypes showed preferential accumulation in 10- and 20-d postanthesis fibers, respectively.

Gossypium hirsutum cellulose synthase catalytic subunit 4 (GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To understand the molecular mechanisms involved in transcriptional regulation of GhCesA4, β-glucuronidase (GUS) activity regulated by a GhCesA4 promoter (-2574/+56) or progressively deleted promoters were determined in both cotton tissues and transgenic Arabidopsis. The spatial regulation of GhCesA4 expression was similar between cotton tissues and transgenic Arabidopsis. GUS activity regulated by the GhCesA4 promoter (-2574/+56) was found in trichomes and root vascular tissues in both cotton and transgenic Arabidopsis. The -2574/-1824 region was responsible for up-regulation of GhCesA4 expression in trichomes and root vascular tissues in transgenic Arabidopsis. The -1824/-1355 region negatively regulated GhCesA4 expression in most Arabidopsis vascular tissues. For vascular expression in stems and leaves, the -898/-693 region was required. The -693/-320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis roots. Exogenous phytohormonal treatments on transgenic Arabidopsis revealed that phytohormones may be involved in the differential regulation of GhCesA4 during cotton fiber development.

Anionic isoperoxidases from cotton cotyledonary leaf tissue have been purified to electrophoretic homogeneity by chromatography on ion exchange, lectin affinity, and gel filtration matrices. The enzyme was stable and active at a wide range of temperatures with optimal activity at 55°C. Maximum activity was observed when the pH was adjusted from pH 5.5 to pH 7.5. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels indicated a relative molecular mass of 56 000 Da when the sample was treated with a disulfide reducing agent and heated. The electrophoretic mobility was altered when either the disulfide reducing agent or heat treatment was omitted. Isoelectric focusing under native conditions resolved the activity into three isozymes with isoelectric points of 4.2, 4.4 and 4.6. The K m of peroxidase for 4-aminoantipyrine and o -dianisidine was 350 and 36 μM, respectively. Enzyme activity was inhibited by elevated concentrations of H 2 O 2 and by disulfide reducing agents. Known inhibitors of peroxidase activity such as potassium azide, sodium cyanide and sodium sulfite inhibited cotton anionic peroxidases. The absorption spectrum showed maxima at 280 and 402 nm, indicating the presence of a heme group in the active enzyme. The extinction coefficient at 402 nm was 1.12 × 10 5 mol −1 cm −1 .

Rapid and dynamic changes in the expression of small RNAs are seen during ovule and fiber development in allotetraploid cotton., Background Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression. Results Here we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers. Conclusions Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

Gene expression profiles of developing cotton (Gossypium hirsutum L.) fibers from two near-isogenic lines (NILs) that differ in fiber-bundle strength, short-fiber content, and in fewer than two genetic loci were compared using an oligonucleotide microarray. Fiber gene expression was compared at five time points spanning fiber elongation and secondary cell wall (SCW) biosynthesis. Fiber samples were collected from field plots in a randomized, complete block design, with three spatially distinct biological replications for each NIL at each time point. Microarray hybridizations were performed in a loop experimental design that allowed comparisons of fiber gene expression profiles as a function of time between the two NILs. Overall, developmental expression patterns revealed by the microarray experiment agreed with previously reported cotton fiber gene expression patterns for specific genes. Additionally, genes expressed coordinately with the onset of SCW biosynthesis in cotton fiber correlated with gene expression patterns of other SCW-producing plant tissues. Functional classification and enrichment analysis of differentially expressed genes between the two NILs revealed that genes associated with SCW biosynthesis were significantly up-regulated in fibers of the high-fiber quality line at the transition stage of cotton fiber development. For independent corroboration of the microarray results, 15 genes were selected for quantitative reverse transcription PCR analysis of fiber gene expression. These analyses, conducted over multiple field years, confirmed the temporal difference in fiber gene expression between the two NILs. We hypothesize that the loci conferring temporal differences in fiber gene expression between the NILs are important regulatory sequences that offer the potential for more targeted manipulation of cotton fiber quality.

