An analysis of lncRNAs related to fiber quality and the discovery of their target genes in a Gossypium hirsutum line with Gossypium mustelinum introgression.

KEY MESSAGE: Analysis of fiber quality lncRNAs and their target genes from a pair of Gossypium mustelinum near-isogenic lines provide new prospects for improving the fiber quality of Upland cotton. Long noncoding RNAs (lncRNAs) are an important part of genome transcription and play roles in a wide range of biological processes in plants. In this research, a pair of near-isogenic cotton lines, namely, a Gossypium mustelinum introgression line (IL9) with outstanding fiber quality and its recurrent Upland cotton parent (PD94042), were used as the experimental materials. Cotton fibers were selected for lncRNA sequencing at 17 and 21 days post-anthesis. A total of 2693 differentially expressed genes were identified. In total, 5841 lncRNAs were ultimately screened, from which 163 differentially expressed lncRNAs were identified. Target genes of the lncRNAs were predicted by two different methods: cis and trans. Some of the target genes were related to cell components, membrane components, plant hormone signal transduction and catalytic metabolism, and the results indicated that there might also be important effects on the development of fiber. Four differentially expressed target genes related to fiber quality (Gomus.D05G015100, Gomus.A05G281300, Gomus.A12G023400 and Gomus.A10G226800) were screened through gene function annotation, and the functions of these four genes were verified through virus-induced gene silencing (VIGS). Compared to the negative controls, plants in which any of these four genes were silenced showed significant reductions in fiber strength. In addition, the plants in which the Gomus.A12G023400 gene was silenced showed a significant reduction in fiber uniformity, whereas the plants in which Gomus.A05G281300 was silenced showed a significant increase in fiber fineness as measured via micronaire. Our results showed that these genes play different roles during fiber development, impacting fiber quality.

Biotechnological and Biocontrol Approaches for Mitigating Postharvest Diseases Caused by Fungal Pathogens and Their Mycotoxins in Fruits: A Review.

Postharvest diseases caused by fungal pathogens are significant contributors to the postharvest losses of fruits. Moreover, some fungal pathogens produce mycotoxins, which further compromise the safety and quality of fruits. In this review, the potential of biotechnological and biocontrol approaches for mitigating postharvest diseases and mycotoxins in fruits is explored. The review begins by discussing the impact of postharvest diseases on fruit quality and postharvest losses. Next, it provides an overview of major postharvest diseases caused by fungal pathogens. Subsequently, it delves into the role of biotechnological approaches in controlling these diseases. The review also explored the application of biocontrol agents, such as antagonistic yeasts, bacteria, and fungi, which can suppress pathogen growth. Furthermore, future trends and challenges in these two approaches are discussed in detail. Overall, this review can provide insights into promising biotechnological and biocontrol strategies for managing postharvest diseases and mycotoxins in fruits.

Dynamic characteristics and functional analysis provide new insights into the role of GauERF105 for resistance against Verticillium dahliae in cotton.

BACKGROUND: The cotton industry suffers significant yield losses annually due to Verticillium wilt, which is considered the most destructive disease affecting the crop. However, the precise mechanisms behind this disease in cotton remain largely unexplored. METHODS: Our approach involved utilizing transcriptome data from G. australe which was exposed to Verticillium dahliae infection. From this data, we identified ethylene-responsive factors and further investigated their potential role in resistance through functional validations via Virus-induced gene silencing (VIGS) in cotton and overexpression in Arabidopsis. RESULTS: A total of 23 ethylene response factors (ERFs) were identified and their expression was analyzed at different time intervals (24 h, 48 h, and 72 h post-inoculation). Among them, GauERF105 was selected based on qRT-PCR expression analysis for further investigation. To demonstrate the significance of GauERF105, VIGS was utilized, revealing that suppressing GauERF105 leads to more severe infections in cotton plants compared to the wild-type. Additionally, the silenced plants exhibited reduced lignin deposition in the stems compared to the WT plants, indicating that the silencing of GauERF105 also impacts lignin content. The overexpression of GauERF105 in Arabidopsis confirmed its pivotal role in conferring resistance against Verticillium dahliae infection. Our results suggest that WT possesses higher levels of the oxidative stress markers MDA and H2O2 as compared to the overexpressed lines. In contrast, the activities of the antioxidant enzymes SOD and POD were higher in the overexpressed lines compared to the WT. Furthermore, DAB and trypan staining of the overexpressed lines suggested a greater impact of the disease in the wild-type compared to the transgenic lines. CONCLUSIONS: Our findings provide confirmation that GauERF105 is a crucial candidate in the defense mechanism of cotton against Verticillium dahliae invasion, and plays a pivotal role in this process. These results have the potential to facilitate the development of germplasm resistance in cotton.

