Suppressing a ß-1,3-glucanase gene expression increases the seed and fibre yield in cotton.

Seeds are initiated from the carpel margin meristem (CMM) and high seed yield is top one of breeding objectives for many crops. ß-1,3-glucanases play various roles in plant growth and developmental processes; however, whether it participates in CMM development and seed formation remains largely unknown. Here, we identified a ß-1,3-glucanase gene (GLU19) as a determinant of CMM callose deposition and seed yield in cotton. GLU19 was differentially expressed in carpel tissues between Gossypium barbadense (Gb) and Gossypium hirsutum (Gh). Based on resequencing data, one interspecies-specific InDel in the promoter of GLU19 was further detected. The InDel was involved in the binding site of the CRABS CLAW (CRC) transcription factor, a regulator of carpel development. We found that the CRC binding affinity to the GLU19 promoter of G. barbadense was higher than that of G. hirsutum. Since G. barbadense yields fewer seeds than G. hirsutum, we speculated that stronger CRC binding to the GLU19 promoter activated higher expression of GLU19 which in turn suppressed seed production. Consistent with this hypothesis was that the overexpression of GhGLU19 caused reduced seed number, boll weight and less callose formation in CMM. Conversely, GhGLU19-knockdown (GhGLU19-KD) cotton led to the opposite phenotypes. By crossing GhGLU19-KD lines with several G. hirsutum and G. barbadense cotton accessions, all F1 and F2 plants carrying GhGLU19-KD transgenic loci exhibited higher seed yield than control plants without the locus. The increased seed effect was also found in the down-regulation of Arabidopsis orthologs lines, indicating that this engineering strategy may improve the seed yield in other crops.

Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

MYB30-INTERACTING E3 LIGASE 1 regulates LONELY GUY 5-mediated cytokinin metabolism to promote drought tolerance in cotton.

Ubiquitination plays important roles in modulating the abiotic stress tolerance of plants. Drought seriously restricts agricultural production, but how ubiquitination participates in regulating drought tolerance remains largely unknown. Here, we identified a drought-inducible gene, MYB30-INTERACTING E3 LIGASE 1 (GhMIEL1), which encodes a RING E3 ubiquitin ligase in cotton (Gossypium hirsutum). GhMIEL1 was strongly induced by polyethylene glycol (PEG-6000) and the phytohormone abscisic acid (ABA). Overexpression and knockdown of GhMIEL1 in cotton substantially enhanced and reduced drought tolerance, respectively. GhMIEL1 interacted with the MYB transcription factor GhMYB66 and could ubiquitinate and degrade it in vitro. GhMYB66 directly bound to the LONELY GUY 5 (GhLOG5) promoter, a gene encoding cytokinin riboside 5'-monophosphate phosphoribohydrolase, to repress its transcription. Overexpression of GhMIEL1 and silencing of GhMYB66 altered the homeostasis of cytokinin of plant roots, increased total root length and number of root tips, and enhanced plant drought tolerance. Conversely, silencing GhLOG5 decreased total root length and number of root tips and reduced plant drought tolerance. Our studies reveal that the GhMIEL1-GhMYB66-GhLOG5 module positively regulates drought tolerance in cotton, which deepens our understanding of plant ubiquitination-mediated drought tolerance and provides insights for improving drought tolerance.

Combined genome and transcriptome analysis of elite fiber quality in Gossypium barbadense.

Gossypium barbadense, which is one of several species of cotton, is well known for its superior fiber quality. However, the genetic basis of its high-quality fiber remains largely unexplored. Here, we resequenced 269 G. barbadense accessions. Phylogenetic structure analysis showed that the set of accessions was clustered into 3 groups: G1 and G2 mainly included modern cultivars from Xinjiang, China, and G3 was related to widely introduced accessions in different regions worldwide. A genome-wide association study of 5 fiber quality traits across multiple field environments identified a total of 512 qtls (main-effect QTLs) and 94 qtlEs (QTL-by-environment interactions) related to fiber quality, of which 292 qtls and 57 qtlEs colocated with previous studies. We extracted the genes located in these loci and performed expression comparison, local association analysis, and introgression segment identification. The results showed that high expression of hormone-related genes during fiber development, introgressions from Gossypium hirsutum, and the recombination of domesticated elite allelic variation were 3 major contributors to improve the fiber quality of G. barbadense. In total, 839 candidate genes with encoding region variations associated with elite fiber quality were mined. We confirmed that haplotype GB_D03G0092H traced to G. hirsutum introgression, with a 1-bp deletion leading to a frameshift mutation compared with GB_D03G0092B, significantly improved fiber quality. GB_D03G0092H is localized in the plasma membrane, while GB_D03G0092B is in both the nucleus and plasma membrane. Overexpression of GB_D03G0092H in Arabidopsis (Arabidopsis thaliana) significantly improved the elongation of longitudinal cells. Our study systematically reveals the genetic basis of the superior fiber quality of G. barbadense and provides elite segments and gene resources for breeding high-quality cotton cultivars.

