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SUMMARY: Verticillium dahliae, a soil‐borne fungal pathogen, compromises host innate immunity by secreting a plethora of effectors, thereby facilitating host colonization and causing substantial yield and quality losses. The mechanisms underlying the modulation of cotton immunity by V. dahliae effectors are predominantly unexplored. In this study, we identified that the V. dahliae effector Vd6317 inhibits plant cell death triggered by Vd424Y and enhances PVX viral infection in Nicotiana benthamiana. Attenuation of Vd6317 significantly decreased the virulence of V. dahliae, whereas ectopic expression of Vd6317 in Arabidopsis and cotton enhanced susceptibility to V. dahliae infection, underscoring Vd6317's critical role in pathogenicity. We observed that Vd6317 targeted the Arabidopsis immune regulator AtNAC53, thereby impeding its transcriptional activity on the defense‐associated gene AtUGT74E2. Arabidopsis nac53 and ugt74e2 mutants exhibited heightened sensitivity to V. dahliae compared to wild‐type plants. A mutation at the conserved residue 193L of Vd6317 abrogated its interaction with AtNAC53 and reduced the virulence of V. dahliae, which was partially attributable to a reduction in Vd6317 protein stability. Our findings unveil a hitherto unrecognized regulatory mechanism by which the V. dahliae effector Vd6317 directly inhibits the plant transcription factor AtNAC53 activity to suppress the expression of AtUGT74E2 and plant defense. Significance Statement: Verticillium dahliae effector Vd6317 regulates plant immunity. [ABSTRACT FROM AUTHOR]

Summary: Heavy metal pollution poses a significant risk to human health and wreaks havoc on agricultural productivity. Phytoremediation, a plant‐based, environmentally benign, and cost‐effective method, is employed to remove heavy metals from contaminated soil, particularly in agricultural or heavy metal‐sensitive lands. However, the phytoremediation capacity of various plant species and germplasm resources display significant genetic diversity, and the mechanisms underlying these differences remain hitherto obscure. Given its potential benefits, genetic improvement of plants is essential for enhancing their uptake of heavy metals, tolerance to harmful levels, as well as overall growth and development in contaminated soil. In this study, we uncover a molecular cascade that regulates cadmium (Cd2+) tolerance in cotton, involving GhRCD1, GhbHLH12, GhMYB44, and GhHMA1. We identified a Cd2+‐sensitive cotton T‐DNA insertion mutant with disrupted GhRCD1 expression. Genetic knockout of GhRCD1 by CRISPR/Cas9 technology resulted in reduced Cd2+ tolerance in cotton seedlings, while GhRCD1 overexpression enhanced Cd2+ tolerance. Through molecular interaction studies, we demonstrated that, in response to Cd2+ presence, GhRCD1 directly interacts with GhbHLH12. This interaction activates GhMYB44, which subsequently activates a heavy metal transporter, GhHMA1, by directly binding to a G‐box cis‐element in its promoter. These findings provide critical insights into a novel GhRCD1–GhbHLH12–GhMYB44–GhHMA1 regulatory module responsible for Cd2+ tolerance in cotton. Furthermore, our study paves the way for the development of elite Cd2+‐tolerant cultivars by elucidating the molecular mechanisms governing the genetic control of Cd2+ tolerance in cotton. [ABSTRACT FROM AUTHOR]

This article, published in the Plant Biotechnology Journal, discusses the use of host-induced gene silencing (HIGS) to target a gene in the cotton plant that confers resistance to Verticillium wilt, a devastating disease caused by the fungus Verticillium dahliae. The researchers investigated the expression pattern and function of a gene called VdEXG, which encodes an exoglucanase enzyme. They found that silencing VdEXG in the cotton plant led to increased resistance to V. dahliae infection. Additionally, they identified an interacting protein called GhRD21A that plays a role in enhancing cotton resistance to the fungus. These findings provide a new strategy for breeding wilt-resistant cotton cultivars. [Extracted from the article]

MYB transcription factors play important roles during abiotic stress responses in plants. However, little is known about the accurate systematic analysis of MYB genes in the four cotton species, Gossypium hirsutum, G. barbadense, G. arboreum and G. raimondii. Herein, we performed phylogenetic analysis and showed that cotton MYBs and Arabidopsis MYBs were clustered in the same subfamilies for each species. The identified cotton MYB s were distributed unevenly on chromosomes in various densities for each species, wherein genome-wide tandem and segment duplications were the main driving force of MYB family expansion. Synteny analysis suggested that the abundant collinearity pairs of MYB s were identified between G. hirsutum and the other three species, and that they might have undergone strong purification selection. Characteristics of conserved motifs, along with their consensus sequence, promoter cis elements and gene structure, revealed that MYB proteins might be highly conserved in the same subgroups for each species. Subsequent analysis of differentially expressed genes and expression patterns indicated that most GhMYB s might be involved in response to drought (especially) and salt stress, which was supported by the expression levels of nine GhMYB s using real-time quantitative PCR. Finally, we performed a workflow that combined virus-induced gene silencing and the heterologous transformation of Arabidopsis , which confirmed the positive roles of GhMYB s under drought conditions, as validated by determining the drought-tolerant phenotypes, damage index and/or water loss rate. Collectively, our findings not only expand our understanding of the relationships between evolution and function of MYB genes, but they also provide candidate genes for cotton breeding. [ABSTRACT FROM AUTHOR]

