GhUBC10-2 mediates GhGSTU17 degradation to regulate salt tolerance in cotton (Gossypium hirsutum).

Ubiquitin-conjugating enzyme (UBC) is a crucial component of the ubiquitin-proteasome system, which contributes to plant growth and development. While some UBCs have been identified as potential regulators of abiotic stress responses, the underlying mechanisms of this regulation remain poorly understood. Here, we report a cotton (Gossypium hirsutum) UBC gene, GhUBC10-2, which negatively regulates the salt stress response. We found that the gain of function of GhUBC10-2 in both Arabidopsis (Arabidopsis thaliana) and cotton leads to reduced salinity tolerance. Additionally, GhUBC10-2 interacts with glutathione S-transferase (GST) U17 (GhGSTU17), forming a heterodimeric complex that promotes GhGSTU17 degradation. Intriguingly, GhUBC10-2 can be self-polyubiquitinated, suggesting that it possesses E3-independent activity. Our findings provide new insights into the PTM of plant GST-mediated salt response pathways. Furthermore, we found that the WRKY transcription factor GhWRKY13 binds to the GhUBC10-2 promoter and suppresses its expression under salt conditions. Collectively, our study unveils a regulatory module encompassing GhWRKY13-GhUBC10-2-GhGSTU17, which orchestrates the modulation of reactive oxygen species homeostasis to enhance salt tolerance.

Identification and molecular evolution of the GLX genes in 21 plant species: a focus on the Gossypium hirsutum.

BACKGROUND: The glyoxalase system includes glyoxalase I (GLXI), glyoxalase II (GLXII) and glyoxalase III (GLXIII), which are responsible for methylglyoxal (MG) detoxification and involved in abiotic stress responses such as drought, salinity and heavy metal. RESULTS: In this study, a total of 620 GLX family genes were identified from 21 different plant species. The results of evolutionary analysis showed that GLX genes exist in all species from lower plants to higher plants, inferring that GLX genes might be important for plants, and GLXI and GLXII account for the majority. In addition, motif showed an expanding trend in the process of evolution. The analysis of cis-acting elements in 21 different plant species showed that the promoter region of the GLX genes were rich in phytohormones and biotic and abiotic stress-related elements, indicating that GLX genes can participate in a variety of life processes. In cotton, GLXs could be divided into two groups and most GLXIs distributed in group I, GLXIIs and GLXIIIs mainly belonged to group II, indicating that there are more similarities between GLXII and GLXIII in cotton evolution. The transcriptome data analysis and quantitative real-time PCR analysis (qRT-PCR) show that some members of GLX family would respond to high temperature treatment in G.hirsutum. The protein interaction network of GLXs in G.hirsutum implied that most members can participate in various life processes through protein interactions. CONCLUSIONS: The results elucidated the evolutionary history of GLX family genes in plants and lay the foundation for their functions analysis in cotton.

Identification of MYB gene family and functional analysis of GhMYB4 in cotton (Gossypium spp.).

Myeloblastosis (MYB) transcription factors (TFs) form a large gene family involved in a variety of biological processes in plants. Little is known about their roles in the development of cotton pigment glands. In this study, 646 MYB members were identified in Gossypium hirsutum genome and phylogenetic classification was analyzed. Evolution analysis revealed assymetric evolution of GhMYBs during polyploidization and sequence divergence of MYBs in G. hirustum was preferentially happend in D sub-genome. WGCNA (weighted gene co-expression network analysis) showed that four modules had potential relationship with gland development or gossypol biosynthesis in cotton. Eight differentially expressed GhMYB genes were identified by screening transcriptome data of three pairs of glanded and glandless cotton lines. Of these, four were selected as candidate genes for cotton pigment gland formation or gossypol biosynthesis by qRT-PCR assay. Silencing of GH_A11G1361 (GhMYB4) downregulated expression of multiple genes in gossypol biosynthesis pathway, indicating it could be involved in gossypol biosynthesis. The potential protein interaction network suggests that several MYBs may have indirect interaction with GhMYC2-like, a key regulator of pigment gland formation. Our study was the systematic analysis of MYB genes in cotton pigment gland development, providing candidate genes for further study on the roles of cotton MYB genes in pigment gland formation, gossypol biosynthesis and future crop plant improvement.