Cotton peroxisome-localized lysophospholipase counteracts the toxic effects of Verticillium dahliae NLP1 and confers wilt resistance.

Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis-and-ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant-specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP-producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome-localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity-related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over-accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP-producing microbial pathogens.

LIPID TRANSFER PROTEIN4 regulates cotton ceramide content and activates fiber cell elongation.

Cell elongation is a fundamental process for plant growth and development. Studies have shown lipid metabolism plays important role in cell elongation; however, the related functional mechanisms remain largely unknown. Here, we report that cotton (Gossypium hirsutum) LIPID TRANSFER PROTEIN4 (GhLTP4) promotes fiber cell elongation via elevating ceramides (Cers) content and activating auxin-responsive pathways. GhLTP4 was preferentially expressed in elongating fibers. Over-expression and down-regulation of GhLTP4 led to longer and shorter fiber cells, respectively. Cers were greatly enriched in GhLTP4-overexpressing lines and decreased dramatically in GhLTP4 down-regulating lines. Moreover, auxin content and transcript levels of indole-3-acetic acid (IAA)-responsive genes were significantly increased in GhLTP4-overexpressing cotton fibers. Exogenous application of Cers promoted fiber elongation, while NPA (N-1-naphthalic acid, a polar auxin transport inhibitor) counteracted the promoting effect, suggesting that IAA functions downstream of Cers in regulating fiber elongation. Furthermore, we identified a basic helix-loop-helix transcription factor, GhbHLH105, that binds to the E-box element in the GhLTP4 promoter region and promotes the expression of GhLTP4. Suppression of GhbHLH105 in cotton reduced the transcripts level of GhLTP4, resulting in smaller cotton bolls and decreased fiber length. These results provide insights into the complex interactions between lipids and auxin-signaling pathways to promote plant cell elongation.

A cell wall-localized ß-1,3-glucanase promotes fiber cell elongation and secondary cell wall deposition.

ß-1,3-glucanase functions in plant physiological and developmental processes. However, how ß-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a ß-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of ß-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to <1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze ß-1,3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction, and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber SCW-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

Carotenoid sequestration protein FIBRILLIN participates in CmOR-regulated ß-carotene accumulation in melon.

Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, ß-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high ß-carotene melon variety and its isogenic line low-ß mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops.

Thiamine functions as a key activator for modulating plant health and broad-spectrum tolerance in cotton.

Global climate changes cause an increase of abiotic and biotic stresses that tremendously threaten the world's crop security. However, studies on broad-spectrum response pathways involved in biotic and abiotic stresses are relatively rare. Here, by comparing the time-dependent transcriptional changes and co-expression analysis of cotton (Gossypium hirsutum) root tissues under abiotic and biotic stress conditions, we discovered the common stress-responsive genes and stress metabolism pathways under different stresses, which included the circadian rhythm, thiamine and galactose metabolism, carotenoid, phenylpropanoid, flavonoid, and zeatin biosynthesis, and the mitogen-activated protein kinase signaling pathway. We found that thiamine metabolism was an important intersection between abiotic and biotic stresses; the key thiamine synthesis genes, GhTHIC and GhTHI1, were highly induced at the early stage of stresses. We confirmed that thiamine was crucial and necessary for cotton growth and development, and its deficiency could be recovered by exogenous thiamine supplement. Furthermore, we revealed that exogenous thiamine enhanced stress tolerance in cotton via increasing calcium signal transduction and activating downstream stress-responsive genes. Overall, our studies demonstrated that thiamine played a crucial role in the tradeoff between plant health and stress resistance. The thiamine deficiency caused by stresses could transiently induce upregulation of thiamine biosynthetic genes in vivo, while it could be totally salvaged by exogenous thiamine application, which could significantly improve cotton broad-spectrum stress tolerance and enhance plant growth and development.

Suppression of GhGLU19 encoding ß-1,3-glucanase promotes seed germination in cotton.