Summary: Plant somatic embryogenesis (SE) is a multifactorial developmental process where embryos that can develop into whole plants are produced from somatic cells rather than through the fusion of gametes. The molecular regulation of plant SE, which involves the fate transition of somatic cells into embryogenic cells, is intriguing yet remains elusive.We deciphered the molecular mechanisms by which GhRCD1 interacts with GhMYC3 to regulate cell fate transitions during SE in cotton. While silencing of GhMYC3 had no discernible effect on SE, its overexpression accelerated callus formation, and proliferation.We identified two of GhMYC3 downstream SE regulators, GhMYB44 and GhLBD18. GhMYB44 overexpression was unconducive to callus growth but bolstered EC differentiation. However, GhLBD18 can be triggered by GhMYC3 but inhibited by GhMYB44, which positively regulates callus growth. On top of the regulatory cascade, GhRCD1 antagonistically interacts with GhMYC3 to inhibit the transcriptional function of GhMYC3 on GhMYB44 and GhLBD18, whereby a CRISPR‐mediated rcd1 mutation expedites cell fate transition, resembling the effects of GhMYC3 overexpression. Furthermore, we showed that reactive oxygen species (ROS) are involved in SE regulation.Our findings elucidated that SE homeostasis is maintained by the tetrapartite module, GhRCD1–GhMYC3–GhMYB44–GhLBD18, which acts to modulate intracellular ROS in a temporal manner. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene‐edited cotton plants through either particle bombardment or Agrobacterium‐mediated transformation. Here, we developed a shoot apical meristem (SAM) cell‐mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2–3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP‐ and RUBY‐positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9‐mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene‐edited plants for genetic improvement of recalcitrant cotton varieties. [ABSTRACT FROM AUTHOR]

Subtilases (SBTs), which belong to the serine peptidases, control plant development by regulating cell wall properties and the activity of extracellular signaling molecules, and affect all stages of the life cycle, such as seed development and germination, and responses to biotic and abiotic environments. In this study, 146 Gossypium hirsutum, 138 Gossypium barbadense, 89 Gossypium arboreum and 84 Gossypium raimondii SBTs were identified and divided into six subfamilies. Cotton SBTs are unevenly distributed on chromosomes. Synteny analysis showed that the members of SBT1 and SBT4 were expanded in cotton compared to Arabidopsis thaliana. Co-expression network analysis showed that six Gossypium arboreum SBT gene family members were in a network, among which five SBT1 genes and their Gossypium hirsutum and Arabidopsis thaliana direct homologues were down-regulated by salt treatment, indicating that the co-expression network might share conserved functions. Through co-expression network and annotation analysis, these SBTs may be involved in the biological processes of auxin transport, ABA signal transduction, cell wall repair and root tissue development. In summary, this study provides valuable information for the study of SBT genes in cotton and excavates SBT genes in response to salt stress, which provides ideas for cotton breeding for salinity resistance. [ABSTRACT FROM AUTHOR]

Summary: Cotton's fundamental requirements for long periods of growth and specific seasonal temperatures limit the global arable areas that can be utilized to cultivate cotton. This constraint can be alleviated by breeding for early‐maturing varieties. By delaying the sowing dates without impacting the boll‐opening time, early‐maturing varieties not only mitigate the yield losses brought on by unfavorable weathers in early spring and late autumn but also help reducing the competition between cotton and other crops for arable land, thereby optimizing the cropping system. This review presents studies and breeding efforts for early‐maturing cotton, which efficiently pyramid early maturity, high‐quality, multiresistance traits, and suitable plant architecture by leveraging pleiotropic genes. Attempts are also made to summarize our current understanding of the molecular mechanisms underlying early maturation, which involves many pathways such as epigenetic, circadian clock, and hormone signaling pathways. Moreover, new avenues and effective measures are proposed for fine‐scale breeding of early‐maturing crops to ensure the healthy development of the agricultural industry. [ABSTRACT FROM AUTHOR]

Cotton is one of the Uyghur medical materials in China and is rich in flavonoids. Flavonoids have important pharmacological effects. The yield of flavonoids in traditional extraction methods is low, which affects the development of flavonoids. Therefore, it is urgent to optimize the extraction techniques. The yield of flavonoids in cotton flowers was effectively improved by response surface methodology, and the highest yield of flavonoids reached 5.66%, and the optimal extraction process conditions were obtained. The DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, superoxide anion free radical scavenging rate, and reducing ability were tested to reflect the antioxidant capacity of flavonoids. The flavonoids had an excellent antioxidant effect. Cell experiments suggested that the flavonoids had the effect of protecting glutamate-induced damage to HT-22 cells. The results of this study provide a theoretical basis for the extraction of cotton flowers flavonoids and the comprehensive evaluation of antioxidant products, as well as the extraction of other plant flavonoids. [ABSTRACT FROM AUTHOR]

