GhGRF4/GhARF2-GhGASA24 module regulates fiber cell wall thickness by modulating cellulose biosynthesis in upland cotton (Gossypium hirsutum).

Fiber elongation rate is an essential characteristic of cotton fiber in the textile industry, yet it has been largely overlooked in genetic studies. Gibberellins (GAs) and auxin (IAA) are recognized for their role in directing numerous developmental processes in plants by influencing cell differentiation and elongation. However, the degree to which GA-IAA interaction governs cellular elongation in cotton fiber cells remains to be fully understood. In this study, we identified a causal gene, Gibberellic Acid-Stimulated in Arabidopsis 24 (GhGASA24), that appears to be responsible for fiber elongation rate via regulating fiber cell wall thickness. Subsequent experiments revealed that GhGASA24 influences cell wall formation by promoting the expression of GhCesA8 and GhCesA10. Our findings suggest that Auxin Response Factor 2 (GhARF2) regulates fiber elongation rate by directly binding to the AuxRE elements in GhGASA24 promoter. In addition, we identified Growth Regulation Factor 4 (GhGRF4) as a transcription factor that interacts with GhARF2 to form a heterodimer complex, which also transcriptionally activates GhGASA24. Intriguingly, GhGRF4 regulates GhARF2 expression by directly binding to its promoter, thereby acting as a cascade regulator to enhance the transcriptional levels of GhGASA24. We propose that the GhGRF4/GhARF2-GhGASA24-GhCesAs module may contribute to fiber cell wall thickness by modulating cellulose biosynthesis, and provide a theoretical basis for improvement of fiber quality.

GhMAX2 Contributes to Auxin-Mediated Fiber Elongation in Cotton (Gossypium hirsutum).

Strigolactones (SLs) represent a new group of phytohormones that play a pivotal role in the regulation of plant shoot branching and the development of adventitious roots. In cotton (Gossypium hirsutum, Gh), SLs play a crucial role in the regulation of fiber cell elongation and secondary cell wall thickness. However, the underlying molecular mechanisms of SL signaling involved in fiber cell development are largely unknown. In this study, we report two SL-signaling genes, GhMAX2-3 and GhMAX2-6, which positively regulate cotton fiber elongation. Further protein-protein interaction and degradation assays showed that the repressor of the auxin cascade GhIAA17 serves as a substrate for the F-box E3 ligase GhMAX2. The in vivo ubiquitination assay suggested that GhMAX2-3 and GhMAX2-6 ubiquitinate GhIAA17 and coordinately degrade GhIAA17 with GhTIR1. The findings of this investigation offer valuable insights into the roles of GhMAX2-mediated SL signaling in cotton and establish a solid foundation for future endeavors aimed at optimizing cotton plant cultivation.

Strigolactone-gibberellin crosstalk mediated by a distant silencer fine-tunes plant height in upland cotton.

Optimal plant height is crucial in modern agriculture, influencing lodging resistance and facilitating mechanized crop production. Upland cotton (Gossypium hirsutum) is the most important fiber crop globally; however, the genetic basis underlying plant height remains largely unexplored. In this study, we conducted a genome-wide association study to identify a major locus controlling plant height (PH1) in upland cotton. This locus encodes gibberellin 2-oxidase 1A (GhPH1) and features a 1133-bp structural variation (PAVPH1) located approximately 16 kb upstream. The presence or absence of PAVPH1 influences the expression of GhPH1, thereby affecting plant height. Further analysis revealed that a gibberellin-regulating transcription factor (GhGARF) recognizes and binds to a specific CATTTG motif in both the GhPH1 promoter and PAVPH1. This interaction downregulates GhPH1, indicating that PAVPH1 functions as a distant upstream silencer. Intriguingly, we found that DWARF53 (D53), a key repressor of the strigolactone (SL) signaling pathway, directly interacts with GhGARF to inhibit its binding to targets. Moreover, we identified a previously unrecognized gibberellin-SL crosstalk mechanism mediated by the GhD53-GhGARF-GhPH1/PAVPH1 module, which is crucial for regulating plant height in upland cotton. These findings shed light on the genetic basis and gene interaction network underlying plant height, providing valuable insights for the development of semi-dwarf cotton varieties through precise modulation of GhPH1 expression.

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.

Exploring the regulatory role of non-coding RNAs in fiber development and direct regulation of GhKCR2 in the fatty acid metabolic pathway in upland cotton.

