Cotton Cotyledons Research Articles

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

The long non-coding RNA GhlncRNA149.1 improves cotton defense response to aphid damage as a positive regulator

Long non-coding RNAs (lncRNAs) play crucial roles in various plant biological regulatory processes and defense responses to all sorts of biotic and abiotic stresses. However, there are limited studies on the function and mechanism of lncRNAs in plant defense responses to phytophagous insects. In this research, a lncRNA GhlncRNA149.1 was identified from upland cotton (Gossypium hirsutum L.). Depending on the position relationship between lncRNA and mRNA, we identified CC-NBS-LRR family gene GhA01G0129 as the target gene of GhlncRNA149.1. Expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were significantly down-regulated in cotton cotyledons upon cotton aphid infestation. The expression level of GhlncRNA149.1 was significantly down-regulated after the induction of methyl jasmonate and salicylic acid, whereas expression level of its target gene GhA01G0129 was significantly up-regulated. Overexpression of GhlncRNA149.1 in cotton cotyledons enhanced the tolerance of cotton plants to cotton aphid feeding. Expectedly, silencing GhlncRNA149.1 increased susceptibility of cotton plants to cotton aphids. The expression level of GhA01G0129, the target gene of GhlncRNA149.1, was significantly up-regulated in cotton plants overexpressing GhlncRNA149.1, and significantly down-regulated in cotton plants silencing GhlncRNA149.1. The activity and expression levels of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were up-regulated in cotton plants transiently overexpressing GhlncRNA149.1, while opposite results were obtained in cotton plants silencing GhlncRNA149.1. In addition, similar results were obtained in the expression levels of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), enhanced disease susceptibility 1 (EDS1) and non-expression of pathogenesis-related gene 1 (NPR1) in cotton plants transiently overexpressing GhlncRNA149.1 and silencing GhlncRNA149.1. These results collectively suggest that GhlncRNA149.1 improve the plant defense response to aphid attack, and thus has potential for enforcing cotton aphid prevention and control.

Discovery of a secreted Verticillium dahliae protease that cleaves cotton CRR1 and induces plant cell death

Domain of unknown function 26 (DUF26) is a non-catalytic protein domain found only in land plant proteins. These proteins are often associated with defense. Cotton (Gossypium hirsutum L) CRR1 is a secreted protein consisting of two DUF26 domains connected by a linker. In this study we report that CRR1 is cleaved by an alkaline subtilase secreted by Verticillium dahliae, a hemi-biotrophic fungal pathogen. Recombinant CRR1 was converted from a 30 kDa glycoprotein into products of ∼15 kDa (CRR1-P) when incubated with secreted protein extracts from V. dahliae cultures. Using this activity as a guide, the protease was purified, and its tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. It was identified as alkaline subtilase G2X826_VERDV, which we named Vd-DUMP for V. dahliaeDUF26 modifying protein. This identification was confirmed by producing active recombinant Vd-DUMP in the yeast Pichia pastoris. Biochemical analysis indicated that Vd-DUMP cleaves CRR1 between the DUF26 domains. Infiltration of Vd-DUMP into Arabidopsis (Arabidopsis thaliana) leaves or cotton cotyledons induced cell death, a response that was absent when Vd-DUMP was chemically inactivated prior to infiltration. This study identifies plant DUF26 proteins, associated with defense, as substrates for alkaline subtilases secreted by fungi that are known to function as elicitors and effectors.

Expression of Modified Snowdrop Lectin (Galanthus nivalis Agglutinin) Protein Confers Aphids and Plutella xylostella Resistance in Arabidopsis and Cotton.

Cotton is a major fiber crop in the world that can be severely infested by pests in agricultural fields. Identifying new insect-resistance genes and increasing the expression of known insect-resistance genes are imperative in cultivated cotton. Galanthus nivalis agglutinin (GNA), a lectin that is toxic to both chewing and sucking pests, is mainly expressed in monocotyledons. It is necessary to improve the expression of the GNA protein and to test whether the lectin confers insect resistance to dicotyledons plants. We report a modified GNA gene (ASGNA) via codon optimization, its insertion into Arabidopsis thaliana, and transient expression in cotton to test its efficacy as an insect-resistance gene against cotton aphids and Plutella xylostella. The amount of ASGNA in transgenic plants reached approximately 6.5 μg/g of fresh weight. A feeding bioassay showed that the survival rate of aphids feeding on the leaves of ASGNA transgenic plants was lower than those of aphids feeding on the leaves of non-optimized GNA (NOGNA) transgenic plants and wild-type plants. Meanwhile, the fertility rate was 36% when fed on the ASGNA transgenic plants, while the fertility was 70% and 95% in NOGNA transgenic plants and wild-type plants. Correspondingly, the highest mortality of 55% was found in ASGNA transgenic lines, while only 35% and 20% mortality was observed in NOGNA transgenic plants and wild-type plants, respectively. Similar results were recorded for aphids feeding on cotton cotyledons with transient expression of ASGNA. Taken together, the results show that ASGNA exhibited high insecticidal activity towards sap-sucking insects and thus is a promising candidate gene for improving insect resistance in cotton and other dicotyledonous plants.