Interpretation of convolutional neural networks reveals crucial sequence features involving in transcription during fiber development.

BACKGROUND: Upland cotton provides the most natural fiber in the world. During fiber development, the quality and yield of fiber were influenced by gene transcription. Revealing sequence features related to transcription has a profound impact on cotton molecular breeding. We applied convolutional neural networks to predict gene expression status based on the sequences of gene transcription start regions. After that, a gradient-based interpretation and an N-adjusted kernel transformation were implemented to extract sequence features contributing to transcription. RESULTS: Our models had approximate 80% accuracies, and the area under the receiver operating characteristic curve reached over 0.85. Gradient-based interpretation revealed 5' untranslated region contributed to gene transcription. Furthermore, 6 DOF binding motifs and 4 transcription activator binding motifs were obtained by N-adjusted kernel-motif transformation from models in three developmental stages. Apart from 10 general motifs, 3 DOF5.1 genes were also detected. In silico analysis about these motifs' binding proteins implied their potential functions in fiber formation. Besides, we also found some novel motifs in plants as important sequence features for transcription. CONCLUSIONS: In conclusion, the N-adjusted kernel transformation method could interpret convolutional neural networks and reveal important sequence features related to transcription during fiber development. Potential functions of motifs interpreted from convolutional neural networks could be validated by further wet-lab experiments and applied in cotton molecular breeding.

Molecular cloning and characterization of GhERF105, a gene contributing to the regulation of gland formation in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Gossypium hirsutum L. (cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear. RESULTS: In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing (VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. CONCLUSIONS: These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate 'glandless-seed' and 'glanded-plant' cotton cultivar.

Genome-wide identification and expression analysis of GL2-interacting-repressor (GIR) genes during cotton fiber and fuzz development.

MAIN CONCLUSION: GL2-interacting-repressor (GIR) family members may contribute to fiber/fuzz formation via a newly discovered unique pathway in Gossypium arboreum. There are similarities between cotton fiber development and the formation of trichomes and root hairs. The GL2-interacting-repressors (GIRs) are crucial regulators of root hair and trichome formation. The GaFzl gene, annotated as GaGIR1, is negatively associated with trichome development and fuzz initiation. However, there is relatively little available information regarding the other GIR genes in cotton, especially regarding their effects on cotton fiber development. In this study, 21 GIR family genes were identified in the diploid cotton species Gossypium arboreum; these genes were divided into three groups. The GIR genes were characterized in terms of their phylogenetic relationships, structures, chromosomal distribution and evolutionary dynamics. These GIR genes were revealed to be unequally distributed on 12 chromosomes in the diploid cotton genome, with no GIR gene detected on Ga06. The cis-acting elements in the promoter regions were predicted to be responsive to light, phytohormones, defense activities and stress. The transcriptomic data and qRT-PCR results revealed that most GIR genes were not differentially expressed between the wild-type control and the fuzzless mutant line. Moreover, 14 of 21 family genes were expressed at high levels, indicating these genes may play important roles during fiber development and fuzz formation. Furthermore, Ga01G0231 was predominantly expressed in root samples, suggestive of a role in root hair formation rather than in fuzz initiation and development. The results of this study have enhanced our understanding of the GIR genes and their potential utility for improving cotton fiber through breeding.

Gene Expression Correlation Analysis Reveals MYC-NAC Regulatory Network in Cotton Pigment Gland Development.

Plant NAC (NAM, ATAF1/2, and CUC2) family is involved in various development processes including Programmed Cell Death (PCD) associated development. However, the relationship between NAC family and PCD-associated cotton pigment gland development is largely unknown. In this study, we identified 150, 153 and 299 NAC genes in newly updated genome sequences of G. arboreum, G. raimondii and G. hirsutum, respectively. All NAC genes were divided into 8 groups by the phylogenetic analysis and most of them were conserved during cotton evolution. Using the vital regulator of gland formation GhMYC2-like as bait, expression correlation analysis screened out 6 NAC genes which were low-expressed in glandless cotton and high-expressed in glanded cotton. These 6 NAC genes acted downstream of GhMYC2-like and were induced by MeJA. Silencing CGF1(Cotton Gland Formation1), another MYC-coding gene, caused almost glandless phenotype and down-regulated expression of GhMYC2-like and the 6 NAC genes, indicating a MYC-NAC regulatory network in gland development. In addition, predicted regulatory mechanism showed that the 6 NAC genes were possibly regulated by light, various phytohormones and transcription factors as well as miRNAs. The interaction network and DNA binding sites of the 6 NAC transcription factors were also predicted. These results laid the foundation for further study of gland-related genes and gland development regulatory network.

