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Cottonseed is an important source of protein, oil, and minerals for human health and livestock feed. Therefore, understanding the physiological and genetic traits influencing the nutrient content is critical. To our knowledge, there is no information available on the effects of leaf shape—curly leaf (CRL)—on cottonseed protein, oil, and minerals. Therefore, the objective of the current research was to investigate the effect of the curly leaf trait on cottonseed protein, oil, and minerals in cotton lines differing in leaf shape. Our hypothesis was that since leaf shape is known to be associated with nutrient uptake, assimilation, and photosynthesis process, leaf shape can influence seed protein, oil, and minerals. A two-year field experiment using two curly leaf lines (Uzbek CRL and DP 5690 CRL) and one normal leaf (DP 5690 wild type) line was conducted in 2014 and 2015 in Stoneville, MS, USA. The experiment was a randomized complete block design with three replicates. The results showed that both Uzbek CRL and DP 5690 wild type lines had higher seed oil, and nutrients N, P, K, and Mg than DP 5690 CRL. Calcium was higher in DP 5690 CRL for two years and protein was only higher than the parents in 2015. Consistent significant positive and negative correlations between some nutrients were observed, which may be due to environmental conditions, especially heat. This indicates that curly leaf trait may partially regulate the accumulation of these nutrients in seeds. The results demonstrated that leaf shape trait—curly leaf—can affect cottonseed nutritional qualities. This research is important to breeders for cotton selection for high seed oil or protein, and to other researchers to further understand the genetic impact of leaf shapes on seed nutritional quality. It is also important for scientists to use leaf shape as a tool for physiological, biochemical, and morphological research related to leaf development.

Information about the effects of phenotype traits on cottonseed protein, oil, and nutrients is scarce. The objective of this research was to investigate the effects of leaf color trait on seed nutrition in near-isogenic Gossypium hirsutum cotton expressing green (G) and yellow (Y) leaf color phenotypes. Our hypothesis was that leaf color can influence the accumulation of nutrients in seeds. Sets of isogenic lines were: DES 119 (G) and DES 119 (Y); DP 5690 (G) and DP 5690 (Y); MD 51ne (G) and MD 51ne (Y); SG 747 (G) and SG 747 (Y). Each NIL set is 98.44 % identical. Parent line SA 30 (P) was used as the control. The experiment was repeated for two years (2014 and 2015). The results showed that, in 2014, seed oil in DES 119 (G) and SG 747 (G) were significantly higher than their equivalent yellow lines. Green lines showed higher content of phosphorus compared with yellow lines. Higher levels of Cu, Fe, Mn, Ni, and Zn were recorded in DES 119 (G) and MD 51ne (G). In 2015, seed protein, oil, C, N, P, B, Cu, and Fe were higher in green lines than in yellow lines. There was a significant correlation between protein and nutrients, and between oil and nutrients in 2015, but not in 2014 as the temperature was warmer in 2015 than in 2014. This research demonstrated that leaf color can alter seed composition and mineral nutrition under certain environmental growing conditions such as temperature.

Palmer amaranth (Amaranthus palmeri S. Wats.) is a major weed problem of cotton (Gossypium hirsutum L.) production systems in the southern United States. Hyperspectral remote sensing has shown promise as a tool for crop weed discrimination, and there is a growing interest in using this technology for identifying weeds in cotton production systems. Information is lacking on differentiating Palmer amaranth from cotton with an okra leaf structure based on canopy hyperspectral reflectance properties. Two greenhouse studies were conducted to compare canopy hyperspectral reflectance profiles of Palmer amaranth to canopy hyperspectral reflectance profiles of okra and super-okra leaf cotton and to identify optimal regions of the electromagnetic spectrum for their discrimination. Ground-based hyperspectral measurements of the plant canopies were obtained with a spectroradiometer (400 - 2350 nm range). Analysis of variance (ANOVA, p ≤ 0.05), Dunnett’s test (p ≤ 0.05), and difference and sensitivity measurements were tabulated to determine the optimal wavebands for Palmer amaranth and cotton discrimination. Results were inconsistent for Palmer amaranth and okra leaf cotton separation. Optimal wavebands for distinguishing Palmer amaranth from super-okra leaf cotton were observed in the shortwave infrared region (2000 nm and 2180 nm) of the optical spectrum. Ground-based and airborne sensors can be tuned into the shortwave infrared bands identified in this study, facilitating application of remote sensing technology for Palmer amaranth discrimination from super-okra leaf cotton and implementation of the technology as a decision support tool in cotton weed management programs.

