GhJUB1_3-At positively regulate drought and salt stress tolerance under control of GhHB7, GhRAP2-3 and GhRAV1 in Cotton.

Climate change severely affects crop production. Cotton is one of the primary fiber crops in the world and its production is susceptible to various environmental stresses, especially drought and salinity. Development of stress tolerant genotypes is the only way to escape from these environmental constraints. We identified sixteen homologs of the Arabidopsis JUB1 gene in cotton. Expression of GhJUB1_3-At was significantly induced in the temporal expression analysis of GhJUB1 genes in the roots of drought tolerant (H177) and susceptible (S9612) cotton genotypes under drought. The silencing of the GhJUB1_3-At gene alone and together with its paralogue GhJUB1_3-Dt reduced the drought tolerance in cotton plants. The transgenic lines exhibited tolerance to the drought and salt stress as compared to the wildtype (WT). The chlorophyll and relative water contents of wildtype decreased under drought as compared to the transgenic lines. The transgenic lines showed decreased H2O2 and increased proline levels under drought and salt stress, as compared to the WT, indicating that the transgenic lines have drought and salt stress tolerance. The expression analysis of the transgenic lines and WT revealed that GAI was upregulated in the transgenic lines in normal conditions as compared to the WT. Under drought and salt treatment, RAB18 and RD29A were strongly upregulated in the transgenic lines as compared to the WT. Conclusively, GhJUB1_3-At is not an auto activator and it is regulated by the crosstalk of GhHB7, GhRAP2-3 and GhRAV1. GhRAV1, a negative regulator of abiotic stress tolerance and positive regulator of leaf senescence, suppresses the expression of GhJUB1_3-At under severe circumstances leading to plant death.

Genome-wide analysis and prediction of chloroplast and mitochondrial RNA editing sites of AGC gene family in cotton (Gossypium hirsutum L.) for abiotic stress tolerance.

BACKGROUND: Cotton is one of the topmost fiber crops throughout the globe. During the last decade, abrupt changes in the climate resulted in drought, heat, and salinity. These stresses have seriously affected cotton production and significant losses all over the textile industry. The GhAGC kinase, a subfamily of AGC group and member of serine/threonine (Ser/Thr) protein kinases group and is highly conserved among eukaryotic organisms. The AGC kinases are compulsory elements of cell development, metabolic processes, and cell death in mammalian systems. The investigation of RNA editing sites within the organelle genomes of multicellular vascular plants, such as Gossypium hirsutum holds significant importance in understanding the regulation of gene expression at the post-transcriptional level. METHODS: In present work, we characterized twenty-eight GhAGC genes in cotton and constructed phylogenetic tree using nine different species from the most primitive to the most recent. RESULTS: In sequence logos analyses, highly conserved amino acid residues were found in G. hirsutum, G. arboretum, G. raimondii and A. thaliana. The occurrence of cis-acting growth and stress-related elements in the promoter regions of GhAGCs highlight the significance of these factors in plant development and abiotic stress tolerance. Ka/Ks levels demonstrated that purifying selection pressure resulting from segmental events was applied to GhAGC with little functional divergence. We focused on identifying RNA editing sites in G. hirsutum organelles, specifically in the chloroplast and mitochondria, across all 28 AGC genes. CONCLUSION: The positive role of GhAGCs was explored by quantifying the expression in the plant tissues under abiotic stress. These findings help in understanding the role of GhAGC genes under abiotic stresses which may further be used in cotton breeding for the development of climate smart varieties in abruptly changing climate.

CDPK protein in cotton: genomic-wide identification, expression analysis, and conferring resistance to heat stress.

