Comprehensive identification and systematical characterization of BRX gene family and the functional of GhBRXL5A in response to salt stress.

BACKGROUND: BRVIS RADIX (BRX) family is a small gene family with the highly conserved plant-specific BRX domains, which plays important roles in plant development and response to abiotic stress. Although BRX protein has been studied in other plants, the biological function of cotton BRX-like (BRXL) gene family is still elusive. RESULT: In this study, a total of 36 BRXL genes were identified in four cotton species. Whole genome or segmental duplications played the main role in the expansion of GhBRXL gene family during evolutionary process in cotton. These BRXL genes were clustered into 2 groups, α and ß, in which structural and functional conservation within same groups but divergence among different groups were found. Promoter analysis indicated that cis-elements were associated with the phytohormone regulatory networks and the response to abiotic stress. Transcriptomic analysis indicated that GhBRXL2A/2D and GhBRXL5A/5D were up/down-regulated in response to the different stress. Silencing of GhBRXL5A gene via virus-induced gene silencing (VIGS) improved salt tolerance in cotton plants. Furthermore, yeast two hybrid analysis suggested homotypic and heterotypic interactions between GhBRXL1A and GhBRXL5D. CONCLUSIONS: Overall, these results provide useful and valuable information for understanding the evolution of cotton GhBRXL genes and their functions in salt stress.

A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are a class of peptide hormones that have been shown in previous studies to play an important role in regulating the development and response to abiotic stress in model plants. However, their role in cotton is not well understood. In this study, we identified 54, 59, 34, and 35 CEP genes from Gossypium hirsutum (2n = 4x = 52, AD1), G. barbadense (AD2), G. arboreum (2n = 2X = 26, A2), and G. raimondii (2n = 2X = 26, D5), respectively. Sequence alignment and phylogenetic analyses indicate that cotton CEP proteins can be categorized into two subgroups based on the differentiation of their CEP domain. Chromosomal distribution and collinearity analyses show that most of the cotton CEP genes are situated in gene clusters, suggesting that segmental duplication may be a critical factor in CEP gene expansion. Expression pattern analyses showed that cotton CEP genes are widely expressed throughout the plant, with some genes exhibiting specific expression patterns. Ectopic expression of GhCEP46-D05 in Arabidopsis led to a significant reduction in both root length and seed size, resulting in a dwarf phenotype. Similarly, overexpression of GhCEP46-D05 in cotton resulted in reduced internode length and plant height. These findings provide a foundation for further investigation into the function of cotton CEP genes and their potential role in cotton breeding.

Genome-Wide Identification of Calmodulin-Binding Protein 60 Gene Family and the Function of GhCBP60B in Cotton Growth and Development and Abiotic Stress Response.

The calmodulin-binding protein 60 (CBP60) family is a gene family unique to plants, and its members play a crucial role in plant defense responses to pathogens and growth and development. Considering that cotton is the primary source of natural cotton textile fiber, the functional study of its CBP60 gene family members is critical. In this research, we successfully identified 162 CBP60 members from the genomes of 21 species. Of these, 72 members were found in four cotton species, divided into four clades. To understand the function of GhCBP60B in cotton in depth, we conducted a detailed analysis of its sequence, structure, cis-acting elements, and expression patterns. Research results show that GhCBP60B is located in the nucleus and plays a crucial role in cotton growth and development and response to salt and drought stress. After using VIGS (virus-induced gene silencing) technology to conduct gene silencing experiments, we found that the plants silenced by GhCBP60B showed dwarf plants and shortened stem nodes, and the expression of related immune genes also changed. In further abiotic stress treatment experiments, we found that GhCBP60B-silenced plants were more sensitive to drought and salt stress, and their POD (peroxidase) activity was also significantly reduced. These results imply the vital role of GhCBP60B in cotton, especially in regulating plant responses to drought and salt stress. This study systematically analyzed CBP60 gene family members through bioinformatics methods and explored in depth the biological function of GhCBP60B in cotton. These research results lay a solid foundation for the future use of the GhCBP60B gene to improve cotton plant type and its drought and salt resistance.