Palmer amaranth (Amaranthus palmeri S. Wats.) invasion negatively impacts cotton (Gossypium hirsutum L.) production systems throughout the United States. The objective of this study was to evaluate canopy hyperspectral narrowband data as input into the random forest machine learning algorithm to distinguish Palmer amaranth from cotton. The study focused on differentiating the Palmer amaranth from cotton near-isogenic lines with bronze, green, and yellow leaves. A spectroradiometer was used to acquire hyperspectral reflectance measurements of Palmer amaranth and cotton canopies for two separate dates, December 12, 2016, and May 14, 2017. Data were collected from plants that were grown in a greenhouse. The spectral data were aggregated to twenty-four hyperspectral narrowbands proposed for study of vegetation and agriculture crops. Those bands were tested by the conditional inference version of random forest (cforest) to differentiate the Palmer amaranth from cotton. Classifications were binary: Palmer amaranth and cotton bronze, Palmer amaranth and cotton green, and Palmer amaranth and cotton yellow. Classification accuracies were verified with overall, user’s, and producer’s accuracy. For the two dates combined, overall accuracy ranged from 77.8% to 88.9%. The highest overall accuracies were observed for the Palmer amaranth versus the cotton yellow classification (88.9%, December 12, 2016; 83.3%, May 14, 2017). Producer’s and user’s accuracies range was 66.7% to 94.4%. Errors were predominately attributed to cotton being misclassified as Palmer amaranth. The overall results indicated that cforest has moderate to strong potential for differentiating Palmer amaranth from cotton when it used hyperspectral narrowbands known to be useful for vegetation and agricultural surveys as input variables. This research further supports using hyperspectral narrowband data and cforest as decision support tools in cotton production systems.

To implement strategies to control Palmer amaranth (Amaranthus palmeri S. Wats.) and redroot pigweed (Amaranthus retroflexus L.) infestations in cotton (Gossypium hirsutum L.) production systems, managers need effective techniques to identify the weeds. Leaf light reflectance measurements have shown promise as a tool to distinguish crops from weeds. Studies have targeted plants with green leaves. This study focused on using leaf hyperspectral reflectance data to develop spectral profiles of Palmer amaranth, redroot pigweed, and cotton and to determine regions of the light spectrum most sensitive for pigweed and cotton discrimination. The study focused on cotton near-isogenic lines created to have bronze, green, or yellow colored leaves. Reflectance measurements within the 400 to 2500 nm spectral range were obtained from cotton and weed plants grown in a greenhouse in 2015 and 2016. Two scenarios were evaluated for the comparison: (1) Palmer amaranth versus cotton lines and (2) redroot pigweed versus cotton lines. Statistical significance (p ≤ 0.05) was determined with analysis of variance (ANOVA) and Dunnett’s test. Sensitivity measurements were tabulated to determine the optimal region of the light spectrum for weed and cotton line discrimination. Optimal bands for weed and cotton separation were 600 to 700 nm (both weeds versus cotton bronze and cotton yellow), 710 nm (Palmer amaranth versus cotton green), and 1460 nm (redroot pigweed versus cotton green). Spectral bands were identified for separating Palmer amaranth and redroot pigweed from cotton lines with bronze, green, and yellow leaves. Ground-based and airborne sensors can be tuned into the regions of spectrum identified, facilitating using remote sensing technology for Palmer amaranth and redroot pigweed identification in cotton production systems.

Fuzzless cotton often has traits desirable to the cotton industry, including longer fibers, reduced short fiber content, fewer neps, and improved ginning efficiency. This two-year field study described yield and fiber properties of five pairs of fuzzy and fuzzless near-isogenic lines, developed from fuzzless parent Gossypium hirsutum SA 243 crossed with five fuzzy seeded parents, and processed using saw or roller gins. Yield and fiber data were collected from a two-year field study. The fuzzless lines generally had longer and stronger fibers, lower immature fiber content, higher maturity ratios, reduced short fiber content, higher fineness and micronaire values, and lower lint percentages compared to their fuzzy counterparts. Longer fibers were found in saw ginned samples, and roller ginned samples had higher percentages of short fibers and fewer neps. The fuzzless lines had improved fiber properties useful to the cotton industry, but poor lint yields of only 7% to 14% in these lines limits their utility ...

Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li 2 ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species. Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li 2) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li 2 mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D5 genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li 2 from its NIL. Next, we sequenced total DNA from pools of F2 plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F2 population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li 2 fiber cells.

Gossypium hirsutum flowers are easily emasculated by splitting the staminal column with the fingernail and removing the corolla and androecium. However, any damage to the ovary is considered detrimental to successful boll formation and damaged flowers are typically discarded. This study evaluated boll retention after different emasculation treatments. Removal of the membrane surrounding the ovary reduces boll retention compared to self-pollinated flowers, and showed a similar response across 18 genotypes. Damage to the ovary wall reduced boll retention compared to emasculate flowers without ovary damage. Damaged flowers could be cross-pollinated to produce additional bolls as compared to discarding the flowers. Key words: Breeding, emasculation, Gossypium hirsutum, hybridization

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.

Haploid seed-producing semigamy in cotton is controlled by a single incompletely dominant gene (Se). However, its molecular mechanism is currently poorly understood. In this study, the mRNA differential display (DD) was employed to compare gene expression between non-semigametic Pima S-1 (Gossypium barbadense) and its naturally occurring semigametic mutant 57–4. Of the 70 DD cDNA fragments isolated from a total of 3,000 cDNA fragments, some exhibited semigamy-specific expression across anthers and/or ovules. Of the 61 cDNA fragments cloned and sequenced, nine showed high homology to genes encoding for α-tubulin, SPK1, Rab11, sucrose synthase, isocitrate lyase, phosphoethanolamine N-methyltransferase, phospholipase D, MADS 7, and WRKY 2. From cDNA–single-strand conformational polymorphism using primers designed from DD sequences, qualitative expression differences in six genes between the semigametic and non-semigametic genotypes were validated. However, a primer pair designed from an α-tubulin gene sequence yielded PCR products with quantitative difference in band intensity, which is linked to the semigamy gene at a recombination frequency of 0.316 in a (57–4 × Pima S-1) × Pima S-1 BC1F1 population. Its expression in the semigametic 57–4 anthers was suppressed by 2-fold as compared to that in Pima S-1 using a quantitative reverse transcription PCR analysis. This study identified several novel genes that were missed in prior microarray analysis, and their potential roles with regards to reproductive biology are discussed.