BACKGROUND: In eudicots, germination begins with water uptake by the quiescent dry seed and is greatly related to the permeability of micropyle enriched callose layers. Once imbibition starts, seeds undergo a cascade of physiological, biochemical, and molecular events to initiate cellular activities. However, the effects of callose on water uptake and following seed metabolic events during germination are largely unknown. Cotton (Gossypium hirsutum) is a eudicot plant with natural fiber and edible oil production for humans. Here, we addressed this question by examining the role of GhGLU19, a gene encoding ß-1,3-glucanase, in cotton seed germination. RESULTS: GhGLU19 belongs to subfamily B and was expressed predominately in imbibed seeds and early seedlings. Compared to wild type, GhGLU19-suppressing and GhGLU19-overexpressing transgenic cotton lines showed the higher and lower seed germination percentage, respectively. Callose was enriched more at inner integument (ii) than that in embryo and seed coat in cotton seeds. In GhGLU19-suppressing lines, callose at ii of cotton seeds was greatly increased and brought about a prolonged water uptake process during imbibition. Both proteomic and transcriptomic analysis revealed that contrary to GhGLU19-overexpressing lines, the glycolysis and pyruvate metabolism was decreased, and abscisic acid (ABA) biosynthesis related genes were downregulated in imbibed seeds of GhGLU19-suppressing lines. Also, endogenous ABA was significantly decreased in GhGLU19-suppressing line while increased in GhGLU19-overexpressing line. CONCLUSIONS: Our results demonstrate that suppression of GhGLU19 improves cotton seed germination via accumulating callose of inner integument, modulating glycolysis and pyruvate metabolism, and decreasing ABA biosynthesis. This study provides a potential way for improving germination percentage in cotton seed production, and other eudicot crops.

Genome-wide association analysis reveals genetic variations and candidate genes associated with salt tolerance related traits in Gossypium hirsutum.

BACKGROUND: Cotton is more resistant to salt and drought stresses as compared to other field crops, which makes itself as a pioneer industrial crop in saline-alkali lands. However, abiotic stresses still negatively affect its growth and development significantly. It is therefore important to breed salt tolerance varieties which can help accelerate the improvement of cotton production. The development of molecular markers linked to causal genes has provided an effective and efficient approach for improving salt tolerance. RESULTS: In this study, a genome-wide association study (GWAS) of salt tolerance related traits at seedling stage was performed based on 2 years of phenotype identification for 217 representative upland cotton cultivars by genotyping-by-sequencing (GBS) platform. A total of 51,060 single nucleotide polymorphisms (SNPs) unevenly distributed among 26 chromosomes were screened across the cotton cultivars, and 25 associations with 27 SNPs scattered over 12 chromosomes were detected significantly (-log10p > 4) associated with three salt tolerance related traits in 2016 and 2017. Among these, the associations on chromosome A13 and D08 for relative plant height (RPH), A07 for relative shoot fresh matter weight (RSFW), A08 and A13 for relative shoot dry matter weight (RSDW) were expressed in both environments, indicating that they were likely to be stable quantitative trait loci (QTLs). A total of 12 salt-induced candidate genes were identified differentially expressed by the combination of GWAS and transcriptome analysis. Three promising genes were selected for preliminary function verification of salt tolerance. The increase of GH_A13G0171-silenced plants in salt related traits under salt stress indicated its negative function in regulating the salt stress response. CONCLUSIONS: These results provided important genetic variations and candidate genes for accelerating the improvement of salt tolerance in cotton.

Genome-wide association analysis reveals quantitative trait loci and candidate genes involved in yield components under multiple field environments in cotton (Gossypium hirsutum).