Cotton fiber holds immense importance as the primary raw material for the textile industry. Consequently, comprehending the regulatory mechanisms governing fiber development is pivotal for enhancing fiber quality. Our study aimed to construct a regulatory network of competing endogenous RNAs (ceRNAs) and assess the impact of non-coding RNAs on gene expression throughout fiber development. Through whole transcriptome data analysis, we identified differentially expressed genes (DEGs) regulated by non-coding RNA (ncRNA) that were predominantly enriched in phenylpropanoid biosynthesis and the fatty acid elongation pathway. This analysis involved two contrasting phenotypic materials (J02-508 and ZRI015) at five stages of fiber development. Additionally, we conducted a detailed analysis of genes involved in fatty acid elongation, including KCS, KCR, HACD, ECR, and ACOT, to unveil the factors contributing to the variation in fatty acid elongation between J02-508 and ZRI015. Through the integration of histochemical GUS staining, dual luciferase assay experiments, and correlation analysis of expression levels during fiber development stages for lncRNA MSTRG.44818.23 (MST23) and GhKCR2, we elucidated that MST23 positively regulates GhKCR2 expression in the fatty acid elongation pathway. This identification provides valuable insights into the molecular mechanisms underlying fiber development, emphasizing the intricate interplay between non-coding RNAs and protein-coding genes.

Identification and characterization of candidate genes for primary root length in Asiatic cotton (Gossypium arboreum L.).

KEY MESSAGE: One major gene controlling primary root length (PRL) in Gossypium arboreum is identified and this research provides a theoretical basis for root development for cotton. Primary root elongation is an essential process in plant root system structure. Here, we investigated the primary root length (PRL) of 215 diploid cotton (G. arboreum) accessions at 5, 8, 10, 15 days after sowing. A Genome-wide association study was performed for the PRL, resulting in 49 significant SNPs associated with 32 putative candidate genes. The SNP with the strongest signal (Chr07_8047530) could clearly distinguish the PRLs between accessions with two haplotypes. GamurG is the only gene that showed higher relative expression in the long PRL genotypes than the short PRL genotypes, which indicated it was the most likely candidate gene for regulating PRL. Moreover, the GamurG-silenced cotton seedlings showed a shorter PRL, while the GamurG-overexpressed Arabidopsis exhibited a significantly longer PRL. Our findings provide insight into the regulation mechanism of cotton root growth and will facilitate future breeding programs to optimize the root system structure in cotton.

Genomic loci associated with leaf abscission contribute to machine picking and environmental adaptability in upland cotton (Gossypium hirsutum L.).

INTRODUCTION: Defoliation by applying defoliants before machine picking is an important agricultural practice that enhances harvesting efficiency and leads to increased raw cotton purity. However, the fundamental characteristics of leaf abscission and the underlying genetic basis in cotton are not clearly understood. OBJECTIVES: In this study, we aimed to (1) reveal the phenotypic variations in cotton leaf abscission, (2) discover the whole-genome differentiation sweeps and genetic loci related to defoliation, (3) identify and verify the functions of key candidate genes associated with defoliation, and (4) explore the relationship between haplotype frequency of loci and environmental adaptability. METHODS: Four defoliation-related traits of 383 re-sequenced Gossypium hirsutum accessions were investigated in four environments. The genome-wide association study (GWAS), linkage disequilibrium (LD) interval genotyping and functional identification were conducted. Finally, the haplotype variation related to environmental adaptability and defoliation traits was revealed. RESULTS: Our findings revealed the fundamental phenotypic variations of defoliation traits in cotton. We showed that defoliant significantly increased the defoliation rate without incurring yield and fiber quality penalties. The strong correlations between defoliation traits and growth period traits were observed. A genome-wide association study of defoliation traits identified 174 significant SNPs. Two loci (RDR7 on A02 and RDR13 on A13) that significantly associated with the relative defoliation rate were described, and key candidate genes GhLRR and GhCYCD3;1, encoding a leucine-rich repeat (LRR) family protein and D3-type cell cyclin 1 protein respectively, were functional verified by expression pattern analysis and gene silencing. We found that combining of two favorable haplotypes (HapRDR7 and HapRDR13) improved sensitivity to defoliant. The favorable haplotype frequency generally increased in high latitudes in China, enabling adaptation to the local environment. CONCLUSION: Our findings lay an important foundation for the potentially broad application of leveraging key genetic loci in breeding machine-pickable cotton.

Physiological and transcriptional analyses reveal formation of memory under recurring drought stresses in seedlings of cotton (Gossypium hirsutum).

