Tolerant Cotton Research Articles

Regulating drought tolerance in cotton by the expression of a specific allele of heat shock protein 70

Plant-specific heat shock protein 70 s (HSP70s) play crucial roles in response to various environmental stimuli. GhHSP70-26, an HSP70 member, is involved in the response to multiple abiotic stresses in cotton (Gossypium hirsutum). This study explored the relationship between the genetic diversity of GhHSP70–26 and drought resistance in cotton to aid crop breeding. Indel sites at −650 bp upstream of the GhHSP70–26 promoter were discovered in cotton and designated an M-650 marker. The M-650 marker was significantly associated with the effective boll numbers (M-650-In360 > M-650-Del) in 110 types of cotton under drought stress conditions for three years. The relative expression levels of GhHSP70–26 was linearly correlated with comprehensive drought resistance in cotton seedlings. The gene expression levels were enhanced in BC2F2(pGhHSP70–26In360) of cotton and the transgenic lines pGhHSP70–26In360:GhHSP70–26 of Arabidopsis, and drought resistance was also enhanced. After PEG, ABA and SA treatments, the transgenic lines of M-650-In360 promoter showed higher GUS expression activity than the M-650-del promoter in Arabidopsis. The GhFLZ, GhABF3, and GhRVE8 transcription factors could regulate to In360 bp of pGhHSP70–26 to activate high levels of gene expression. These results provide important genetic information to study the natural variation of drought tolerance in cotton, and the loci or exceptional promoters identified can be used as direct targets for cotton trait improvement and genetic engineering.

Evaluation of Bi-Parental Mating System out Puts in Two Crosses for Drought Tolerance in Egyptian Cotton (Gossipum Barbadense L.)

Evaluation of Bi-Parental Mating System out Puts in Two Crosses for Drought Tolerance in Egyptian Cotton (Gossipum Barbadense L.)

Function analysis of GhWRKY53 regulating cotton resistance to verticillium wilt by JA and SA signaling pathways.

WRKY transcription factors (TFs) play an important role in regulating the mechanism of plant self-defense. However, the function of most WRKY TFs in upland cotton (Gossypium hirsutum) is still unknown. Hence, studying the molecular mechanism of WRKY TFs in the resistance of cotton to Verticillium dahliae is of great significance to enhancing cotton disease resistance and improving its fiber quality. In this study, Bioinformatics has been used to characterize the cotton WRKY53 gene family. we analyzed the GhWRKY53 expression patterns in different resistant upland cotton cultivars treated with salicylic acid (SA) and methyl jasmonate (MeJA). Additionally, GhWRKY53 was silenced using a virus-induced gene silencing (VIGS) to determine the contribution of GhWRKY53 to V. dahliae resistance in cotton. The result showed that GhWRKY53 mediated SA and MeJA signal transduction pathways. After VIGS of the GhWRKY53, the ability of cotton to resist V. dahliae decreased, indicating that the GhWRKY53 could be involved in the disease resistance mechanism of cotton. Studies on the levels of SA and jasmonic acid (JA) and their related pathway genes demonstrated that the silencing of GhWRKY53 inhibited the SA pathway and activated the JA pathway, thereby reducing the resistance of plants to V. dahliae. In conclusion, GhWRKY53 could change the tolerance of upland cotton to V. dahliae by regulating the expression of SA and JA pathway-related genes. However, the interaction mechanism between JA and SA signaling pathways in cotton in response to V. dahliae requires further study.

Roles of S-Adenosylmethionine and Its Derivatives in Salt Tolerance of Cotton.

Salinity is a major abiotic stress that restricts cotton growth and affects fiber yield and quality. Although studies on salt tolerance have achieved great progress in cotton since the completion of cotton genome sequencing, knowledge about how cotton copes with salt stress is still scant. S-adenosylmethionine (SAM) plays important roles in many organelles with the help of the SAM transporter, and it is also a synthetic precursor for substances such as ethylene (ET), polyamines (PAs), betaine, and lignin, which often accumulate in plants in response to stresses. This review focused on the biosynthesis and signal transduction pathways of ET and PAs. The current progress of ET and PAs in regulating plant growth and development under salt stress has been summarized. Moreover, we verified the function of a cotton SAM transporter and suggested that it can regulate salt stress response in cotton. At last, an improved regulatory pathway of ET and PAs under salt stress in cotton is proposed for the breeding of salt-tolerant varieties.