Comprehensive identification and functional characterization of GhpPLA gene family in reproductive organ development.

BACKGROUND: Phospholipases As (PLAs) are acyl hydrolases that catalyze the release of free fatty acids in phospholipids and play multiple functions in plant growth and development. The three families of PLAs are: PLA1, PLA2 (sPLA), and patatin-related PLA (pPLA). The diverse functions that pPLAs play in the growth and development of a broad range of plants have been demonstrated by prior studies. METHODS: Genome-wide analysis of the pPLA gene family and screening of genes for expression verification and gene silencing verification were conducted. Additionally, pollen vitality testing, analysis of the pollen expression pattern, and the detection of POD, SOD, CAT, MDA, and H2O2 were performed. RESULT: In this study, 294 pPLAs were identified from 13 plant species, including 46 GhpPLAs that were divided into three subfamilies (I-III). Expression patterns showed that the majority of GhpPLAs were preferentially expressed in the petal, pistil, anther, and ovule, among other reproductive organs. Particularly, GhpPLA23 and GhpPLA44, were found to be potentially important for the reproductive development of G. hirsutum. Functional validation was demonstrated by VIGS which showed that reduced expression levels of GhpPLA23 and GhpPLA44 in the silenced plants were associated with a decrease in pollen activity. Moreover, a substantial shift in ROS and ROS scavengers and a considerable increase in POD, CAT, SOD, and other physiological parameters was found out in these silenced plants. Our results provide plausibility to the hypothesis that GhpPLA23 and GhpPLA44 had a major developmental impact on cotton reproductive systems. These results also suggest that pPLAs are important for G. hirsutum's reproductive development and suggest that they could be employed as potential genes for haploid induction. CONCLUSIONS: The findings of the present research indicate that pPLA genes are essential for the development of floral organs and sperm cells in cotton. Consequently, this family might be important for the reproductive development of cotton and possibly for inducing the plant develop haploid progeny.

Characteristic of GEX1 genes reveals the essential roles for reproduction in cotton.

GEX1 (gamete expressed 1) proteins are critical membrane proteins conserved among flowering plants that are involved in the nuclear fusion and embryonic development. Herein, we identified the 32 GEX1 proteins from representative land plants. In cotton, GEX1 genes expressed in various tissues across all stages of the life cycle, especially in pollen. Subcellular localization indicated the position of GhGEX1 protein was localized in the endoplasmic reticulum. Experimental research has demonstrated that GhGEX1 has the potential to improve the partial abortion phenotype in Arabidopsis. CRISPR/Cas9-mediated knockout of GhGEX1 exhibited the seed abortion. Paraffin section of the ovule revealed that the polar nuclear fusion of ghgex1 plants remains at a standstill when the wild type has developed into a normal embryo. Comparative transcriptome analysis showed that the DEGs of reproductive-related processes and membrane-related processes were repressed in the pollen of knockout lines. The predicted protein interactions showed that GhGEX1 probably functioned through interactions with proteins related to reproduction and membrane. From all these investigations, it was possible to conclude that the GEX1 proteins are evolutionarily conserved in flowering plants and elucidated the pivotal roles during fertilization and early embryonic development in cotton.

GhCKX14 responding to drought stress by modulating antioxi-dative enzyme activity in Gossypium hirsutum compared to CKX family genes.