Keywords: Cotton; Pink fibre; Betalain; Synthetic biology EN Cotton Pink fibre Betalain Synthetic biology 674 676 3 03/28/23 20230401 NES 230401 The vast majority of the world's cotton varieties produce white fibre, which is then coloured with synthetic dyes during textile processing to fulfil the diverse needs of consumers. However, it remains unknown if the betalain pathway can be engineered into cotton plants to alter the colour of cotton fibre without compromising fibre quality. As illustrated in Figures 1m,n,p,t, the fibre length and strength of the transgenic fibres at 45 DPA and mature stage were comparable to those of the WT fibre. [Extracted from the article]

Plant auxin is essential in plant growth and development. However, the molecular mechanisms of auxin involvement in plant immunity are unclear. Here, we addressed the function of the cotton (Gossypium hirsutum) miR393-TIR1 module in plant defense against Verticillium dahliae infection via auxin perception and signaling. GhTIR1 was directedly cleaved by ghr-miR393 according to mRNA degradome data, 5′-RACE analysis, and a GUS reporter assay. Ghr-miR393 knockdown significantly increased plant susceptibility to V. dahliae compared to the control, while ghr-miR393 overexpression and GhTIR1 knockdown significantly increased plant resistance. External indole-3-acetic acid (IAA) application significantly enhanced susceptibility to V. dahliae in ghr-miR393 knockdown and control plants compared to mock treatment, and only slightly increased susceptibility in overexpressing ghr-miR393 and GhTIR1-silenced plants. Application of external PEO-IAA (an auxin antagonist) had a contrary trend with IAA application. Based on yeast two-hybrid and bimolecular fluorescence complementation assays, GhTIR1 interacted with GhIAA14 in the nucleus, and GhIAA14 knockdown reduced plant resistance to V. dahliae infection. The results suggested that the ghr-miR393-GhTIR1 module regulates plant defense via auxin perception and signaling. Additionally, simultaneous knockdown of GhTIR1 and GhICS1 significantly increased plant susceptibility to V. dahliae compared to the control, indicating that salicylic acid (SA) accumulation is vital for the ghr-miR393-GhTIR1 module to regulates plant resistance. Transcriptome data also demonstrated that GhTIR1 knockdown significantly downregulated expression of auxin-related genes and upregulated expression of SA-related genes. Overall, the ghr-miR393-GhTIR1 module participates in plant response to V. dahliae infection via IAA perception and signaling partially depending on the SA defense pathway. [ABSTRACT FROM AUTHOR]

During somatic embryogenesis (SE), somatic cells initiate embryogenic development under appropriate conditions. Uniconazole, a plant growth regulator, was found to inhibit the proliferation of callus but promoted the conversion of callus into an embryogenic callus (EC) in cotton. The supplementation of uniconazole in the culture medium significantly suppressed the endogenous auxin [indole acetic acid (IAA)] level in callus tissues in both the callus initiation and proliferation stage but enhanced the abscisic acid (ABA) level only in the callus proliferation stage. Exogenous ABA and uniconazole showed cooperative effects on promoting the differentiation rate of callus into EC. These findings were verified by RNA-seq analysis, which elucidated that the genes involved in the IAA biosynthesis, metabolism, and signaling, and ABA metabolism pathways were regulated by uniconazole during the callus development and SE. Overall, the results suggest that uniconazole could modulate callus proliferation and callus differentiation rate by regulating the endogenous levels of IAA and ABA. [ABSTRACT FROM AUTHOR]

Patatin-like proteins (PLPs) have non-specific lipid acyl hydrolysis (LAH) activity, which can hydrolyze membrane lipids into fatty acids and lysophospholipids. The vital role of PLPs in plant growth and abiotic stress has been well documented. However, the function of PLPs in plant defense responses against pathogens is still poorly understood. Here, we isolated and identified a novel cotton (Gossypium hirsutum) PLP gene GhPLP2. The expression of GhPLP2 was induced upon treatment with Verticillium dahliae , the signaling molecules jasmonic acid (JA) and ethylene (ETH) in cotton plants. Subcellular localization revealed that GhPLP2 was localized to the plasma membrane. GhPLP2 -silenced cotton plants were more susceptible to infection by V. dahliae , while the overexpression of GhPLP2 in Arabidopsis enhanced its resistance to V. dahliae , which was apparent as mild symptoms, and a decrease in the disease index and fungal biomass. The hypersensitive response, deposition of callose, and H2O2 accumulation triggered by V. dahliae elicitor were reduced in GhPLP2 -silenced cotton plants. The overexpression of GhPLP2 in Arabidopsis resulted in the accumulation of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) and facilitated the biosynthesis of JA and JA-mediated defensive responses. GhPLP2 silencing in cotton plants consistently reduced the accumulation of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) and suppressed the biosynthesis of JA and the defensive responses mediated by JA. These results indicate that GhPLP2 is involved in the resistance of cotton to V. dahliae by maintaining fatty acid metabolism pools for JA biosynthesis and activating the JA signaling pathway. [ABSTRACT FROM AUTHOR]