Genomic Dynamics and Functional Insights under Salt Stress in Gossypium hirsutum L.

The changing climate is intensifying salt stress globally. Salt stress is a menace to cotton crop quality and yield. The seedling, germination, and emergence phases are more prone to the effects of salt stress than other stages. Higher levels of salt can lead to delayed flowering, a reduced number of fruiting positions, shedding of fruits, decreased boll weight, and yellowing of fiber, all of which have an adverse effect on the yield and quality of the seed cotton. However, sensitivity toward salt stress is dependent on the salt type, cotton growth phase, and genotype. As the threat of salt stress continues to grow, it is crucial to gain a comprehensive understanding of the mechanisms underlying salt tolerance in plants and to identify potential avenues for enhancing the salt tolerance of cotton. The emergence of marker-assisted selection, in conjunction with next-generation sequencing technologies, has streamlined cotton breeding efforts. This review begins by providing an overview of the causes of salt stress in cotton, as well as the underlying theory of salt tolerance. Subsequently, it summarizes the breeding methods that utilize marker-assisted selection, genomic selection, and techniques for identifying elite salt-tolerant markers in wild species or mutated materials. Finally, novel cotton breeding possibilities based on the approaches stated above are presented and debated.

Development and identification of molecular markers of GhHSP70-26 related to heat tolerance in cotton

Heat stress significantly affect plant growth and development, which is an important factor contributing to crop yield loss. However, heat shock proteins (HSPs) in plants can effectively alleviate cell damage caused by heat stress. In order to rapidly and accurately cultivate heat-tolerant cotton varieties, this study conducted correlation analysis between heat tolerance index and insertion/deletion (In/Del) sites of GhHSP70-26 promoter in 39 cotton materials, so as to find markers related to heat tolerance function of cotton, which can be used in molecular marker-assisted breeding. The results showed the natural variation allele (Del22 bp) type at −1590 bp upstream of GhHSP70-26 promoter (haplotype2, Hap2) in cotton (Gossypium spp.) promoted GhHSP70-26 expression under heat stress. The relative expression level of GhHSP70-26 of M−1590−Del22 cotton materials were significantly higher than that of M−1590−In type cotton materials under heat stress (40 ℃). Also, M−1590−Del22 material had lower conductivity and less cell damage after heat stress, indicating that it is a heat resistant cotton material. The Hap1 (M−1590−In) promoter was mutated into Hap1del22, and Hap1 and Hap1del22 were fused with GUS to transform Arabidopsis thaliana. Furthermore, Hap1del22 promoter had higher induction activity than Hap1 under heat stress and abscisic acid (ABA) treatment in transgenic Arabidopsis thaliana. Further analysis confirmed that M−1590−Del22 was the dominant heat-resistant allele. In summary, these results identify a key and previously unknown natural variation in GhHSP70-26 with respect to heat tolerance, providing a valuable functional molecular marker for genetic breeding of cotton and other crops with heat tolerance.

Identification and characterization of the LDAP family revealed GhLDAP2_Dt enhances drought tolerance in cotton.

Lipid droplet-associated proteins (LDAPs) play essential roles in tissue growth and development and in drought stress responses in plants. Cotton is an important fiber and cash crop; however, the LDAP family has not been characterized in cotton. In this study, a total of 14, six, seven, and seven genes were confirmed as LDAP family members in Gossypium hirsutum, Gossypium raimondii, Gossypium arboreum, and Gossypium stocksii, respectively. Additionally, expansion in the LDAP family occurred with the formation of Gossypium, which is mirrored in the number of LDAPs found in five Malvaceae species (Gossypioides kirkii, Bombax ceiba, Durio zibethinus, Theobroma cacao, and Corchorus capsularis), Arabidopsis thaliana, and Carica papaya. The phylogenetic tree showed that the LDAP genes in cotton can be divided into three groups (I, II, and III). The analysis of gene structure and conserved domains showed that LDAPs derived from group I (LDAP1/2/3) are highly conserved during evolution, while members from groups II and III had large variations in both domains and gene structures. The gene expression pattern analysis of LDAP genes showed that they are expressed not only in the reproductive organs (ovule) but also in vegetative organs (root, stem, and leaves). The expression level of two genes in group III, GhLDAP6_At/Dt, were significantly higher in fiber development than in other tissues, indicating that it may be an important regulator of cotton fiber development. In group III, GhLDAP2_At/Dt, especially GhLDAP2_Dt was strongly induced by various abiotic stresses. Decreasing the expression of GhLDAP2_Dt in cotton via virus-induced gene silencing increased the drought sensitivity, and the over-expression of GhLDAP2_Dt led to increased tolerance to mannitol-simulated osmotic stress at the germination stage. Thus, we conclude that GhLDAP2_Dt plays a positive role in drought tolerance.