Patterns of Seed-to-Seedling Transmission of Xanthomonas citri pv. malvacearum, the Causal Agent of Cotton Bacterial Blight.

Cotton bacterial blight (CBB), caused by Xanthomonas citri pv. malvacearum, was a major disease of cotton in the United States in the early part of the twentieth century. The reemergence of CBB revealed many gaps in our understanding of this important disease. In this study, we employed a wild-type (WT) field isolate of X. citri pv. malvacearum from Georgia (U.S.A.) to generate a nonpathogenic hrcV mutant lacking a functional type-III secretion system (T3SS-). We tagged the WT and T3SS- strains with an auto-bioluminescent Tn7 reporter and compared colonization patterns of CBB-susceptible and CBB-resistant cotton seedlings using macroscopic image analysis and bacterial load enumeration. WT and T3SS- X. citri pv. malvacearum strains colonized cotton cotyledons of CBB-resistant and CBB-susceptible cotton cultivars. However, X. citri pv. malvacearum populations were significantly higher in CBB-susceptible seedlings inoculated with the WT strain. Additionally, WT and T3SS- X. citri pv. malvacearum strains systemically colonized true leaves, although at different rates. Finally, we observed that seed-to-seedling transmission of X. citri pv. malvacearum may involve systemic spread through the vascular tissue of cotton plants. These findings yield novel insights into potential X. citri pv. malvacearum reservoirs for CBB outbreaks.

Transcriptomic analyses show that 24-epibrassinolide (EBR) promotes cold tolerance in cotton seedlings.

In plants, brassinosteroids (BRs) are a class of steroidal hormones that are involved in numerous physiological responses. However, the function of BRs in cold tolerance in cotton has not been explored. In this study, cotton seedlings were treated with five concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/L) of 24-Epibrassinolide (EBR) at 4°C. We measured the electrolyte leakage, malondialdehyde (MDA) content, proline content, and net photosynthesis rate (Pn) of the seedlings, which showed that EBR treatment increased cold tolerance in cotton in a dose-dependent manner, and that 0.2 mg/L is an optimum concentration for enhancing cold tolerance. The function of EBR in cotton cotyledons was investigated in the control 0 mg/L (Cold+water) and 0.2 mg/L (Cold+EBR) treatments using RNA-Seq. A total of 4,001 differentially expressed genes (DEGs), including 2,591 up-regulated genes and 1,409 down-regulated genes were identified. Gene Ontology (GO) and biochemical pathway enrichment analyses showed that EBR is involved in the genetic information process, secondary metabolism, and also inhibits abscisic acid (ABA) and ethylene (ETH) signal transduction. In this study, physiological experiments showed that EBR can increase cold tolerance in cotton seedlings, and the comprehensive RNA-seq data shed light on the mechanisms through which EBR increases cold tolerance in cotton seedlings.

Ergosterol-targeting fusion antifungal peptide significantly increases the Verticillium wilt resistance of cotton.

SummaryIncreasing the targeting ability of antifungal proteins towards specific components of fungal cells has the potential to improve their antifungal activity and reduce harmful effects to nontarget cells. To obtain effective disease resistance genes against cotton Verticillium wilt, we constructed several fusion genes, in which binding domains targeting chitin, sphingolipid or ergosterol in the fungal cell wall or cell membrane were individually fused to the antifungal peptide BbAFP1 from entomopathogenic fungus Beauveria bassiana. Transient expression of fusion genes in cotton cotyledons indicated that the BbAFP1::ErBD fusion peptide with an ergosterol binding domain exhibited better disease resistance against V. dahliae than wild‐type BbAFP1 and other fusion genes. BbAFP1::ErBD and BbAFP1 transgenic cotton were obtained and verified by Southern and Western blotting. Compared with BbAFP1‐expressing cotton, BbAFP1::ErBD‐expressing cotton showed higher disease resistance against V. dahliae, with smaller lesion areas (0.07 cm2 vs. 0.16 cm2) on the leaves and a lower disease index (23.9 vs. 34.5). Overexpression of BbAFP1::ErBD by transgenic tobacco also showed enhanced disease resistance against V. dahliae compared with that of the wild‐type gene. These results indicated that construction of fusion antifungal peptides that target fungal cells is a powerful strategy to obtain new anti‐disease genes, and the obtained fusion gene BbAFP1::ErBD has the potential to defend against plant fungal diseases.

