Molecular traits of MAPK kinases and the regulatory mechanism of GhMAPKK5 alleviating drought/salt stress in cotton.

Mitogen-activated protein kinase kinases (MAPKKs) play a critical role in the mitogen-activated protein kinase (MAPK) signaling pathway, transducing external stimuli into intracellular responses and enabling plant adaptation to environmental challenges. Most research has focused on the model plant Arabidopsis (Arabidopsis thaliana). The systematic analysis and characterization of MAPKK genes across different plant species, particularly in cotton (Gossypium hirsutum), are somewhat limited. Here, we identified MAPKK family members from 66 different species, which clustered into 5 different sub-groups, and MAPKKs from four cotton species clustered together. Through further bioinformatic and expression analysis, GhMAPKK5 was identified as the most responsive MAPKK member to salt and drought stress among the 23 MAPKKs identified in Gossypium hirsutum. Silencing GhMAPKK5 in cotton through virus-induced gene silencing (VIGS) led to quicker wilting under salt and drought conditions, while overexpressing GhMAPKK5 in Arabidopsis enhanced root growth and seed germination under these stresses, demonstrating GhMAPKK5's positive role in stress tolerance. Transcriptomics and Yeast-Two-Hybrid assays revealed a MAPK cascade signal module comprising GhMEKK (Mitogen-activated protein kinase kinase kinases)3/8/31-GhMAPKK5-GhMAPK11/23. This signaling cascade may play a role in managing drought and salt stress by regulating transcription factor genes, such as WRKYs, which are involved in the biosynthesis and transport pathways of ABA, proline, and RALF. This study is highly important for further understanding the regulatory mechanism of MAPKK in cotton, contributing to its stress tolerance and offering potential in targets for genetic enhancement.

JrPPO1/2 play distinct roles in regulating walnut fruit browning by different spatiotemporal expression and enzymatic characteristics.

Polyphenol oxidase (PPO) activity drives walnut fruit browning, but the roles of its only two-family genes, JrPPO1 and JrPPO2, remain unclear. This study explores the spatiotemporal expression and enzymatic characteristics of JrPPO1 and JrPPO2 in walnut. Treatment with the PPO activator CuSO4 and H2O2 accelerated fruit browning and up-regulated JrPPO1/2 expression, whereas treatment with the PPO inhibitor ascorbic acid delayed browning, down-regulating JrPPO1 and up-regulating JrPPO2 expression. Compared to mJrPPO1, mJrPPO2 can exhibited better enzyme activity at higher temperatures (47 °C) and in more acidic environments (pH 4.25). mJrPPO2 exhibited a higher substrate specificity over mJrPPO1, and the preferred substrates are catechol, chlorogenic acid, and epicatechin. Additionally, mJrPPO2 adapted better to low concentration of oxygen (as low as 1.0% O2) and slightly elevated CO2 levels compared to mJrPPO1. Subcellular localization and spatiotemporal expression patterns showed that JrPPO1 is only expressed in green tissues and located in chloroplasts, while JrPPO2 is also located in chloroplasts, partly associated with membranes, and is expressed in both green and non-green tissues. Silencing JrPPO1/2 with virus-induced gene silencing (VIGS) reduced fruit browning, maintained higher total phenols, and decreased MDA production. Notably, silencing JrPPO1 had a greater impact on browning than JrPPO2, indicating JrPPO1's greater contribution to PPO activity and fruit browning in walnut fruits. Consequently, JrPPO1 can be effectively regulated both at the molecular level and by manipulating environmental conditions, to achieve the objective of controlling fruit browning.

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.

Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol.

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62-91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues.

The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

Genome-wide identification of TBL gene family and functional analysis of GhTBL84 under cold stress in cotton.

Cotton fiber, the mainstay of the world's textile industry, is formed by the differentiation of epidermal cells on the outer peridium of the ovule. The TBL gene family is involved in the regulation of epidermal hair development as well as response to abiotic stress. However, the function of TBL genes in cotton has not been systematically studied yet. Here, we identified 131 and 130 TBL genes in TM-1 (Gossypium hirsutum) and Hai7124 (Gossypium barbadense), respectively. Phylogenetic, gene structure, expression pattern and cis-element of promoter analysis were performed and compared. Single gene association analysis indicated that more TBL genes related to fiber quality traits were found in G. barbadense, whereas more genes associated with yield traits were found in G. hirsutum. One gene, GhTBL84 (GH_D04G0930), was induced by treatment at 4°C for 12 and 24 h in G. hirsutum and silencing of the GhTBL84 gene by VIGS technology in TM-1 can significantly improve the resistance of cotton seedlings to low temperature stress. In sum, our study conducted a genome-wide identification and comparative analysis of TBL family genes in G. hirsutum and G. barbadense and demonstrated a group of TBL genes significantly associated with fiber quality and excavated cold stress responsive gene, such as GhTBL84, providing a theoretical basis for further improving cotton agronomic traits.

Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress.

BACKGROUND: Despite its known significance in plant abiotic stress responses, the role of the RAV gene family in the response of Capsicum annuum to chilling stress remains largely unexplored. RESULTS: In this study, we identified and characterized six members of the CaRAV gene subfamily in pepper plants through genome-wide analysis. Subsequently, the CaRAV subfamily was classified into four branches based on homology with Arabidopsis thaliana, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs, whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT‒PCR analysis revealed that CaRAV1, CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAV gene family. Under normal growth conditions, the MDA content and SOD enzyme activity of the silenced plants were slightly greater than those of the control plants, and the REC activity was significantly greater than that of the control plants. The levels of MDA and electrolyte leakage were greater in the silenced plants after they were exposed to chilling stress, and the POD and CAT enzyme activities were significantly lower than those in the control, which was particularly evident under repeated chilling stress. In addition, the relative expression of CaPOD and CaCAT was greater in V2 plants upon repeated chilling stress, especially CaCAT was significantly greater in V2 plants than in the other two silenced plants, with 3.29 and 1.10 increases within 12 and 24 h. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress. CONCLUSIONS: Silencing of key members of the CaRAV gene family results in increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly under repeated chilling stress. This study provides valuable information for understanding the classification and putative functions of RAV transcription factors in pepper plants.

Terpene synthases GhTPS6 and GhTPS47 participate in resistance to Verticillium dahliae in upland cotton.

Terpene synthases (TPSs) are enzymes responsible for catalyzing the production of diverse terpenes, the largest class of secondary metabolites in plants. Here, we identified 107 TPS gene loci encompassing 92 full-length TPS genes in upland cotton (Gossypium hirsutum L.). Phylogenetic analysis showed they were divided into six subfamilies. Segmental duplication and tandem duplication events contributed greatly to the expansion of TPS gene family, particularly the TPS-a and TPS-b subfamilies. Expression profile analysis screened out that GhTPSs may mediate the interaction between cotton and Verticillium dahliae. Three-dimensional structures and subcellular localizations of the two selected GhTPSs, GhTPS6 and GhTPS47, which belong to the TPS-a subfamily, demonstrated similarity in protein structures and nucleus and cytoplasm localization. Virus-induced gene silencing (VIGS) of the two GhTPSs yielded plants characterized by increased wilting and chlorosis, more severe vascular browning, and higher disease index than control plants. Additionally, knockdown of GhTPS6 and GhTPS47 led to the down-regulation of cotton terpene synthesis following V. dahliae infection, indicating that these two genes may positively regulate resistance to V. dahliae through the modulation of disease-resistant terpene biosynthesis. Overall, our study represents a comprehensive analysis of the G. hirsutum TPS gene family, revealing their potential roles in defense responses against Verticillium wilt.

A review on strategies for crop improvement against drought stress through molecular insights.

The demand for food goods is rising along with the world population growth, which is directly related to the yield of agricultural crops around the world. However, a number of environmental factors, including floods, salinity, moisture, and drought, have a detrimental effect on agricultural production around the world. Among all of these stresses, drought stress (DS) poses a constant threat to agricultural crops and is a significant impediment to global agricultural productivity. Its potency and severity are expected to increase in the future years. A variety of techniques have been used to generate drought-resistant plants in order to get around this restriction. Different crop plants exhibit specific traits that contribute to drought resistance (DR), such as early flowering, drought escape (DE), and leaf traits. We are highlighting numerous methods that can be used to overcome the effects of DS in this review. Agronomic methods, transgenic methods, the use of sufficient fertilizers, and molecular methods such as clustered regularly interspaced short palindromic repeats (CRISPRs)-associated nuclease 9 (Cas9), virus-induced gene silencing (VIGS), quantitative trait loci (QTL) mapping, microRNA (miRNA) technology, and OMICS-based approaches make up the majority of these techniques. CRISPR technology has rapidly become an increasingly popular choice among researchers exploring natural tolerance to abiotic stresses although, only a few plants have been produced so far using this technique. In order to address the difficulties imposed by DS, new plants utilizing the CRISPR technology must be developed.

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.

PaWOX3 and PaWOX3B Regulate Flower Number and The Lip Symmetry of Phalaenopsis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decrease in mature flower and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium orthologue, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' can not be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to control lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip.

Jasmonate ZIM Domain Protein (JAZ) Gene SLJAZ15 Increases Resistance to Orobanche aegyptiaca in Tomato.