Improved reconstruction and comparative analysis of chromosome 12 to rectify Mis-assemblies in Gossypium arboreum.

BACKGROUND: Genome sequencing technologies have been improved at an exponential pace but precise chromosome-scale genome assembly still remains a great challenge. The draft genome of cultivated G. arboreum was sequenced and assembled with shotgun sequencing approach, however, it contains several misassemblies. To address this issue, we generated an improved reassembly of G. arboreum chromosome 12 using genetic mapping and reference-assisted approaches and evaluated this reconstruction by comparing with homologous chromosomes of G. raimondii and G. hirsutum. RESULTS: In this study, we generated a high quality assembly of the 94.64 Mb length of G. arboreum chromosome 12 (A_A12) which comprised of 144 scaffolds and contained 3361 protein coding genes. Evaluation of results using syntenic and collinear analysis of reconstructed G. arboreum chromosome A_A12 with its homologous chromosomes of G. raimondii (D_D08) and G. hirsutum (AD_A12 and AD_D12) confirmed the significant improved quality of current reassembly as compared to previous one. We found major misassemblies in previously assembled chromosome 12 (A_Ca9) of G. arboreum particularly in anchoring and orienting of scaffolds into a pseudo-chromosome. Further, homologous chromosomes 12 of G. raimondii (D_D08) and G. arboreum (A_A12) contained almost equal number of transcription factor (TF) related genes, and showed good collinear relationship with each other. As well, a higher rate of gene loss was found in corresponding homologous chromosomes of tetraploid (AD_A12 and AD_D12) than diploid (A_A12 and D_D08) cotton, signifying that gene loss is likely a continuing process in chromosomal evolution of tetraploid cotton. CONCLUSION: This study offers a more accurate strategy to correct misassemblies in sequenced draft genomes of cotton which will provide further insights towards its genome organization.

Genome-wide identification of the expansin gene family reveals that expansin genes are involved in fibre cell growth in cotton.

BACKGROUND: Expansins (EXPs), a group of proteins that loosen plant cell walls and cellulosic materials, are involved in regulating cell growth and diverse developmental processes in plants. However, the biological functions of this gene family in cotton are still unknown. RESULTS: In this paper, we identified a total of 93 expansin genes in Gossypium hirsutum. These genes were classified into four subfamilies, including 67 GhEXPAs, 8 GhEXPBs, 6 GhEXLAs, and 12 GhEXLBs, and divided into 15 subgroups. The 93 expansin genes are distributed over 24 chromosomes, excluding Ghir_A02 and Ghir_D06. All GhEXP genes contain multiple exons, and each GhEXP protein has multiple conserved motifs. Transcript profiling and qPCR analysis revealed that the expansin genes have distinct expression patterns among different stages of cotton fibre development. Among them, 3 genes (GhEXPA4o, GhEXPA1A, and GhEXPA8h) were highly expressed in the initiation stage, 9 genes (GhEXPA4a, GhEXPA13a, GhEXPA4f, GhEXPA4q, GhEXPA8f, GhEXPA2, GhEXPA8g, GhEXPA8a, and GhEXPA4n) had high expression during the fast elongation stage, and GhEXLA1c and GhEXLA1f were preferentially expressed in the transition stage of fibre development. CONCLUSIONS: Our results provide a solid basis for further elucidation of the biological functions of expansin genes in relation to cotton fibre development and valuable genetic resources for future crop improvement.

Fine mapping and identification of the fuzzless gene GaFzl in DPL972 (Gossypium arboreum).