BACKGROUND: Cytokinin oxidase/dehydrogenase (CKX) plays a vital role in response to abiotic stress through modulating the antioxidant enzyme activities. Nevertheless, the biological function of the CKX gene family has yet to be reported in cotton. RESULT: In this study, a total of 27 GhCKXs were identified by the genome-wide investigation and distributed across 18 chromosomes. Phylogenetic tree analysis revealed that CKX genes were clustered into four clades, and most gene expansions originated from segmental duplications. The CKXs gene structure and motif analysis displayed remarkably well conserved among the four groups. Moreover, the cis-acting elements related to the abiotic stress, hormones, and light response were identified within the promoter regions of GhCKXs. Transcriptome data and RT-qPCR showed that GhCKX genes demonstrated higher expression levels in various tissues and were involved in cotton's abiotic stress and phytohormone response. The protein-protein interaction network indicates that the CKX family probably participated in redox regulation, including oxidoreduction or ATP levels, to mediate plant growth and development. Functionally identified via virus-induced gene silencing (VIGS) found that the GhCKX14 gene improved drought resistance by modulating the antioxidant-related activitie. CONCLUSIONS: In this study, the CKX gene family members were analyzed by bioinformatics, and validates the response of GhCKX gene to various phytohormone treatment and abiotic stresses. Our findings established the foundation of GhCKXs in responding to abiotic stress and GhCKX14 in regulating drought resistance in cotton.

Identification, Classification and Characterization Analysis of FBXL Gene in Cotton.

F-box/LR (FBXL), Leucine-rich repeats in F-box proteins, belongs to the Skp1-Cullin1-F-box protein (SCF) E3 ligase family. FBXL genes play important roles in plant growth, such as plant hormones, responses to environmental stress, and floral organ development. Here, a total of 518 FBXL genes were identified and analyzed in six plant species. Phylogenetic analysis showed that AtFBXLs, VvFBXLs, and GrFBXLs were clustered into three subfamilies (Ⅰ-Ⅲ). Based on the composition of the F-box domain and carboxyl-terminal amino acid sequence, FBXL proteins were classified into three types (Type-A/-B/-C). Whole-genome duplication (WGD) along with tandem duplications and segmental contributed to the expansion of this gene family. The result indicates that four cotton species are also divided into three subfamilies. FBXLs in cotton were classified into three clades by phylogenetic and structural analyses. Furthermore, expression analyses indicated that the expression patterns of GhFBXLs in different cotton tissues were different. The highly expressed of GH_A07G2363 in 5-8 mm anthers, indicates that this gene might play a role in the reproductive process, providing candidate genes for future studies on cotton fertility materials. This study provides an original functional opinion and a useful interpretation of the FBXL protein family in cotton.

Evolutionary Relationships and Divergence of Filamin Gene Family Involved in Development and Stress in Cotton (Gossypium hirsutum L.).

Filamin protein is characterized by an N-terminal actin-binding domain that is followed by 24 Ig (immunoglobulin)-like repeats, which act as hubs for interactions with a variety of proteins. In humans, this family has been found to be involved in cancer cell invasion and metastasis and can be involved in a variety of growth signal transduction processes, but it is less studied in plants. Therefore, in this study, 54 Filamin gene family members from 23 plant species were investigated and divided into two subfamilies: FLMN and GEX2. Subcellular localization showed that most of the Filamin gene family members were located in the cell membrane. A total of 47 Filamin gene pairs were identified, most of which were whole-genome copies. Through the analyses of cis-acting elements, expression patterns and quantitative fluorescence, it was found that GH_ A02G0519 and GH_ D02G0539 are mainly expressed in the reproductive organs of upland cotton, and their interacting proteins are also related to the fertilization process, whereas GH_A02G0216 and GH_D02G0235 were related to stress. Thus, it is speculated that two genes of the GEX2 subfamily (GH_A02G0519 and GH_D02G0539) may be involved in the reproductive development of cotton and may affect the fertilization process of cotton. This study provides a theoretical basis for the further study of the cotton Filamin gene family.

Systematical Characterization of the Cotton Di19 Gene Family and the Role of GhDi19-3 and GhDi19-4 as Two Negative Regulators in Response to Salt Stress.