Verticillium wilt is caused by the soil‐borne vascular pathogen Verticillium dahliae, and affects a wide range of economically important crops, including upland cotton (Gossypium hirsutum). Previous studies showed that expression levels of BIN2 were significantly down‐regulated during infestation with V. dahliae. However, the underlying molecular mechanism of BIN2 in plant regulation against V. dahliae remains enigmatic. Here, we characterized a protein kinase GhBIN2 from Gossypium hirsutum, and identified GhBIN2 as a negative regulator of resistance to V. dahliae. The Verticillium wilt resistance of Arabidopsis and cotton were significantly enhanced when BIN2 was knocked down. Constitutive expression of BIN2 attenuated plant resistance to V. dahliae. We found that BIN2 regulated plant endogenous JA content and influenced the expression of JA‐responsive marker genes. Further analysis revealed that BIN2 interacted with and phosphorylated JAZ family proteins, key repressors of the JA signalling pathway in both Arabidopsis and cotton. Spectrometric analysis and site‐directed mutagenesis showed that BIN2 phosphorylated AtJAZ1 at T196, resulting in the degradation of JAZ proteins. Collectively, these results show that BIN2 interacts with JAZ proteins and plays a negative role in plant resistance to V. dahliae. Thus, BIN2 may be a potential target gene for genetic engineering against Verticillium wilt in crops. [ABSTRACT FROM AUTHOR]

Summary: Branching determines cotton architecture and production, but the underlying regulatory mechanisms remain unclear. Here, we report that the miR164‐GhCUC2 (CUP‐SHAPED COTYLEDON2) module regulates lateral shoot development in cotton and Arabidopsis. We generated OE‐GhCUC2m (overexpression GhCUC2m) and STTM164 (short tandem target mimic RNA of miR164) lines in cotton and heterologous expression lines for gh‐miR164, GhCUC2 and GhCUC2m in Arabidopsis to study the mechanisms controlling lateral branching. GhCUC2m overexpression resulted in a short‐branch phenotype similar to STTM164. In addition, heterologous expression of GhCUC2m led to decreased number and length of branches compared with wild type, opposite to the effects of the OE‐gh‐pre164 line in Arabidopsis. GhCUC2 interacted with GhBRC1 and exhibited similar negative regulation of branching. Overexpression of GhBRC1 in the brc1‐2 mutant partially rescued the mutant phenotype and decreased branch number. GhBRC1 directly bound to the NCED1 promoter and activated its transcription, leading to local abscisic acid (ABA) accumulation and response. Mutation of the NCED1 promoter disrupted activation by GhBRC1. This finding demonstrates a direct relationship between BRC1 and ABA signalling and places ABA downstream of BRC1 in the control of branching development. The miR164‐GhCUC2‐GhBRC1‐GhNCED1 module provides a clear regulatory axis for ABA signalling to control plant architecture. [ABSTRACT FROM AUTHOR]

Background: Many of genome features which could help unravel the often complex post-speciation evolution of closely related species are obscured because of their location in chromosomal regions difficult to accurately characterize using standard genome analysis methods, including centromeres and repeat regions. Results: Here, we analyze the genome evolution and diversification of two recently diverged sister cotton species based on nanopore long-read sequence assemblies and Hi-C 3D genome data. Although D genomes are conserved in gene content, they have diversified in gene order, gene structure, gene family diversification, 3D chromatin structure, long-range regulation, and stress-related traits. Inversions predominate among D genome rearrangements. Our results support roles for 5mC and 6mA in gene activation, and 3D chromatin analysis showed that diversification in proximal-vs-distal regulatory-region interactions shape the regulation of defense-related-gene expression. Using a newly developed method, we accurately positioned cotton centromeres and found that these regions have undergone obviously more rapid evolution relative to chromosome arms. We also discovered a cotton-specific LTR class that clarifies evolutionary trajectories among diverse cotton species and identified genetic networks underlying the Verticillium tolerance of Gossypium thurberi (e.g., SA signaling) and salt-stress tolerance of Gossypium davidsonii (e.g., ethylene biosynthesis). Finally, overexpression of G. thurberi genes in upland cotton demonstrated how wild cottons can be exploited for crop improvement. Conclusions: Our study substantially deepens understanding about how centromeres have developed and evolutionarily impacted the divergence among closely related cotton species and reveals genes and 3D genome structures which can guide basic investigations and applied efforts to improve crops. [ABSTRACT FROM AUTHOR]