BACKGROUND: Numerous quantitative trait loci (QTLs) and candidate genes associated with yield-related traits have been identified in cotton by genome-wide association study (GWAS) analysis. However, most of the phenotypic data were from a single or few environments, and the stable loci remained to be validated under multiple field environments. RESULTS: Here, 242 upland cotton accessions collected from different origins were continuously investigated for phenotypic data of four main yield components, including boll weight (BW) and lint percentage (LP) under 13 field environments, and boll number per plant (BN) and seed index (SI) under 11 environments. Correlation analysis revealed a positive correlation between BN and LP, BW and SI, while SI had a negative correlation with LP and BN. Genetic analysis indicated that LP had the highest heritability estimates of 94.97%, followed by 92.08% for SI, 86.09% for BW, and 72.92% for BN, indicating LP and SI were more suitable traits for genetic improvement. Based on 56,010 high-quality single nucleotide polymorphisms (SNPs) and GWAS analysis, a total of 95 non-redundant QTLs were identified, including 12 of BN, 23 of BW, 45 of LP, and 33 of SI, respectively. Of them, 10 pairs of homologous QTLs were detected between A and D sub-genomes. We also found that 15 co-located QTLs with more than two traits and 12 high-confidence QTLs were detected under more than six environments, respectively. Further, two NET genes (GH_A08G0716 and GH_A08G0783), located in a novel QTL hotspot (qtl24, qtl25 and qlt26) were predominately expressed in early fiber development stages, exhibited significant correlation with LP and SI. The GH_A07G1389 in the stable qtl19 region encoded a tetratricopeptide repeat (TPR)-like superfamily protein and was a homologous gene involved in short fiber mutant ligon lintless-y (Liy), implying important roles in cotton yield. CONCLUSIONS: The present study provides a foundation for understanding the regulatory mechanisms of yield components and may enhance yield improvement through molecular breeding in cotton.

Genome-wide association analysis reveals loci and candidate genes involved in fiber quality traits in sea island cotton (Gossypium barbadense).

BACKGROUND: Sea island cotton (Gossypium barbadense) has markedly superior high quality fibers, which plays an important role in the textile industry and acts as a donor for upland cotton (G. hirsutum) fiber quality improvement. The genetic characteristics analysis and the identification of key genes will be helpful to understand the mechanism of fiber development and breeding utilization in sea island cotton. RESULTS: In this study, 279 sea island cotton accessions were collected from different origins for genotyping and phenotyping fiber quality traits. A set of 6303 high quality single nucleotide polymorphisms (SNPs) were obtained by high-density CottonSNP80K array. The population characteristics showed that the sea island cotton accessions had wide genetic diversity and were clustered into three groups, with Group1 closely related to Menoufi, an original sea island cotton landrace, and Group2 and Group3 related to widely introduced accessions from Egypt, USA and Former Soviet Union. Further, we used 249 accessions and evaluated five fiber quality traits under normal and salt environments over 2 years. Except for fiber uniformity (FU), fiber length (FL) and fiber elongation (FE) were significantly decreased in salt conditions, while fiber strength (FS) and fiber micronaire (MIC) were increased. Based on 6303 SNPs and genome-wide association study (GWAS) analysis, a total of 34 stable quantitative trait loci (QTLs) were identified for the five fiber quality traits with 25 detected simultaneously under normal and salt environments. Gene Ontology (GO) analysis indicated that candidate genes in the 25 overlapped QTLs were enriched mostly in "cellular and biological process". In addition, "xylem development" and "response to hormone" pathways were also found. Haplotype analyses found that GB_A03G0335 encoding an E3 ubiquitin-protein ligase in QTL TM6004 had SNP variation (A/C) in gene region, was significantly correlated with FL, FS, FU, and FE, implying a crucial role in fiber quality. CONCLUSIONS: The present study provides a foundation for genetic diversity of sea island cotton accessions and will contribute to fiber quality improvement in breeding practice.

Genome-wide association reveals genetic variation of lint yield components under salty field conditions in cotton (Gossypium hirsutum L.).

BACKGROUND: Salinity is one of the most significant environmental factors limiting the productivity of cotton. However, the key genetic components responsible for the reduction in cotton yield in saline-alkali soils are still unclear. RESULTS: Here, we evaluated three main components of lint yield, single boll weight (SBW), lint percentage (LP) and boll number per plant (BNPP), across 316 G. hirsutum accessions under four salt conditions over two years. Phenotypic analysis indicated that LP was unchanged under different salt conditions, however BNPP decreased significantly and SBW increased slightly under high salt conditions. Based on 57,413 high-quality single nucleotide polymorphisms (SNPs) and genome-wide association study (GWAS) analysis, a total of 42, 91 and 25 stable quantitative trait loci (QTLs) were identified for SBW, LP and BNPP, respectively. Phenotypic and QTL analysis suggested that there was little correlation among the three traits. For LP, 8 stable QTLs were detected simultaneously in four different salt conditions, while fewer repeated QTLs for SBW or BNPP were identified. Gene Ontology (GO) analysis indicated that their regulatory mechanisms were also quite different. Via transcriptome profile data, we detected that 10 genes from the 8 stable LP QTLs were predominantly expressed during fiber development. Further, haplotype analyses found that a MYB gene (GhMYB103), with the two SNP variations in cis-regulatory and coding regions, was significantly correlated with lint percentage, implying a crucial role in lint yield. We also identified that 40 candidate genes from BNPP QTLs were salt-inducible. Genes related to carbohydrate metabolism and cell structure maintenance were rich in plants grown in high salt conditions, while genes related to ion transport were active in plants grown in low salt conditions, implying different regulatory mechanisms for BNPP at high and low salt conditions. CONCLUSIONS: This study provides a foundation for elucidating cotton salt tolerance mechanisms and contributes gene resources for developing upland cotton varieties with high yields and salt stress tolerance.