GhCLC5/16 genes participate in the regulation of Cl−-salinity tolerance in seedlings of different upland cotton cultivars

For plants or crops exposed to salinity stress, chloride channels (CLCs) play crucial functions in the regulation of anion (such as Cl− and NO3−) absorption, transport and distribution or anion homeostasis. In this study, how the physiological functions and molecular mechanisms of GhCLC5/16 genes affect Cl− -salinity tolerance in two upland cotton cultivars (the salt-tolerant cv. Lu7619 and the salt-sensitive cv. Xin51) was investigated using transformations of the yeast Δgef1 mutant, soybean hairy-root, Arabidopsis atclc-b mutant, and virus-induced gene silencing (VIGS) cotton plants. The results showed that, the target GhCLC5/16 genes, with the higher NaCl-induced upregulation of expression in the roots of Lu7619 plants, could complement the Cl−-sensitive phenotype of the yeast Δgef1 mutant. Under NaCl stress, compared to empty vector (EV) plants, GhCLC5/16-overexpressed soybean hairy-root composite plants (hrGhCLC5/16-OE) displayed enhanced NaCl tolerance with superior growth, lower relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in roots and leaves. Compared to the atclc-b mutant, atclc-b-GhCLC5 and atclc-b-GhCLC16 Arabidopsis seedlings displayed improved NaCl tolerance with less reduced root length, higher leaf chlorophyll content, lower shoot MDA content and REL values. GhCLC5/16-VIGS cotton plants displayed mitigated salt damage with significantly higher plant height and fresh weight (FW) than that of EV plants. The enhanced NaCl tolerance or mitigated salt damage of these plants, even with the salt-tolerant cv. Lu7619 (characterized by high GhCLC5/16 expression in the roots, whole plants, or GhCLC5/16-silencing in the leaves), occurs because the plants can accumulate more Cl− in the roots under NaCl stress, reduce the transport and accumulation of Cl− to shoots (especially the leaves), simultaneously enhance the NO3− content in the shoots/leaves, and thus significantly decrease the Cl−/NO3− ratio in the shoots/leaves. Therefore, GhCLC5/16 genes may be crucial members of the GhCLC family that contribute to the varied tolerance to Cl−-salinity observed in different cotton cultivars. The results may provide new perspectives and valuable candidate gene resources for molecular breeding of chloride-salinity tolerance in cotton and other crops.

Combined transcriptomic and metabolomic analyses elucidate key salt-responsive biomarkers to regulate salt tolerance in cotton

BackgroundCotton is an important industrial crop and a pioneer crop for saline-alkali land restoration. However, the molecular mechanism underlying the cotton response to salt is not completely understood.MethodsHere, we used metabolome data and transcriptome data to analyze the salt tolerance regulatory network of cotton and metabolic biomarkers.ResultsIn this study, cotton was stressed at 400 m M NaCl for 0 h, 3 h, 24 h and 48 h. NaCl interfered with cotton gene expression, altered metabolite contents and affected plant growth. Metabolome analysis showed that NaCl stress increased the contents of amino acids, sugars and ABA, decreased the amount of vitamin and terpenoids. K-means cluster analysis of differentially expressed genes showed that the continuously up-regulated genes were mainly enriched in metabolic pathways such as flavonoid biosynthesis and amino acid biosynthesis.ConclusionThe four metabolites of cysteine (Cys), ABA(Abscisic acid), turanose, and isopentenyladenine-7-N-glucoside (IP7G) were consistently up-regulated under salt stress, which may indicate that they are potential candidates for cotton under salt stress biomarkers. Combined transcriptome and metabolome analysis revealed accumulation of cysteine, ABA, isopentenyladenine-7-N-glucoside and turanose were important for salt tolerance in cotton mechanism. These results will provide some metabolic insights and key metabolite biomarkers for salt stress tolerance, which may help to understanding of the metabolite response to salt stress in cotton and develop a foundation for cotton to grow better in saline soil.