Drought-induced 19 (Di19) protein is a Cys2/His2 (C2H2) type zinc-finger protein, which plays a crucial role in plant development and in response to abiotic stress. This study systematically investigated the characteristics of the GhDi19 gene family, including the member number, gene structure, chromosomal distribution, promoter cis-elements, and expression profiles. Transcriptomic analysis indicated that some GhDi19s were up-regulated under heat and salt stress. Particularly, two nuclear localized proteins, GhDi19-3 and GhDi19-4, were identified as being in potential salt stress responsive roles. GhDi19-3 and GhDi19-4 decreased sensitivity under salt stress through virus-induced gene silencing (VIGS), and showed significantly lower levels of H2O2, malondialdehyde (MDA), and peroxidase (POD) as well as significantly increased superoxide dismutase (SOD) activity. This suggested that their abilities were improved to effectively reduce the reactive oxygen species (ROS) damage. Furthermore, certain calcium signaling and abscisic acid (ABA)-responsive gene expression levels showed up- and down-regulation changes in target gene-silenced plants, suggesting that GhDi19-3 and GhDi19-4 were involved in calcium signaling and ABA signaling pathways in response to salt stress. In conclusion, GhDi19-3 and GhDi19-4, two negative transcription factors, were found to be responsive to salt stress through calcium signaling and ABA signaling pathways.

Expression Patterns Divergence of Reciprocal F1 Hybrids Between Gossypium hirsutum and Gossypium barbadense Reveals Overdominance Mediating Interspecific Biomass Heterosis.

Hybrid breeding has provided an impetus to the process and achievement of a higher yield and quality of crops. Interspecific hybridization is critical for resolving parental genetic diversity bottleneck problems. The reciprocal interspecific hybrids and their parents (Gossypium hirsutum and Gossypium barbadense) have been applied in this study to elucidate the transcription regulatory mechanism of early biomass heterosis. Phenotypically, the seed biomass, plant height over parent heterosis, leaf area over parent heterosis, and fresh and dry biomass were found to be significantly higher in hybrids than in parents. Analysis of leaf areas revealed that the one-leaf stage exhibits the most significant performance in initial vegetative growth vigor and larger leaves in hybrids, increasing the synthesis of photosynthesis compounds and enhancing photosynthesis compound synthesis. Comparative transcriptome analysis showed that transgressive down-regulation (TDR) is the main gene expression pattern in the hybrids (G. hirsutum × G. barbadense, HB), and it was found that the genes of photosystem I and Adenosine triphosphate (ATP)-binding may promote early growth vigor. Transgressive up-regulation (TUR) is the major primary gene expression pattern in the hybrids (G. barbadense × G. hirsutum, BH), and photosystem II-related genes mediated the performance of early biomass heterosis. The above results demonstrated that overdominance mediates biomass heterosis in interspecific hybrid cotton and the supervisory mechanism divergence of hybrids with different females. Photosynthesis and other metabolic process are jointly involved in controlling early biomass heterosis in interspecific hybrid cotton. The expression pattern data of transcriptome sequencing were supported using the qRT-PCR analysis. Our findings could be useful in theoretical and practical studies of early interspecific biomass heterosis, and the results provide potential resources for the theoretical and applied research on early interspecific biomass heterosis.

Genome-wide Characterization of the JmjC Domain-Containing Histone Demethylase Gene Family Reveals GhJMJ24 and GhJMJ49 Involving in Somatic Embryogenesis Process in Cotton.

The Jumonji C (JmjC) domain-containing protein family, an important family of histone demethylase in plants, can directly reverse histone methylation and play important roles in various growth and development processes. In the present study, 51 JmjC genes (GhJMJs) were identified by genome-wide analysis in upland cotton (Gossypium hirsutum), which can be categorized into six distinct groups by phylogenetic analysis. Extensive syntenic relationship events were found between G. hirsutum and Theobroma cacao. We have further explored the putative molecular regulatory mechanisms of the JmjC gene family in cotton. GhJMJ24 and GhJMJ49 were both preferentially expressed in embryogenic callus compared to nonembryogenic callus in cotton tissue culture, which might be regulated by transcription factors and microRNAs to some extent. Further experiments indicated that GhJMJ24 and GhJMJ49 might interact with SUVH4, SUVH6, DDM1, CMT3, and CMT1 in the nucleus, potentially in association with demethylation of H3K9me2. Taken together, our results provide a foundation for future research on the biological functions of GhJMJ genes in cotton, especially in somatic embryogenesis in cotton tissue culture, which is crucial for the regeneration of transgenic plants.