Plants are frequently subjected to a range of environmental stresses, including drought, salinity, cold, pathogens, and herbivore attacks. To survive in such conditions, plants have evolved a novel adaptive mechanism known as 'stress memory'. The formation of stress memories necessitates coordinated responses at the cellular, genetic/genomic, and epigenetic levels, involving altered physiological responses, gene activation, hyper-induction and chromatin modification. Cotton (Gossypium spp.) is an important economic crop with numerous applications and high economic value. In this study, we establish G. hirsutum drought memory following cycles of mild drought and re-watering treatments and analyzed memory gene expression patterns. Our findings reveal the physiological, biochemical, and molecular mechanisms underlying drought stress memory formation in G. hirsutum. Specifically, H3K4me3, a histone modification, plays a crucial role in regulating [+ /+ ] transcriptional memory. Moreover, we investigated the intergenerational inheritance of drought stress memory in G. hirsutum. Collectively, our data provides theoretical guidance for cotton breeding.

Transcriptomic analysis reveals the beneficial effects of salt priming on enhancing defense responses in upland cotton under successive salt stress.

Priming-mediated stress tolerance in plants stimulates defense mechanisms and enables plants to cope with future stresses. Seed priming has been proven effective for tolerance against abiotic stresses; however, underlying genetic mechanisms are still unknown. We aimed to assess upland cotton genotypes and their transcriptional behaviors under salt priming and successive induced salt stress. We pre-selected 16 genotypes based on previous studies and performed morpho-physiological characterization, from which we selected three genotypes, representing different tolerance levels, for transcriptomic analysis. We subjected these genotypes to four different treatments: salt priming (P0), salt priming with salinity dose at 3-true-leaf stage (PD), salinity dose at 3-true-leaf stage without salt priming (0D), and control (CK). Although the three genotypes displayed distinct expression patterns, we identified common differentially expressed genes (DEGs) under PD enriched in pathways related to transferase activity, terpene synthase activity, lipid biosynthesis, and regulation of acquired resistance, indicating the beneficial role of salt priming in enhancing salt stress resistance. Moreover, the number of unique DEGs associated with G. hirsutum purpurascens was significantly higher compared to other genotypes. Coexpression network analysis identified 16 hub genes involved in cell wall biogenesis, glucan metabolic processes, and ribosomal RNA binding. Functional characterization of XTH6 (XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE) using virus-induced gene silencing revealed that suppressing its expression improves plant growth under salt stress. Overall, findings provide insights into the regulation of candidate genes in response to salt stress and the beneficial effects of salt priming on enhancing defense responses in upland cotton.

Genome-wide analysis of the serine carboxypeptidase-like protein family reveals Ga09G1039 is involved in fiber elongation in cotton.

The Gossypium is a model genus for understanding polyploidy and the evolutionary pattern of inheritance. This study aimed to investigate the characteristics of SCPLs in different cotton species and their role in fiber development. A total of 891 genes from one typical monocot and ten dicot species were naturally divided into three classes based on phylogenetic analysis. The SCPL gene family in cotton has undergone intense purifying selection with some functional variation. Segmental duplication and whole genome duplication were shown to be the two main reasons for the increase in the number of genes during cotton evolution. The identification of Gh_SCPL genes exhibiting differential expression in particular tissues or response to environmental stimuli provides a new measure for the in-depth characterization of selected genes of importance. Ga09G1039 was involved in the developmental process of fibers and ovules, and it is significantly different from proteins from other cotton species in terms of phylogenetic, gene structure, conserved protein motifs and tertiary structure. Overexpression of Ga09G1039 significantly increased the length of stem trichomes. Ga09G1039 may be a serine carboxypeptidase protein with hydrolase activity, according to functional region, prokaryotic expression, and western blotting analysis. The results provide a comprehensive overview of the genetic basis of SCPLs in Gossypium and further our knowledge in understanding the key aspects of SCPLs in cotton with their potential role in fiber development and stress resistance.

Silencing of GhORP_A02 enhances drought tolerance in Gossypium hirsutum.

BACKGROUND: ORP (Oxysterol-binding protein-related proteins) genes play a role in lipid metabolism, vesicular transferring and signaling, and non-vesicular sterol transport. However, no systematic identification and analysis of ORP genes have been reported in cotton. RESULT: In this study, we identified 14, 14, 7, and 7 ORP genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis showed that all ORP genes could be classified into four groups. Gene structure and conserved motif analysis suggest that the function of this gene family was conserved. The Ka/Ks analysis showed that this gene family was exposed to purifying selection during evolution. Transcriptome data showed that four ORP genes, especially GhORP_A02, were induced by abiotic stress treatment. The cis-acting elements in the ORP promoters were responsive to phytohormones and various abiotic stresses. The silenced plants of GhORP_A02 were more sensitive to drought stress when compared to control. CONCLUSION: The major finding of this study shed light on the potential role of ORP genes in abiotic stress and provided a fundamental resource for further analysis in cotton.

