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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.

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.

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.

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.

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.

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.

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).

The length of cotton fiber is an important agronomic trait characteristic that directly affects the quality of yarn and fabric. The cotton (Gossypium hirsutum L.) fiber mutation, Ligon lintless-2, is controlled by a single dominant gene (Li 2 ) and results in extremely shortened lint fibers on mature seeds with no visible pleiotropic effects on vegetative growth and development. The Li 2 mutant phenotype provides an ideal model system to study fiber elongation. To understand metabolic processes involved in cotton fiber elongation, changes in metabolites and transcripts in the Li 2 mutant fibers were compared to wild-type fibers during development. Principal component analysis of metabolites from GC-MS data separated Li 2 mutant fiber samples from WT fiber samples at the WT elongation stage, indicating that the Li 2 mutation altered the metabolome of the mutant fibers. The observed alterations in the Li 2 metabolome included significant reductions in the levels of detected free sugars, sugar alcohols, sugar acids, and sugar phosphates. Biological processes associated with carbohydrate biosynthesis, cell wall loosening, and cytoskeleton were also down-regulated in Li 2 fibers. Gamma-aminobutyric acid, known as a signaling factor in many organisms, was significantly elevated in mutant fibers. Higher accumulation of 2-ketoglutarate, succinate, and malate suggested higher nitrate assimilation in the Li 2 line. Transcriptional activation of genes involved in nitrogen compound metabolism along with changes in the levels of nitrogen transport amino acids suggested re-direction of carbon flow into nitrogen metabolism in Li 2 mutant fibers. This report provides the first comprehensive analysis of metabolite and transcript changes in response to the Li 2 mutation in elongating fibers. A number of factors associated with cell elongation found in this study will facilitate further research in understanding metabolic processes of cotton fiber elongation.

Background Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li 1 ) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li 1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. Results Physical and morphological measurements of the Li 1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li 1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li 1 gene. Conclusions The early termination of fiber elongation in the Li 1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li 1 mutant were identified. Molecular markers closely associated with the Li 1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.