KEY MESSAGE: The fuzzless gene GaFzl was fine mapped to a 70-kb region containing a GIR1 gene, Cotton_A_11941, responsible for the fuzzless trait in Gossypium arboreum DPL972. Cotton fiber is the most important natural textile resource. The fuzzless mutant DPL972 (Gossypium arboreum) provides a useful germplasm resource to explore the molecular mechanism underlying fiber and fuzz initiation and development. In our previous research, the fuzzless gene in DPL972 was identified as a single dominant gene and named GaFzl. In the present study, we fine mapped this gene using F2 and BC1 populations. By combining traditional map-based cloning and next-generation sequencing, we mapped GaFzl to a 70-kb region containing seven annotated genes. RNA-Sequencing and re-sequencing analysis narrowed these candidates to two differentially expressed genes, Cotton_A_11941 and Cotton_A_11942. Sequence alignment uncovered no variation in coding or promoter regions of Cotton_A_11942 between DPL971 and DPL972, whereas two single-base mutations in the promoter region and a TTG insertion in the coding region were detected in Cotton_A_11941 in DPL972. Cotton_A_11941 encoding a homologous gene of GIR1 (GLABRA2-interacting repressor) in Arabidopsis thaliana is thus the candidate gene most likely responsible for the fuzzless trait in DPL972. Our findings should lead to a better understanding of cotton fuzz formation, thereby accelerating marker-assisted selection during cotton breeding.

Correction to: Fine mapping and identification of the fuzzless gene GaFzl in DPL972 (Gossypium arboreum).

Unfortunately, Figs. 5 and 6 were interchanged in the results section. Figures should swap positions, whereas the legends should stay in the given order.

Recent insights into cotton functional genomics: progress and future perspectives.

Functional genomics has transformed from futuristic concept to well-established scientific discipline during the last decade. Cotton functional genomics promise to enhance the understanding of fundamental plant biology to systematically exploit genetic resources for the improvement of cotton fibre quality and yield, as well as utilization of genetic information for germplasm improvement. However, determining the cotton gene functions is a much more challenging task, which has not progressed at a rapid pace. This article presents a comprehensive overview of the recent tools and resources available with the major advances in cotton functional genomics to develop elite cotton genotypes. This effort ultimately helps to filter a subset of genes that can be used to assemble a final list of candidate genes that could be employed in future novel cotton breeding programme. We argue that next stage of cotton functional genomics requires the draft genomes refinement, re-sequencing broad diversity panels with the development of high-throughput functional genomics tools and integrating multidisciplinary approaches in upcoming cotton improvement programmes.

A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

Cotton transposon-related variome reveals roles of transposon-related variations in modern cotton cultivation.

INTRODUCTION: Transposon plays a vital role in cotton genome evolution, contributing to the expansion and divergence of genomes within the Gossypium genus. However, knowledge of transposon activity in modern cotton cultivation is limited. OBJECTIVES: In this study, we aimed to construct transposon-related variome within Gossypium genus and reveal role of transposon-related variations during cotton cultivation. In addition, we try to identify valuable transposon-related variations for cotton breeding. METHODS: We utilized graphical genome construction to build up the graphical transposon-related variome. Based on the graphical variome, we integrated t-test, eQTL analysis and Mendelian Randomization (MR) to identify valuable transposon activities and elite genes. In addition, a convolutional neural network (CNN) model was constructed to evaluate epigenomic effects of transposon-related variations. RESULTS: We identified 35,980 transposon activities among 10 cotton genomes, and the diversity of genomic and epigenomic features was observed among 21 transposon categories. The graphical cotton transposon-related variome was constructed, and 9,614 transposon-related variations with plasticity in the modern cotton cohort were used for eQTL, phenotypic t-test and Mendelian Randomization. 128 genes were identified as gene resources improving fiber length and strength simultaneously. 4 genes were selected from 128 genes to construct the elite gene panel whose utility has been validated in a natural cotton cohort and 2 accessions with phenotypic divergence. Based on the eQTL analysis results, we identified transposon-related variations involved in cotton's environmental adaption and human domestication, providing evidence of their role in cotton's adaption-domestication cooperation. CONCLUSIONS: The cotton transposon-related variome revealed the role of transposon-related variations in modern cotton cultivation, providing genomic resources for cotton molecular breeding.

Fingerprint Finder: Identifying Genomic Fingerprint Sites in Cotton Cohorts for Genetic Analysis and Breeding Advancement.