Transcriptome Analysis Revealed that GhPP2C43-A Negatively Regulates Salinity Tolerance in an Introgression Line from a Semi-Wild Upland Cotton.

Salt damage is a major threat to sustainable cotton production owing to the limited arable land in China, which is mainly occupied by the production of staple food crops. Salt-stress-tolerant cotton varieties are lacking in production, and the mechanisms underpinning salt stress tolerance in cotton remain enigmatic. Here, DM37, an intraspecific introgression line from Gossypium hirsutum race yucatanense acc TX-1046 into the G. hirsutum acc TM-1 background, was found to be highly tolerant to salt stress. Its seed germination rate and germination potential were significantly higher than those of the recipient TM-1 under salt stress. Physiological analysis showed that DM37 had a higher proline content and peroxidase activity and lower Na+/K+ ratios at the seedling stage, which is consistent with a higher seedling survival rate after durable salt stress. Furthermore, comparative transcriptome analysis revealed that responsive patterns to salt stress in DM37 were different from those in TM-1. Weighted correlation network analysis demonstrated that co-expression modules associated with salt stress in DM37 also differed from those in TM-1. From this analysis, GhPP2C43-A, a phosphatase gene, was found to exhibit negative regulation of salt stress tolerance verified by virus-induced gene silencing and the genration of transgenic Arabidopsis. Gene expression showed that GhPP2C43-A in TM-1 was induced by durable salt stress but not in DM37, probably attributable to a variation in the cis-element in its promoter, thereby conferring different salt stress tolerance. These results provide new genes/germplasms from semi-wild cotton in salt-stress-tolerant cotton breeding, as well as new insight into the mechanisms underpinning salt stress tolerance in cotton.

Genome-wide identification and functional analysis of ICE genes reveal that Gossypium thurberi “GthICE2” is responsible for cold and drought stress tolerance

Cold stress has been found to have a negative impact on cotton growth and annual production. To address this issue, the utilization of cold-tolerant gene resources from wild species of Gossypium is crucial for genetic improvements in cultivated cotton. ICE (inducer of CBF expression) are the key regulators of cold tolerance in plants, however, there is relatively little information on ICE genes in cotton. Herein, we performed comprehensive bioinformatics analyses of the ICE gene family in eight cotton species. Phylogenetic analysis showed that 52 ICE genes were clustered into four subgroups. Cis-regulatory elements analysis suggests that the expression of ICE genes might be regulated by light, plant hormones, and various environment stresses. Higher expression of GthICE2 was observed in leaves as compared to roots and stems, in response to cold, drought, and exogenous hormone ABA. Furthermore, overexpression of GthICE2 in A. thaliana led to higher germination and survival rates, longer root length, lower ion leakage, and induction under cold and drought stress. Histochemical staining showed that oxidative damage in transgenic lines was much lower compared to wild-type plants. Lower MDA contents and higher SOD and POD activities were observed in overexpressed plants. Y1H and LUC assays revealed that GthICE2 might activate the expression of GthCBF4, a cold-responsive gene, by connecting with the MYC cis-element present in the promoter of GthCBF4. GthICE2 confers cold and drought stress tolerance in cotton. Our findings add significantly to the existing knowledge regarding cold stress tolerance and helps to elucidate cold response mechanisms in cotton.

GhMYB44 enhances stomatal closure to confer drought stress tolerance in cotton and Arabidopsis

MYB genes play crucial roles in plant response to abiotic stress. However, the function of MYB genes in cotton during abiotic stress is less well elucidated. Here, we found an R2R3-type MYB gene, GhMYB44, was induced by simulated drought (PEG6000) and ABA in three cotton varieties. After drought stress, the GhMYB44-silenced plants showed substantial changes at the physiological level, including significantly increased malondialdehyde content and decreased SOD activity. Silencing the GhMYB44 gene increased stomatal aperture and water loss rate, reduced plant drought tolerance. Transgenic Arabidopsis thaliana over-expressed GhMYB44 (GhMYB44-OE) enhanced resistance to mannitol-simulated osmotic stress. The stomatal aperture of the GhMYB44-OE Arabidopsis was significantly smaller than those of the wild type (WT), and the GhMYB44-OE Arabidopsis increased tolerance to drought stress. Transgenic Arabidopsis had higher germination rate under ABA treatment compared to WT, and the transcript levels of AtABI1, AtPP2CA and AtHAB1 were suppressed in GhMYB44-OE plants, indicating a potential role of GhMYB44 in the ABA signal pathway. These results showed that GhMYB44 acts as a positive regulator in plant response to drought stress, potentially useful for engineering drought-tolerant cotton.