A cotton α1,3-/4-fucosyltransferase-encoding gene, FucT4, plays an important role in cell elongation and is significantly associated with fiber quality.

Fucosylation, one of the key posttranslational modifications, plays an important role in plants. It is involved in the development, signal transduction, reproduction, and disease resistance. α1,3-/4-Fucosyltransferase is responsible for transferring L-fucose from GDP-L-fucose to the N-glycan to exert fucosylational functions. However, the roles of the fucosyltransferase gene in cotton remain unknown. This study provided a comprehensive investigation of its possible functions. A genome-wide analysis identified four, four, eight, and eight FucT genes presented in the four sequenced cotton species, diploid Gossypium raimondii, G. arboreum, tetraploid G. hirsutum acc. TM-1, and G. barbadense cv. H7124, respectively. These FucTs were classified into two groups, with FucT4 homologs alone as a group. We isolated FucT4 in TM-1 and H7124, and named it GhFucT4 and GbFucT4, respectively. Quantitative RT-PCR and transcriptome data demonstrated that GhFucT4 had the highest expression levels in fibers among all GhFucT genes. Association studies and QTL co-localization supported the possible involvement of GhFucT4 in cotton fiber development. GhFucT4 and GbFucT4 shared high sequence identities, and FucT4 had higher expression in H7124 fiber tissues compared with TM-1. Furthermore, ectopic expression of FucT4 in transgenic Arabidopsis promoted root cell elongation, upregulated expression of genes related to cell wall loosening, and led to longer primary root. These results collectively indicate that FucT4 plays an important role in promoting cell elongation and modulating fiber development, which could be utilized to improve fiber quality traits in cotton breeding.

GbCYP86A1-1 from Gossypium barbadense positively regulates defence against Verticillium dahliae by cell wall modification and activation of immune pathways.

Suberin acts as stress-induced antipathogen barrier in the root cell wall. CYP86A1 encodes cytochrome P450 fatty acid ω-hydroxylase, which has been reported to be a key enzyme for suberin biosynthesis; however, its role in resistance to fungi and the mechanisms related to immune responses remain unknown. Here, we identified a disease resistance-related gene, GbCYP86A1-1, from Gossypium barbadense cv. Hai7124. There were three homologs of GbCYP86A1 in cotton, which are specifically expressed in roots and induced by Verticillium dahliae. Among them, GbCYP86A1-1 contributed the most significantly to resistance. Silencing of GbCYP86A1-1 in Hai7124 resulted in severely compromised resistance to V. dahliae, while heterologous overexpression of GbCYP86A1-1 in Arabidopsis improved tolerance. Tissue sections showed that the roots of GbCYP86A1-1 transgenic Arabidopsis had more suberin accumulation and significantly higher C16-C18 fatty acid content than control. Transcriptome analysis revealed that overexpression of GbCYP86A1-1 not only affected lipid biosynthesis in roots, but also activated the disease-resistant immune pathway; genes encoding the receptor-like kinases (RLKs), receptor-like proteins (RLPs), hormone-related transcription factors, and pathogenesis-related protein genes (PRs) were more highly expressed in the GbCYP86A1-1 transgenic line than control. Furthermore, we found that when comparing V. dahliae -inoculated and noninoculated plants, few differential genes related to disease immunity were detected in the GbCYP86A1-1 transgenic line; however, a large number of resistance genes were activated in the control. This study highlights the role of GbCYP86A1-1 in the defence against fungi and its underlying molecular immune mechanisms in this process.

Suppressing a Putative Sterol Carrier Gene Reduces Plasmodesmal Permeability and Activates Sucrose Transporter Genes during Cotton Fiber Elongation.

Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D, a putative sterol carrier protein gene from elongating cotton (Gossypium hirsutum) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D-suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D, which encodes a PD-targeting ß-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs, leading to the switch from symplasmic to apoplasmic pathways.

Combining genome-wide and transcriptome-wide analyses reveal the evolutionary conservation and functional diversity of aquaporins in cotton.