Three virescent mutants and two wild-type lines of upland cotton were evaluated for chlorophyll (Chl), carotenoids (Car), carbon exchange ratio (CER), water use efficiency (WUE), chlorophyll fluorescence, and growth. Lines 30 and 174 were allelic for the virescent locus and had reduced ratios of ChlTot, Chla, Chlb, CER, and WUE compared to DP90ne (in both 1995 and 1996). Line 174 had a higher CER than line 30 in both 1995 and 1996. The CER values of line 174 were similar to those of line 31. The only differences in fluorescent measurements were found in initial fluorescence (F0) values, reflecting the concentrations of Chl. Of the three plant growth indicators, only plant height showed consistent statistical differences during growth. Reduced plant height was associated with lower chlorophyll concentrations. The number of main-stem nodes was slightly affected, with statistical differences occurring on only two dates—41 and 82 days after planting. The data indicate that these virescence alleles negatively ...

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609 bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the α-factor secretion sequence in pPICZαA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5 kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.

Segregating populations were developed to evaluate the inheritance of the fiberless seed phenotype of upland cotton (Gossypium hirsutum L.) line SL1-7-1. We report the inheritance of fuzzy, fuzzless and fiberless seed from crosses of SL1-7-1 with wildtype DP5690, Mexican fuzzless seed UA 3-3 (accession 143), Ballard fuzzless seed (accession 243), and MD17. Results from the F1, F2 and F2:3 progeny derived from the SL1-7-1 X DP5690 indicated that the expression of the fiberless phenotype fit a three loci model with one locus being the dominant fuzzless seed allele N 1 . The other two loci were tested to verify whether they were allelic to either recessive fuzzless seed alleles n 2 or n 3 . Using the segregation ratios of the F2 progeny derived from the 143 X SL1-7-1 cross and F2-derived F3 families from SL1-7-1 X DP5690 with fuzzy seed (lacked N 1 ), it is proposed that SL1-7-1 lacks the recessive n 2 allele, but contains the n 3 allele in the genotype of SL1-7-1. The third locus was previously not characterized and has been designated as fl 1 (fiberless), therefore, the genotype for the fiberless phenotype of SL1-7-1 is N 1 N 1 fl 1 fl 1 n 3 n 3 . Fiberless lines MD17 X SL1-7-1 were crossed to verify similarities in genotypes between line and the genotype model predictability. Various combinations of the homozygous and heterozygous expression of N 1 , n 2 , n 3 and fl 1 allele produced plants with lower lint percentages.

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf (2) from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf (2) gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf(2) rf(2)) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf(2) rf(2)) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf(2) pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf(2) to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.

Fifteen fuzzless seed lines in “obsolete” backgrounds of Upland cotton (Gossypium hirsutum L.) were obtained from the National Cotton Germplasm Collection and evaluated for fuzzless seed genotype, lint percent, and lint quality. Fourteen of these fifteen lines were found to be homozygous for the dominant fuzzless seed allele N 1. Only one line was homozygous for the recessive fuzzless seed allele n 2. The measured lint percent of each line was very stable through time, however, large variability existed between many of the N 1 lines ranging from 0.7 to 23.6% lint. The lint percent for the n 2 line was 24.4%. Scanning electron microscopy was used to differentiate patterns of lint initiation on 1 day post anthesis ovules. General patterns included: first, lint initiation restricted to the chalazal end of the seed crest; second, lint initiation along the seed crest and laterally around the chalazal end of the ovule; and third, lint initiation covered all but the micropylar end of the ovule. Lint quality was evaluated for each line using the Advanced Fiber Information System (AFIS) which included measurements of length, maturity, and fineness. The means of each measurement varied among the fuzzless seed lines with significant differences between fiber length, short fiber content, immature fiber content, fineness, and maturity ratio. No correlation was found between lint percent and any of the lint quality measurements. These lines will provide a valuable resource for the study of fiber initiation and lint quality.