Orobanche aegyptiaca Pers. is a holoparasitic plant that severely reduces tomato (Solanum lycopersicum L.) production in China. However, there is a lack of effective control methods and few known sources of genetic resistance. In this study, we focused on key genes in the JAZ family, comparing the JAZ family in Arabidopsis thaliana (L. Heynh.) to the tomato genome. After identifying the JAZ family members in S. lycopersicum, we performed chromosomal localization and linear analysis with phylogenetic relationship analysis of the JAZ family. We also analyzed the gene structure of the JAZ gene family members in tomato and the homology of the JAZ genes among the different species to study their relatedness. The key genes for O. aegyptiaca resistance were identified using VIGS (virus-induced gene silencing), and the parasitization rate of silenced tomato plants against O. aegyptiaca increased by 47.23-91.13%. The genes were localized in the nucleus by subcellular localization. Heterologous overexpression in A. thaliana showed that the key gene had a strong effect on the parasitization process of O. aegyptiaca, and the overexpression of the key gene reduced the parasitization rate of O. aegyptiaca 1.69-fold. Finally, it was found that the SLJAZ15 gene can positively regulate the hormone content in tomato plants and affect plant growth and development, further elucidating the function of this gene.

Heat shock protein HvHSP16.9 from wild barley enhances tolerance to salt stress.

Heat shock proteins (HSPs) are known to play a crucial role in the response of plants to environmental stress, particularly heat stress. Nevertheless, the function of HSPs in salt stress tolerance in plants, especially in barley, remains largely unexplored. Here, we aimed to investigate and compare the salt tolerance mechanisms between wild barley EC_S1 and cultivated barley RGT Planet through a comprehensive analysis of physiological parameters and transcriptomic profiles. Results demonstrated that the number of differentially expressed genes (DEGs) in EC_S1 was significantly higher than in RGT Planet, indicating that wild barley gene regulation is more adaptive to salt stress. KEGG enrichment analysis revealed that DEGs were mainly enriched in the processes of photosynthesis, plant hormone signal transduction, and reactive oxygen species metabolism. Furthermore, the application of weighted gene correlation network analysis (WGCNA) enabled the identification of a set of key genes, including small heat shock protein (sHSP), Calmodulin-like proteins (CML), and protein phosphatases 2C (PP2C). Subsequently, a novel sHSP gene, HvHSP16.9 encoding a protein of 16.9 kDa, was cloned from wild barley, and its role in plant response to salt stress was elucidated. In Arabidopsis, overexpression of HvHSP16.9 increased the salt tolerance. Meanwhile, barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) of HvHSP16.9 significantly reduced the salt tolerance in wild barley. Overall, this study offers a new theoretical framework for comprehending the tolerance and adaptation mechanisms of wild barley under salt stress. It provides valuable insights into the salt tolerance function of HSP, and identifies new candidate genes for enhancing cultivated barley varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01455-4.

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.

Genome-wide expression analysis of LACS gene family implies GhLACS25 functional responding to salt stress in cotton.

BACKGROUND: Long-chain acyl-coenzyme A synthetase (LACS) is a type of acylating enzyme with AMP-binding, playing an important role in the growth, development, and stress response processes of plants. RESULTS: The research team identified different numbers of LACS in four cotton species (Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum). By analyzing the structure and evolutionary characteristics of the LACS, the GhLACS were divided into six subgroups, and a chromosome distribution map of the family members was drawn, providing a basis for further research classification and positioning. Promoter cis-acting element analysis showed that most GhLACS contain plant hormones (GA, MeJA) or non-biological stress-related cis-elements. The expression patterns of GhLACS under salt stress treatment were analyzed, and the results showed that GhLACS may significantly participate in salt stress response through different mechanisms. The research team selected 12 GhLACSs responsive to salt stress for tissue expression analysis and found that these genes are expressed in different tissues. CONCLUSIONS: There is a certain diversity of LACS among different cotton species. Analysis of promoter cis-acting elements suggests that GhLACS may be involved in regulating plant growth, development and stress response processes. GhLACS25 was selected for in-depth study, which confirmed its significant role in salt stress response through virus-induced gene silencing (VIGS) and induced expression in yeast cells.

GhiPLATZ17 and GhiPLATZ22, zinc-dependent DNA-binding transcription factors, promote salt tolerance in upland cotton.