The Evolution and Expression Profiles of EC1 Gene Family during Development in Cotton.

Fertilization is essential to sexual reproduction of flowering plants. EC1 (EGG CELL 1) proteins have a conserved cysteine spacer characteristic and play a crucial role in double fertilization process in many plant species. However, to date, the role of EC1 gene family in cotton is fully unknown. Hence, detailed bioinformatics analysis was explored to elucidate the biological mechanisms of EC1 gene family in cotton. In this study, we identified 66 genes in 10 plant species in which a total of 39 EC1 genes were detected from cotton genome. Phylogenetic analysis clustered the identified EC1 genes into three families (I-III) and all of them contain Prolamin-like domains. A good collinearity was observed in the synteny analysis of the orthologs from cotton genomes. Whole-genome duplication was determined to be one of the major impetuses for the expansion of the EC1 gene family during the process of evolution. qRT-PCR analysis showed that EC1 genes were highly expressed in reproductive tissues under multiple stresses, signifying their potential role in enhancing stress tolerance or responses. Additionally, gene interaction networks showed that EC1 genes may be involved in cell stress and response transcriptional regulator in the synergid cells and activate the expression of genes required for pollen tube guidance. Our results provide novel functional insights into the evolution and functional elucidation of EC1 gene family in cotton.

Cotton DMP gene family: characterization, evolution, and expression profiles during development and stress.

Members of DOMAIN OF UNKNOWN FUNCTION 679 membrane protein (DMP) gene family, a type of plant-specific membrane proteins, have been proposed to function in various physiological processes such as reproductive development and senescence in plants. Here, a total of 174 DMP genes were identified and analyzed in 16 plant species (including 58 DMPs in four cotton species). Phylogenetic analysis showed that these DMPs could be clustered into five subfamilies (I-V). 137 duplicated cotton gene pairs were identified and most duplicate events were formed by whole-genome duplication (WGD)/segmental duplications. Expression analysis revealed that most of cotton DMPs were mainly expressed in the reproductive organs (the sepal, petal, pistil and anther) and the fiber of secondary cell wall stage. GhDMPs promoter regions containing the different cis-elements also showed different responses to abiotic stress. In addition, gene interaction networks showed that DMPs, as an endomembrane system, were involved in plant senescence process and flower reproductive development. We speculated GhDMP8-A/-D, GbDMP8-A/-D could be used as some candidate gene for inducing cotton haploid. This genome-wide study provides a systematic analysis of the cotton DMP gene family, and further insights towards understanding the potential functions of candidate genes.

Evolution and Functional Divergence of SUN Genes in Plants.

SUN-domain containing proteins are crucial nuclear membrane proteins involved in a plethora of biological functions, including meiosis, nuclear morphology, and embryonic development, but their evolutionary history and functional divergence are obscure. In all, 216 SUN proteins from protists, fungi, and plants were divided into two monophyletic clades (Cter-SUN and Mid-SUN). We performed comprehensive evolutionary analyses, investigating the characteristics of different subfamilies in plants. Mid-SUNs further evolved into two subgroups, SUN3 and SUN5, before the emergence of the ancestor of angiosperms, while Cter-SUNs retained one subfamily of SUN1. The two clades were distinct from each other in the conserved residues of the SUN domain, the TM motif, and exon/intron structures. The gene losses occurred with equal frequency between these two clades, but duplication events of Mid-SUNs were more frequent. In cotton, SUN3 proteins are primarily expressed in petals and stamens and are moderately expressed in other tissues, whereas SUN5 proteins are specifically expressed in mature pollen. Virus-induced knock-down and the CRISPR/Cas9-mediated knockout of GbSUN5 both showed higher ratios of aborted seeds, although pollen viability remained normal. Our results indicated divergence of biological function between SUN3 and SUN5, and that SUN5 plays an important role in reproductive development.