Analysis of the biochemical factors required for improved cotton fiber growth is aided by a collection of near isogenic mutants. The cotton mutants (Gossypium hir sutum L. Texas Marker-1 background ) tested in this study include immature fiber, pilose, and naked seed. Fiber physical properties for each mutant were determined for two years of greenhouse-grown and one year of field-grown cotton plants. Yarn spun from 50-gram fiber samples was subjected to physical properties testing. A com parison of open-end and ring spinning methods was made for two of the fiber samples. The comparison of nep content in yam and fiber between the mutants and Texas Marker-1 control lines shows one mutant (naked seed) with a substantial reduction in biological nep content. Another mutant, pilose, has a substantial increase in bio logical neps over the control fiber. Quantitative data on fiber physical properties ob tained from this study support the mostly descriptive information previously reported for these mutants lines.

Cotton fibers (cottonseed trichomes) are a well-established model system for investigating cellulose biosynthesis. Cotton ovules, excised from ovaries prior to fertilization, will continue to develop and produce trichomes in vitro under defined conditions when auxin and gibberellic acid are present. Our team is also using cotton fiber development in vitro as a model to investigate plant cell differentiation at the single-cell level and to identify the regulatory components of the transition from cell elongation to secondary wall biogenesis. These two developmental phases determine key properties of fiber quality and yield. Fiber cells elongate to 1-1.5 cm in culture and then produce a thick, cellulosic secondary cell wall. During the transition from cell expansion to secondary wall thickening, the rate of cellulose biosynthesis rises nearly 100-fold. A similar increase in cellulose biosynthetic capacity occurs in fibers produced in vitro by ovule cultures. Although the genes for the cellulose synthase catalytic subunit, CesA, were first described from cotton fiber, little is known about

The number of cotton (Gossypium sp.) ovule epidermal cells differentiating into fiber initials is an important factor affecting cotton yield and fiber quality. Despite extensive efforts in determining the molecular mechanisms regulating fiber initial differentiation, only a few genes responsible for fiber initial differentiation have been discovered. To identify putative genes directly involved in the fiber initiation process, we used a cotton ovule culture technique that controls the timing of fiber initial differentiation by exogenous phytohormone application in combination with comparative expression analyses between wild type and three fiberless mutants. The addition of exogenous auxin and gibberellins to pre-anthesis wild type ovules that did not have visible fiber initials increased the expression of genes affecting auxin, ethylene, ABA and jasmonic acid signaling pathways within 1 h after treatment. Most transcripts expressed differentially by the phytohormone treatment in vitro were also differentially expressed in the ovules of wild type and fiberless mutants that were grown in planta. In addition to MYB25-like, a gene that was previously shown to be associated with the differentiation of fiber initials, several other differentially expressed genes, including auxin/indole-3-acetic acid (AUX/IAA) involved in auxin signaling, ACC oxidase involved in ethylene biosynthesis, and abscisic acid (ABA) 8'-hydroxylase an enzyme that controls the rate of ABA catabolism, were co-regulated in the pre-anthesis ovules of both wild type and fiberless mutants. These results support the hypothesis that phytohormonal signaling networks regulate the temporal expression of genes responsible for differentiation of cotton fiber initials in vitro and in planta.

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.