KEY MESSAGE: The utilization of transcriptome analysis, functional validation, VIGS, and DAB techniques have provided evidence that GhiPLATZ17 and GhiPLATZ22 play a pivotal role in improving the salt tolerance of upland cotton. PLATZ (Plant AT-rich sequences and zinc-binding proteins) are known to be key regulators in plant growth, development, and response to salt stress. In this study, we comprehensively analyzed the PLATZ family in ten cotton species in response to salinity stress. Gossypium herbaceum boasts 25 distinct PLATZ genes, paralleled by 24 in G. raimondii, 25 in G. arboreum, 46 in G. hirsutum, 48 in G. barbadense, 43 in G. tomentosum, 67 in G. mustelinum, 60 in G. darwinii, 46 in G. ekmanianum, and a total of 53 PLATZ genes attributed to G. stephensii. The PLATZ gene family shed light on the hybridization and allopolyploidy events that occurred during the evolutionary history of allotetraploid cotton. Ka/Ks analysis suggested that the PLATZ gene family underwent intense purifying selection during cotton evolution. Analysis of synteny and gene collinearity revealed a complex pattern of segmental and dispersed duplication events to expand PLATZ genes in cotton. Cis-acting elements and gene expressions revealed that GhiPLATZ exhibited salt stress resistance. Transcriptome analysis, functional validation, virus-induced gene silencing (VIGS), and diaminobenzidine staining (DAB) demonstrated that GhiPLATZ17 and GhiPLATZ22 enhance salt tolerance in upland cotton. The study can potentially advance our understanding of identifying salt-resistant genes in cotton.

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

Comparative analysis, diversification, and functional validation of plant nucleotide-binding site domain genes.

Nucleotide-binding site (NBS) domain genes are one of the superfamily of resistance genes involved in plant responses to pathogens. The current study identified 12,820 NBS-domain-containing genes across 34 species covering from mosses to monocots and dicots. These identified genes are classified into 168 classes with several novel domain architecture patterns encompassing significant diversity among plant species. Several classical (NBS, NBS-LRR, TIR-NBS, TIR-NBS-LRR, etc.) and species-specific structural patterns (TIR-NBS-TIR-Cupin_1-Cupin_1, TIR-NBS-Prenyltransf, Sugar_tr-NBS etc.) were discovered. We observed 603 orthogroups (OGs) with some core (most common orthogroups; OG0, OG1, OG2, etc.) and unique (highly specific to species; OG80, OG82, etc.) OGs with tandem duplications. The expression profiling presented the putative upregulation of OG2, OG6, and OG15 in different tissues under various biotic and abiotic stresses in susceptible and tolerant plants to cotton leaf curl disease (CLCuD). The genetic variation between susceptible (Coker 312) and tolerant (Mac7) Gossypium hirsutum accessions identified several unique variants in NBS genes of Mac7 (6583 variants) and Coker312 (5173 variants). The protein-ligand and proteins-protein interaction showed a strong interaction of some putative NBS proteins with ADP/ATP and different core proteins of the cotton leaf curl disease virus. The silencing of GaNBS (OG2) in resistant cotton through virus-induced gene silencing (VIGS) demonstrated its putative role in virus tittering. The presented study will be further helpful in understanding the plant adaptation mechanism.

Effect of cold stress on photosynthetic physiological characteristics and molecular mechanism analysis in cold-resistant cotton (ZM36) seedlings.

Low temperature and cold damage seriously hinder the growth, development, and morphogenesis of cotton seedlings. However, the response mechanism of cotton seedlings under cold stress still lacks research. In this study, transcriptome sequencing, gas exchange parameters, and rapid chlorophyll fluorescence parameters were analyzed in leaves of cold-tolerant upland cotton variety "ZM36" under different temperature stress [25°C (T25, CK), 15°C (T15), 10°C (T10), and 4°C (T4)]. The results showed that the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), PSII potential maximum photochemical efficiency (Fv/Fm), and performance index (PIabs) of cotton leaves significantly decreased, and the intercellular CO2 concentration (Ci) and Fo/Fm significantly increased under cold stress. The transcriptome sequencing analysis showed that a total of 13,183 DEGs were involved in the response of cotton seedlings at each temperature point (T25, T15, T10, and T4), mainly involving five metabolic pathways-the phosphatidylinositol signaling system, photosynthesis, photosynthesis antenna protein, carbon fixation in photosynthetic organisms, and carotenoid synthesis. The 1,119 transcription factors were discovered among all the DEGs. These transcription factors involve 59 families, of which 15.8% of genes in the NAC family are upregulated. Through network regulatory analysis, the five candidate genes GhUVR8 (GH_A05G3668), GhPLATZ (GH_A09G2161), GhFAD4-1 (GH_A01G0758), GhNFYA1 (GH_A02G1336), and GhFAD4-2 (GH_D01G0766) were identified in response to cold stress. Furthermore, suppressing the expression level of GhPLATZ by virus-induced gene silencing led to the reduction of low temperature resistance, implying GhPLATZ as a positive regulator of low temperature tolerance. The findings of the study revealed a piece of the complex response mechanism of the cold-tolerant variety "ZM36" to different cold stresses and excavated key candidate genes for low temperature response, which provided support for accelerating the selection and breeding of cotton varieties with low temperature tolerance.