Germin-like proteins (GLPs) are a diverse and ubiquitous family of plant glycoproteins belonging to the cupin super family; they play considerable roles in plant responses against various abiotic and biotic stresses. Here, we provide evidence that GLP2 protein from cotton (Gossypium hirsutum) functions in plant defense responses against Verticillium dahliae, Fusarium oxysporum and oxidative stress. Purified recombinant GhGLP2 exhibits superoxide dismutase (SOD) activity and inhibits spore germination of pathogens. Virus-induced silencing of GhGLP2 in cotton results in increased susceptibility to pathogens, plants exhibited severe wilt on leaves, enhanced vascular browning and suppressed callose deposition. Transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing GhGLP2 showed significant resistance to V. dahliae and F. oxysporum, with reduced mycelia growth, increased callose deposition and cell wall lignification at infection sites on leaves. The enhanced tolerance of GhGLP2-transgenic Arabidopsis to oxidative stress was investigated by methyl viologen and ammonium persulfate treatments, along with increased H2O2 production. Further, the expression of several defense-related genes (PDF1.2, LOX2, and VSP1) or oxidative stress-related genes (RbohD, RbohF) was triggered by GhGLP2. Thus, our results confirmed the involvement of GhGLP2 in plant defense response against Verticillium and Fusarium wilt pathogens and stress conditions. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) and transcriptional regulators are important switches in transcriptional networks. In recent years, the transcriptional regulator TIE1 (TCP interactor containing EAR motif protein 1) was identified as a nuclear transcriptional repressor which regulates leaf development and controls branch development. However, the function and regulatory network of GhTIE1 has not been studied in cotton. Here, we demonstrated that GhTIE1 is functionally conserved in controlling shoot branching in cotton and Arabidopsis. Overexpression of GhTIE1 in Arabidopsis leads to higher bud vigor and more branches, while silencing GhTIE1 in cotton reduced bud activity and increased branching inhibition. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that GhTIE1 directly interacted with subclass II TCPs (GhBRC1, GhBRC2, and GhTCP13) in vivo and in vitro. Overexpression of GhBRC1 , GhBRC2 , and GhTCP13 in mutant brc1 -2 partially rescued the mutant phenotype and decreased the number of branches, showing that these TCPs are functionally redundant in controlling branching. A transient dual-luciferase reporter assay indicated that GhTIE1 repressed the protein activity of GhBRC1 and GhTCP13, and thereby decreased the expression of their target gene GhHB21. Gene expression level analysis in GhTIE1 -overexpressed and silenced plants also proved that GhTIE1 regulated shoot branching via repressing the activity of BRC1 , HB21 , HB40 , and HB53. Our data reveals that shoot branching can be controlled via modulation of the activity of the TIE1 and TCP proteins and provides a theoretical basis for cultivating cotton varieties with ideal plant types. [ABSTRACT FROM AUTHOR]

Germin-like proteins (GLPs) are water-soluble plant glycoproteins belonging to the cupin superfamily. The important role of GLPs in plant responses against various abiotic and biotic stresses, especially pathogens, is well validated. However, little is known about cotton GLPs in relation to fungal pathogens. Here, a novel GLP gene was isolated from Gossypium hirsutum and designated as GhABP19. The expression of GhABP19 was upregulated in cotton plants inoculated with Verticillium dahliae and Fusarium oxysporum and in response to treatment with jasmonic acid (JA) but was suppressed in response to salicylic acid treatment. A relatively small transient increase in GhABP19 was seen in H2O2 treated samples. The three-dimensional structure prediction of the GhABP19 protein indicated that the protein has three histidine and one glutamate residues responsible for metal ion binding and superoxide dismutase (SOD) activity. Purified recombinant GhABP19 exhibits SOD activity and could inhibit growth of V. dahliae , F. oxysporum , Rhizoctonia solani , Botrytis cinerea , and Valsa mali in vitro. To further verify the role of GhABP19 in fungal resistance, GhABP19 -overexpressing Arabidopsis plants and GhABP19 -silenced cotton plants were developed. GhABP19-transgenic Arabidopsis lines showed much stronger resistance to V. dahliae and F. oxysporum infection than control (empty vector) plants did. On the contrary, silencing of GhABP19 in cotton conferred enhanced susceptibility to fungal pathogens, which resulted in necrosis and wilt on leaves and vascular discoloration in GhABP19 -silenced cotton plants. The H2O2 content and endogenous SOD activity were affected by GhABP19 expression levels in Arabidopsis and cotton plants after inoculation with V. dahliae and F. oxysporum , respectively. Furthermore, GhABP19 overexpression or silencing resulted in activation or suppression of JA-mediated signaling, respectively. Thus, GhABP19 plays important roles in the regulation of resistance to verticillium and fusarium wilt in plants. These modulatory roles were exerted by its SOD activity and ability to activate the JA pathway. All results suggest that GhABP19 was involved in plant disease resistance. [ABSTRACT FROM AUTHOR]

Members of the NF-YB transcription factor gene family play important roles in diverse processes related to plant growth and development, such as seed development, drought tolerance, and flowering time. However, the function of NF-YB genes in cotton remains unclear. A total of 23, 24, and 50 NF-YB genes were identified in Gossypium arboreum (G. arboreum), Gossypium raimondii (G. raimondii), and G. hirsutum, respectively. A systematic phylogenetic analysis was carried out in G. arboretum, G. raimondii, G. hirsutum, Arabidopsis thaliana, cacao, rice and, sorghum, where the 150 NF-YB genes were divided into five groups (α–ε). Of these groups, α is the largest clade, and γ contains the LEC1 type NF-YB proteins. Syntenic analyses revealed that paralogues of NF-YB genes in G. hirsutum exhibited good collinearity. Owing to segmental duplication within the A sub-genome (At) and D sub-genome (Dt), there was an expanded set of NF-YB genes in G. hirsutum. Furthermore, we investigated the structures of exons, introns, and conserved motifs of NF-YB genes in upland cotton. Most of the NF-YB genes had only one exon, and the genes from the same clade exhibited a similar motif pattern. Expression data show that most NF-YB genes were expressed ubiquitously, and only a few genes were highly expressed in specific tissues, as confirmed by quantitative real-time PCR (qRT-PCR) analysis. The overexpression of GhDNF-YB22 gene, predominantly expressed in embryonic tissues, indicates that GhDNF-YB22 may affect embryogenesis in cotton. This study is the first comprehensive characterization of the GhNF-YB gene family in cotton, and showed that NF-YB genes could be divided into five clades. The duplication events that occurred over the course of evolution were the major impetus for NF-YB gene expansion in upland cotton. Collectively, this work provides insight into the evolution of NF-YB in cotton and further our knowledge of this commercially important species. [ABSTRACT FROM AUTHOR]