Role of Molecular Breeding Tools in Enhancing the Breeding of Drought-Resilient Cotton Genotypes: An Updated Review

Drought stress is an inevitable factor that disturbs the production of plants by altering morphological, physiological, biochemical, and molecular functions. Breeding for drought tolerance requires a complete understanding of the molecular factors controlling stress-responsive pathways. The plant responds to drought stress by adopting four mechanisms: avoidance, escape, tolerance, and recovery. Traditional plant-breeding tools have been employed to increase tolerance in cotton, but the complexity of drought tolerance has limited the use of these breeding methods. The plant adopts several key strategies against drought stress, such as activating the signaling network and activating molecular factors. Cotton breeders have been engaged in elucidating the molecular mechanisms of drought tolerance in cotton using significant molecular tools such as quantitative trait loci (QTL) mapping, transcription factor (TFs) analysis, transcriptome analysis, genome-wide association studies (GWAS), genetic engineering, and CRISPR/Cas9. Breeders have studied the functional description of genes and the interacting pathways accountable for controlling drought tolerance in cotton. Hundreds of genes/QTL have been identified, and many have been cloned for drought tolerance in cotton; however, a complete understanding of these traits still needs more study. This review presents a detailed overview of molecular tools, their application for improving drought tolerance in cotton, and their prospects. This review will help future researchers to conduct further studies to develop drought-tolerant cotton genotypes that can thrive under conditions of water scarcity.

Differential responses of contrasting low phosphorus tolerant cotton genotypes under low phosphorus and drought stress

BackgroundDrought is one of the main reasons for low phosphorus (P) solubility and availability.AimsThe use of low P tolerant cotton genotypes might be a possible option to grow in drought conditions.MethodsThis study investigates the tolerance to drought stress in contrasting low P-tolerant cotton genotypes (Jimian169; strong tolerant to low P and DES926; weak tolerant to low P). In hydroponic culture, the drought was artificially induced with 10% PEG in both cotton genotypes followed by low (0.01 mM KH2PO4) and normal (1 mM KH2PO4) P application.ResultsThe results showed that under low P, PEG-induced drought greatly inhibited growth, dry matter production, photosynthesis, P use efficiency, and led to oxidative stress from excessive malondialdehyde (MDA) and higher accumulation of reactive oxygen species (ROS), and these effects were more in DES926 than Jimian169. Moreover, Jimian169 alleviated oxidative damage by improving the antioxidant system, photosynthetic activities, and an increase in the levels of osmoprotectants like free amino acids, total soluble proteins, total soluble sugars, and proline.ConclusionsThe present study suggests that the low P-tolerant cotton genotype can tolerate drought conditions through high photosynthesis, antioxidant capacity, and osmotic adjustment.

Micro-Climatic effect on Cotton Yield, quality, Bt toxin & GT Gene

Unsuitable change in climatic conditions cause decline in quality and yield of major crops. Plant growth is directly affected if temperature, rainfall or humidity are not optimum. A multi-location and multi season evaluation of climatic effects on quality and yield may produce a reliable data for future breeding. A set of 39 upcoming varieties of cotton were evaluated on six different Micro-climatic locations of Punjab i.e. Multan, Bahawalpur, Sahiwal, Rahimyar khan, Vehari and Faisalabad in a triplicated trial. The experiment was repeated next year on same locations. Data for three key environmental factors such as temperature, rainfall and humidity was recorded at each station. The crop was analyzed for yield, fiber length, fiber strength and fiber fineness. The genotypes were also evaluated for Bt toxin and Glyphosate tolerance gene (GTG). The analysis revealed that high temperature has negative effect on yield, Bt expression, fineness, uniformity and GTG. Precipitation and humidity had positive effect on fiber fineness and uniformity, whereas, negative effect of both environmental factors was recorded for fiber length and strength. Increase in precipitation at early cropping stage was associated with increase in yield whereas higher humidity has negative impact on yield. As compared to high average temperature and number of days above 400C, cotton yield is more sensitive to heat waves (maximum temperature). Varieties with high temperature tolerance in cotton should be breed for climate change scenario.