BACKGROUND: Calcium-dependent protein kinase (CDPK) plays a key role in cotton tolerance to abiotic stress. However, its role in cotton heat stress tolerance is not well understood. Here, we characterize the GhCDPK gene family and their expression profiles with the aim of identifying CDPK genes associated with heat stress tolerance. RESULTS: This study revealed 48 GhCDPK members in the cotton genome, distributed on 18 chromosomes. Tree phylogenetic analysis showed three main clustering groups of the GhCDPKs. Cis-elements revealed many abiotic stress and phytohormone pathways conserved promoter regions. Similarly, analysis of the transcription factor binding sites (TFBDS) in the GhCDPK genes showed many stress and hormone related sites. The expression analysis based on qRT-PCR showed that GhCDPK16 was highly responsive to high-temperature stress. Subsequent protein-protein interactions of GhCDPK16 revealed predictable interaction with ROS generating, calcium binding, and ABA signaling proteins. Overexpression of GhCDPK16 in cotton and Arabidopsis improved thermotolerance by lowering ROS compound buildup. Under heat stress, GhCDPK16 transgenic lines upregulated heat-inducible genes GhHSP70, GHSP17.3, and GhGR1, as demonstrated by qRT-PCR analysis. Contrarily, GhCDPK16 knockout lines in cotton exhibited an increase in ROS accumulation. Furthermore, antioxidant enzyme activity was dramatically boosted in the GhCDPK16-ox transgenic lines. CONCLUSIONS: The collective findings demonstrated that GhCDPK16 could be a viable gene to enhance thermotolerance in cotton and, therefore, a potential candidate gene for improving heat tolerance in cotton.

Gene pyramiding improved cell membrane stability under heat stress in cotton (Gossypium hirsutum L.).

Climate change has been drastically affecting cotton not only in Pakistan but also all over the world. Normally cotton is known as heat tolerant when compared with other crops, but if the high temperature occurs during flowering period the yield decreases significantly. Marker assisted gene pyramiding provides a sustainable solution to improve heat tolerance. A total of seven genotypes were developed by a series of crossing seven tolerant genotypes over the period of three years. Tolerant genotypes were selected by screening for important transcription factors (GHSP26, HSP3, HSFA2, DREB1A, HSP101, DREB2A, GhNAC2, HSPCB, GhWRKY41, TPS, GbMYB5, ANNAT8, GhMPK17, GhMKK1, GhMKK3, GhMPK2, HSC70, APX1 and GhPP2A1). The seven genotypes were evaluated under normal and heat stress in a multi-year trial. The traits related to heat tolerance, such as cell membrane stability, relative water content, excised leaf water loss, plant height, number of nodes, internodal length, number of buds, number of bolls and leaf area was observed under normal and heat stress conditions. The developed genotypes showed improvement in cell membrane stability and relative water content under heat stress. The genotypes [(VH-305×MNH-886)×MNH-1035)×NIAB-78)], [(MNH-1035×MNH-886)×MNH-886)×SM-431] and [(MNH-1035×MNH-886)×MNH-886)×SS-32] depicted heat tolerance and could be used as heat tolerant material for variety development in breeding programs.

Genome­wide analysis of cotton SCAMP genes and functional characterization of GhSCAMP2 and GhSCAMP4 in salt tolerance.

BACKGROUND: Secretory carrier membrane proteins (SCAMPs) form a family of integral membrane proteins and play a crucial role in mediating exocytosis in both animals and plants. While SCAMP genes have been studied in several plant species, their functions in cotton, particularly in response to abiotic stress, have not yet been reported. RESULTS: In this study, a total of 53 SCAMP genes were identified in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense. These genes were classified into five groups based on a phylogenetic analysis with SCAMPs from Arabidopsis thaliana. The main factor driving the expansion of the SCAMP gene family in G. hirsutum is tandem and segmental duplication events. Using MEME, in addition to the conserved SCAMP domain, we identified 3-13 other domains in each GhSCAMP. The cis-element analysis suggested that GhSCAMPs were widely involved in cotton growth and development, and responses to abiotic stresses. RNA sequencing (RNA-Seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results showed that most GhSCAMPs were expressed highly in many tissues and had differential expression responses to drought, cold, and heat stresses. Knock-down of GhSCAMP2 and GhSCAMP4 by virus-induced gene silencing (VIGS) lead to a salt-sensitive phenotype and had a lower content of CAT, POD, and SOD. CONCLUSIONS: This study identified SCAMP genes in four cotton species, enhancing our understanding of the potential biological functions of SCAMPs. Additionally, we demonstrated that GhSCAMP2 and GhSCAMP4 positively regulate cotton tolerance to salt stress.