High temperature stress influences pollen grains development in upland cotton (Gossypium hirsutum L.) anthers, resulting in anthers with an abnormal structure and pollen grains with a low germination rate. To examine the thermotolerance mechanisms of pollen grains in upland cotton, we observed pollen germination rates, pollen grain ultrastructure, anther structure, and the expression of heat shock protein (HSP) genes in pollen grains after the plants were continuously exposed to high temperatures (36/30°C) in a phytotron for 8 h every day over a period of 10 d. After the high‐temperature treatment, the pollen germination percentage of the heat‐sensitive cultivar was reduced compared with the heat‐tolerant cultivar, and the structure of indehiscent anthers and the ultrastructure of pollen grains in the heat‐sensitive cultivar were more abnormal than that in the heat‐tolerant cultivar. There were more abnormal mitochondria, endoplasmic reticulum, and vacuoles as well as fewer starch granules in the pollen grains of the heat‐sensitive cultivar compared with the heat‐tolerant cultivar. The deformation of the pollen grain architecture may influence the pollen germination rate. The expression of the GhHSP90, GhHSP2, GhHSP7, and GhHSP8 genes was up‐regulated to a greater extent in the heat‐sensitive pollen grains compared with the heat‐tolerant pollen during and after the high temperature period. The up‐regulation of HSP gene expression may protect pollen grains from damage under high temperature stress (36/30°C). The structure of the anthers, ultrastructure of the pollen grains, and expression of HSP genes are related to the thermotolerance of pollen grains. These results provide a new approach for screening upland cotton cultivars for thermotolerance. [ABSTRACT FROM AUTHOR]

Brassinosteroid (BR), a growth-promoting phytohormone, regulates many plant growth processes including cell development. However, the mechanism by which BR regulates fiber growth is poorly understood. Cotton (Gossypium hirsutum) fibers are an ideal single-cell model in which to study cell elongation due to their length. Here we report that BR controls cotton fiber elongation by modulating very-long-chain fatty acid (VLCFA) biosynthesis. BR deficiency reduces the expression of 3-ketoacyl-CoA synthases (GhKCSs), the rate-limiting enzymes involved in VLCFA biosynthesis, leading to lower saturated VLCFA contents in pagoda1 (pag1) mutant fibers. In vitro ovule culture experiments show that BR acts upstream of VLCFAs. Silencing of BRI1-EMS-SUPPRESOR 1.4 (GhBES1.4), encoding a master transcription factor of the BR signaling pathway, significantly reduces fiber length, whereas GhBES1.4 overexpression produces longer fibers. GhBES1.4 regulates endogenous VLCFA contents and directly binds to BR RESPONSE ELEMENTS (BRREs) in the GhKCS10_At promoter region, which in turn regulates GhKCS10_At expression to increase endogenous VLCFA contents. GhKCS10_At overexpression promotes cotton fiber elongation, whereas GhKCS10_At silencing inhibits cotton fiber growth, supporting a positive regulatory role for GhKCS10_At in fiber elongation. Overall, these results uncover a mechanism of fiber elongation through crosstalk between BR and VLCFAs at the single-cell level. [ABSTRACT FROM AUTHOR]

Cotton is a paramount cash crop around the globe. Among all abiotic stresses, drought is a leading cause of cotton growth and yield loss. However, the molecular link between drought stress and vascular growth and development is relatively uncharted. Here, we validated a crucial role of GhWOX4, a transcription factor, modulating drought stress with that of vasculature growth in cotton. Knock-down of GhWOX4 decreased the stem width and severely compromised vascular growth and drought tolerance. Conversely, ectopic expression of GhWOX4 in Arabidopsis enhanced the tolerance to drought stress. Comparative RNAseq analysis revealed auxin responsive protein (AUX/IAA), abscisic acid (ABA), and ethylene were significantly induced. Additionally, MYC-bHLH, WRKY, MYB, homeodomain, and heat-shock transcription factors (HSF) were differentially expressed in control plants as compared to GhWOX4-silenced plants. The promotor zone of GhWOX4 was found congested with plant growth, light, and stress response related cis-elements. differentially expressed genes (DEGs) related to stress, water deprivation, and desiccation response were repressed in drought treated GhWOX4-virus-induced gene silencing (VIGS) plants as compared to control. Gene ontology (GO) functions related to cell proliferation, light response, fluid transport, and flavonoid biosynthesis were over-induced in TRV: 156-0 h/TRV: 156-1 h (control) in comparison to TRV: VIGS-0 h/TRV: VIGS-1 h (GhWOX4-silenced) plants. This study improves our context for elucidating the pivotal role of GhWOX4 transcription factors (TF), which mediates drought tolerance, plays a decisive role in plant growth and development, and is likely involved in different regulatory pathways in cotton. [ABSTRACT FROM AUTHOR]