Our previous research, conducted under well-watered conditions without fertilizer application, showed that fuzziness cottonseed trait resulted in cottonseed nutrition differences between fuzzy (F) and fuzzless (N) cottonseed. Under water stress conditions, B mobility is further limited, inhibiting B movement within the plant, affecting seed nutrition (quality). Therefore, we hypothesized that both foliar B and water stress can affect B mobility, altering cottonseed protein, oil, and mineral nutrition. The objective of the current research was to evaluate the effects of the fuzziness seed trait on boron (B) and seed nutrition under water stress and foliar B application using near-isogenic cotton lines (NILs) grown in a repeated greenhouse experiment. Plants were grown under-well watered conditions (The soil water potential was kept between -15 to -20 kPa, considered field capacity) and water stress conditions (soil water potential between -100 and -150 kPa, stressed conditions). Foliar B was applied at a rate of 1.8 kg B ha(-1) as H3BO3. Under well-watered conditions without B the concentrations of seed oil in N lines were higher than in F lines, and seed K and N levels were lower in N lines than in F lines. Concentrations of K, N, and B in leaves were higher in N lines than in F lines, opposing the trend in seeds. Water-stress resulted in higher seed protein concentrations, and the contribution of cell wall (structural) B to the total B exceeded 90%, supporting the structural role of B in plants. Foliar B application under well-watered conditions resulted in higher seed protein, oil, C, N, and B in only some lines. This research showed that cottonseed nutrition differences can occur due to seed fuzziness trait, and water stress and foliar B application can alter cottonseed nutrition.

A cDNA encoding metallothionein (MT) was isolated from a library constructed with poly A(+) RNA purified from 48 h etiolated cotton (Gossypium hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). The cotton MT3 protein sequence is 76.2, 69.8, 66.7, 60.3 and 33.5% identical to MT3 from Carica papaya, Rubus idaeus, Ribes nigrum, Citrus unshiu, and Gossypium hirsutum type I MT, respectively. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 9 residues to the MT3 (after Factor Xa digestion site) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein began inhibiting bacterial growth after 1 h. Addition of Cu (1 muM to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferentially binds Cu.

Mapping-by-sequencing and SNP marker analysis were used to fine map the Ligon-lintless-1 ( Li 1 ) short fiber mutation in tetraploid cotton to a 255-kb region that contains 16 annotated proteins. The Ligon-lintless-1 (Li 1 ) mutant of cotton (Gossypium hirsutum L.) has been studied as a model for cotton fiber development since its identification in 1929; however, the causative mutation has not been identified yet. Here we report the fine genetic mapping of the mutation to a 255-kb region that contains only 16 annotated genes in the reference Gossypium raimondii genome. We took advantage of the incompletely dominant dwarf vegetative phenotype to identify 100 mutants (Li 1 /Li 1 ) and 100 wild-type (li 1 /li 1 ) homozygotes from a mapping population of 2567 F2 plants, which we bulked and deep sequenced. Since only homozygotes were sequenced, we were able to use a high stringency in SNP calling to rapidly narrow down the region harboring the Li 1 locus, and designed subgenome-specific SNP markers to test the population. We characterized the expression of all sixteen genes in the region by RNA sequencing of elongating fibers and by RT-qPCR at seven time points spanning fiber development. One of the most highly expressed genes found in this interval in wild-type fiber cells is 40-fold under-expressed at the day of anthesis (DOA) in the mutant fiber cells. This gene is a major facilitator superfamily protein, part of the large family of proteins that includes auxin and sugar transporters. Interestingly, nearly all genes in this region were most highly expressed at DOA and showed a high degree of co-expression. Further characterization is required to determine if transport of hormones or carbohydrates is involved in both the dwarf and lintless phenotypes of Li 1 plants.

Segregating populations were developed to evaluate the inheritance of the fuzzless seed phenotypes in upland cotton (Gossypium hirsutum L.). Accession 143 of the Mississippi Obsolete Variety Collection (MOVC) has a fuzzless seed phenotype. This line carries the n 2 locus which is recessive to the seed fuzz phenotype. Data from the F 2 , BC 1 F 1 , F 2 : 3 , and BC 1 F 2 populations of DP 5690 x 143 fit a two-loci model for expression of the recessive fuzzless seed phenotype. Fuzzless seeds were obtained in n 2 n 2 plants when a second recessive locus (n3) was present. The dominant N 3 allele found in DP 5690 confers the fuzzy seed phenotype in homozygous n 2 plants. Accession 243 of the MOVC carries the N 1 locus, which is dominant to the presence of seed coat fuzz. No variation from expected ratios was observed in the F 2 , BC 1 F 1 , F 2 : 3 , and BC 1 F 2 populations of the DP 5690 x 243 cross. The N 3 allele had no apparent effect on the expression of the N 1 locus. In a cross between accessions 243 x 143, a few plants were observed which were completely devoid of lint and fuzz fiber (fiberless). A fiberless line was developed from one of these fiberless plants. This line was designated MD 17 fiberless. In a cross between DP 5690 x MD 17 fiberless, we demonstrated that at least three loci were involved in the expression of the fiberless phenotype. The involvement of n 2 and n 3 in the expression of this fiberless phenotype was demonstrated in the F 2 progeny of the cross between 143 x MD 17 fiberless. This is the first demonstration that N 1 , n 2 , and n 3 interacted to produce fiberless seed.