A Retrotransposon Insertion in GhMML3_D12 Is Likely Responsible for the Lintless Locus li3 of Tetraploid Cotton.

Cotton (Gossypium) seed fibers can be divided into lint (long) or fuzz (very short). Using fiberless (fuzzless-lintless) mutants, the lint initiation gene Li3 was identified by map-based cloning. The gene is an R2R3-MYB transcription factor located on chromosome D12 (GhMML3_D12). Sequence analysis revealed that li3 is a loss-of-function allele containing a retrotransposon insertion in the second exon that completely blocks the gene's expression. The genetic loci n2 and n3 underlying the recessive fuzzless phenotype in Gossypium hirsutum were also mapped. The genomic location of n3 overlapped with that of the dominant fuzzless locus N1 , and n3 appeared to be a loss-of-function allele caused by a single nucleotide polymorphism (SNP) mutation in the coding region of GhMML3_A12. The n2 allele was found to be co-located with li3 and originated from G. babardense. n2 and li3 are possibly the multiple alleles of the GhMML3_D12 gene. Genetic analysis showed that Li3 and N3 are a pair of homologs with additive effects for the initiation of fibers (fuzz or lint). In addition, the presence of another locus was speculated, and it appeared to show an inhibitory effect on the expression of GhMML3. These findings provide new information about the genetic factors affecting the initiation of fibers in cotton.

Characterization and transcriptome analysis of a dominant genic male sterile cotton mutant.

BACKGROUND: Male sterility is an efficient trait for hybrid seed production and germplasm innovation. Until now, most studies on male sterility were on cytoplasmic and recessive genic sterility, with few on dominant genic male sterility, especially in cotton, due to lack of such mutant. RESULTS: We discovered a natural male sterile (MS) Sea Island cotton (G. barbadense) mutant. Genetic analysis showed the mutation was caused by a dominant mutation in a single nuclear gene. Comparative cytological observation of anther sections from MS and wild-type (WT) uncovered cellular differences in anther at and after the tetrad stage of pollen mother cells (PMC). In the MS anthers, the outer wall of pollen grains was free of spinules, the tapetum was vacuolated and showed delayed degradation, consequently, no functional pollen grains. Comparison of transcriptomes from meiosis, tetrad, mononuclear and binuclear pollen, and pollen maturation stages identified 13,783 non-redundant differentially expressed genes (DEGs) between MS and WT. Based on the number of DEGs, analyses of enriched GO terms and KEGG pathways, it was evident that significant transcriptomic changes occurred at and after the tetrad stage, consistent with cytological observation, and that the major differences were on metabolism of starch, sucrose, ascorbate, aldarate, alanine, aspartate and glutamate, and biosynthesis of cutin, suberine and wax. WGCNA analysis identified five modules containing 920 genes highly related to anther development, especially the greenyellow module with 54 genes that was highly associated with PMC meiosis and tetrad formation. A NAC transcription factor (Gh_D11G2469) was identified as a hub gene for this module, which warrants further functional characterization. CONCLUSIONS: We demonstrated that the MS trait was controlled by a single dominant nuclear gene and caused by delayed tapetum degradation at the tetrad stage. Comparative transcriptome analysis and gene network construction identified DEGs, enriched GO terms and metabolic pathways, and hub genes potentially associated with anther development and the MS trait. These results contribute to our understanding of dominant genic male sterility (DGMS) and provided source for innovation of cotton germplasm.

Open-Bud Duplicate Loci Are Identified as MML10s, Orthologs of MIXTA-Like Genes on Homologous Chromosomes of Allotetraploid Cotton.