Fertile grounds: exploring male sterility in cotton and its marker development.

The high cost of producing conventional hybrid cotton seeds led to more research efforts on cotton male sterility systems. There is a lack of studies on cytology, histology, morphological variation, yield, and altered restorer backgrounds to identify and develop male sterility markers in cotton hybrids. Hybrid cotton can be efficiently produced by exploiting genetic male sterility. Among the 19 Genetic Male Sterility (GMS) genes discovered, the lines with ms5ms6 genes are mostly utilised to establish successful hybrid cotton in India. Molecular markers closely associated with the MS alleles are identified to facilitate the efficient and rapid backcrossing of male-sterility genes into elite lines or cultivars by marker-assisted backcrossing. The majority of the markers which are random DNA markers (RDMs), are probably lost, when recombination occurs. In contradiction, molecular markers (functional markers, or FMs) within the genic region can be identified and employed in crops for diverse traits, if prospective characteristic genes are known. In this review, the mechanism of male sterility, its gene expression level, and the need for functional markers for the male sterility trait in cotton have been put forward.

Evaluation of memory drought stress effects on storage compounds seedlings of cotton (Gossypium hirsutum) and in-silico analysis of glutathione reductase.

In breeding programs, stress memory in plants can develop drought stress tolerance. Memory stress, as an approach, can keep stress data by activating tolerance mechanisms. This research was conducted to evaluate some physiologically effective mechanisms in inducing memory drought stress in the seeds that were exposed to water stress three times in four treatments including rainfed, 33%, 66%, and 100% of field capacity (FC). After the production of the seeds, the third-generation seeds were placed under different irrigation treatments, seed and seedling traits, starch to carbohydrate ratio in seed, protein concentration and glutathione reductase were investigatied in a factorial format based on a randomized complete block design with three replications. Results showed that percentage of changes from the lowest to the highest value for traits including seed vigor, seed endosperm weight, seed coat weight, accelerated aging, cold test, seedling biomass and seedling length were 25, 37, 65, 65, 55, 77, 55, 65 and 79, respectively and germination uniformity was 3.9 times higher than the lowest amount. According to the deterioration percentage, seed vigor and the percentage of seed germination in cold test data, it can be reported that seed production by 100% FC was not appropriate for rainfed plots. However, considering the the appropriate results in the percentage of germination for a cold test, germination uniformity percentage, and the lowest accelerated aging seeds, seed production under the rainfed conditions with 33% FC watering can be recommended. In-silico analysis was coducted on Glutathione reductase (GR) enzymes in Gossypium hirsutum. It is clear that GR has a Redox-active site and NADPH binding, and it interacts with Glutathione S transferase (GST). So, memory drought stress through inducing physiological drought tolerance mechanisms such as starch-to-carbohydrate ratio and GR can determine the suitable pattern for seed production for rainfed and low rainfall regions in a breeding program. Our study thus illustrated that seed reprduction under 33% FC equipped cotton with the tolerance against under draught stress from the seedling stage. This process is done through activating glutathione reductase and balancing the ratio of starch to carbohydrates concentration.

N6-methyladenosine RNA modification regulates photoperiod sensitivity in cotton.