Transcriptomic Profiling of Young Cotyledons Response to Chilling Stress in Two Contrasting Cotton (Gossypium hirsutum L.) Genotypes at the Seedling Stage.

Young cotyledons of cotton seedlings are most susceptible to chilling stress. To gain insight into the potential mechanism of cold tolerance of young cotton cotyledons, we conducted physiological and comparative transcriptome analysis of two varieties with contrasting phenotypes. The evaluation of chilling injury of young cotyledons among 74 cotton varieties revealed that H559 was the most tolerant and YM21 was the most sensitive. The physiological analysis found that the ROS scavenging ability was lower, and cell membrane damage was more severe in the cotyledons of YM21 than that of H559 under chilling stress. RNA-seq analysis identified a total of 44,998 expressed genes and 19,982 differentially expressed genes (DEGs) in young cotyledons of the two varieties under chilling stress. Weighted gene coexpression network analysis (WGCNA) of all DEGs revealed four significant modules with close correlation with specific samples. The GO-term enrichment analysis found that lots of genes in H559-specific modules were involved in plant resistance to abiotic stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that pathways such as plant hormone signal transduction, MAPK signaling, and plant–pathogen interaction were related to chilling stress response. A total of 574 transcription factors and 936 hub genes in these modules were identified. Twenty hub genes were selected for qRT-PCR verification, revealing the reliability and accuracy of transcriptome data. These findings will lay a foundation for future research on the molecular mechanism of cold tolerance in cotyledons of cotton.

Comparison of WeatherMax<sup>®</sup> and Engame<sup>TM</sup> Formulations of Glyphosate on Cotyledon Surface Structure, Chlorophyll A Fluorescence and Shikimate Levels in Isogenic Cotton Cultivars Differing in Roundup Resistance

The effects of Roundup WeatherMax® and EngameTM formulations of glyphosate were investigated on the cotyledons of glyphosate resistant (GR) and glyphosate sensitive (GS) isogenic cotton cultivars. EngameTM is a mixture of glyphosate and 1-aminomethanamide dihydrogen tetraoxosulfate (AMADS). Fully expanded cotton cotyledons treated with EngameTM or AMADS developed surface lesions within 2 hours after treatment whereas surfactant-treated control or WeatherMax®-treated tissues did not develop lesions. The EngameTM and AMADS damage appeared as depressions which were confirmed by scanning electron microscopy. Light micrographs of cross sections through the depressions revealed collapsed and compressed epidermal and mesophyll cells with congealed cytoplasmic contents in the palisade and spongy mesophyll cells. Changes to photosynthetic electron transport were evident at 4 hours after treatment (HAT) in all treatments as revealed by chlorophyll A fluorescence. In GR cotton, the fluorescence perturbations decreased with time such that at 72 HAT EngameTM-treated cotyledons could not be distinguished from the surfactant-or WeatherMax®-treated plants. The GS cotton continued to show progressive decreases in the fluorescence parameters Fv/Fm and performance index (PI) to 72 HAT. Shikimate levels increased following glyphosate treatment in glyphosate sensitive cotton and EngameTM caused a two-to three-fold greater increase in shikimate compared to WeatherMax®. These results indicate that the EngameTM-based glyphosate formulation involved structural tissue damage which likely increased glyphosate uptake and subsequently increased inhibition of photosynthesis and the shikimate pathway.

Alternaria leaf spot of cotton seedlings grown in New South Wales, Australia is predominantly associated with Alternaria alternata

Alternaria leaf spot (ALS) of cotton, especially on cotyledons is generally considered a minor disease in Australia. However, severe disease outbreaks were recorded in multiple cotton crops in southern New South Wales (NSW) in the 2017/18 season. Due to ALS being considered a minor disease, causal species have not been formally characterised in Australia. In this study, putative small-spored Alternaria alternata was predominantly recovered from diseased cotyledons sampled from ALS hot spot fields in southern NSW as well as other locations across NSW. Species status of recovered isolates as A. alternata was confirmed through morphology and sequence analyses of the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1) regions. Phylogenetic analyses of the two regions both individually or in combination resulted in a well-supported group of 69 isolates (83%) clustering with the ex-type specimen of A. alternata CBS 916.96, thus confirming the preliminary morphological identifications. In artificially inoculated assays on 15-day-old cotton seedlings, a subset of selected A. alternata isolates produced similar ALS symptoms at 7 days after inoculation (DAI) as seen in the field. Koch’s postulate was fulfilled by re-isolating A. alternata from infected ALS cotyledons at 21 DAI. This is the first main stream published report of A. alternata causing ALS on cotton cotyledons in NSW, Australia.