Exploring the nano-wonders: unveiling the role of Nanoparticles in enhancing salinity and drought tolerance in plants.

Plants experience diverse abiotic stresses, encompassing low or high temperature, drought, water logging and salinity. The challenge of maintaining worldwide crop cultivation and food sustenance becomes particularly serious due to drought and salinity stress. Sustainable agriculture has significant promise with the use of nano-biotechnology. Nanoparticles (NPs) have evolved into remarkable assets to improve agricultural productivity under the robust climate alteration and increasing drought and salinity stress severity. Drought and salinity stress adversely impact plant development, and physiological and metabolic pathways, leading to disturbances in cell membranes, antioxidant activities, photosynthetic system, and nutrient uptake. NPs protect the membrane and photosynthetic apparatus, enhance photosynthetic efficiency, optimize hormone and phenolic levels, boost nutrient intake and antioxidant activities, and regulate gene expression, thereby strengthening plant's resilience to drought and salinity stress. In this paper, we explored the classification of NPs and their biological effects, nanoparticle absorption, plant toxicity, the relationship between NPs and genetic engineering, their molecular pathways, impact of NPs in salinity and drought stress tolerance because the effects of NPs vary with size, shape, structure, and concentration. We emphasized several areas of research that need to be addressed in future investigations. This comprehensive review will be a valuable resource for upcoming researchers who wish to embrace nanotechnology as an environmentally friendly approach for enhancing drought and salinity tolerance.

Characterization of WOX genes revealed drought tolerance, callus induction, and tissue regeneration in Gossypium hirsutum.

Cotton is an important natural fiber crop; its seeds are the main oil source. Abiotic stresses cause a significant decline in its production. The WUSCHEL-related Homeobox (WOX) genes have been involved in plant growth, development, and stress responses. However, the functions of WOX genes are less known in cotton. This study identified 39, 40, 21, and 20 WOX genes in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii, respectively. All the WOX genes in four cotton species could be classified into three clades, which is consistent with previous research. The gene structure and conserved domain of all WOX genes were analyzed. The expressions of WOX genes in germinating hypocotyls and callus were characterized, and it was found that most genes were up-regulated. One candidate gene Gh_ A01G127500 was selected to perform the virus-induced gene silencing (VIGS) experiment, and it was found that the growth of the silenced plant (pCLCrVA: GhWOX4_A01) was significantly inhibited compared with the wild type. In the silenced plant, there is an increase in antioxidant activities and a decrease in oxidant activities compared with the control plant. In physiological analysis, the relative electrolyte leakage level and the excised leaf water loss of the infected plant were increased. Still, both the relative leaf water content and the chlorophyll content were decreased. This study proved that WOX genes play important roles in drought stress and callus induction, but more work must be performed to address the molecular functions of WOX genes.

Identification and Characterization of the Growth-Regulating Factors-Interacting Factors in Cotton.

Growth-regulating factors-interacting factors (GIFs) are a type of transcription co-activators in plants, playing crucial roles in plants' growth, development, and stress adaptation. Here, a total of 35 GIF genes were identified and clustered into two groups by phylogenetic analysis in four cotton genus. The gene structure and conserved domain analysis proved the conservative characteristics of GIF genes in cotton. The function of GIF genes was evaluated in two cotton accessions, Ji A-1-7 (33xi) and King, which have larger and smaller lateral root numbers, respectively. The results showed that the expression of GhGIF4 in Ji A-1-7 (33xi) was higher than that in King. The enzyme activity and microstructure assay showed a higher POD activity, lower MDA content, and more giant cells of the lateral root emergence part phenotype in Ji A-1-7 (33xi) than in King. A mild waterlogging assay showed the GIF genes were down-regulated in the waterlogged seedling. Further confirmation of the suppression of GhGIF4 in cotton plants further confirmed that GhGIF4 could reduce the lateral root numbers in cotton. This study could provide a basis for future studies of the role of GIF genes in upland cotton.

Integrating Genome-wide association and whole transcriptome analysis to reveal genetic control of leaf traits in Gossypium arboreum L.