The methylation of N6-methyladenosine (m6A) involves writers, erasers, and readers, acting synergistically in posttranscriptional regulation. These processes influence various biological processes, including plant floral transition. However, the specific role of m6A modifications in photoperiod sensitivity in cotton (Gossypium hirsutum) remains obscure. To elucidate this, in this study, we conducted transcriptome-wide m6A sequencing during critical flowering transition stages in the photoperiod-sensitive wild G. hirsutum var. yucatanense (yucatanense) and the photoperiod-insensitive cultivated cotton G. hirsutum acc. TM-1 (TM-1). Our results revealed significant variations in m6A methylation of 2 cotton varieties, with yucatanense exhibiting elevated m6A modification levels compared with TM-1 under long-day conditions. Notably, distinct m6A peaks between TM-1 and yucatanense correlated significantly with photoperiod sensitivity. Moreover, our study highlighted the role of the demethylase G. hirsutum ALKB homolog 5 (GhALKBH5) in modulating m6A modification levels. Silencing GhALKBH5 led to a decreased mRNA level of key photoperiodic flowering genes (GhADO3, GhAGL24, and GhFT1), resulting in delayed bud emergence and flowering. Reverse transcription quantitative PCR analyses confirmed that silencing GhADO3 and GhAGL24 significantly downregulated the expression of the floral integrator GhFT1. Collectively, our findings unveiled a transcriptional regulatory mechanism in which GhALKBH5-mediated m6A demethylation of crucial photoperiodic flowering transcripts modulated photoperiod sensitivity in cotton.

Silencing of GhSINAT5 Reduces Drought Resistance and Salt Tolerance in Cotton.

The SEVEN IN ABSENTIA (SINA) E3 ubiquitin ligase is widely involved in drought and salt stress in plants. However, the biological function of the SINA proteins in cotton is still unknown. This study aimed to reveal the function of GhSINAT5 through biochemical, genetic and molecular approaches. GhSINAT5 is expressed in several tissues of cotton plants, including roots, stems, leaves and cotyledons, and its expression levels are significantly affected by polyethylene glycol, abscisic acid and sodium chloride. When GhSINAT5 was silenced in cotton plants, drought and salinity stress occurred, and the length, area and volume of the roots significantly decreased. Under drought stress, the levels of proline, superoxide dismutase, peroxidase and catalase in the GhSINAT5-silenced cotton plants were significantly lower than those in the non-silenced control plants, whereas the levels of hydrogen peroxide and malondialdehyde were greater. Moreover, the expression of stress-related genes in silenced plants under drought stress suggested that GhSINAT5 may play a positive role in the plant response to drought and salt stress by regulating these stress response-related genes. These findings not only deepen our understanding of the mechanisms of drought resistance in cotton but also provide potential targets for future improvements in crop stress resistance through genetic engineering.

Gossypium arboreum PPD2 facilitates root architecture development to increase plant resilience to salt stress.

The jasmonic acid (JA) signaling pathway plays an important role in plant responses to abiotic stresses. The PEAPOD (PPD) and jasmonate ZIM-domain (JAZ) protein in the JA signaling pathway belong to the same family, but their functions in regulating plant defense against salt stress remain to be elucidated. Here, Gossypium arboreum PPD2 was overexpressed in Arabidopsis thaliana and systematically silenced in cotton for exploring its function in regulating plant defense to salt stress. The GaPPD2-overexpressed Arabidopsis thaliana plants significantly increased the tolerance to salt stress compared to the wild type in both medium and soil, while the GaPPD2-silenced cotton plants showed higher sensitivity to salt stress than the control in pots. The antioxidant activities experiment showed that GaPPD2 may mitigate the accumulation of reactive oxygen species by promoting superoxide dismutase accumulation, consequently improving plant resilience to salt stress. Through the exogenous application of MeJA (methy jasmonate) and the protein degradation inhibitor MG132, it was found that GaPPD2 functions in plant defense against salt stress and is involved in the JA signaling pathway. The RNA-seq analysis of GaPPD2-overexpressed A. thaliana plants and receptor materials showed that the differentially expressed genes were mainly enriched in antioxidant activity, peroxidase activity, and plant hormone signaling pathways. qRT-PCR results demonstrated that GaPPD2 might positively regulate plant defense by inhibiting GH3.2/3.10/3.12 expression and activating JAZ7/8 expression. The findings highlight the potential of GaPPD2 as a JA signaling component gene for improving the cotton plant resistance to salt stress and provide insights into the mechanisms underlying plant responses to environmental stresses.