In the plant response to pathogen infection, many genes' expression is temporally induced, while few spatially induced expression genes have been reported. Here, we show that GhBOP1 can autonomously expand expression from restrained tissue when Gossypium hirsutum plants are attacked by Verticillium dahliae, which is considered to be spatially induced expression. Loss- and gain-of-function analyses show that GhBOP1 is a positive regulator in the modulation of plant resistance to V. dahliae. Yeast two-hybrid assays, luciferase complementation imaging and GUS reporting show that GhBOP1 interaction with GhTGA3 promotes its activation activity, regulating the expression of down-stream defence-related genes. Moreover, the induced spatial expression of GhBOP1 is accompanied by GhBP1 repression. Both antagonistically regulate the lignin biosynthesis, conferring cotton plants enhanced resistance to V. dahliae. Taken together, these results demonstrate that GhBOP1 is an economic positive regulator participating in plant defence through both the GhBOP1-GhTGA3 module and lignin accumulation. Zhennan Zhang, Peng Wang, Xiaoli Luo et al. show that BOP1 from cotton plants is able to induce gene expression in tissues affected by the fungal pathogen Verticillium dahlia, resulting in increased disease resistance. They propose a model involving TGA3 and lignin accumulation. [ABSTRACT FROM AUTHOR]

In plants, MYB transcription factors play diverse roles in growth, development, and response to abiotic and biotic stresses. However, the signaling processes of these transcription factors in defense against pathogen attacks remain largely unknown. This study isolated a novel R2R3-type MYB transcription factor GhODO1 from cotton (Gossypium hirsutum) and functionally characterized its positive role in tolerance to Verticillium dahliae. GhODO1 was induced by V. dahliae and jasmonic acid (JA) and transient expression of fused GhODO1-GFP in onion epidermal cells showed that GhODO1 protein was localized in the cell nucleus. Knockdown of GhODO1 significantly reduced the resistance of cotton to V. dahliae , whereas GhODO1 ectopic overexpression in Arabidopsis conferred enhanced resistance to V. dahliae. Lignin deposition was significantly decreased in GhODO1 -silenced cotton plants after V. dahliae inoculation and mock treatment. The expression levels of genes and activities of enzymes involved in lignin biosynthesis were reduced in GhODO1 -silenced cotton plants compared to the TRV:00. Yeast one-hybrid assays revealed that GhODO1 protein interacts with the promoters of lignin biosynthesis-related genes Gh4CL1 and GhCAD3 , directly activates their expression, and enhances total lignin accumulation. Moreover, GhODO1 silencing compromised JA-mediated defense signaling and JA accumulation. These results show that GhODO1 is involved in cotton resistance to V. dahliae by involving the lignin biosynthesis and the JA signaling pathway. • An R2R3 MYB protein GhODO1 from Gossypium hirsutum was functionally characterized. • The loss- and gain-of-function analyses show that GhODO1 is a positive regulator in the modulation of plant resistance to V. dahliae. • GhODO1 binds promoter of Gh4CL1 and GhCAD3 , activating lignin biosynthesis, which results in enhanced resistance of plants against V. dahliae. • GhODO1 is involved in the JA signaling pathway in cotton plants upon V. dahliae infection. • This study provides evidence for the application of GhODO1 protein to promote cotton germplasm innovation with sustainable disease resistance. [ABSTRACT FROM AUTHOR]

• GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. • GhACIF1 confers resistance to Verticillium dahliae. • GhACIF1 mediates hypersensitive and system acquired immune response triggered by elicitor PevD1. Avr9/Cf-9-INDUCED F-BOX1 (ACIF1) was first identified during screening of Avr9/Cf-9-elicited genes in tobacco. Further analysis revealed that ACIF1 was required for hypersensitive responses triggered by various elicitors in tobacco and tomato, indicating that it may be involved in various disease resistance. Here, we cloned its cotton (Gossypium hirsutum) homolog GhACIF1 , which encodes an F-box protein. We show that GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. Disease resistance assays show that GhACIF1 enhances resistance to Verticillium dahliae in Arabidopsis plants, while silencing of GhACIF1 confers sensitivity to V. dahliae in cotton. Further analysis show that PevD1 elicitor activates hypersensitive and acquired immune response mediated by GhACIF1. Collectively, these results indicate that GhACIF1 contributes to protection against V. dahliae infection. [ABSTRACT FROM AUTHOR]

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton ( Gossypium hirsutum ), named GhBEE1-Like . Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding. [ABSTRACT FROM AUTHOR]