BACKGROUND: Aquaporins (AQPs) are integral membrane proteins from a larger family of major intrinsic proteins (MIPs) and function in a huge variety of processes such as water transport, plant growth and stress response. The availability of the whole-genome data of different cotton species allows us to study systematic evolution and function of cotton AQPs on a genome-wide level. RESULTS: Here, a total of 53, 58, 113 and 111 AQP genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. A comprehensive analysis of cotton AQPs, involved in exon/intron structure, functional domains, phylogenetic relationships and gene duplications, divided these AQPs into five subfamilies (PIP, NIP, SIP, TIP and XIP). Comparative genome analysis among 30 species from algae to angiosperm as well as common tandem duplication events in 24 well-studied plants further revealed the evolutionary conservation of AQP family in the organism kingdom. Combining transcriptome analysis and Quantitative Real-time PCR (qRT-PCR) verification, most AQPs exhibited tissue-specific expression patterns both in G. raimondii and G. hirsutum. Meanwhile, a bias of time to peak expression of several AQPs was also detected after treating G. davidsonii and G. hirsutum with 200 mM NaCl. It is interesting that both PIP1;4 h/i/j and PIP2;2a/e showed the highly conserved tandem structure, but differentially contributed to tissue development and stress response in different cotton species. CONCLUSIONS: These results demonstrated that cotton AQPs were structural conservation while experienced the functional differentiation during the process of evolution and domestication. This study will further broaden our insights into the evolution and functional elucidation of AQP gene family in cotton.

RNA-seq analysis reveals alternative splicing under salt stress in cotton, Gossypium davidsonii.

BACKGROUND: Numerous studies have focused on the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the post-transcriptional level. RESULTS: Using a diploid D genome wild salinity-tolerant cotton species, Gossypium davidsonii, we analyzed alternative splicing (AS) of genes related to salt stress by comparing high-throughput transcriptomes from salt-treated and well-watered roots and leaves. A total of 14,172 AS events were identified involving 6798 genes, of which intron retention (35.73%) was the most frequent, being detected in 3492 genes. Under salt stress, 1287 and 1228 differential alternative splicing (DAS) events were identified in roots and leaves, respectively. These DAS genes were associated with specific functional pathways, such as "responses to stress", "metabolic process" and "RNA splicing", implying that AS represents an important pathway of gene regulation in response to salt stress. Several salt response genes, such as pyrroline-5-carboxylate synthase (P5CS), K+ channel outward (KCO1), plasma membrane intrinsic protein (PIP) and WRKY33 which were involved in osmotic balance, ion homeostasis, water transportation and transcriptional regulation, respectively, were identified with differential alternative splicing under salt stress. Moreover, we revealed that 13 genes encoding Ser/Arg-rich (SR) proteins related to AS regulation were differentially alternatively spliced under salt stress. CONCLUSION: This study first provide a comprehensive view of AS in G. davidsonii, and highlight novel insights into the potential roles of AS in plant responses to salt stress.

Divergence and evolution of cotton bHLH proteins from diploid to allotetraploid.

BACKGROUND: Polyploidy is considered a major driving force in genome expansion, yielding duplicated genes whose expression may be conserved or divergence as a consequence of polyploidization. RESULTS: We compared the genome sequences of tetraploid cotton (Gossypium hirsutum) and its two diploid progenitors, G. arboreum and G. raimondii, and found that the bHLH genes were conserved over the polyploidization. Oppositely, the expression of the homeolgous gene pairs was diversified. The biased homeologous proportion for bHLH family is significantly higher (64.6%) than the genome wide homeologous expression bias (40%). Compared with cacao (T. cacao), orthologous genes only accounted for a small proportion (41.7%) of whole cotton bHLHs family. The further Ks analysis indicated that bHLH genes underwent at least two distinct episodes of whole genome duplication: a recent duplication (1.0-60.0 million years ago, MYA, 0.005 < Ks < 0.312) and an old duplication (> 60.0 MYA, 0.312 < Ks < 3.0). The old duplication event might have played a key role in the expansion of the bHLH family. Both recent and old duplicated pairs (68.8%) showed a divergent expression profile, indicating specialized functions. The expression diversification of the duplicated genes suggested it might be a universal feature of the long-term evolution of cotton. CONCLUSIONS: Overview of cotton bHLH proteins indicated a conserved and divergent evolution from diploids to allotetraploid. Our results provided an excellent example for studying the long-term evolution of polyploidy.