Genome Editing in Cotton with the CRISPR/Cas9 System.

Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton (Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1, and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.

Global analysis of DNA methylation in young (J1) and senescent (J2) Gossypium hirsutum L. cotyledons by MeDIP-Seq.

DNA methylation is an important epigenetic modification regulating gene expression, genomic imprinting, transposon silencing and chromatin structure in plants and plays an important role in leaf senescence. However, the DNA methylation pattern during Gossypium hirsutum L. cotyledon senescence is poorly understood. In this study, global DNA methylation patterns were compared between two cotyledon development stages, young (J1) and senescence (J2), using methylated DNA immunoprecipitation (MeDIP-Seq). Methylated cytosine occurred mostly in repeat elements, especially LTR/Gypsy in both J1 and J2. When comparing J1 against J2, there were 1222 down-methylated genes and 623 up-methylated genes. Methylated genes were significantly enriched in carbohydrate metabolism, biosynthesis of other secondary metabolites and amino acid metabolism pathways. The global DNA methylation level decreased from J1 to J2, especially in gene promoters, transcriptional termination regions and regions around CpG islands. We further investigated the expression patterns of 9 DNA methyltransferase-associated genes and 2 DNA demethyltransferase-associated genes from young to senescent cotyledons, which were down-regulated during cotyledon development. In this paper, we first reported that senescent cotton cotyledons exhibited lower DNA methylation levels, primarily due to decreased DNA methyltransferase activity and which also play important role in regulating secondary metabolite process.

The Establishment of Agrobacterium -mediated Transient Expres-sion System in Cotton Cotyledons

The Establishment of <i>Agrobacterium</i> -mediated Transient Expres-sion System in Cotton Cotyledons

Foraging on Individual Leaves by an Intracellular Feeding Insect Is Not Associated with Leaf Biomechanical Properties or Leaf Orientation

Nearly all herbivorous arthropods make foraging-decisions on individual leaves, yet systematic investigations of the adaptive significance and ecological factors structuring these decisions are rare with most attention given to chewing herbivores. This study investigated why an intracellular feeding herbivore, Western flower thrips (WFT) Frankliniella occidentalis Pergande, generally avoids feeding on the adaxial leaf surface of cotton cotyledons. WFT showed a significant aversion to adaxial-feeding even when excised-cotyledons were turned up-side (abaxial-side ‘up’), suggesting that negative-phototaxis was not a primary cause of thrips foraging patterns. No-choice bioassays in which individual WFT females were confined to either the abaxial or adaxial leaf surface showed that 35% fewer offspring were produced when only adaxial feeding was allowed, which coincided with 32% less plant feeding on that surface. To test the hypothesis that leaf biomechanical properties inhibited thrips feeding on the adaxial surface, we used a penetrometer to measure two variables related to the ‘toughness’ of each leaf surface. Neither variable negatively co-varied with feeding. Thus, while avoiding the upper leaf surface was an adaptive foraging strategy, the proximate cause remains to be elucidated, but is likely due, in part, to certain leaf properties that inhibit feeding.

Comparative Transcriptomic Analysis of Developing Cotton Cotyledons and Embryo Axis

BackgroundAs a by product of higher value cotton fibre, cotton seed has been increasingly recognised to have excellent potential as a source of additional food, feed, biofuel stock and even a renewable platform for the production of many diverse biological molecules for agriculture and industrial enterprises. The large size difference between cotyledon and embryo axis that make up a cotton seed results in the under-representation of embryo axis gene transcript levels in whole seed embryo samples. Therefore, the determination of gene transcript levels in the cotyledons and embryo axes separately should lead to a better understanding of metabolism in these two developmentally diverse tissues.ResultsA comparative study of transcriptome changes between cotton developing cotyledon and embryo axis has been carried out. 17,384 unigenes (20.74% of all the unigenes) were differentially expressed in the two adjacent embryo tissues, and among them, 7,727 unigenes (44.45%) were down-regulated and 9,657 unigenes (55.55%) were up-regulated in cotyledon.ConclusionsOur study has provided a comprehensive dataset that documents the dynamics of the transcriptome at the mid-maturity of cotton seed development and in discrete seed tissues, including embryo axis and cotyledon tissues. The results showed that cotton seed is subject to many transcriptome variations in these two tissue types and the differential gene expression between cotton embryo axis and cotyledon uncovered in our study should provide an important starting point for understanding how gene activity is coordinated during seed development to make a seed. Further, the identification of genes involved in rapid metabolite accumulation stage of seed development will extend our understanding of the complex molecular and cellular events in these developmental processes and provide a foundation for future studies on the metabolism, embryo differentiation of cotton and other dicot oilseed crops.