Leaves are important organs for crop photosynthesis and transpiration, and their morphological characteristics can directly reflect the growth state of plants. Accurate measurement of leaf traits and mining molecular markers are of great significance to the study of cotton growth. Here, we performed a Genome-wide association study on 7 leaf traits in 213 Asian cotton accessions. 32 significant SNPs and 44 genes were identified. A field experiment showed significant difference in leaf hair and leaf area between DPL971 and its natural mutant DPL972. We also compared the leaf transcriptome difference between DPL971 and DPL972, and found a batch of differentially expressed genes and non-coding RNAs (including lncRNAs, microRNAs, and circRNAs). After integrating the GWAS and transcriptome results, we finally selected two coding genes (Ga03G2383 and Ga05G3412) and two microRNAs (hbr-miR156, unconservative_Chr03_contig343_2364) as the candidate for leaf traits. Those findings will provide important genomic resources for cotton leaf improvement breeding.

Deltapine 15 contributes to the genomic architecture of modern upland cotton cultivars.

Foundation parents play a critical role in the genetic constituents of the derived genotypes. Deltapine-15 (DLP-15), introduced to China in 1950, is one of the most commonly used parents for early breeding programs in China. However, the formation and inheritance patterns of genomic constituents have not been studied. Therefore, this study aimed at understanding and exploring the genomic architecture of 146 DLP-15 derived cultivars with a common foundation parent DLP-15. Population structure based on sequencing data clustered genotypes into two groups (G1 and G2) supported by principal component analysis. Further exploration led to the identification of Chr-A08 with significantly differentiated regions between two groups. Moreover, we identified genome-wide identity by descent (IBD) segments (840 segments) to understand the genomic inheritance pattern in DLP-15 derived cultivars, spanning the 20-95 Mb region on Chr-A08. Interestingly, Chr-A08 depicted a unique inheritance pattern from DLP-15 to its derived cultivars. IBD-segment-based haplotype analysis suggested significant differences among the two groups. Phenotypic trait association with DLP-derived haplotypes concerning Chr-A08 suggested a significant increase in yield and fiber quality. Furthermore, distinguished IBD segments overlapped with previously reported QTLs concerning fiber yield and quality. Our results systematically identified genomic signatures transmitted from the foundation parent DLP-15 to its derived cultivars and provided a basis for further exploiting excellent haplotypes associated with DLP-15.

Genome-wide association study for seedling biomass-related traits in Gossypium arboreum L.

BACKGROUND: Seedling stage plant biomass is usually used as an auxiliary trait to study plant growth and development or stress adversities. However, few molecular markers and candidate genes of seedling biomass-related traits were found in cotton. RESULT: Here, we collected 215 Gossypium arboreum accessions, and investigated 11 seedling biomass-related traits including the fresh weight, dry weight, water content, and root shoot ratio. A genome-wide association study (GWAS) utilizing 142,5003 high-quality SNPs identified 83 significant associations and 69 putative candidate genes. Furthermore, the transcriptome profile of the candidate genes emphasized higher expression of Ga03G1298, Ga09G2054, Ga10G1342, Ga11G0096, and Ga11G2490 in four representative cotton accessions. The relative expression levels of those five genes were further verified by qRT-PCR. CONCLUSIONS: The significant SNPs, candidate genes identified in this study are expected to lay a foundation for studying the molecular mechanism for early biomass development and related traits in Asian cotton.

Genetic Factors Underlying Single Fiber Quality in A-Genome Donor Asian Cotton (Gossypium arboreum).

The study of A-genome Asian cotton as a potential fiber donor in Gossypium species may offer an enhanced understanding of complex genetics and novel players related to fiber quality traits. Assessment of individual fibers providing classified fiber quality information to the textile industry is Advanced Fiber Information System (AFIS) in the recent technological era. Keeping the scenario, a diverse collection of 215 Asiatic cotton accessions were evaluated across three agro-ecological zones of China. Genome-Wide Association Studies (GWAS) was performed to detect association signals related to 17 AFIS fiber quality traits grouped into four categories viz: NEPs, fiber length, maturity, and fineness. Significant correlations were found within as well as among different categories of various traits related to fiber quality. Fiber fineness has shown a strong correlation to all other categories, whereas these categories are shown interrelationships via fiber-fineness. A total of 7,429 SNPs were found in association with 17 investigated traits, of which 177 were selected as lead SNPs. In the vicinity of these lead SNPs, 56 differentially expressed genes in various tissues/development stages were identified as candidate genes. This compendium connecting trait-SNP-genes may allow further prioritization of genes in GWAS loci to enable mechanistic studies. These identified quantitative trait nucleotides (QTNs) may prove helpful in fiber quality improvement in Asian cotton through marker-assisted breeding as well as in reviving eroded genetic factors of G. hirsutum via introgression breeding.