An insight into heat stress response and adaptive mechanism in cotton.

The growing worldwide population is driving up demand for cotton fibers, but production is hampered by unpredictable temperature rises caused by shifting climatic conditions. Numerous research based on breeding and genomics have been conducted to increase the production of cotton in environments with high and low-temperature stress. High temperature (HT) is a major environmental stressor with global consequences, influencing several aspects of cotton plant growth and metabolism. Heat stress-induced physiological and biochemical changes are research topics, and molecular techniques are used to improve cotton plants' heat tolerance. To preserve internal balance, heat stress activates various stress-responsive processes, including repairing damaged proteins and membranes, through various molecular networks. Recent research has investigated the diverse reactions of cotton cultivars to temperature stress, indicating that cotton plant adaptation mechanisms include the accumulation of sugars, proline, phenolics, flavonoids, and heat shock proteins. To overcome the obstacles caused by heat stress, it is crucial to develop and choose heat-tolerant cotton cultivars. Food security and sustainable agriculture depend on the application of genetic, agronomic, and, biotechnological methods to lessen the impacts of heat stress on cotton crops. Cotton producers and the textile industry both benefit from increased heat tolerance. Future studies should examine the developmental responses of cotton at different growth stages, emphasize the significance of breeding heat-tolerant cultivars, and assess the biochemical, physiological, and molecular pathways involved in seed germination under high temperatures. In a nutshell, a concentrated effort is required to raise cotton's heat tolerance due to the rising global temperatures and the rise in the frequency of extreme weather occurrences. Furthermore, emerging advances in sequencing technologies have made major progress toward successfully se sequencing the complex cotton genome.

Calcium-Dependent Protein Kinase GhCDPK16 Exerts a Positive Regulatory Role in Enhancing Drought Tolerance in Cotton.

Cotton is essential for the textile industry as a primary source of natural fibers. However, environmental factors like drought present significant challenges to its cultivation, adversely affecting both production levels and fiber quality. Enhancing cotton's drought resilience has the potential to reduce yield losses and support the growth of cotton farming. In this study, the cotton calcium-dependent protein kinase GhCDPK16 was characterized, and the transcription level of GhCDPK16 was significantly upregulated under drought and various stress-related hormone treatments. Physiological analyses revealed that the overexpression of GhCDPK16 improved drought stress resistance in Arabidopsis by enhancing osmotic adjustment capacity and boosting antioxidant enzyme activities. In contrast, silencing GhCDPK16 in cotton resulted in increased dehydration compared with the control. Furthermore, reduced antioxidant enzyme activities and downregulation of ABA-related genes were observed in GhCDPK16-silenced plants. These findings not only enhanced our understanding of the biological functions of GhCDPK16 and the mechanisms underlying drought stress resistance but also underscored the considerable potential of GhCDPK16 in improving drought resilience in cotton.

H3K36 methyltransferase GhKMT3;1a and GhKMT3;2a promote flowering in upland cotton.