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

BackgroundHerbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds. Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches. At present, no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism (GMO) regulation law. We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate (EMS) mutagenesis, offering an alternative choice for weed management.ResultsSeeds of an elite cotton cultivar Lumianyan 37 (Lu37) were treated with EMS, and a mutant Lu37-1 showed strong tolerance to imidazolinone (IMI) herbicides was identified. A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase (ALS) (Gh_D10G1253) in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton. The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species. Cleaved amplified polymorphic sequence (CAPS) markers were developed to identify the ALS mutant allele. The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.ConclusionThe nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identified mutation in cotton. This substitution mutation has also been identified in the orthologous ALS genes in other crops. This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.Graphical abstract

The transcription factor GhWRKY70 from gossypium hirsutum enhances resistance to verticillium wilt via the jasmonic acid pathway

BackgroundThe WRKY transcription factors play significant roles in plant growth, development, and defense responses. However, in cotton, the molecular mechanism of most WRKY proteins and their involvement in Verticillium wilt tolerance are not well understood.ResultsGhWRKY70 is greatly up-regulated in cotton by Verticillium dahliae. Subcellular localization suggests that GhWRKY70 is only located in the nucleus. Transcriptional activation of GhWRKY70 further demonstrates that GhWRKY70 function as a transcriptional activator. Transgenic Arabidopsis plants overexpressing GhWRKY70 exhibited better growth performance and higher lignin content, antioxidant enzyme activities and jasmonic acid (JA) levels than wild-type plants after infection with V. dahliae. In addition, the transgenic Arabidopsis resulted in an enhanced expression level of AtAOS1, a gene related to JA synthesis, further leading to a higher JA accumulation compared to the wild type. However, the disease index (DI) values of the VIGS-treated cotton plants with TRV:WRKY70 were also significantly higher than those of the VIGS-treated cotton plants with TRV:00. The chlorophyll and lignin contents of TRV:WRKY70 plants were significantly lower than those of TRV:00 plants. GhAOS1 expression and JA abundance in TRV:WRKY70 plants were decreased. The GhWRKY70 protein was confirmed to bind to the W-box element in the promoter region of GhAOS by yeast one-hybrid assay and transient expression.ConclusionThese results indicate that the GhWRKY70 transcription factor is a positive regulator in Verticillium wilt tolerance of cotton, and may promote the production of JA via regulation of GhAOS1 expression.

Priming with sodium nitroprusside and hydrogen peroxide increases cotton seed tolerance to salinity and water deficit during seed germination and seedling development

A major difficulty in cotton crops is high-quality seed production, which interferes with plant vigor and establishment. Techniques such as seed physiological conditioning have been reported due to their ability to improve stress tolerance in seeds and plants. Considering this, we carried out this research to evaluate the use of signaling molecules to increase cotton plant stress tolerance. Seeds were conditioned on aerated solutions of indoleacetic acid (100 µM), hydrogen peroxide (100 µM), chitosan (0.75 mM), melatonin (0.2 mM), sodium nitroprusside (100 µM), and pure water (hydropriming), at 20 °C for 24 h. Then seeds were washed with running water and dried in an oven with forced air circulation at 25 °C for 24 h. Treated seeds or nontreated seeds were placed to germinate under salinity (10 dS m−1 NaCl) and water deficit by using polyethylene glycol (PEG 6000) at − 0.6 MPa, and with nonstressed condition (water). The solutions mentioned were used to moisten the Germitest® paper roll at 2.5 times its weight. We also analyzed the activity of the enzymes superoxide dismutase, catalase, and ascorbate peroxidase, as well as the content of proline, hydrogen peroxide, and lipid peroxidation. Physiological conditioning with signaling molecules effectively improved cotton seed germination under stress, especially to hydrogen peroxide and sodium nitroprusside, which resulted in better performance on the parameters of germination and seedling growth. As stress conditions induced oxidative stress, as observed by lipid peroxidation, we found that physiological conditioning improved the seed antioxidant system, with hydrogen peroxide and sodium nitroprusside resulting in better performance. Our results highlighted the potential of sodium nitroprusside and hydrogen peroxide as inducers of stress tolerance in cotton.