Tolerance to drought in plants is not a simple trait, but a complex of mechanisms working in combination to avoid or to resist water deficit. Genotypes that differ in tolerance to water deficit may show qualitative and quantitative differences in gene expression when submitted to drought periods. Four cotton (Gossypium hirsutum L.) genotypes (Siokra L-23, Stoneville 506, CS 50 and T-1521) with contrasting responses to water deficit stress were studied using the Differential Display (DD) technique to identify and isolate genes which may differ among them. Fifty-two cDNA fragments differentially expressed during water deficit were isolated, cloned and sequenced. Search of gene bank databases showed that two cDNA clones, A12B15-6 and A12B13-1, have high homology with a heat shock protein that binds to calmodulin found in Nicotiana tabacum (2.9e-32 P(N)) and with an Arabidopsis thaliana trehalose-6-phosphate synthase enzyme (9.0e-37 P(N)), respectively. One of the presumed functions of heat shock proteins is related to prevention of protein denaturation during cellular dehydration. Trehalose-6-phosphate synthase is involved in the production of trehalose, a disaccharide known to osmotically protect cell membranes during dehydration. The HSP homologue was found to be differentially expressed during the drought period in two drought tolerant genotypes but not in drought-sensitive genotypes. The trehalose-6-phosphate synthase homologue was also up-regulated during water deficit stress, however, all four genotypes were induced to express this homologue. Ribonuclease protection assays confirmed these results. This is an important finding since there are only few reports of trehalose presence in higher plants and none in cotton. Tolerância à seca em plantas não é uma característica simples, mas sim um complexo de mecanismos trabalhando em combinação para evitar ou tolerar períodos de déficit hídrico. Genótipos que diferem em tolerância à déficits hídricos apresentam diferenças qualitativas e quantitativas de expressão gênica. Quatro genótipos de algodão (Gossypium hirsutum L.) (Siokra L-23, Stoneville 506, CS 50 e T-1521) que apresentam distintas respostas quando submetidos ao déficit hídrico, foram estudados pelo uso da técnica "Differential Display -- (DD)". Com esta técnica é possivel identificar e isolar genes diferencialmente expressos nestes genótipos. Cinquenta e dois fragmentos de DNA complementar (DNAc) foram isolados, clonados e seqüenciados. Análises em bancos de dados mostraram que dois clones de DNAc, A12B15-6 e A12B13-1 tem alta homologia com uma proteína de choque térmico que adere a calmodulin, e é encontrada em Nicotiana tabacum (2.9e-32 P(N)) e com a enzima trehalose-6-fosfato sintase de Arabidopsis thaliana (9.0e-37 P(N)), respectivamente. Uma das funções propostas para as proteínas de choque térmico (HSP) é a de prevenir a desnaturação de proteínas durante a desidratação celular. A enzima trehalose-6-fosfato sintase esta envolvida na produção de trehalose, um disacarídio conhecido por estar envolvido na proteção de membranas celulares durante a desidratação. O homólogo da HSP foi encontrado diferencialmente expresso durante o período de seca em dois genótipos tolerantes à seca, mas não nos genótipos sensíveis. O homólogo da enzima trehalose-6-fosfato sintase foi também induzido durante o déficit hídrico, entretanto, todos os quatro genótipos expressaram este homólogo. Ensaios protegidos de ribonucleases confirmaram os resultados.

Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li 2 ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species. Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D5) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li 2) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li 2 mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D5 genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li 2 from its NIL. Next, we sequenced total DNA from pools of F2 plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F2 population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li 2 fiber cells.

Cotton fiber walls (1–2 days post anthesis) are distinctly bilayered compared to those of nonfiber epidermal cells, with a more electron-opaque outer layer and a less electron-opaque, more finely fibrillar inner layer. When probed with antibodies and affinity probes to various saccharides, xyloglucans and cellulose are found exclusively in the inner layer and de-esterified pectins and extensin exclusively in the outer layer. Ovular epidermal cells that do not differentiate into fibers have no pectin sheath, but are labelled throughout with antixyloglucan and cellulase-gold probes. Middle lamellae between adjacent cells were clearly labelled with the antibodies to de-esterified pectins, however. Similarly, cell walls of leaf trichomes have a bilayered wall strongly enriched in pectin, whereas other epidermal cells are not bilayered and are pectin poor. These data indicate that one of the early markers of fiber and trichome cells from other epidermal cells involves the production of a pectin layer. The de-esterified pectins present in the ensheathing layer may allow for expansion and elongation of the fiber cells that does not occur in the other epidermal cells without such a sheath or may even be a consequence of the elongation process.

Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-grown cotton (Gossypium hirsutum L.) seedling cDNA library. Nucleotide sequence analysis revealed two different CA isoforms designated GhCA1 and GhCA2. The encoded polypeptides possess N-terminal serine/threonine-rich regions indicative of plastid transit peptides, and approximately 80% sequence identity to other plant plastidial beta-CAs. The GhCA1 cDNA encodes a nearly complete preprotein of 323 amino acids with a molecular mass of 34.9 kDa and a predicted mature protein of 224 amino acids with a molecular mass of 24.3 kDa. Eleven nucleotide differences within ORFs of GhCA1 and GhCA2 result in 5 conservative amino acid substitutions. The 3' GhCA2 untranslated region contains five additional substitutions and one single nucleotide addition. GhCA1 clones, nearly full-length or with 70% of the transit peptide deleted, were expressed as LacZ alpha fusion proteins in E. coli. Lysates of these strains contained 9-fold higher levels of CA activity as compared to untransformed controls and this activity was inhibited by CA-specific inhibitors. Sulfanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombinant CA activity approximately 50%, 65%, and 75%, respectively. In plant tissue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively. Although CA activity was bighest in extracts of mature cotton leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in the cotyledons of dark-grown cotton seedlings. Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated seeds, suggesting a role for CA in postgerminative growth.