BACKGROUND: The SET domain group (SDG) genes encode histone lysine methyltransferases, which regulate gene transcription by altering chromatin structure and play pivotal roles in plant flowering determination. However, few studies have investigated their role in the regulation of flowering in upland cotton. RESULTS: A total of 86 SDG genes were identified through genome-wide analysis in upland cotton (Gossypium hirsutum). These genes were unevenly distributed across 25 chromosomes. Cluster analysis revealed that the 86 GhSDGs were divided into seven main branches. RNA-seq data and qRT‒PCR analysis revealed that lysine methyltransferase 3 (KMT3) genes were expressed at high levels in stamens, pistils and other floral organs. Using virus-induced gene silencing (VIGS), functional characterization of GhKMT3;1a and GhKMT3;2a revealed that, compared with those of the controls, the GhKMT3;1a- and GhKMT3;2a-silenced plants exhibited later budding and flowering and lower plant heightwere shorter. In addition, the expression of flowering-related genes (GhAP1, GhSOC1 and GhFT) significantly decreased and the expression level of GhSVP significantly increased in the GhKMT3;1a- and GhKMT3;2a-silenced plants compared with the control plants. CONCLUSION: A total of 86 SDG genes were identified in upland cotton, among which GhKMT3;1a and GhKMT3;2a might regulate flowering by affecting the expression of GhAP1, GhSOC1, GhFT and GhSVP. These findings will provide genetic resources for advanced molecular breeding in the future.

Integrative physiology and transcriptome sequencing reveal differences between G. hirsutum and G. barbadense in response to salt stress and the identification of key salt tolerance genes.

BACKGROUND: Soil salinity is one of the major abiotic stresses that threatens crop growth. Cotton has some degree of salt tolerance, known as the "pioneer crop" of saline-alkali land. Cultivation of cotton is of great significance to the utilization of saline-alkali land and the development of cotton industry. Gossypium hirsutum and G. barbadense, as two major cotton species, are widely cultivated worldwide. However, until recently, the regulatory mechanisms and specific differences of their responses to salt stress have rarely been reported. RESULTS: In this study, we comprehensively compared the differences in the responses of G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124 to salt stress. The results showed that Hai7124 exhibited better growth than did TM-1 under salt stress, with greater PRO content and antioxidant capability, whereas TM-1 only presented greater K+ content. Transcriptome analysis revealed significant molecular differences between the two cotton species in response to salt stress. The key pathways of TM-1 induced by salt are mainly related to growth and development, such as porphyrin metabolism, DNA replication, ribosome and photosynthesis. Conversely, the key pathways of Hai7124, such as plant hormone signal transduction, MAPK signaling pathway-plant, and phenylpropanoid biosynthesis, are mainly related to plant defense. Further comparative analyses of differentially expressed genes (DEGs) revealed that antioxidant metabolism, abscisic acid (ABA) and jasmonic acid (JA) signalling pathways were more strongly activated in Hai7124, whereas TM-1 was more active in K+ transporter-related genes and ethylene (ETH) signalling pathway. These differences underscore the various molecular strategies adopted by the two cotton species to navigate through salt stress, and Hai7124 responded more strongly to salt stress, which explains the potential reasons for the greater salt tolerance of Hai7124. Finally, we identified 217 potential salt tolerance-related genes, 167 of which overlapped with the confidence intervals of significant SNPs identified in previous genome-wide association studies (GWASs), indicating the high reliability of these genes. CONCLUSIONS: These findings provide new insights into the differences in the regulatory mechanisms of salt tolerance between G. hirsutum and G. barbadense, and identify key candidate genes for salt tolerance molecular breeding in cotton.

Multistage temporal transcriptomic atlas unveils major contributor to reproductive phase in upland cotton.