The CCCH-Type Zinc-Finger Protein GhC3H20 Enhances Salt Stress Tolerance in Arabidopsis thaliana and Cotton through ABA Signal Transduction Pathway

The CCCH zinc-finger protein contains a typical C3H-type motif widely existing in plants, and it plays an important role in plant growth, development, and stress responses. In this study, a CCCH zinc-finger gene, GhC3H20, was isolated and thoroughly characterized to regulate salt stress in cotton and Arabidopsis. The expression of GhC3H20 was up-regulated under salt, drought, and ABA treatments. GUS activity was detected in the root, stem, leaves, and flowers of ProGhC3H20::GUS transgenic Arabidopsis. Compared with the control, the GUS activity of ProGhC3H20::GUS transgenic Arabidopsis seedlings under NaCl treatment was stronger. Through the genetic transformation of Arabidopsis, three transgenic lines of 35S-GhC3H20 were obtained. Under NaCl and mannitol treatments, the roots of the transgenic lines were significantly longer than those of the wild-type (WT) Arabidopsis. The leaves of the WT turned yellow and wilted under high-concentration salt treatment at the seedling stage, while the leaves of the transgenic Arabidopsis lines did not. Further investigation showed that compared with the WT, the content of catalase (CAT) in the leaves of the transgenic lines was significantly higher. Therefore, compared with the WT, overexpression of GhC3H20 enhanced the salt stress tolerance of transgenic Arabidopsis. A virus-induced gene silencing (VIGS) experiment showed that compared with the control, the leaves of pYL156-GhC3H20 plants were wilted and dehydrated. The content of chlorophyll in pYL156-GhC3H20 leaves was significantly lower than those of the control. Therefore, silencing of GhC3H20 reduced salt stress tolerance in cotton. Two interacting proteins (GhPP2CA and GhHAB1) of GhC3H20 have been identified through a yeast two-hybrid assay. The expression levels of PP2CA and HAB1 in transgenic Arabidopsis were higher than those in the WT, and pYL156-GhC3H20 had expression levels lower than those in the control. GhPP2CA and GhHAB1 are the key genes involved in the ABA signaling pathway. Taken together, our findings demonstrate that GhC3H20 may interact with GhPP2CA and GhHAB1 to participate in the ABA signaling pathway to enhance salt stress tolerance in cotton.

Genome-wide identification and expression analysis of the HVA22 gene family in cotton and functional analysis of GhHVA22E1D in drought and salt tolerance.

The HVA22 family of genes, induced by abscisic acid and stress, encodes a class of stress response proteins with a conserved TB2/DP1/HVA22 domain that are unique among eukaryotes. Previous studies have shown that HVA22s play an important role in plant responses to abiotic stresses. In the present study, 34, 32, 16, and 17 HVA22s were identified in G. barbadense, G. hirsutum, G. arboreum, and G. raimondii, respectively. These HVA22 genes were classified into nine subgroups, randomly distributed on the chromosomes. Synteny analysis showed that the amplification of the HVA22s were mainly due to segmental duplication or whole genome replication (WGD). Most HVA22s promoter sequences contain a large number of drought response elements (MYB), defense and stress response elements (TC-rich repeats), and hormone response elements (ABRE, ERE, SARE, etc.), suggesting that HVA22s may respond to adversity stresses. Expression profiling demonstrated that most GhHVA22s showed a constitutive expression pattern in G. hirsutum and could respond to abiotic stresses such as salt, drought, and low temperature. Overexpression of GhHVA22E1D (GH_D07G0564) in Arabidopsis thaliana enhances salt and drought tolerance in Arabidopsis. Virus-induced gene silencing of GhHVA22E1D reduced salt and drought tolerance in cotton. This indicates that GhHVA22E1D plays an active role in the plant response to salt stress and drought stress. GhHVA22E1D may act in plant response to adversity by altering the antioxidant capacity of plants. This study provides valuable information for the functional genomic study of the HVA22 gene family in cotton. It also provides a reference for further elucidation of the functional studies of HVA22 in plant resistance to abiotic stress response.