Genome-wide identification and expression analysis of terpene synthases in Gossypium species in response to gossypol biosynthesis.

Cottonseed is an invaluable resource, providing protein, oil, and abundant minerals that significantly contribute to the well-being and nutritional needs of both humans and livestock. However, cottonseed also contains a toxic substance called gossypol, a secondary metabolite in Gossypium species that plays an important role in cotton plant development and self-protection. Herein, genome-wide analysis and characterization of the terpene synthase (TPS) gene family identified 304 TPS genes in Gossypium. Bioinformatics analysis revealed that the gene family was grouped into six subgroups TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g. Whole-genome, segmental, and tandem duplication contributed to the evolution of TPS genes. According to the analysis of selection pressure, it was predicted that TPS genes experience predominantly negative selection, with positive selection occurring subsequently. RT-qPCR analysis in TM-1 and CRI-12 lines revealed GhTPS48 gene as the candidate gene for silencing experiments. To summarize, comprehensive genome-wide studies, RT-qPCR, and gene silencing experiments have collectively demonstrated the involvement of the TPS gene family in the biosynthesis of gossypol in cotton.

Genome-wide identification of the geranylgeranyl pyrophosphate synthase (GGPS) gene family involved in chlorophyll synthesis in cotton.

BACKGROUND: Geranylgeranyl pyrophosphate synthase (GGPS) is a structural enzyme of the terpene biosynthesis pathway that is involved in regulating plant photosynthesis, growth and development, but this gene family has not been systematically studied in cotton. RESULTS: In the current research, genome-wide identification was performed, and a total of 75 GGPS family members were found in four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii. The GGPS genes were divided into three subgroups by evolutionary analysis. Subcellular localization prediction showed that they were mainly located in chloroplasts and plastids. The closely related GGPS contains a similar gene structure and conserved motif, but some genes are quite different, resulting in functional differentiation. Chromosome location analysis, collinearity and selection pressure analysis showed that many fragment duplication events occurred in GGPS genes. Three-dimensional structure analysis and conservative sequence analysis showed that the members of the GGPS family contained a large number of α-helices and random crimps, and all contained two aspartic acid-rich domains, DDxxxxD and DDxxD (x is an arbitrary amino acid), suggesting its key role in function. Cis-regulatory element analysis showed that cotton GGPS may be involved in light response, abiotic stress and other processes. A GGPS gene was silenced successfully by virus-induced gene silencing (VIGS), and it was found that the chlorophyll content in cotton leaves decreased significantly, suggesting that the gene plays an important role in plant photosynthesis. CONCLUSIONS: In total, 75 genes were identified in four Gossypium species by a series of bioinformatics analysis. Gene silencing from GGPS members of G. hirsutum revealed that GGPS plays an important regulatory role in photosynthesis. This study provides a theoretical basis for the biological function of GGPS in cotton growth and development.

Transcriptome, proteome and functional characterization reveals salt stress tolerance mechanisms in upland cotton (Gossypium hirsutum L.).

Uncovering the underlying mechanism of salt tolerance is important to breed cotton varieties with improved salt tolerance. In this study, transcriptome and proteome sequencing were performed on upland cotton (Gossypium hirsutum L.) variety under salt stress, and integrated analysis was carried out to exploit salt-tolerance genes in cotton. Enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed on differentially expressed genes (DEGs) obtained from transcriptome and proteome sequencing. GO enrichment was carried out mainly in the cell membrane, organelle, cellular process, metabolic process, and stress response. The expression of 23,981 genes was changed in physiological and biochemical processes such as cell metabolism. The metabolic pathways obtained by KEGG enrichment included glycerolipid metabolism, sesquiterpene and triterpenoid biosynthesis, flavonoid production, and plant hormone signal transduction. Combined transcriptome and proteome analysis to screen and annotate DEGs yielded 24 candidate genes with significant differential expression. The quantitative real-time polymerase chain reaction (qRT-PCR) validation of the candidate genes showed that two genes (Gh_D11G0978 and Gh_D10G0907) responded significantly to the induction of NaCl, and these two genes were further selected as target genes for gene cloning and functional validation through virus-induced gene silencing (VIGS). The silenced plants exhibited early wilting with a greater degree of salt damage under salt treatment. Moreover, they showed higher levels of reactive oxygen species (ROS) than the control. Therefore, we can infer that these two genes have a pivotal role in the response to salt stress in upland cotton. The findings in this research will facilitate the breeding of salt tolerance cotton varieties that can be grown on saline alkaline lands.