Although the fibers of cotton (Gossypium hirsutum L.) are single cells with a secondary wall composed primarily of cellulose, the cell-wall polymers of the fibers are technically difficult to characterize with respect to molecular weights. This limitation hinders understanding how the fiber wall composition changes during development, particularly with respect to genotypic variations, and how the molecular composition is related to physical properties. We analyzed cell-wall polymers from cotton fibers (cultivar, Texas Marker-1) at several developmental stages (8–60 days post-anthesis; DPA) by gel-permeation chromatography of components soluble in dimethyl acetamide and lithium chloride. This procedure solubilizes fiber cell-wall components directly without prior extraction or derivatization, processes that could lead to degradation of high-molecular-weight components. Cellwall polymers from fibers at primary cell-wall stages had lower molecular weights than the cellulose from fibers at the secondary wall stages; however, the high-molecularweight cellulose characteristic of mature cotton was detected as early as 8 DPA. High-molecular-weight material decreased during the period of 10–18 DPA with concomitant increase in lower-molecular-weight wall components, possibly indicating hydrolysis during the later stages of elongation.

The presence of cotton ( Gossypium hirsutum L.) fiber transcripts coding for a germin-like protein (GLP) was revealed by differential display analysis in which early stages of cotton fiber development between a wild type line, Texas Marker-1 (TM1) and a near isogenic mutant, Naked Seed (N1) were compared. Transcripts of the cotton GLP ( GhGLP1) accumulated specifically in TM1, but did not accumulate in the mutant although the GhGLP1 gene was present in both lines. The deduced protein sequence of GhGLP1 is similar to Prunus persica auxin-binding proteins, a barley ADP-glucose pyrophosphatase/phosphodiesterase and two different classes of hydrogen peroxide-producing enzymes: wheat germin oxalate oxidase and moss extracellular Mn-superoxide dismutase. Cotton GLPs constitute a multigene family like those of Arabidopsis, rice, soybean, and barley. GhGLP1 transcripts accumulated to their highest levels during the period of fiber expansion, followed by a sharp decline when the rate of cell expansion decreased. While germins and GLPs appear to be involved in defense mechanisms in some plants, both biotic and abiotic stress down-regulated the expression of GhGLP1. Numerous functions have been proposed for dicot GLPs. However, to date, there is little direct evidence for how these proteins function in vivo. The association of maximal GhGLP1 expression with stages of maximal cotton fiber elongation suggests that some GLPs may be important for cell wall expansion.

Secondary wall thickness is considered to be a function of maturity in cotton fibers. Normal fibers are formed from a single epidermal cell of a fertilized ovule. Development of these cells into fibers occurs in two, possibly overlapping, stages. Fibers begin to elongate at anthesis, and elongation continues for approximately 20 days postanthesis (DPA). As this elongation period ends, deposition of secondary wall begins, and continues for 25-30 days until the boll “matures” and opens. The exact period of elongation and secondary thickening is dependent on factors such as variety, growing temperature, and light level. Between six and nine weeks, wall thickening terminates as bolls begin to open.

Growth and development of cotton fibers in a developmental mutant, Ligon-lintless, and its near isogenic wild type, Texas Marker-1, were compared in ovule and cell suspension cultures. In both organ and cell cultures the pattern of growth of fiber cells from the two genotypes mimicked the pattern ofin vivo growth. The timing of fiber cell initiation soon after anthesis in Ligon-lintless suggests that the fiber cells on this mutant are analogous to the commercially important lint fibers. Length distributions of elongated cells from cell suspension culture of Ligon-lintless and Texas Marker-1 indicate that the length attained in culture is affected by the genotype of the explant tissue.

The biosynthesis of cotton (Gossypium hirsutum L. ‘Stoneville 208′) peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [35S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [35S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.

The lectin, wheat germ agglutinin (WGA), is synthesized de novo by developing wheat ( Triticum aestivum , L.) embryos but is not synthesized or localized in developing endosperm as shown by radioimmunoassay. Young embryos removed from the grain and cultured on a defined medium germinate precociously and concomitantly cease WGA synthesis. In vitro precocious germination of young embryos is reversibly inhibited by low levels (1–100 μ M ) of the plant growth substance abscisic acid (ABA). Embryos inhibited from germinating by this growth regulator not only continue synthesizing WGA, but do so at an accelerated rate when compared with embryos left associated with the grain.