Genomic data in Gossypium provide numerous data resources for the cotton genomics community. However, to fill the gap between genomic analysis and breeding field work, detecting the featured genomic items of a subset cohort is essential for geneticists. We developed FPFinder v1.0 software to identify a subset of the cohort's fingerprint genomic sites. The FPFinder was developed based on the term frequency-inverse document frequency algorithm. With the short-read sequencing of an elite cotton pedigree, we identified 453 pedigree fingerprint genomic sites and found that these pedigree-featured sites had a role in cotton development. In addition, we applied FPFinder to evaluate the geographical bias of fiber-length-related genomic sites from a modern cotton cohort consisting of 410 accessions. Enriching elite sites in cultivars from the Yangtze River region resulted in the longer fiber length of Yangze River-sourced accessions. Apart from characterizing functional sites, we also identified 12,536 region-specific genomic sites. Combining the transcriptome data of multiple tissues and samples under various abiotic stresses, we found that several region-specific sites contributed to environmental adaptation. In this research, FPFinder revealed the role of the cotton pedigree fingerprint and region-specific sites in cotton development and environmental adaptation, respectively. The FPFinder can be applied broadly in other crops and contribute to genetic breeding in the future.

GhHDZ76, a cotton HD-Zip transcription factor, involved in regulating the initiation and early elongation of cotton fiber development in G. hirsutum.

In this study, the whole HD-Zip family members of G. hirsutum were identified, and GhHDZ76 was classified into the HD-Zip IV subgroup. GhHDZ76 was predominantly expressed in the 0-5 DPA of fiber development stage and localized in the nucleus. Overexpression of GhHDZ76 significantly increased the length and density of trichomes in Arabidopsis thaliana. The fiber length of GhHDZ76 knockout lines by CRISPR/Cas9 was significantly shorter than WT at the early elongation and mature stage, indicating that GhHDZ76 positively regulate the fiber elongation. Scanning electron microscopy showed that the number of ovule surface protrusion of 0 DPA of GhHDZ76 knockout lines was significantly lower than WT, suggesting that GhHDZ76 can also promote the initiation of fiber development. The transcript level of GhWRKY16, GhRDL1, GhEXPA1 and GhMYB25 genes related to fiber initiation and elongation in GhHDZ76 knockout lines were significantly decreased. Yeast two-hybrid and Luciferase complementation imaging (LCI) assays showed that GhHDZ76 can interact with GhWRKY16 directly. As a transcription factor, GhHDZ76 has transcriptional activation activity, which could bind to L1-box elements of the promoters of GhRDL1 and GhEXPA1. Double luciferase reporter assay showed that the GhWRKY16 could enhance the transcriptional activity of GhHDZ76 to pGhRDL1, but it did not promote the transcriptional activity of GhHDZ76 to pGhEXPA1. GhHDZ76 protein may also promote the transcriptional activity of GhWRKY16 to the downstream target gene GhMYB25. Our results provided a new gene resource for fiber development and a theoretical basis for the genetic improvement of cotton fiber quality.

Genome-wide analysis elucidates the roles of GhHMA genes in different abiotic stresses and fiber development in upland cotton.

The heavy metal-binding domain is involved in heavy metal transporting and plays a significant role in plant detoxification. However, the functions of HMAs are less well known in cotton. In this study, a total of 143 GhHMAs (heavy metal-binding domain) were detected by genome-wide identification in G. hirsutum L. All the GhHMAs were classified into four groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GhHMA genes was highly conserved. 212 paralogous GhHMA gene pairs were identified, and the segmental duplications were the main role to the expansion of GhHMAs. The Ka/Ks values suggested that the GhHMA gene family has undergone purifying selection during the long-term evolutionary process. GhHMA3 and GhHMA75 were located in the plasma membrane, while GhHMA26, GhHMA117 and GhHMA121 were located in the nucleus, respectively. Transcriptomic data and qRT-PCR showed that GhHMA26 exhibited different expression patterns in each tissue and during fiber development or under different abiotic stresses. Overexpressing GhHMA26 significantly promoted the elongation of leaf trichomes and also improved the tolerance to salt stress. Therefore, GhHMA26 may positively regulate fiber elongation and abiotic stress. Yeast two-hybrid assays indicated that GhHMA26 and GhHMA75 participated in multiple biological functions. Our results suggest some genes in the GhHMAs might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GhHMA genes in cotton.

Comparative Transcriptome Analysis Revealed Key Genes Regulating Gossypol Synthesis in Tetraploid Cultivated Cotton.

Tetraploid cultivated cotton (Gossypium spp.) produces cottonseeds rich in protein and oil. Gossypol and related terpenoids, stored in the pigment glands of cottonseeds, are toxic to human beings and monogastric animals. However, a comprehensive understanding of the genetic basis of gossypol and gland formation is still lacking. We performed a comprehensive transcriptome analysis of four glanded versus two glandless tetraploid cultivars distributed in Gossypium hirsutum and Gossypium barbadense. A weighted gene co-expression network analysis (WGCNA) based on 431 common differentially expressed genes (DEGs) uncovered a candidate module that was strongly associated with the reduction in or disappearance of gossypol and pigment glands. Further, the co-expression network helped us to focus on 29 hub genes, which played key roles in the regulation of related genes in the candidate module. The present study contributes to our understanding of the genetic basis of gossypol and gland formation and serves as a rich potential source for breeding cotton cultivars with gossypol-rich plants and gossypol-free cottonseed, which is beneficial for improving food safety, environmental protection, and economic gains of tetraploid cultivated cotton.

Identification and Characterization of Phytocyanin Family Genes in Cotton Genomes.

Phytocyanins (PCs) are a class of plant-specific blue copper proteins that have been demonstrated to play a role in electron transport and plant development. Through analysis of the copper ligand residues, spectroscopic properties, and domain architecture of the protein, PCs have been grouped into four subfamilies: uclacyanins (UCs), stellacyanins (SCs), plantacyanins (PLCs), and early nodulin-like proteins (ENODLs). The present study aimed to identify and characterise the PCs present in three distinct cotton species (Gossypium hirsutum, Gossyium arboreum, and Gossypium raimondii) through the identification of 98, 63, and 69 genes respectively. We grouped PCs into four clades by using bioinformatics analysis and sequence alignment, which exhibit variations in gene structure and motif distribution. PCs are distributed across all chromosomes in each of the three species, with varying numbers of exons per gene and multiple conserved motifs, and with a minimum of 1 and maximum of 11 exons found on one gene. Transcriptomic data and qRT-PCR analysis revealed that two highly differentiated PC genes were expressed at the fibre initiation stage, while three highly differentiated PCs were expressed at the fibre elongation stage. These findings serve as a foundation for further investigations aimed at understanding the contribution of this gene family in cotton fibre production.

Functional characterization of the GhNRT2.1e gene reveals its significant role in improving nitrogen use efficiency in Gossypium hirsutum.

Background: Nitrate is the primary type of nitrogen available to plants, which is absorbed and transported by nitrate transporter 2 (NRT2) at low nitrate conditions. Methods: Genome-wide identification of NRT2 genes in G. hirsutum was performed. Gene expression patterns were revealed using RNA-seq and qRT-PCR. Gene functions were characterized using overexpression in A. thaliana and silencing in G. hirsutum. Protein interactions were verified by yeast two-hybrid and luciferase complementation imaging (LCI) assays. Results: We identified 14, 14, seven, and seven NRT2 proteins in G. hirsutum, G. barbadense, G. raimondii, and G. arboreum. Most NRT2 proteins were predicted in the plasma membrane. The NRT2 genes were classified into four distinct groups through evolutionary relationships, with members of the same group similar in conserved motifs and gene structure. The promoter regions of NRT2 genes included many elements related to growth regulation, phytohormones, and abiotic stresses. Tissue expression pattern results revealed that most GhNRT2 genes were specifically expressed in roots. Under low nitrate conditions, GhNRT2 genes exhibited different expression levels, with GhNRT2.1e being the most up-regulated. Arabidopsis plants overexpressing GhNRT2.1e exhibited increased biomass, nitrogen and nitrate accumulation, nitrogen uptake and utilization efficiency, nitrogen-metabolizing enzyme activity, and amino acid content under low nitrate conditions. In addition, GhNRT2.1e-silenced plants exhibited suppressed nitrate uptake and accumulation, hampered plant growth, affected nitrogen metabolism processes, and reduced tolerance to low nitrate. The results showed that GhNRT2.1e could promote nitrate uptake and transport under low nitrate conditions, thus effectively increasing nitrogen use efficiency (NUE). We found that GhNRT2.1e interacts with GhNAR2.1 by yeast two-hybrid and LCI assays. Discussion: Our research lays the foundation to increase NUE and cultivate new cotton varieties with efficient nitrogen use.