Corrigendum: Genome-wide identification and expression analysis elucidates the potential role of PFK gene family in drought stress tolerance and sugar metabolism in cotton.

[This corrects the article DOI: 10.3389/fgene.2022.922024.].

Overexpression of cotton GhNAC072 gene enhances drought and salt stress tolerance in transgenic Arabidopsis.

BACKGROUND: Crops face several environmental stresses (biotic and abiotic), thus resulting in severe yield losses. Around the globe abiotic stresses are the main contributors of plant damages, primarily drought and salinity. Many genes and transcription factors are involved in abiotic and biotic stress responses. NAC TF (Transcription Factors) improves tolerance to stresses by controlling the physiological and enzyme activities of crops. RESULTS: In current research, GhNAC072 a highly upregulated TF in RNA-Seq was identified as a hub gene in the co-expression network analysis (WGCNA). This gene was transformed to Arabidopsis thaliana to confirm its potential role in drought and salt stress tolerance. Significant variations were observed in the morpho-physiological traits with high relative leaf water contents, chlorophyll contents, higher germination and longer root lengths of the overexpressed lines and low excised leaf loss and ion leakage as compared to the wildtype plants. Besides, overexpressed lines have higher amounts of antioxidants and low oxidant enzyme activities than the wildtype during the period of stress exposure. CONCLUSIONS: In summary, the above analysis showed that GhNAC072 might be the true candidate involved in boosting tolerance mechanisms under drought and salinity stress.

Genome-Wide Identification and Expression Analysis Elucidates the Potential Role of PFK Gene Family in Drought Stress Tolerance and Sugar Metabolism in Cotton.

Drought has been identified as a major threat for global crop production worldwide. Phosphofructokinase (PFK) is vital for sugar metabolism. During phosphorylation, plants have two enzymes: ATP-dependent phosphofructokinase (PFK) and pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP). Genome-wide identification led to the identification of 80 PFK genes, 26 genes in G. hirsutum and G. barbadense, and 14 genes in G. arboreum and G. raimondii. Phylogenetic, gene structure, and motif analyses showed that PFK genes were grouped into two main categories, namely, PFK and PFP, with 18 and 8 genes in the allotetraploid species and 10 PFK and 4 PFP genes in the diploid species, respectively. Using the RNA-seq expressions of 26 genes from GhPFK, a co-expression network analysis was performed to identify the hub genes. GhPFK04, GhPFK05, GhPFK09, GhPFK11, GhPFK13, GhPFK14, and GhPFK17 in leaves and GhPFK02, GhPFK09, GhPFK11, GhPFK15, GhPFK16, and GhPFK17 in root tissues were found as hub genes. RT-qPCR analysis validated the expressions of identified hub genes. Interestingly, GhPFK11 and GhPFK17 were identified as common hub genes, and these might be the true candidate genes involved in the drought stress tolerance. In the KEGG enrichment analysis, amino acids such as L-valine, L-histidine, L-glutamine, L-serine, L-homoserine, L-methionine, L-cysteine, and gluconic acid were significantly upregulated, whereas sugars, mainly fructose-1-phosphate, D-mannitol, D-sorbitol, dulcitol, and lactose, were significantly downregulated during drought stress. Genome-wide analysis paves the way for a deeper understanding of the PFK genes and establishes the groundwork for future research into PFK's role in enhancing drought stress tolerance and sugar metabolism in cotton.

Late embryogenesis abundant gene LEA3 (Gh_A08G0694) enhances drought and salt stress tolerance in cotton.

