Comparative transcriptome and lipidome reveal that a low K^(+) signal effectively alleviates the effect induced by Ca^(2+) deficiency in cotton fibers

Calcium(Ca^(2+))plays an important role in determining plant growth and development because it maintains cell wall and membrane integrity.Therefore,understanding the role of Ca^(2+)in carbon and lipid metabolism could provide insights into the dynamic changes in cell membranes and cell walls during the rapid elongation of cotton fibers.In the present study,we found that the lack of Ca^(2+)promoted fiber elongation and rapid ovule expansion,but it also caused tissue browning in the ovule culture system.RNA-sequencing revealed that Ca^(2+)deficiency induced cells to be highly oxidized,and the expression of genes related to carbon metabolism and lipid metabolism was activated significantly.All gene members of nine key enzymes involved in glycolysis were up-regulated,and glucose was significantly reduced in Ca^(2+)deficiency-treated tissues.Ca^(2+)deficiency adjusted the flowing of glycolysis metabolic.However,low K^(+)recovered the expression levels of glycolysis genes and glucose content caused by Ca^(2+)deficiency.Electrospray ionizationtandem mass spectrometry technology was applied to uncover the dynamic profile of lipidome under Ca^(2+)and K^(+)interacted conditions.Ca^(2+)deficiency led to the decrease of fatty acid(FA),diacylglycerol(DAG),glycolipid and the significant increase of triacylglycerol(TAG),phospholipid phosphatidylethanolamine(PE),phosphatidylglycerol(PG),and PC(phosphatidylcholine).Low K^(+)restored the contents of FA,phospholipids,and glycolipids,effectively relieved the symptoms caused by Ca^(2+)deficiency,and recovered the development of fiber cells.This study revealed dynamic changes in transcript and metabolic levels and uncovered the signaling interaction of Ca^(2+)deficiency and low K^(+)in glycolysis and lipid metabolism during fiber development.

Preparation of TiO_2 Nanoparticles Coated Cotton Fibers at Low Temperature and Their Photocatalytic Activity

TiO_2 nanoparticles coated cotton fiber composite was successfully prepared by using a sol-gel method at low temperature(about 100℃) using tetrabutyl-titanate [Ti(OBu)_4] as raw material.The preparation of the TiO_2 colloid and the composite were described.The properties of resulting materials were characterized by SEM and XRD,the photocatalytic degradation performance was tested using methylene blue(MB) as the target pollutant in aqueous solution.The results showed that the amorphous TiO_2 nanoparticles were distributed evenly on the outer surfaces of cotton fibers,which shows efficient photocatalytic properties when exposed to UV light,the degradation rate of MB reached 95.35% under the conditions of catalyst dosage 2.5 g/L,MB concentration 50 mg/L,irradiation time 120 min,and pH 10,and the photocatalytic activity of TiO_2/cotton fibers remained above 90% of its activity as-prepared after being used four times,the degradation rate of MB could reach 88.78% when irradiation time was 120 min.The photocatalytic degradation of MB could be properly described by the first-order kinetic law.By comparison of the removal rates of MB with and without UV light,it could be affirmed that the disappearance of MB was due to photodegradation rather than adsorption on cotton fibers.

Identification of new cotton fiber-quality QTL by multiple genomic analyses and development of markers for genomic breeding

Cotton fiber is one of the main raw materials for the textile industry.In recent years,many cotton fiber quality QTL have been identified,but few were applied in breeding.In this study,a genome wide association study(GWAS)of fiber-quality traits in 265 upland cotton breeding intermediate lines(GhBreeding),combined with genome-wide selective sweep analysis(GSSA)and genomic selection(GS),revealed 25 QTL.Most of these QTL were ignored by only using GWAS.The CRISPR/Cas9 mutants of GhMYB_D13 had shorter fiber,which indicates the credibility of QTL to a certain extent.Then these QTL were verified in other cotton natural populations,5 stable QTL were found having broad potential for application in breeding.Additionally,among these 5 stable QTL,superior genotypes of 4 showed an enrichment in most improved new varieties widely cultivated currently.These findings provide insights for how to identify more QTL through combined multiple genomic analysis to apply in breeding.

Cotton pads-derived carbon materials/reduced graphene oxide modified with polypyrrole for electrode of supercapacitors

This study investigates the influence of electropolymerization conditions on the deposition of polypyrrole(PPy)onto cotton-derived carbon fiber(CF)modified with reduced graphene oxide(rGO)for supercapacitors applications using an experimental/theorical approach.The surface modification of CF by rGO and/or by PPy electrodeposited at 10,25 and 50 mV s^(-1) was thoroughly examined physicochemical and electrochemically.Composite electrodes comprising CF-rGo-PPy,synthesized via electropolymerization at 25 mV s^(-1),demonstrated a remarkable increase in capacitance,showcasing~742 F g^(-1) compared to 153 F g^(-1) for CF.SEM,N_(2)-surface area,XPS,and TD-DFT approach revealed that the higher capacitance observed in CF-rGo-PPy electrodes underscores the influence of morphology and charged nitrogen species on the electrochemical performance of these modified electrodes.Notably,this electrode material achieves a specific capacitance retention of~96%of their initial capacitance after 10000 cycles at 0.5 A g^(-1) measured in a two-electrodes cell configuration.This work also discusses the influence of the scan rate used for pyrrole electropolymerization on the pseudocapacitance contribution of PPy and its possible effect on the porosity of the material.These results highlight the importance of appropriate electropolymerization conditions that allow obtaining the synergistic effect between CF,rGO and PPy.

Facile and scalable preparation of ZIF-67 decorated cotton fibers as recoverable and efficient adsorbents for removal of malachite green

Recently,metal–organic frameworks(MOFs)have received considerable attention as highly efficient adsorbents for dye wastewater remediation.However,the immobilization of MOFs on the substrate surfaces to fabricate easy recy-clable adsorbents via a facile route is still a challenge.In this work,ZIF-67/cotton fibers as adsorbents for dye removal were prepared in a large-scale using a simple coordination replication method.The successful fabrication of the ZIF-67/cotton fibers was confirmed by FTIR,XRD,XPS,SEM and BET analysis,respectively.As expected,the as-prepared ZIF-67/cotton fibers exhibited high adsorption capacity of 3787 mg/g towards malachite green(MG).Meanwhile,the adsorption kinetics and isotherm obeyed the pseudo-second-order kinetics and Langmuir model,respectively.Moreover,its removal efficiency towards MG was not significantly influenced by the pH and ionic strength of aque-ous solution.Most importantly,the ZIF-67/cotton fibers can remove MG from synthetic effluents,and it can be easily regenerated without filtration or centrifugation processes,with the regeneration efficiency remaining over 90%even after 10 cycles.Additionally,the ZIF-67/cotton fibers presented excellent antimicrobial performance against E.coli and S.aureus.Hence,the distinctive features of the as-prepared ZIF-67/cotton fibers make it promisingly applicable for the colored wastewater treatment.

Solid State NMR Applied to Evaluate Fibers Cotton

The evaluation of cotton fibers from Nuclear Magnetic Resonance (NMR) in solid state using high and low-field showed to be a good alternative to evaluate these materials. The main objective of this work was to use the solid-state NMR techniques to accompany the fibers nature, size and morphology to have a direct method to evaluate cotton fibers maturity. We could conclude that the solid-state NMR techniques are good source to evaluate the molecular behavior of cotton short and long fibers and give important information on sample’s morphology, indicating that this technique can be used to distinguish the size of cotton fibers.

Molecular and Biochemical Evidence for Phenylpropanoid Synthesis and Presence of Wall-linked Phenolics in Cotton Fibers

The mature cotton （Gossypium hirsutum L.） fiber is a single cell with a typically thickened secondary cell wall. The aim of this research was to use molecular, spectroscopic and chemical techniques to investigate the possible occurrence of previously overlooked accumulation of phenolics during secondary cell wall formation in cotton fibers. Relative quantitative reverse transcription-polymerase chain reaction analysis showed that GhCAD6 and GhCAD1 were predominantly expressed among seven gene homologs, only GhCAD6 was up-regulated during secondary wall formation in cotton fibers. Phylogenic analysis revealed that GhCAD6 belonged to Class I and was proposed to have a major role in monolignol biosynthesis, and GhCAD1 belonged to Class III and was proposed to have a compensatory mechanism for monolignol biosynthesis. Amino acid sequence comparison showed that the cofactor binding sites of GhCADs were highly conserved with high similarity and identity to bona fide cinnamyl alcohol dehydrogenases. The substrate binding site of GhCAD1 is different from GhCAD6. This difference was confirmed by the different catalytic activities observed with the enzymes. Cell wall auto-fluorescence, Fourier transform infrared spectroscopy （FTIR）, high-performance liquid chromatography （HPLC） and chemical analyses confirmed that phenolic compounds were bound to the cell walls of mature cotton fibers. Our findings may suggest a potential for genetic manipulation of cotton fiber properties, which are of central importance to agricultural, cotton processing and textile industries.

Molecular Cloning and Characterization of a β-Galactosidase Gene Expressed Preferentially in Cotton Fibers

β-galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes in plants that is thought to be involved in metabolism of cell wall polysaccharides. We reported herein the isolation of a fulllength cDNA encoding a typical β-galactosidase protein, designated GhGal1 (Gossypium hirsutum L.galactosidase), of 843 amino acids with a predicted molecular mass of nearly 94.8 kDa. In addition to a glycosyl hydrolase family 35 domain and a putative signal peptide, an unusual characteristic of GhGal 1 is that, at the C-terminus of the enzyme, a domain was found that is structurally related to a sea urchin egg lectin (SUEL-lectin) with D-galactose- and L-rhamnose-binding domains. Based on results from Southern blot, we estimated that there would be two copies of the GhGal1 gene per haploid genome of G. hirsutum.The transcripts of GhGal1 were regulated spatially and temporally and were present in very high abundance at the elongation stage of the cotton fiber. The expression pattern suggests that the GhGal1 gene could be involved in metabolism of the primary cell wall.

Investigation of Working Bodies of the Device for Separation of Fibers Suitable for Spinning from Cotton Waste

It is known that fiber wastes (lint, down and seeds) produced at ginneries contain fibers that are suitable for spinning and can be used in industry, and their separation significantly increases the level of fiber production (1.9% - 2.5%). Based on these analyzes, the study aimed to create a new device that separates long fibers from lint and down. As a result, the amount of fiber output in the enterprise will increase and the enterprise will have significant economic benefits. In addition, the introduction of the device will prevent the addition of long fibers (longer than 16 mm) that can be used in the textile industry to the waste. This article focuses on the creation of a fiber separation device suitable for the treatment and spinning of fibrous waste produced in ginneries. The study theoretically examined the main working bodies of the fiber separation device from waste. Theoretical research is devoted to the study of the strength of the main working body of the fiber separation device−the separating saw drum and its shaft. In the study, the sawdust drum is a more stressed steel coating, and it was found that the strength reserve of this drum is [δТ] = 2.03 (where δТ = 0.8 - 2.5) was found to be. As a result of calculating the resistance of the saw drum shaft to stiffness and vibration, it was determined that the shafts are resistant to vibration under periodic loading and that the oscillation frequency along its axis through the critical rotation frequency is vcr=10.3 Gts.

Fiber-specific increase of carotenoid content promotes cotton fiber elongation by increasing abscisic acid and ethylene biosynthesis

Cotton fiber is a raw material for the global textile industry and fiber quality is essential to its industrial application.Carotenoids are plant secondary metabolites that may serve as dietary components,regulate light harvesting,and scavenge reactive oxygen species.Although carotenoids accumulate predominantly in rapidly elongating cotton fibers,their roles in cotton fiber development remain poorly understood.In this study,a fiber-specific promoter proSCFP was applied to drive the expression of GhOR1Del,a positive regulator of carotenoid accumulation,to upregulate the carotenoid level in cotton fiber in planta.Fiber length,strength,and fineness were increased in proSCFP:GhOR1Del transgenic cotton and abscisic acid(ABA)and ethylene contents were increased in elongating fibers.The ABA downstream regulator GhbZIP27a stimulated the expression of the ethylene synthase gene GhACO3 by binding to its promoter,suggesting that ABA promoted fiber elongation by increasing ethylene production.These findings suggest the involvement of carotenoids and ABA signaling in promoting cotton fiber elongation and provide a strategy for improving cotton fiber quality.

Flavanone and flavonoid hydroxylase genes regulate fiber color formation in naturally colored cotton

Using naturally colored cotton(NCC)can eliminate dyeing,printing and industrial processing,and reduce sewage discharge and energy consumption.Proanthocyanidins(PAs),the primary coloration components in brown fibers,are polyphenols formed by oligomers or polymers of flavan-3-ol units derived from anthocyanidins.Three essential structural genes for flavanone and flavonoid hydroxylation encoding flavanone-3-hydroxylase(F3H),flavonoid 3’-hydroxylase(F3’H)and flavonoid 3’5’-hydroxylase(F3’5’H)are initially committed in the flavonoid biosynthesis pathway to produce common precursors.The three genes were all expressed predominantly in developing fibers of NCCs,and their expression patterns varied temporally and spatially among NCC varieties.In GhF3Hi,GhF3’Hi and GhF3’5’Hi silenced lines of NCC varieties XC20 and ZX1,the expression level of the three genes decreased in developing cotton fiber,negatively correlated with anthocyanidin content and fiber color depth.Fiber color depth and type in RNAi lines changed with endogenous gene silencing efficiency and expression pattern,the three hydroxylase genes functioned in fiber color formation.GhF3H showed functional differentiation among NCC varieties and GhF3’H acted in the accumulation of anthocyanin in fiber.Compared with GhF3’H,GhF3’5’H was expressed more highly in brown fiber with a longer duration of expression and caused lighter color of fibers in GhF3’5’H silenced lines.These three genes regulating fiber color depth and type could be used to improve these traits by genetic manipulation.

Assembly and phylogenomic analysis of cotton mitochondrial genomes provide insights into the history of cotton evolution

Cotton is a major crop that provides the most important renewable textile fibers in the world.Studies of the taxonomy and evolution of cotton species have received wide attentions,not only due to cotton’s economic value but also due to the fact that Gossypium is an ideal model system to study the origin,evolution,and cultivation of polyploid species.Previous studies suggested the involvement of mitochondrial genome editing sites and copy number as well as mitochondrial functions in cotton fiber elongation.Whereas,with only a few mitogenomes assembled in the cotton genus Gossypium,our knowledge about their roles in cotton evolution and speciation is still scarce.To close this gap,here we assembled 20 mitogenomes from 15 cotton species spanning all the cotton clades(A–G,K,and AD genomes)and 5 cotton relatives using short and long sequencing reads.Systematic analyses uncovered a high level of mitochondrial gene sequence conservation,abundant sequence repeats and many insertions of foreign sequences,as well as extensive structural variations in cotton mitogenomes.The sequence repeats and foreign sequences caused significant mitogenome size inflation in Gossypium and its close relative Kokia in general,while there is no significant difference between the lint and fuzz cotton mitogenomes in terms of gene content,RNA editing,and gene expression level.Interestingly,we further revealed the specific presence and expression of two novel mitochondrial open reading frames(ORFs)in lint-fiber cotton species.Finally,these structural features and novel ORFs help us gain valuable insights into the history of cotton evolution and polyploidization and the origin of species producing long lint fibers from a mitogenomic perspective.

NaOH/Urea Swelling Treatment and Hydrothermal Degradation of Waste Cotton Fiber

In this study,waste cotton fabric was used as cellulose raw material and pretreated in aqueous NaOH/urea solution system to investigate the effect of NaOH/urea pretreatment solution on the hydrolysis of cotton fiber.The cotton fiber was pretreated with different conditions of aqueous NaOH/urea solution,and the pretreated cotton fiber was hydrolyzed under the same conditions as the original cotton fiber.The results of characterization analysis showed that water retention value of pretreated cotton fiber was higher than that of unpretreated sample.Moreover,the cotton fiber presented both a convoluted structure and a coarser surface,XRD results suggested that the crystallinity degree of cellulose decreased dramatically,more cellulose II appeared,and the hydrogen bond is broken.Among the different pretreatment conditions,the pretreatment effect was the best when the reaction temperature was 0°C,the solid-liquid ratio was 2:50,and the NaOH/urea ratio was 7:12.The hydrolysis experiments of pretreated and unpretreated cotton fibers showed that when the hydrothermal temperature was 230°C,the heat preservation was 2 h,and the hydrochloric acid concentration was 5 wt.%,the glucose yield reached 29.99%.H+could catalyze the hydrolysis of cotton fiber more effectively due to damage to crystal structure and hydrogen bonds.

Cloning and expression of two sterol C-24 methyltransferase genes from upland cotton(Gossypium hirsuturm L.)

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. Two kinds of intermediates, sitosterol and campesterol, play a crucial role in cell elongation, cellulose biosynthesis, and accumulation. To illuminate the effects of sitosterol and campesterol on the development of cotton （Gossypiurn hirsuturm L.） fibers through screening cotton fiber EST database and contigging the candidate ESTs, two key genes GhSMT2-1 and GhSMT2-2 controlling the sitosterol biosynthesis were cloned from developing fibers of upland cotton cv. Xuzhou 142. The full length of GhSMT2-1 was 1,151 bp, including an 8 bp 5＇-untranslated region （UTR）, a 1,086 bp open reading frame （ORF）, and a 57 bp 3＇-UTR. GhSMT2-1 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40 kDa. The full length of GhSMT2-2 was 1,166 bp, including an 18 bp 5＇-UTR, a 1,086 bp ORF, and a 62 bp 3＇-UTR. GhSMT2-2 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40 kDa. The two deduced amino acid sequences had high homology with the SMT2 from Arabidopsis thaliana and Nicotiana tabacurn. Furthermore, the typical conserved structures characterized by the sterol C-24 methyltransferase, such as region I （LDVGCGVGGPMRAI）, region II （IEATCHAP）, and region III （YEWGWGQSFHF）, were present in both deduced proteins. Southern blotting analysis indicated that GhSMT2-1 or GhSMT2-2 was a single copy in upland cotton genome. Quantitative real-time RT-PCR analysis revealed that the highest expression levels of both genes were detected in 10 DPA （day post anthesis） fibers, while the lowest levels were observed in cotyledon and leaves. The expression level of GhSMT2-1 was 10 times higher than that of GhSMT2-2 in all the organs and tissues detected. These results indicate that the homologue of sterol C-24 methyltransferase gene was cloned from upland cotton and both GhSMT2 genes play a crucial role in fiber elongation. The role of GhSMT2-1 may be more important than that of GhSMT2-2.

The bHLH transcription factor GhPAS1 mediates BR signaling to regulate plant development and architecture in cotton

Cotton (Gossypium spp.) is the most important natural textile fiber crop in the world.The ideal plant architecture of cotton is suitable for mechanical harvesting and productivity in modern agricultural production.However,cotton genes regulating plant development and architecture have not been fully identified.We identified a basic helix-loop-helix (b HLH) transcription factor,GhPAS1 (PAGODA1 SUPPRESSOR1) in G.hirsutum (Upland cotton).GhPAS1 was located in the nucleus and showed a strong transcription activation effect.Tissue-specific expression patterns showed that GhPAS1 was highly expressed in floral organs,followed by high expression in early stages of ovule development and rapid fiber elongation.GhPAS1 overexpression in Arabidopsis and BRZ (brassinazole,BR biosynthesis inhibitor) treatment indicated that GhPAS1 positively regulates and responds to the BR (brassinosteroid) signaling pathway and promotes cell elongation.GhPAS1 overexpression in Arabidopsis mediated plant development in addition to increasing plant biomass.Virus-induced gene silencing of GhPAS1 indicated that down-regulation of GhPAS1 inhibited cotton growth and development,as plant height,fruit branch length,and boll size of silenced plants were lower than in control plants.Fiber length and seed yield were also lower in silenced plants.We conclude that GhPAS1,a b HLH transcription factor,regulates plant development and architecture in cotton.These findings may help breeders and researchers develop cotton cultivars with desirable agronomic characteristics.

Brassinosteroids and Auxin Down-Regulate DELLA Genes in Fiber Initiation and Elongation of Cotton

Plant hormones play important roles in cotton fiber growth and development.However,the interaction of phytohormones is largely unknown in fiber cells up to now.DELLA proteins are critical component in GA （gibberellic acid） signal transduction,which are also regulated by other phytohormones,such as auxin and ethylene.To understand the regulation of DELLA genes in cotton fiber growth and development,we cloned four DELLA genes from upland cotton fibers （Gossypium hirsutum L.）,named GhGAI1,GhGAI2,GhGAI3,and GhGAI4.Alignment of the four predicted proteins with other reported DELLA proteins in various species displayed that they shared conserved domains and high homology.Expression profiles of the four GhGAIs in various tissues and organs as well as cotton fibers in different stages displayed that GhGAI1 has higher transcriptional levels than other GhGAIs in all detected samples.Furthermore,the expression level of GhGAI1 was significantly reduced in 0 dpa （day post anthesis） ovules by addition of IAA and epi-BL,and exogenous epi-BL decreased GhGAI1 level in 7 dpa fiber.Similarly,the levels of the other three GhGAIs in 0 dpa ovules and 7 dpa fibers were also regulated by applied phytohormones.In addition,the levels of GhGAI1 were higher in Xuzhou142 fl mutant （fuzzless-lintless） than in FL （Gossypium hirsutum vs.Xuzhou 142） from-1 to 3 dpa ovules,suggesting that GhGAI1 engaged in cotton fiber cell initiation.These results indicated that DELLA genes are involved in the process of fiber cell initiation and elongation regulated by different phytohormones.

Effects of Increased Night Temperature on Cellulose Synthesis and the Activity of Sucrose Metabolism Enzymes in Cotton Fiber

Temperature is one of the key factors that influence cotton fiber synthesis at the late growth stage of cotton. In this paper, using two early-maturing cotton varieties as experimental materials, night temperature increase was stimulated in the field using far-infrared quartz tubes set in semi-mobile incubators and compared with the normal night temperatures （control） in order to investigate the effects of night temperature on the cotton fiber cellulose synthesis during secondary wall thickening. The results showed that the activity of sucrose synthase （SuSy） and sucrose phosphate synthase （SPS） quickly increased and remained constant during the development of cotton fiber, while the activity of acid invertase （AI） and alkaline invertase （NI） decreased, increased night temperatures prompted the rapid transformation of sugar, and all the available sucrose fully converted into cellulose. With night temperature increasing treatment, an increase in SuSy activity and concentration of sucrose indicate more sucrose converted into UDPG （uridin diphosphate-glucose） during the early and late stages of cotton fiber development. Furthermore, SPS activity and the increased concentration of fructose accelerated fructose degradation and reduced the inhibition of fructose to SuSy; maintaining higher value of allocation proportion of invertase and sucrose during the early development stages of cotton fiber, which was propitious to supply a greater carbon source and energy for cellulose synthesis. Therefore, the minimum temperature in the nightime was a major factor correlated with the activity of sucrose metabolism enzymes in cotton fiber. Consequently, soluble sugar transformation and cellulose accumulation were closely associated with the minimum night temperature.

Preparation and Characterization of Carbon Microspheres From Waste Cotton Textiles By Hydrothermal Carbonization

Carbon microspheres were prepared from waste cotton fibers by hydrothermal carbonization(HTC)with the addition of copper sulphate in this work.The important influence factors,temperature,concentration of copper sulphate,resident time were explored here.The smooth and regular carbon microspheres could be formed at 330°C with 0.15 wt%copper sulphate after 6 h from waste cotton fibers.The crystal structures of cotton fibers were destructed in a short resident time with 0.15 wt%copper sulphate from SEM images and XRD patterns of solid products.This strategy provides a new,mild and efficient method to prepare carbon microspheres from waste cotton fibers by HTC.FTIR spectra verified that the abundant functional groups existed on the surface of synthesized carbon microspheres.From XPS and element analysis results,the copper sulphate participated in the forming process of carbon microspheres indeed.The presence of copper sulphate in the carbon microspheres provided a possibility for the application in antibacterial field.Besides,the catalytic mechanism of copper sulphate on the hydrolysis and carbonization of waste cotton fibers were also discussed.In conclusion,the copper sulphate is an efficient agent for preparing carbon microspheres by HTC from waste cotton fibers.

Identification of Differentially Expressed Genes Associated with Cotton Fiber Development in a Chromosomal Substitution Line(CS-B22sh)

One of the impediments in the genetic improvement of cotton fiber is the paucity of information about genes associated with fiber development.Availability of chromosome arm substitution line CS-

Cloning and expression analysis of GhDET3, a vacuolar H^+-ATPase subunit C gene, from cotton

Vacuolar H^+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.