CHANGING PROTEIN PATTERNS DURING COTTON COTYLEDON EMBRYOGENESIS AND GERMINATION AS SHOWN BY IN VIVO AND IN VITRO SYNTHESIS

The rise and fall of different populations of mRNA during the embryogenesis of cotton cotyledons have been followed by cataloging (a) the extant protein population, (b) the in vivo synthesis of proteins and (c) the in vitro synthesis of proteins from extracted mRNA, all at intervals during embryogenesis. Evidence is presented for the existence of three mRNA subsets, all under different regulatory regimes, one of which may be induced by the regulator abscisic acid (ABA).

RNA complexity during seed formation and germination.

ABSTRACT The sequence composition and quantity of cotton cotyledon mRNAs present after 24 h of germination were evaluated by physical techniques, in vitro translation in wheat germ extracts, and hybridization with complementary DNA. The results indicate that most of the 25,000 individual mRNAs exist in both the poly(A)plus mRNA and the poly(A)minus mRNA populations in similar concentrations. Polysomal and nonpolysomal mRNA populations also appear to be virtually identical. The cotyledon mRNAs present during cotyledon development were compared by hybridization of poly(A)plus mRNA from each of three stages with DNA complementary to RNAs present in the other stages. Dramatic changes in the concentration of most of the abundant mRNA sequences and many of the less abundant mRNA sequences occur during cotyledon development. Most of the about 25,000 rare mRNA sequences appear to be common to all three stage mRNAs. A summary is presented of the mRNA sequences in other plant issues, as reported from other laborato...

A novel, conditional, lesion mimic phenotype in cotton cotyledons due to the expression of an endochitinase gene from Trichoderma virens

We have observed a novel, lesion mimic phenotype (LMP) in the cotyledons of cotton seedlings expressing an endochitinase gene from Trichoderma virens. This phenotype, however, is conditional and is elicited only when the transgenic seedlings are germinating on a medium that is devoid of mineral nutrients. The LMP manifests itself around the 5th day in the form of scattered, dry necrotic lesions on the cotyledons. The severity of the LMP is correlated with the level of transgene activity. Production of reactive oxygen species and activities of certain defense related enzymes and genes were substantially higher in the cotyledons of seedlings that were growing under mineral nutrient stress. Molecular and biochemical analyses indicated significantly higher-level activities of certain defense-related genes/enzymes at the onset of the phenotype. Treatment with methyl jasmonate can induce LMP in the cotyledons of wild-type (WT) seedlings similar to that observed in the endochitinase-expressing seedlings grown on nutrient-free medium. On the other hand, salicylic acid (SA), its functional analog, benzo(1,2,3) thiadiazole-7-carbothioic acid (BTH), and ibuprofen can rescue the LMP induced by the seedling-growth on nutrient-deficient medium. Nutrient deficiency-induced activation of a defense response appears to be the contributing factor in the development of LMP in endochitinase-expressing cotton seedlings.

Phenological and Growth Interactions between Velvetleaf (Abutilon theophrasti M.) and Cotton (Gossypium hirsutum L.)

The phenology and growth of A. theophrasti in competition with cotton, and the influenced of weed sowing time and weed plant density on phenology and growth were examined in several field studies conducted over the period 2000–2002. A. theophrasti was found to produce three cohorts that emerged in a staggered manner from April 21 to June 20. The first cohort emerged simultaneously with cotton under a plastic cover and the second by the time cotton plants had 5–6 true leaves. Flowering in these two cohorts started at 572–594 growing degree-days (GDD), boll ripening (stage H) at 800–885 GDD and boll drying at 1020–1037 GDD. Velvetleaf and cotton cotyledons were formed simultaneously at all weed sowing densities. Also, plant height of A. theophrasti increased with increasing density, whereas cotton height decreased due to shad and competition.

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of Gossypium hirsutum

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.