Plants have evolved a complex and organized response to abiotic stress that involves physiological and metabolic reprogramming, transcription control, epigenetic regulation, and expressions of thousand interacting genes for instance the late embryogenesis abundant (LEA) proteins are expressed in multiple environmental variables during the plant developmental period, and thus play critical role in enhancing drought and salt stress tolerance. A comprehensive molecular and functional characterization of the LEA3 gene was carried out in cotton under abiotic stress conditions in order to elucidate their functions. Seventy eight genes were identified in cotton, and were clustered into six clades moreover; the LEA genes were more upregulated in the tissues of the tetraploid cotton compared to the diploid type. A key gene, Gh_A08G0694 was the most upregulated, and was knocked in tetraploid cotton, the knocked out significantly increased the susceptibility of cotton plants to salinity and drought stresses, moreover, several ABA/stress-associated genes were down regulated. Similarly, overexpression of the key gene, significantly increased tolerance of the overexpressed plants to drought and salinity stress. The key gene is homologous to GhLEA3 protein, found to have strong interaction to key abiotic stress tolerance genes, voltage-dependent anion channel 1 (VDAC1) and glyceraldehyde-3-phosphate dehydrogenase A (gapA).

Multi-Omics-Based Identification and Functional Characterization of Gh_A06G1257 Proves Its Potential Role in Drought Stress Tolerance in Gossypium hirsutum.

Cotton is one of the most important fiber crops globally. Despite this, various abiotic stresses, including drought, cause yield losses. We used transcriptome profiles to investigate the co-expression patterns of gene networks associated with drought stress tolerance. We identified three gene modules containing 3,567 genes highly associated with drought stress tolerance. Within these modules, we identified 13 hub genes based on intramodular significance, for further validation. The yellow module has five hub genes (Gh_A07G0563, Gh_D05G0221, Gh_A05G3716, Gh_D12G1438, and Gh_D05G0697), the brown module contains three hub genes belonging to the aldehyde dehydrogenase (ALDH) gene family (Gh_A06G1257, Gh_A06G1256, and Gh_D06G1578), and the pink module has five hub genes (Gh_A02G1616, Gh_D12G2599, Gh_D07G2232, Gh_A02G0527, and Gh_D07G0629). Based on RT-qPCR results, the Gh_A06G1257 gene has the highest expression under drought stress in different plant tissues and it might be the true candidate gene linked to drought stress tolerance in cotton. Silencing of Gh_A06G1257 in cotton leaves conferred significant sensitivity in response to drought stress treatments. Overexpression of Gh_A06G1257 in Arabidopsis also confirms its role in drought stress tolerance. L-valine, Glutaric acid, L-proline, L-Glutamic acid, and L-Tryptophan were found to be the most significant metabolites playing roles in drought stress tolerance. These findings add significantly to existing knowledge of drought stress tolerance mechanisms in cotton.

Progressive Genomic Approaches to Explore Drought- and Salt-Induced Oxidative Stress Responses in Plants under Changing Climate.

Drought and salinity are the major environmental abiotic stresses that negatively impact crop development and yield. To improve yields under abiotic stress conditions, drought- and salinity-tolerant crops are key to support world crop production and mitigate the demand of the growing world population. Nevertheless, plant responses to abiotic stresses are highly complex and controlled by networks of genetic and ecological factors that are the main targets of crop breeding programs. Several genomics strategies are employed to improve crop productivity under abiotic stress conditions, but traditional techniques are not sufficient to prevent stress-related losses in productivity. Within the last decade, modern genomics studies have advanced our capabilities of improving crop genetics, especially those traits relevant to abiotic stress management. This review provided updated and comprehensive knowledge concerning all possible combinations of advanced genomics tools and the gene regulatory network of reactive oxygen species homeostasis for the appropriate planning of future breeding programs, which will assist sustainable crop production under salinity and drought conditions.

Functional Characterization of GhACX3 Gene Reveals Its Significant Role in Enhancing Drought and Salt Stress Tolerance in Cotton.