Photosynthesis is an important component of upland cotton (Gossypium hirsutum L.) yield, but little has been done to increase the photosynthetic performance within the cotton germplasm pool. Part of this dilemma is due to the multi-component aspect of this process and also to lack of information on genetic variation among such components. The objectives of this research were to identify genetic variability in photosynthetic components for six cotton genotypes previously shown to differ in leaf CO2-exchange rates (CER) and to determine if an afternoon decline in photosynthesis altered genotypic differences in CER. CO2-exchange rates were measured at several internal CO2 levels (Ci) to generate CER vs. Ci curves for each genotype and thereby isolate some of the components of photosynthesis. Ribulose 1,5 bisphosphate carboxylase-oxygenase (Rubisco), hydroxypyruvate reductase, malate dehydrogenase, and catalase activities were assayed on leaves used to generate the CER vs. Ci curves. Ambient CER and chlorophyll (Chl) fluorescence measurements were taken before and after solar noon to test for an afternoon decline in photosynthesis. Dixie King, a low ambient CER genotype, exhibited a greater CO2 compensation point, lower carboxylation efficiency, and reduced Photosystem II (PS II) activity than the other genotypes. The carboxylation efficiency of DES 119 was 13% greater than STV 508 and 29% greater than Dixie King, but not different from the other genotypes in 1994. Pee Dee 3 had greater maximum assimilation rate (A) than all other genotypes except STV 213 in 1993. Although no significant genotype by time of day interaction was detected, CER and Chl fluorescence variable to maximum ratio (Fv/Fm) were reduced 8% and 39%, respectively, in the afternoon as compared to the morning. This study demonstrates genetic variations in many of the components of photosynthesis. However, the narrow range of variation in such components for superior photosynthesizing genotypes explains why difficulties are encountered when breeding for increased photosynthesis.

Tonoplast intrinsic proteins (TIPs) have been implicated in the process of cell elongation, such as occurs in the developing cotton fiber. We have isolated a cDNA clone (997 bp in length) from a cotton (Gossypium hirsutum L.) library which putatively encodes a protein of 248 residues (Mr 25079) with 85% identity to Arabidopsis δ-TIP. The derived amino acid sequence included two conserved sequences associated with major intrinsic proteins (SGxHxNPA at residues 78 to 85, NPA residues at 197 to 199) and a cysteine residue at 116 which is reported to bind mercury in Arabidopsis δ-TIP. The polymerase chain reaction was used to generate partial genomic clones of the cotton δ-TIP. In comparison to other genomic TIP sequences, the number (two) and position of the introns were conserved in cotton. Comparing the TIP sequences from cotton revealed two subfamilies, which were consistently distinguished by a Tsp45I restriction site polymorphism. This polymorphism was used to demonstrate that TIP subfamilies were specific to either the A or D genomes of Gossypium. When δ-TIP DNA fragments were amplified from cDNA of fiber 14 days after anthesis, the A and D were found, indicating the presence of δ-TIP transcripts in these elongating cells.

There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using four sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (NF) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (FN) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds. Generally and with few exceptions, seed protein was higher in fuzzy lines that in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality.

Summary Total protein was extracted from ovules during the temporal development of cotton fiber initiation and compared by 2-D PAGE. Comparisons were made between two cultivars of cotton (Gossypium hirsutum L.), i.e., a «normal» fiber-producing line (DPL 5690) and a fiberless line (SL 1-7-1). In all, 37 protein differences were found, and these were categorized into 5 groups. The groups have been designated either V-, D-, S-, B-, or E-types: V for varietal differences, which are proteins found at all ages but occurring in only one line; D for developmental differences unique to DPL 5690; S for developmental differences unique to SL 1-7-1; B (before anthesis) are proteins found in both lines that diminish postanthesis; and E (embryo-genesis) are proteins found in both lines that accumulate postanthesis. Six V-type proteins have been identified, with five of these specific to the fiberless line. Sixteen D-type proteins were identified in which 14 (88 %) accumulated postanthesis. Of the 14 proteins, 5 proteins were specific to the normal line. The data on the 5 proteins indicate that they are involved in early events of fiber development. Eight S-type proteins were identified; however, unlike D-type proteins, only 2 (25 %) accumulate postanthesis. Three B-type proteins were identified, each with the same molecular mass. Four E-type proteins were identified and, along with the B-type, are (probably) involved in ovule growth or associated with the changes taking place in the ovule at fertilization. Overall, these data provide a catalogue of 37 proteins, potentially important to future studies in fiber and ovule developmental physiology.

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 ...