Global crop production is challenged by increasing severity of soil salinization that is exacerbated by the accelerated climate change and agricultural activities. Recently, studies have found that microRNAs (miRNAs) play an important role in response to abiotic stresses, including soil salinity. In our previous studies, we identified cotton mir390a / b / c (ghr-mir390a / b / c) that are responsive to salt stress. In the current study, GhCEPR2 was identified as a target gene of ghr-mir390 that responds to salinity stress. We propose a novel regulatory module comprising ghr-mir390 and GhCEPR2, which was examined by degradome sequencing and verified by 5'RLM-RACE and transient expression in Nicotiana benthamiana. Transgenic over-expression of GhCEPR2 in both Arabidopsis thaliana and cotton led to enhanced tolerance against salinity stress, accompanied by rising proline content and reduction in malondialdehyde concentration. On the other hand, over-expression of ghr-mir390 resulted in increased sensitivity to salt stress, consistent with the observation in GhCEPR2 -silenced cotton. A number of key genes involved in ABA- and salt tolerance-related signaling pathways were found to be up-regulated by ghr-mir390a , but inhibited by GhCEPR2 phosphorylation. Overall, our study may broaden and deepen our understandings on the effect of the ghr-mir390 - GhCEPR2 module in regulating cotton salt tolerance. • mir390 attenuates GhCEPR2 by targeting its mRNA leading to the amelioration of salt tolerance in cotton. • The overexpression of GhCEPR2 could decrease ABA content to enhance plant salt stress resistance. • We propose a new module of mir390-CEPR2 that regulates salt tolerance in plants. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium hirsutum) is the most important fiber crop globally and there is an incentive to improve cotton fiber yield and quality to meet the growing market demand. 1-Aminocyclopropane-1-Carboxylic Acid Oxidase (ACO) has been identified as a rate-limiting enzyme in the biosynthesis of ethylene that is known to play a pivotal regulatory role in fiber development. According to transcriptomic data analysis, GhACO1 is preferentially expressed at fiber elongation stage in a number of G. hirsutum varieties, suggestive of its involvement in regulating fiber development. Ethylene is known to cause premature senescence, and to abrogate its potential negative effects on plant growth by constitutive overexpression of GhACO1 , a fiber-specific promoter E6 that imparts strong transcriptional activity in fiber elongation stage was employed to drive GhACO1 expression to improve fiber quality. The E6::GhACO1 cotton transgenic lines showed substantial improvements in fiber length, fiber strength and fiber yield relative to wild type. Exogenous application of pyrazinecarboxylic acid that is an effective inhibitor of ACO enzyme inhibited fiber elongation in ovule culture, validating that GhACOs may improve fiber quality by increasing ethylene levels. Furthermore, RNA-Seq analysis showed that upregulation of GhACO1 can activate the transcription of genes that are involved in hydrogen peroxide metabolism, cell wall loosening and cytoskeleton arrangement, thereby promoting fiber elongation. These findings suggest that GhACO1 is a key regulator of fiber development, and the application of " E6::GhACO1 " strategy breaks the negative correlation between fiber quality and fiber yield, providing a novel route in formulating fiber improvement strategies in cotton. [Display omitted] • GhACO1 plays a pivotal role in fiber development. • Fiber-specific overexpression of GhACO1 substantially improved fiber quality. • A novel strategy in improving cotton fiber production and fiber quality was provided. [ABSTRACT FROM AUTHOR]

Plant phospholipase D (PLD) and its product phosphatidic acid (PA) function in both abiotic and biotic stress signaling. However, to date, a PLD gene conferring the desired resistance to both biotic and abiotic stresses has not been found in cotton. Here, we isolated and identified a PLD gene GhPLDδ from cotton (Gossypium hirsutum), which functions in Verticillium wilt resistance and salt tolerance. GhPLDδ was highly induced by salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), hydrogen peroxide, PEG 6000, NaCl, and Verticillium dahliae in cotton plants. The positive role of GhPLDδ in regulating plant resistance to V. dahliae was confirmed by loss- and gain-of-function analyses. Upon chitin treatment, accumulation of PA, hydrogen peroxide, JA, SA, and the expression of genes involved in MAPK cascades, JA- and SA-related defense responses were positively related to the level of GhPLDδ in plants. The treatment by exogenous PA could activate the expression of genes related to MAPK, SA, and JA signaling pathways. Moreover, GhPLDδ overexpression enhanced salt tolerance in Arabidopsis as demonstrated by the increased germination rate, longer seedling root, higher chlorophyll content, larger fresh weight, lower malondialdehyde content, and fully expand rosette leaves. Additionally, the PA content and the expression of the genes of the MAPK cascades regulated by PA were increased in GhPLDδ -overexpressed Arabidopsis under salt stress. Taken together, these findings suggest that GhPLDδ and PA are involved in regulating plant defense against both V. dahliae infection and salt stress. • A phospholipase D gene GhPLDδ from Gossypium hirsutum was functionally characterized. • GhPLDδ is a positive regulator in the modulation of plant resistance to V. dahliae. • GhPLDδ confers tolerance to salt in Arabidopsis through upregulation of salt-related genes. • GhPLDδ is involved in ROS, MAPKs, JA and SA signaling pathways after chitin stimulus. • GhPLDδ is a candidate gene to promote cotton germplasm innovation with improved adaptability. [ABSTRACT FROM AUTHOR]