The open-bud (ob) mutants in cotton display abnormal flower buds with the stigma and upper anthers exposed before blooming. This characteristic is potentially useful for the efficient production of hybrid seeds. The recessive inheritance pattern of the ob phenotype in allotetraploid cotton is determined by duplicated recessive loci (ob1ob1ob2ob2). In this study, ob1, which is a MIXTA-like MYB gene on chromosome D13 (MML10_Dt), was identified by map-based cloning. In Gossypium barbadense (Gb) acc. 3-79, a single nucleotide polymorphism (SNP) (G/A) at the splice site of the first intron and an 8-bp deletion in the third exon of MML10_Dt were found, which are the causative mutations at the ob1 loci. A 1783-bp deletion that leads to the loss of the third exon and accounts for the causal variation at the ob2 loci was found in MML10_At of Gossypium hirsutum (Gh) acc. TM-1. The ob phenotype results from the combination of these two loss-of-function loci. Genotyping assays showed that the ob1 and ob2 loci appeared after the formation of allotetraploid cotton and were specific for Gb and Gh, respectively. All Gb lines and most Gh cultivars carry the single corresponding mutant alleles. Genome-wide transcriptome analysis showed that some of the MYB genes and genes related to cell wall biogenesis, trichome differentiation, cytokinin signal transduction, and cell division were repressed in the ob mutants, which may lead to suppression of petal growth. These findings should be of value for breeding superior ob lines in cotton.

Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4⁻100% and 92.9⁻100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.

Identification and Functional Characterization of a Microtubule-Associated Protein, GhCLASP2, From Upland Cotton (Gossypium hirsutum L.).

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins (MAPs) involved in regulation of dynamics of microtubules (MTs) that play an important role in plant growth and development. In this study, we identified cotton CLASP genes and investigated the function of GhCLASP2. GhCLASP2 was mainly expressed in stem and developing fibers, especially in fibers of the secondary cell wall deposition stage. Ectopic expression of GhCLASP2 in Arabidopsis increased the branching number of leaf trichomes and rescued the defective phenotypes of clasp-1. In cotton, overexpression of GhCLASP2 increased fiber strength, probably related to enhanced expression levels of tubulin, cellulose synthase, and expansin genes. Suppression of GhCLASP2 caused shorter internodes and semi-dwarfism, abnormal flower stigma, aborted anthers without pollen grains, and sterility. These changed phenotypes were similar to those observed in the Arabidopsis clasp-1 mutant. GhCLASP2 was co-localized with MTs according to transient experiment. These results suggest that GhCLASP2 functions similarly as AtCLASP, acting as a MAP and controlling cotton growth and development by regulating MTs.

Expression and functional analyses of a Kinesin gene GhKIS13A1 from cotton (Gossypium hirsutum) fiber.

BACKGROUND: Cotton fiber, a natural fiber widely used in the textile industry, is differentiated from single cell of ovule epidermis. A large number of genes are believed to be involved in fiber formation, but so far only a few fiber genes have been isolated and functionally characterized in this developmental process. The Kinesin13 subfamily was found to play key roles during cell division and cell elongation, and was considered to be involved in the regulation of cotton fiber development. RESULTS: The full length of coding sequence of GhKIS13A1 was cloned using cDNA from cotton fiber for functional characterization. Expression pattern analysis showed that GhKIS13A1 maintained a lower expression level during cotton fiber development. Biochemical assay showed that GhKIS13A1 has microtubule binding activity and basal ATPase activity that can be activated significantly by the presence of microtubules. Overexpression of GhKIS13A1 in Arabidopsis reduced leaf trichomes and the percentage of three-branch trichomes, and increased two-branch and shriveled trichomes compared to wild-type. Additionally, the expression of GhKIS13A1 in the Arabidopsis Kinesin-13a-1 mutant rescued the defective trichome branching pattern of the mutant, making its overall trichome branching pattern back to normal. CONCLUSIONS: Our results suggested that GhKIS13A1 is functionally compatible with AtKinesin-13A regarding their role in regulating the number and branching pattern of leaf trichomes. Given the developmental similarities between cotton fibers and Arabidopsis trichomes, it is speculated that GhKIS13A1 may also be involved in the regulation of cotton fiber development.