Flowering is a major developmental transition in plants, but asynchronous flowering hinders the utilization of wild cotton relatives in breeding programs. We performed comparative transcriptomic profiling of early- and late-flowering Gossypium hirsutum genotypes to elucidate genetic factors influencing reproductive timing. Shoot apices were sampled from the photoperiod-sensitive landrace G. hirsutum purpurascens (GhP) and early-maturing variety ZhongMianSuo (ZMS) at five time points following the emergence of sympodial nodes. RNA-sequencing revealed extensive transcriptional differences during floral transition. Numerous flowering-associated genes exhibited genotype-specific expression, including FLOWERING LOCUS T (FT) homologs upregulated in ZMS. FT-interacting factors like SOC1 and CO-like also showed higher expression in ZMS, implicating florigen pathways in early flowering. Additionally, circadian clock and light signalling components were misregulated between varieties, suggesting altered photoperiod responses in GhP. Weighted co-expression network analysis specifically linked a module enriched for circadian-related genes to GhP's late flowering. Through an integrated transcriptome analysis, we defined a regulatory landscape of reproductive phase change in cotton. Differentially expressed genes related to photoperiod, circadian clock, and light signalling likely contribute to delayed flowering in wild cottons. Characterization of upstream flowering regulators will enable modifying photoperiod sensitivity and expand germplasm use for cotton improvement. This study provides candidate targets for elucidating interactive mechanisms that control cotton flowering time across diverse genotypes.

A cotton mitochondrial alternative electron transporter, GhD2HGDH, induces early flowering by modulating GA and photoperiodic pathways.

D-2-hydroxyglutarate dehydrogenase (D2HGDH) is a mitochondrial enzyme containing flavin adenine dinucleotide FAD, existing as a dimer, and it facilitates the specific oxidation of D-2HG to 2-oxoglutarate (2-OG), which is a key intermediate in the tricarboxylic acid (TCA) cycle. A Genome-wide expression analysis (GWEA) has indicated an association between GhD2HGDH and flowering time. To further explore the role of GhD2HGDH, we performed a comprehensive investigation encompassing phenotyping, physiology, metabolomics, and transcriptomics in Arabidopsis thaliana plants overexpressing GhD2HGDH. Transcriptomic and qRT-PCR data exhibited heightened expression of GhD2HGDH in upland cotton flowers. Additionally, early-maturing cotton exhibited higher expression of GhD2HGDH across all tissues than delayed-maturing cotton. Subcellular localization confirmed its presence in the mitochondria. Overexpression of GhD2HGDH in Arabidopsis resulted in early flowering. Using virus-induced gene silencing (VIGS), we investigated the impact of GhD2HGDH on flowering in both early- and delayed-maturing cotton plants. Manipulation of GhD2HGDH expression levels led to changes in photosynthetic pigment and gas exchange attributes. GhD2HGDH responded to gibberellin (GA3) hormone treatment, influencing the expression of GA biosynthesis genes and repressing DELLA genes. Protein interaction studies, including yeast two-hybrid, luciferase complementation (LUC), and GST pull-down assays, confirmed the interaction between GhD2HGDH and GhSOX (Sulfite oxidase). The metabolomics analysis demonstrated GhD2HGDH's modulation of the TCA cycle through alterations in various metabolite levels. Transcriptome data revealed that GhD2HGDH overexpression triggers early flowering by modulating the GA3 and photoperiodic pathways of the flowering core factor genes. Taken together, GhD2HGDH positively regulates the network of genes associated with early flowering pathways.

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

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

Exploring cotton SFR2's conundrum in response to cold stress.

Cotton is an important agricultural crop to many regions across the globe but is sensitive to low-temperature exposure. The activity of the enzyme SENSITIVE TO FREEZING 2 (SFR2) improves cold tolerance of plants and produces trigalactosylsyldiacylglycerol (TGDG), but its role in cold sensitive plants, such as cotton remains unknown. Recently, it was reported that cotton SFR2 produced very little TGDG under normal and cold conditions. Here, we investigate cotton SFR2 activation and TGDG production. Using multiple approaches in the native system and transformation into Arabidopsis thaliana, as well as heterologous yeast expression, we provide evidence that cotton SFR2 activates differently than previously found among other plant species. We conclude with the hypothesis that SFR2 in cotton is not activated in a similar manner regarding acidification or freezing like Arabidopsis and that other regions of SFR2 protein are critical for activation of the enzyme than previously reported.

GhGASA14 regulates the flowering time of upland cotton in response to GA3.

KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.