Background Cotton fiber length is an important quality attribute to the textile industry and longer fibers can be more efficiently spun into yarns to produce superior fabrics. There is typically a negative correlation between yield and fiber quality traits such as length. An understanding of the regulatory mechanisms controlling fiber length can potentially provide a valuable tool for cotton breeders to improve fiber length while maintaining high yields. The cotton (Gossypium hirsutum L.) fiber mutation Ligon lintless-2 is controlled by a single dominant gene (Li 2 ) that results in significantly shorter fibers than a wild-type. In a near-isogenic state with a wild-type cotton line, Li 2 is a model system with which to study fiber elongation. Results Two near-isogenic lines of Ligon lintless-2 (Li 2 ) cotton, one mutant and one wild-type, were developed through five generations of backcrosses (BC5). An F2 population was developed from a cross between the two Li 2 near-isogenic lines and used to develop a linkage map of the Li 2 locus on chromosome 18. Five simple sequence repeat (SSR) markers were closely mapped around the Li 2 locus region with two of the markers flanking the Li 2 locus at 0.87 and 0.52 centimorgan. No apparent differences in fiber initiation and early fiber elongation were observed between the mutant ovules and the wild-type ones. Gene expression profiling using microarrays suggested roles of reactive oxygen species (ROS) homeostasis and cytokinin regulation in the Li 2 mutant phenotype. Microarray gene expression data led to successful identification of an EST-SSR marker (NAU3991) that displayed complete linkage to the Li 2 locus. Conclusions In the field of cotton genomics, we report the first successful conversion of gene expression data into an SSR marker that is associated with a genomic region harboring a gene responsible for a fiber trait. The EST-derived SSR marker NAU3991 displayed complete linkage to the Li 2 locus on chromosome 18 and resided in a gene with similarity to a putative plectin-related protein. The complete linkage suggests that this expressed sequence may be the Li 2 gene.

The ovules of cotton are composed of the immature seed (embryo and seed coat) and developing fiber. By the coincidence of their proximity, fiber and seed are competing sinks fed through a common funiculus. Partitioning of photosynthate between fiber and seed has been altered by plant breeding. Selection for high yield has increased the thickness of the fiber wall, as indicated by increased micronaire readings (Bridge and Meredith, 1983; Wells and Meredith, 1984). Concomitantly, boll and seed size has decreased (Bridge et al., 1971). These changes are not without an agronomic cost. Small seeds have poorer germination and lower seedling survival. Post-harvest problems are caused by small seed as well: small seed will often pass through the gin with the fiber. The agronomic impact of small seed and the economic impact of fiber make a compelling argument for understanding the physiological relationships between fiber and seed development and the physiology associated with specific events of fiber development.

Production of a viable seed is of paramount importance to the survival of a plant species. Many plants, including cotton, amass large reserves of storage protein and oil in their seeds (oilseeds) to use during dormancy, germination, and seedling growth. These reserves are mobilized after imbibition when environmental conditions are favorable for growth. Due to their rich supply of protein and oil, e.g., approximately 80% of the dry weight of cottonseed kernels consists of protein and oil reserves (Doman et al., 1982), oilseeds have also been used as a human food source. The nutritional value of each oilseed variety or species differs; therefore, minor changes in either protein or oil composition could result in major nutritional improvements and increased economic value of the crop. Improving seed quality is not restricted to increasing its nutritional value; it can also be accomplished by increasing seed vigor, viability, cold tolerance, or resistance to disease. A better understanding of the cellular processes involved in the ontogeny of cottonseeds will lead to new approaches in seed improvement. Discussion of developmental events from gametogenesis to seedling growth will be presented and will focus on important processes involved in producing a viable cottonseed and plant.

The cotton fiber is a model system to study cell wall biosynthesis because the fiber cell elongates (∼3 cm in ∼20 days) without mitosis. In this study, developing cotton ovules, examined from 1 day before anthesis (DBA) to 2 days post-anthesis (DPA), that would be difficult to investigate via classical carbohydrate biochemistry were probed using a battery of antibodies that recognize a large number of different wall components. In addition, ovules from these same stages were investigated in three fiberless lines. Most antibodies reacted with at least some component of the ovule, and several of the antibodies reacted specifically with the epidermal layer of cells that may give clues as to the nature of the development of the fibers and the neighboring, nonfiber atrichoblasts. Arabinogalactan proteins (AGPs) labeled the epidermal layers more strongly than other ovular tissue, even at 1 DBA. One of the AGP antibodies, CCRC-M7, which recognizes a 1--6 galactan epitope of AGPs, is lost from the fiber cells by 2 DPA, although labeling in the atrichoblasts remained strong. In contrast, LM5 that recognizes a 1--4 galactan RGI side chain is unreactive with sections until the fibers are produced and only the fibers are reactive. Dramatic changes also occur in the homogalacturonans (HGs). JIM5, which recognizes highly de-esterified HGs, only weakly labels epidermal cells of 1 DBA and 0 DPA ovules, but labeling increases in fibers cells, where a pectinaceous sheath is produced around the fiber cell and stronger reaction in the internal and external walls of the atrichoblast. In contrast, JIM7-reactive, highly esterifed HGs are present at high levels in the epidermal cells throughout development. Fiberless lines displayed similar patterns of labeling to the fibered lines, except that all of the cells had the labeling pattern of atrichoblasts. That is, CCRC-M7 labeled all cells of the fiberless lines, and LM5 labeled no cells at 2 DPA. These data indicate that a number of polysaccharides are unique in quantity or presence in the epidermal cell layers, and some of these might be critical participants in the early stages of initiation and elongation of cotton fibers.