The acyl-coenzyme A oxidase 3 (ACX3) gene involved in the ß-oxidation pathway plays a critical role in plant growth and development as well as stress response. Earlier on, studies focused primarily on the role of ß-oxidation limited to fatty acid breakdown. However, ACX3 peroxisomal ß-oxidation pathways result in a downstream cascade of events that act as a transduction of biochemical and physiological responses to stress. A role that is yet to be studied extensively. In this study, we identified 20, 18, 22, 23, 20, 11, and 9 proteins in Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. arboretum, and G. raimondii genomes, respectively. The tetraploid cotton genome had protein ranging between 18 and 22, while diploids had between 9 and 11. After analyzing the gene family evolution or selection pressure, we found that this gene family undergoes purely segmental duplication both in diploids and tetraploids. W-Box (WRKY-binding site), ABRE, CAAT-Box, TATA-box, MYB, MBS, LTR, TGACG, and CGTCA-motif are abiotic stress cis-regulatory elements identified in this gene family. All these are the binding sites for abiotic stress transcription factors, indicating that this gene is essential. Genes found in G. hirsutum showed a clear response to drought and salinity stress, with higher expression under drought and salt stress, particularly in the leaf and root, according to expression analysis. We selected Gh_DO1GO186, one of the highly expressed genes, for functional characterization. We functionally characterized the GhACX3 gene through overexpression and virus-induced gene silencing (VIGS). Overexpression of this gene enhanced tolerance under stress, which was exhibited by the germination assay. The overexpressed seed growth rate was faster relative to control under drought and salt stress conditions. The survival rate was also higher in overexpressed plants relative to control plants under stress. In contrast, the silencing of the GhACX3 gene in cotton plants resulted in plants showing the stress susceptibility phenotype and reduced root length compared to control. Biochemical analysis also demonstrated that GhACX3-silenced plants experienced oxidative stress while the overexpressed plants did not. This study has revealed the importance of the ACX3 family during stress tolerance and can breed stress-resilient cultivar.

Identification and functional characterization of Gh_D01G0514 (GhNAC072) transcription factor in response to drought stress tolerance in cotton.

Cotton encounters long-term drought stress problems resulting in major yield losses. Transcription factors (TFs) plays an important role in response to biotic and abiotic stresses. The coexpression patterns of gene networks associated with drought stress tolerance were investigated using transcriptome profiles. Applying a weighted gene coexpression network analysis, we discovered a salmon module with 144 genes strongly linked to drought stress tolerance. Based on coexpression and RT-qPCR analysis GH_D01G0514 was selected as the candidate gene, as it was also identified as a hub gene in both roots and leaves with a consistent expression in response to drought stress in both tissues. For validation of GH_D01G0514, Virus Induced Gene Silencing was performed and VIGS plants showed significantly higher excised leaf water loss and ion leakage, while lower relative water and chlorophyll contents as compared to WT (Wild type) and positive control plants. Furthermore, the WT and positive control seedlings showed higher CAT and SOD activities, and lower activities of hydrogen peroxide and MDA enzymes as compared to the VIGS plants. Gh_D01G0514 (GhNAC072) was localized in the nucleus and cytoplasm. Y2H assay demonstrates that Gh_D01G0514 has a potential of auto activation. It was observed that the Gh_D01G0514 was highly upregulated in both tissues based on RNA Seq and RT-qPCR analysis. Thus, we inferred that, this candidate gene might be responsible for drought stress tolerance in cotton. This finding adds significantly to the existing knowledge of drought stress tolerance in cotton and deep molecular analysis are required to understand the molecular mechanisms underlying drought stress tolerance in cotton.

GhGLK1 a Key Candidate Gene From GARP Family Enhances Cold and Drought Stress Tolerance in Cotton.

Drought and low-temperature stresses are the most prominent abiotic stresses affecting cotton. Wild cotton being exposed to harsh environments has more potential to cope with both biotic and abiotic stresses. Exploiting wild cotton material to induce resistant germplasm would be of greater interest. The candidate gene was identified in the BC2F2 population among Gossypium tomentosum and Gossypium hirsutum as wild male donor parent noted for its drought tolerance and the recurrent parent and a high yielding but drought susceptible species by genotyping by sequencing (GBS) mapping. Golden2-like (GLK) gene, which belongs to the GARP family, is a kind of plant-specific transcription factor (TF) that was silenced by virus-induced gene silencing (VIGS). Silencing of GhGLK1 in cotton results in more damage to plants under drought and cold stress as compared with wild type (WT). The overexpression of GhGLK1 in Arabidopsis thaliana showed that the overexpressing plants showed more adaptability than the WT after drought and cold treatments. The results of trypan blue and 3,3'-diaminobenzidine (DAB) staining showed that after drought and cold treatment, the leaf damage in GhGLK1 overexpressed plants was less as compared with the WT, and the ion permeability was also lower. This study suggested that the GhGLK1 gene may be involved in the regulation of drought and cold stress response in cotton. Our current research findings add significantly to the existing knowledge of cold and drought stress tolerance in cotton.