Characterization of the 19 Novel Cotton FLA Genes and Their Expression Profiling in Fiber Development and in Response to Phytohormones and Salt Stress

Fasciclin-like arabinogalactan proteins(FLAs),a subclass of arabinogalactan proteins(AGPs),are usually involved in cell development in plants.To investigate the expression profiling as well

Cloning GhSCFP Gene and Its Function in Cotton Fiber Development

As a major raw material for the textile industry and the most important fiber crop in the world,cotton is of great significance in Chinese economy.The development of cotton fiber can be divided

Expression Profile Analysis of Genes Involved in Brassinosteroid Biosynthesis Pathway in Cotton Fiber Development

Cotton(Gossypium hirsutum L.) is the leading fiber crop and one of the mainstays of the economy in the world.Cotton fibers,as the main product of cotton plants,are unicellular,linear

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-

Genes Expressing Differences in Upland Cotton Fiber Development Between 12 DPA and 16 DPA

Cotton is the major dominant natural fiber crop on the earth.Although some current cotton genetics and breeding programs had made great progresses in cotton lint yield increases and disease resistance(tolerance),fiber quality has little improvement.Global understanding genes expression

A genome-wide identification of the BLH gene family reveals BLH1 involved in cotton fiber development

Background:Cotton is the world’s largest and most important source of renewable natural fiber.BEL1-like homeodomain(BLH)genes are ubiquitous in plants and have been reported to contribute to plant development.However,there is no comprehensive characterization of this gene family in cotton.In this study,32,16,and 18 BLH genes were identified from the G.hirsutum,G.arboreum,and G.raimondii genome,respectively.In addition,we also studied the phylogenetic relationships,chromosomal location,gene structure,and gene expression patterns of the BLH genes.Results:The results indicated that these BLH proteins were divided into seven distinct groups by phylogenetic analysis.Among them,25 members were assigned to 15 chromosomes.Furthermore,gene structure,chromosomal location,conserved motifs,and expression level of BLH genes were investigated in G.hirsutum.Expression profiles analysis showed that four genes(GhBLH1_3,GhBLH1_4,GhBLH1_5,and GhBLH1_6)from BLH1 subfamily were highly expressed during the fiber cell elongation period.The expression levels of these genes were significantly induced by gibberellic acid and brassinosteroid,but not auxin.Exogenous application of gibberellic acid significantly enhanced GhBLH1_3,GhBLH1_4,and GhBLH1_5 transcripts.Expression levels of GhBLH1_3 and GhBLH1_4 genes were significantly increased under brassinosteroid treatment.Conclusions:The BLH gene family plays a very important role in many biological processes during plant growth and development.This study deepens our understanding of the role of the GhBLH1 gene involved in fiber development and will help us in breeding better cotton varieties in the future.

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Comparative analysis of SIMILAR to RCD ONE(SRO)family from tetraploid cotton species and their diploid progenitors depict their significance in cotton growth and development

Background SRO(Similar to RCD1)genes family is largely recognized for their importance in the growth,develop-ment,and in responding to environmental stresses.However,genome-wide identification and functional characteri-zation of SRO genes from cotton species have not been reported so far.Results A total of 36 SRO genes were identified from four cotton species.Phylogenetic analysis divided these genes into three groups with distinct structure.Syntenic and chromosomal distribution analysis indicated uneven distribu-tion of GaSRO,GrSRO,GhSRO,and GbSRO genes on A2,D5 genomes,Gh-At,Gh-Dt,Gb-At,and Gb-Dt subgenomes,respectively.Gene duplication analysis revealed the presence of six duplicated gene pairs among GhSRO genes.In promoter analysis,several elements responsive to the growth,development and hormones were found in GhSRO genes,implying gene induction during cotton growth and development.Several miRNAs responsive to plant growth and abiotic stress were predicted to target 12 GhSRO genes.Organ-specific expression profiling demonstrated the roles of GhSRO genes in one or more tissues.In addition,specific expression pattern of some GhSRO genes dur-ing ovule development depicted their involvement in these developmental processes.Conclusion The data presented in this report laid a foundation for understanding the classification and functions of SRO genes in cotton.

Improvements of Fiber Yield and Fiber Fineness by Expressing the iaaM Gene in Cotton Seed Coat

Cotton,the most important natural fiber crop in the world,is a mainstay in China's economy.However,for over two decades,cotton yields both in China and U.S.have been at a plateau.

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.

Transcriptome Analysis of Ten Days Post Anthesis Elongating Fiber in the Upland Cotton (<i>Gossypium hirsutum</i>) Chromosome Substitution Line CS-B25

A chromosome substitution line, CS-B25, was developed by the substitution of chromosome pair 25 of Gossypium hirsutum TM-1 with the homologous pair of chromosome 25 from G. barbadense, a double haploid Pima 3-79 line. CS-B25 has improved fiber traits compared to its parent TM-1. To explore the molecule mechanisms underlying improved fiber traits, deep sequencing of total RNA was used to compare gene expression in fibers of CS-B25 and TM-1 at 10 days post anthesis (10-DPA). A total of 1872 differentially expressed genes (DEGs) were detected between the two lines, with 1175 up-regulated and 697 down-regulated in CS-B25. Gene Ontology (GO) enrichment analysis of the expression data by Generally Applicable Gene-set Enrichment (GAGE) and ReviGO indicated that the most prevalent Biological Process GO terms associated with DEGs included DNA-templated transcription, response to oxidative stress, and cellulose biosynthesis. Enriched Molecular Function GO terms included structural constituents of cytoskeleton, peroxidase activity, cellulose synthase (UDP-forming) activity, and transcription regulatory region sequence-specific DNA binding factors. GAGE was also used to find enriched KEGG pathways, and the highly represented pathways were Biosynthesis of Amino Acids, Starch and Sucrose Metabolism, Phenylpropanoid Biosynthesis, Protein Processing in Endoplasmic Reticulum, and Plant Hormone Signal Transduction. Many of the identified DEGs are involved in cytoskeleton and cell wall metabolism. The results of gene expression data have provided new insight into the molecular mechanisms of fiber development during the fiber elongation stage and would offer novel candidate genes that may be utilized in cotton fiber quality improvement.

Expression of a cotton MADS-box gene is regulated in anther development and in response to phytohormone signaling

MADS-box gene family encodes a large number and variety of transcription regulators in plants. In this study, a cDNA, GhMADS9, encoding a typical MADS protein with 230 amino acids was isolated from cotton flower cDNA library. Subsequently, a 1,623 bp genomic DNA fragment of GhMADS9 gene was isolated in cotton by PCR. Compared with its cDNA sequence, six introns were found in GhMADS9 gene. Fluorescent microscopy indicated that GhMADS9 protein localized in the nucleus. Transactivation activity assay in yeast cells revealed that GhMADS9 protein did not show transcriptional activation. Quantitative RT-PCR analysis showed that GhMADS9 was specially expressed in cotton anthers. Further in situ hybridization analysis demonstrated that strong expression of GhMADS9 gene was detected in developing pollens, but no or weak signals were found in the other anther tissues. Furthermore, GhMADS9 expression was dramatically up-regulated in anthers with abscisic acid (ABA) treatment, whereas its activity was down-regulated when treated by gibberellin (GA3). Collectively, our results suggest that GhMADS9 is a transcription factor and might be involved in cotton anther/pollen development and in response to ABA and GA3 signaling.

Phytosterol content and the campesterol:sitosterol ratio influence cotton fiber development: role of phytosterols in cell elongation

Phytosterols play an important role in plant growth and development,including cell division,cell elongation,embryogenesis,cellulose biosynthesis,and cell wall formation.Cotton fiber,which undergoes synchronous cell elongation and a large amount of cellulose synthesis,is an ideal model for the study of plant cell elongation and cell wall biogenesis.The role of phytosterols in fiber growth was investigated by treating the fibers with tridemorph,a sterol biosynthetic inhibitor.The inhibition of phytosterol biosynthesis resulted in an apparent suppression of fiber elongation in vitro or in planta.The determination of phytosterol quantity indicated that sitosterol and campesterol were the major phytosterols in cotton fibers;moreover,higher concentrations of these phytosterols were observed during the period of rapid elongation of fibers.Furthermore,the decrease and increase in campesterol:sitosterol ratio was associated with the increase and decease in speed of elongation,respectively,during the elongation stage.The increase in the ratio was associated with the transition from cell elongation to secondary cell wall synthesis.In addition,a number of phytosterol biosynthetic genes were down-regulated in the short fibers of ligon lintless-1mutant,compared to its near-isogenic wild-type TM-1.These results demonstrated that phytosterols play a crucial role in cotton fiber development,and particularly in fiber elongation.

Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis （DPA）, however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate （MeJA）, cold, and abscisic acid （ABA） treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

Rapid Identification of a Candidate Gene Related to Fiber Strength Using a Superior Chromosome Segment Substitution Line from Gossypium hirsutum × Gossypium barbadense via Bulked Segregant RNA-Sequencing

Cotton is the most widely cultivated commercial crop producing natural fiber around the world.As a critical trait for fiber quality,fiber strength principally determined during the secondary wall thickening period.Based on the developed BC5F3:5 CSSLs(chromosome segment substitution lines)from Gossypium hirsutum CCRI36×G.barbadense Hai 1,the superior MBI9915 was chosen to construct the secondary segregated population BC7F2 with its recurrent parent CCRI36,which was subsequently subjected to Bulk segregant RNA-sequencing(BSR-seq)for rapid identification of candidate genes related to fiber strength.A total of 4 fiber-transcriptome libraries were separately constructed and sequenced,including two parents(CCRI36 and MBI9915)and two extreme pools at 20 DPA(days post anathesis).Through multiple comparisons,536 DEGs(differentially expressed genes)were overlapped at 20 DPA.Allelic-polymorphism comparison in mRNA sequences revealed 831 highly probable SNPs between two extreme pools related to fiber strength.Linkage analysis was performed between two extreme pools with SNP-index method.Eighteen correlated regions with 1981 annotation genes were obtained between two pools at 20 DPA,of which 12 common DEGs were similarly identified both between two parents and two pools.One gene(Gh_A07G0837)in the candidate region related to fiber strength was differentially expressed in both parents and extreme pools and involved in fiber strength development through reactive oxygen species(ROS)activity.Co-expression analysis of Gh_A07G0837 showed that Gh_A07G0837 may cooperate with other genes to regulate fiber strength.The reliability of BSR-seq results was validated by the quantitative real-time PCR(qRT-PCR)experiments on 5 common DGEs 20 DPA.Co-expressed analysis results indicated that there were some genes expressed especially low in MBI9915,resulting in good fiber strength.Focusing on bulked segregant analysis on the extreme pools derived from superior CSSL population,this study indicates that BSR-seq can be efficiently applied on rapid identification of candidate genes related to fiber strength,which make contributions to our understanding of fiber quality formation in cotton.

Genome-wide identification and characterization of phospholipase C gene family in cotton (Gossypium spp.)

Phospholipase C (PLC) are important regulatory enzymes involved in several lipid and Ca2+-dependent signaling pathways.Previous studies have elucidated the versatile roles of PLC genes in growth, development and stress responses of many plants, however, the systematic analyses of PLC genes in the important fiber-producing plant, cotton, are still deficient. In this study,through genome-wide survey, we identified twelve phosphatidylinositol-specific PLC (PI-PLC) and nine non-specific PLC (NPC) genes in the allotetraploid upland cotton Gossypium hirsutum and nine PI-PLC and six NPC genes in two diploid cotton G. arboretum and G.raimondii, respectively. The PI-PLC and NPC genes of G. hirsutum showed close phylogenetic relationship with their homologous genes in the diploid cottons and Arabidopsis. Segmental and tandem duplication contributed greatly to the formation of the gene family. Expression profiling indicated that few of the PLC genes are constitutely expressed, whereas most of the PLC genes are preferentially expressed in specific tissues and abiotic stress conditions. Promoter analyses further implied that the expression of these PLC genes might be regulated by MYB transcription factors and different phytohormones.These results not only suggest an important role of phospholipase C members in cotton plant development and abiotic stress response but also provide good candidate targets for future molecular breeding of superior cotton cultivars.

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.

Cotton AnnGh3 Encoding an Annexin Protein is Preferentially Expressed in Fibers and Promotes Initiation and Elongation of Leaf Trichomes in Transgenic Arabidopsis

The annexins are a multifamily of calcium-regulated phospholipid-binding proteins. To investigate the roles of annexins in fiber development, four genes encoding putative annexin proteins were isolated from cotton （Gossypium hirsutum） and designated AnnGh3, AnnGh4, AnnGh5, and AnnGh6. Quantitative reverse transcription-polymerase chain reaction （qRT-PCR） results indicated that AnnGh3, AnnGh4, and AnnGh5 were preferentially expressed in fibers, while the transcripts of AnnGh6 were predominantly accumulated in roots. During fiber development, the transcripts of AnnGh3/4/5 genes were mainly accumulated in rapidly elongating fibers. With fiber cells further developed, their expression activity was dramatically declined to a relatively low level. In situ hybridization results indicated that AnnGh3 and AnnGh5were expressed in initiating fiber cells （0-2 DPA）. Additionally, their expression in fibers was also regulated by phytohormones and [Ca2~]. Subcellular localization analysis discovered that AnnGh3 protein was localized in the cytoplasm. Overexpression of AnnGh3 in Arabidopsis resulted in a significant increase in trichome density and length on leaves of the transgenic plants, suggesting that AnnGh3 may be involved in fiber cell initiation and elongation of cotton.

Response of the enzymes to nitrogen applications in cotton fiber (Gossypium hirsutum L.) and their relationships with fiber strength

To investigate the response of key enzymes to nitrogen (N) rates in cotton fiber and its relationship with fiber strength, experiments were conducted in 2005 and 2006 with cotton cultivars in Nanjing. Three N rates 0, 240 and 480 kgN/hm2, signifying optimum and excessive nitrogen application levels were applied.The activities and the gene expressions of the key enzymes were affected by N, and the characteristics of cellulose accumulation and fiber strength changed as the N rate varied. Beta-1,3-glucanase activity in cotton fiber declined from 9 DPA till boll opening, and the beta-1, 3-glucanase coding gene expression also followed a unimodal curve in 12—24 DPA. In 240 kgN/hm2 condition, the characteristics of enzyme activity and gene expression manner for sucrose synthase and beta-1,3-glucanase in developing cotton fiber were more favorable for forming a longer and more steady cellulose accumulation process, and for high strength fiber development.

Cotton GhMYB7 is predominantly expressed in developing fibers and regulates secondary cell wall biosynthesis in transgenic Arabidopsis

The secondary cell wall in mature cotton fibers contains over 90%cellulose with low quantities of xylan and lignin.However,little is known regarding the regulation of secondary cell wall biosynthesis in cotton fibers.In this study,we characterized an R2R3-MYB transcription factor,Gh MYB7,in cotton.Gh MYB7 is expressed at a high level in developing fibers and encodes a MYB protein that is targeted to the cell nucleus and has transcriptional activation activity.Ectopic expression of Gh MYB7 in Arabidopsis resulted in small,curled,dark green leaves and also led to shorter inflorescence stems.A cross-sectional assay of basal stems revealed that cell wall thickness of vessels and interfascicular fibers was higher in transgenic lines overexpressing Gh MYB7 than in the wild type.Constitutive expression of Gh MYB7 in Arabidopsis activated the expression of a suite of secondary cell wall biosynthesis-related genes(including some secondary cell wall-associated transcription factors),leading to the ectopic deposition of cellulose and lignin.The ectopic deposition of secondary cell walls may have been initiated before the cessation of cell expansion.Moreover,Gh MYB7 was capable of binding to the promoter regions of At SND1 and At Ces A4,suggesting that Gh MYB7 may function upstream of NAC transcription factors.Collectively,these findings suggest that Gh MYB7 is a potential transcriptional activator,which may participate in regulating secondary cell wall biosynthesis of cotton fibers.