Methods were developed for isolating highly-purified peroxisomes under iso-osmotic conditions from 3 plant parts, namely cotyledons of cotton (Gossypium hirsutum L.) seedlings, endosperm of castor bean (Ricinus communis L.) seedlings and leaves of mature spinach (Spinaca oleracea L.) plants. Purification was achieved by sedimentation of the organelles into metrizamide gradients centrifuged in a vertical rotor (VTi 50). Gradients consisted of an upper transition layer (1:1 mixture of homogenizing medium and 0.25 M metrizamide), a linear 0.25–0.76 M metrizamide gradient and a 0.76 M metrizamide pad. Peroxisomes from all 3 plant parts were recovered in a major band at a density ranging from 1.24 to 1.27 g cm−3, which is a density range similar to that for peroxisomes isolated in sucrose gradients. The percent of the total gradient cytochrome c oxidase (mitochondria marker) activity recovered in peroxisome fractions ranged from 1.5% in endosperm to 2.8% in leaves, while a plastid marker (chlorophyll or galactosyl transferase activity) ranged from undetectable in leaf peroxisome fractions to 3.6% in endosperm peroxisome fractions. Intactness of the peroxisomes was judged to be 69%, 89% and 78% for the cotyledon, endosperm and leaf peroxisomes, respectively. Isolated peroxisomes were stable for at least 5 h in metrizamide medium. Microscopic (bright-field and transmission electron microscopy) assessments verified that the peroxisomes were morphologically intact and fractions were essentially free of contaminating organelles. Metrizamide is an excellent iso-osmotic medium for purifying peroxisomes from these plant organs and tissue.

A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.Key words: drought stress, gene annotation, gene mapping, tentative consensus sequence (TC), Xanthomonas campestris.

Cotton fibers are often utilized as a model system to investigate cellulose biosynthesis and cell wall elongation. In this study, we grew cotton fibers in vitro, with ovules dissected at day zero post anthesis as the expiant source, in the presence of three herbicides that inhibit cellulose biosynthesis. Cultures were sampled for electron microscopy and immunocytochemistry 1–2 days after beginning the treatments. After dichlobenil treatment, the fibers were much shorter than the controls and assumed a variety of abnormal shapes, from shortened versions of the control fiber to nearly spherical. The inner layers of the fiber wall often contained juxtaposed electron-translucent and -transparent areas; this layer reacted strongly with antibodies to callose. Cellulase-gold labeling in these newly developed fibers grown in the presence of dichlobenil was present at only about 3% of the control labeling. After treatment with either isoxaben or flupoxam, the fibers assumed spherical shapes and frequently (more than 60% of fibers) exhibited a new cell plate within the fiber, indicating that cell division had occurred, a process that rarely occurred in the controls. Unlike the dichlobenil-treated fibers, fibers grown in the presence of isoxaben or flupoxam contained an extensive accumulation of chiefly deesterified pectins, replacing the entire wall with an elaborated version of the pectin sheath found in control cotton fibers. These data indicate that all three herbicides are effective disrupters of cellulose biosynthesis and cause radical changes in cell wall structure and composition. Moreover, these data indicate that the composition of the walls may influence indirectly cell cycle kinetics, keeping these fiber cells in a more meristematic mode.

The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L.) genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD) technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H) oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription. O objetivo deste estudo foi identificar e isolar genes diferencialmente expressos em quatro genótipos de algodão (Gossypium hirsutum L.) que apresentam respostas distintas ao déficit hídrico. Os genótipos estudados foram Siokra L-23, Stoneville 506, CS 50 e T-1521. As mudanças fisiológicas, morfológicas e de desenvolvimento que as plantas apresentam para suportar a diminuição na disponibilidade de água tem, obrigatoriamente, uma base molecular genética. Utilizou-se a técnica de "Differential Display" para identificar e isolar os genes diferencialmente expressos. RNAs mensageiros diferencialmente expressos durante o déficit hídrico foram identificados, isolados, clonados e seqüenciados. O clone A12B15-5 apresentou homologia com uma NADP(H) oxidase e foi expresso somente durante o período de déficit hídrico em Siokra L-23, um genótipo de algodão tolerante à seca. O uso da técnica "Ribonuclease Protection Assay" confirmou essa expressão.

We have isolated 4 cDNA clones encoding the full-length sequence of the eukaryotic ribosomal protein RL44 from upland cotton (Gossypium hirsutum L.). Sequencing of these clones resulted in the classification of 2 subfamilies of RL44; these subfamilies had coding regions (315 bp) which were 92% identical. RL44-1 (454 bp) and RL44-2 (485 bp) constitute subfamily 1, whereas RL44-3 (913 bp) and RL44-5 (541 bp) constitute subfamily 2. The differences in nucleotide sequences, however, occurred only at third codon positions and the resulting amino acid sequences of the two RL44 subfamilies were identical. The ORF encoded a protein of 105 residues with a Mr = 12029. A bipartite nuclear targeting sequence was identified from residues 29 to 43.

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.

The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation.