GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, play a regulatory role in cotton fiber secondary cell wall biosynthesis.

KEY MESSAGE: GhHB14_D10 and GhREV_D5 regulated secondary cell wall formation and played an important role in fiber development. Cotton serves as an important source of natural fiber, and the biosynthesis of the secondary cell wall plays a pivotal role in determining cotton fiber quality. Nevertheless, the intricacies of this mechanism in cotton fiber remain insufficiently elucidated. This study investigates the functional roles of GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, in secondary cell wall biosynthesis in cotton fibers. Both GhHB14_D10 and GhREV_D5 were found to be localized in the nucleus with transcriptional activation activity. Ectopic overexpression of GhHB14_D10 and GhREV_D5 in Arabidopsis resulted in changed xylem differentiation, secondary cell wall deposition, and expression of genes related to the secondary cell wall. Silencing of GhHB14_D10 and GhREV_D5 in cotton led to enhanced fiber length, reduced cell wall thickness, cellulose contents and expression of secondary cell wall-related genes. Moreover, GhHB14_D10's direct interaction with GhREV_D5, and transcriptional regulation of cellulose biosynthesis genes GhCesA4-4 and GhCesA7-2 revealed their collaborative roles in secondary cell wall during cotton fiber development. Overall, these results shed light on the roles of GhHB14_D10 and GhREV_D5 in secondary cell wall biosynthesis, offering a strategy for the genetic improvement of cotton fiber quality.

Transcriptional and translational landscape fine-tune genome annotation and explores translation control in cotton.

INTRODUCTION: The unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. OBJECTIVES: Despite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored. METHODS: We utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton. RESULTS: Our study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6, a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels. CONCLUSION: Reference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants.

Cell cycle-dependent kinase inhibitor GhKRP6, a direct target of GhBES1.4, participates in BR regulation of cell expansion in cotton.

The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA-seq and DAP-seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle-dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6-silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6-silenced plants compared with the control. The KEGG enrichment results of control and VIGS-GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways - all of which are related to cell expansion. Additionally, some cyclin-dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle-dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle-dependent kinase inhibitor GhKRP6 via GhBES1.4.

N6 -Methyladenosine mRNA modification regulates transcripts stability associated with cotton fiber elongation.

N6 -Methyladenosine (m6 A) is the most abundant methylation modification in eukaryotic mRNA. The discovery of the dynamic and reversible regulatory mechanism of m6 A has greatly promoted the development of m6 A-led epitranscriptomics. However, the characterization of m6 A in cotton fiber is still unknown. Here, we reveal the potential link between m6 A modification and cotton fiber elongation by parallel m6 A-immunoprecipitation-sequencing (m6 A-seq) and RNA-seq analysis of fibers from the short fiber mutants Ligonliness-2 (Li2 ) and wild-type (WT). This study demonstrated a higher level of m6 A in the Li2 mutant, with the enrichment of m6 A modifications in the stop codon, 3'-untranslated region and coding sequence regions than in WT cotton. In the correlation analysis between genes containing differential m6 A modifications and differentially expressed genes, we identified several genes that could potentially regulate fiber elongation, including cytoskeleton, microtubule binding, cell wall and transcription factors (TFs). We further confirmed that the methylation of m6 A affected the mRNA stability of these fiber elongation-related genes including the TF GhMYB44, which showed the highest expression level in the RNA-seq data and m6 A methylation in the m6 A-seq data. Next, the overexpression of GhMYB44 reduces fiber elongation, whereas the silencing of GhMYB44 produces longer fibers. In summary, these results uncover that m6 A methylation regulated the expression of genes related to fiber development by affecting mRNA's stability, ultimately affecting cotton fiber elongation.

Functional characterization of TBL genes revealed the role of GhTBL7 and GhTBL58 in cotton fiber elongation.

TBL (Trichome Birefringence Like) gene family members are involved in trichome initiation and xylan acetylation in several plant species. In our research, we identified 102 TBLs from G. hirsutum. The phylogenetic tree classified TBL genes into five groups. Collinearity analysis of TBL genes indicated 136 paralogous gene pairs in G. hirsutum. Gene duplication indicated that WGD or segmental duplication contributed to the GhTBL gene family expansion. Promoter cis-elements of GhTBLs were related to growth and development, seed-specific regulation, light, and stress responses. GhTBL genes (GhTBL7, GhTBL15, GhTBL21, GhTBL25, GhTBL45, GhTBL54, GhTBL67, GhTBL72, and GhTBL77) exhibited upregulated response under exposure to cold, heat, NaCl, and PEG. GhTBL genes exhibited high expression during fiber development stages. Two GhTBL genes (GhTBL7 and GhTBL58) showed differential expression at 10 DPA fiber, as 10 DPA is a fast fiber elongation stage and fiber elongation is a very important stage of cotton fiber development. Subcellular localization of GhTBL7 and GhTBL58 revealed that these genes reside inside the cell membrane. Promoter GUS activity of GhTBL7 and GhTBL58 exhibited deep staining in roots. To further validate the role of these genes in cotton fiber elongation, we silenced these genes and observed a significant reduction in the fiber length at 10 DPA. In conclusion, the functional study of cell membrane-associated genes (GhTBL7 and GhTBL58) showed deep staining in root tissues and potential function during cotton fiber elongation at 10 DPA fiber.

Brassinosteroids regulate cotton fiber elongation by modulating very-long-chain fatty acid biosynthesis.

Brassinosteroid (BR), a growth-promoting phytohormone, regulates many plant growth processes including cell development. However, the mechanism by which BR regulates fiber growth is poorly understood. Cotton (Gossypium hirsutum) fibers are an ideal single-cell model in which to study cell elongation due to their length. Here we report that BR controls cotton fiber elongation by modulating very-long-chain fatty acid (VLCFA) biosynthesis. BR deficiency reduces the expression of 3-ketoacyl-CoA synthases (GhKCSs), the rate-limiting enzymes involved in VLCFA biosynthesis, leading to lower saturated VLCFA contents in pagoda1 (pag1) mutant fibers. In vitro ovule culture experiments show that BR acts upstream of VLCFAs. Silencing of BRI1-EMS-SUPPRESOR 1.4 (GhBES1.4), encoding a master transcription factor of the BR signaling pathway, significantly reduces fiber length, whereas GhBES1.4 overexpression produces longer fibers. GhBES1.4 regulates endogenous VLCFA contents and directly binds to BR RESPONSE ELEMENTS (BRREs) in the GhKCS10_At promoter region, which in turn regulates GhKCS10_At expression to increase endogenous VLCFA contents. GhKCS10_At overexpression promotes cotton fiber elongation, whereas GhKCS10_At silencing inhibits cotton fiber growth, supporting a positive regulatory role for GhKCS10_At in fiber elongation. Overall, these results uncover a mechanism of fiber elongation through crosstalk between BR and VLCFAs at the single-cell level.

Genomic identification of cotton SAC genes branded ovule and stress-related key genes in Gossypium hirsutum.

SAC genes have been identified to play a variety of biological functions and responses to various stresses. Previously, SAC genes have been recognized in animals and Arabidopsis. For the very first time, we identified 157 SAC genes in eight cotton species including three diploids and five tetraploids with 23 SAC members in G. hirsutum. Evolutionary analysis classified all cotton SAC gene family members into five distinct groups. Cotton SAC genes showed conserved sequence logos and WGD or segmental duplication. Multiple synteny and collinearity analyses revealed gene family expansion and purifying selection pressure during evolution. G. hirsutum SAC genes showed uneven chromosomal distribution, multiple exons/introns, conserved protein motifs, and various growth and stress-related cis-elements. Expression pattern analysis revealed three GhSAC genes (GhSAC3, GhSAC14, and GhSAC20) preferentially expressed in flower, five genes (GhSAC1, GhSAC6, GhSAC9, GhSAC13, and GhSAC18) preferentially expressed in ovule and one gene (GhSAC5) preferentially expressed in fiber. Similarly, abiotic stress treatment verified that GhSAC5 was downregulated under all stresses, GhSAC6 and GhSAC9 were upregulated under NaCl treatment, and GhSAC9 and GhSAC18 were upregulated under PEG and heat treatment respectively. Overall, this study identified key genes related to flower, ovule, and fiber development and important genetic material for breeding cotton under abiotic stress conditions.

A brassinosteroid transcriptional regulatory network participates in regulating fiber elongation in cotton.

Brassinosteroids (BRs) participate in the regulation of plant growth and development through BRI1-EMS-SUPPRESSOR1 (BES1)/BRASSINAZOLE-RESISTANT1 (BZR1) family transcription factors. Cotton (Gossypium hirsutum) fibers are highly elongated single cells, and BRs play a vital role in the regulation of fiber elongation. However, the mode of action on how BR is involved in the regulation of cotton fiber elongation remains unexplored. Here, we generated GhBES1.4 over expression lines and found that overexpression of GhBES1.4 promoted fiber elongation, whereas silencing of GhBES1.4 reduced fiber length. DNA affinity purification and sequencing (DAP-seq) identified 1,531 target genes of GhBES1.4, and five recognition motifs of GhBES1.4 were identified by enrichment analysis. Combined analysis of DAP-seq and RNA-seq data of GhBES1.4-OE/RNAi provided mechanistic insights into GhBES1.4-mediated regulation of cotton fiber development. Further, with the integrated approach of GWAS, RNA-seq, and DAP-seq, we identified seven genes related to fiber elongation that were directly regulated by GhBES1.4. Of them, we showed Cytochrome P450 84A1 (GhCYP84A1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (GhHMG1) promote cotton fiber elongation. Overall, the present study established the role of GhBES1.4-mediated gene regulation and laid the foundation for further understanding the mechanism of BR participation in regulating fiber development.

GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum).

KEY MESSAGE: We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region. Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

Genome-wide characterization and sequence polymorphism analyses of cysteine-rich poly comb-like protein in Glycine max.

Cysteine-rich poly comb-like protein (CPP) is a member of cysteine-rich transcription factors that regulates plant growth and development. In the present work, we characterized twelve CPP transcription factors encoding genes in soybean (Glycine max). Phylogenetic analyses classified CPP genes into six clades. Sequence logos analyses between G. max and G. soja amino acid residues exhibited high conservation. The presence of growth and stress-related cis-acting elements in the upstream regions of GmCPPs highlight their role in plant development and tolerance against abiotic stress. Ka/Ks levels showed that GmCPPs experienced limited selection pressure with limited functional divergence arising from segmental or whole genome duplication events. By using the PAN-genome of soybean, a single nucleotide polymorphism was identified in GmCPP-6. To perform high throughput genotyping, a kompetitive allele-specific PCR (KASP) marker was developed. Association analyses indicated that GmCPP-6-T allele of GmCPP-6 (in exon region) was associated with higher thousand seed weight under both water regimes (well-water and water-limited). Taken together, these results provide vital information to further decipher the biological functions of CPP genes in soybean molecular breeding.

Characterization of Histone H3 Gene Family Reveals That GmHH3-3 is Associated With Higher Seed Weight in Glycine max.

The main function of histone protein is to provide support to the structure of chromosomes. It helps in binding a long thread of DNA into a more condensed shape to fit into the nucleus. From histone variants, histone H3 (HH3) plays a crucial role in plant growth and development. Characterization of histones has not been reported in Glycine max till now. The objective of this study was to characterize the HH3 gene family for molecular breeding of G. max. In this study, 17 HH3 members in G. max were identified by performing local BLASTp using HH3 members from Arabidopsis as a query. Phylogenetic analysis classified HH3 genes in seven clades. Sequence logo analysis among Arabidopsis thaliana, Oryza sativa, and Glycine max showed a higher level of similarity in amino acids. Furthermore, conserveness of G. max HH3 genes was also confirmed by Gene Structure Display. Ten paralogous gene pairs were identified in GmHH3 genes in the Glycine max genome by conducting collinearity analysis. G. max HH3 genes have experienced strong purifying selection pressure, with limited functional divergence originating from the segmental and whole-genome duplication, as evidenced by the Ka/Ks ratio. The KASP marker was developed for GmHH3-3 gene. Genotyping was performed on 46 G. max genotypes. This differentiation was based upon the presence of either GmHH3-3-C or GmHH3-3-T allele in the CDS region. The results showed that G. max accessions containing the GmHH3-3-T allele at respective locus showed higher thousand seed weight than that of those accessions that contain the GmHH3-3-C allele. This research provides the basic information to further decipher the function of HH3 in soybean.

RAD gene family analysis in cotton provides some key genes for flowering and stress tolerance in upland cotton G. hirsutum.

BACKGROUND: RADIALIS (RAD), belongs to the MYB gene family and regulates a variety of functions including floral dorsoventral asymmetry in Antirrhinum majus and development of fruit proteins in Solanum lycopersicum. RAD genes contain an SNF2_N superfamily domain. Here, we comprehensively identified 68 RAD genes from six different species including Arabidopsis and five species of cotton. RESULTS: Phylogenetic analysis classified RAD genes into five groups. Gene structure, protein motifs and conserved amino acid residues indicated that GhRAD genes were highly conserved during the evolutionary process. Chromosomal location information showed that GhRAD genes were distributed unevenly on different chromosomes. Collinearity and selection pressure analysis indicated RAD gene family expansion in G. hirsutum and G. barbadense with purifying selection pressure. Further, various growth and stress related promotor cis-acting elements were observed. Tissue specific expression level indicated that most GhRAD genes were highly expressed in roots and flowers (GhRAD2, GhRAD3, GhRAD4 and GhRAD11). Next, GhRAD genes were regulated by phytohormonal stresses (JA, BL and IAA). Moreover, Ghi-miRN1496, Ghi-miR1440, Ghi-miR2111b, Ghi-miR2950a, Ghi-miR390a, Ghi-miR390b and Ghi-miR7495 were the miRNAs targeting most of GhRAD genes. CONCLUSIONS: Our study revealed that RAD genes are evolutionary conserved and might be involved in different developmental processes and hormonal stress response. Data presented in our study could be used as the basis for future studies of RAD genes in cotton.

Updated role of ABA in seed maturation, dormancy, and germination.

Background: Seed is vital for plant survival and dispersion, however, its development and germination are influenced by various internal and external factors. Abscisic acid (ABA) is one of the most important phytohormones that influence seed development and germination. Until now, impressive progresses in ABA metabolism and signaling pathways during seed development and germination have been achieved. At the molecular level, ABA biosynthesis, degradation, and signaling genes were identified to play important roles in seed development and germination. Additionally, the crosstalk between ABA and other hormones such as gibberellins (GA), ethylene (ET), Brassinolide (BR), and auxin also play critical roles. Although these studies explored some actions and mechanisms by which ABA-related factors regulate seed morphogenesis, dormancy, and germination, the complete network of ABA in seed traits is still unclear. Aim of review: Presently, seed faces challenges in survival and viability. Due to the vital positive roles in dormancy induction and maintenance, as well as a vibrant negative role in the seed germination of ABA, there is a need to understand the mechanisms of various ABA regulators that are involved in seed dormancy and germination with the updated knowledge and draw a better network for the underlying mechanisms of the ABA, which would advance the understanding and artificial modification of the seed vigor and longevity regulation. Key scientific concept of review: Here, we review functions and mechanisms of ABA in different seed development stages and seed germination, discuss the current progresses especially on the crosstalk between ABA and other hormones and signaling molecules, address novel points and key challenges (e.g., exploring more regulators, more cofactors involved in the crosstalk between ABA and other phytohormones, and visualization of active ABA in the plant), and outline future perspectives for ABA regulating seed associated traits.

Identification, evolutionary analysis and functional diversification of RAV gene family in cotton (G. hirsutum L.).

MAIN CONCLUSION: Genome wide analysis, expression pattern analysis, and functional characterization of RAV genes highlight their roles in roots, stem development and hormonal response. RAV (Related to ABI3 and VP1) gene family members have been involved in tissues/organs growth and hormone signaling in various plant species. Here, we identified 247 RAVs from 12 different species with 33 RAV genes from G. hirsutum. Phylogenetic analysis classified RAV genes into four distinct groups. Analysis of gene structure showed that most GhRAVs lack introns. Motif distribution pattern and protein sequence logos indicated that GhRAV genes were highly conserved during the process of evolution. Promotor cis-acting elements revealed that promotor regions of GhRAV genes encode numerous elements related to plant growth, abiotic stresses and phytohormones. Chromosomal location information showed uneven distribution of 33 GhRAV genes on different chromosomes. Collinearity analysis identified 628 and 52 orthologous/ paralogous gene pairs in G. hirsutum and G. barbadense, respectively. Ka/Ks values indicated that GhRAV and GbRAV genes underwent strong purifying selection pressure. Selecton model and codon model selection revealed that GhRAV amino acids were under purifying selection and adaptive evolution exists among GhRAV proteins. Three dimensional structure of GhRAVs indicated the presence of numerous alpha helix and beta-barrels. Expression level revealed that some GhRAV genes exhibited high expression in roots (GhRAV3, GhRAV4, GhRAV11, GhRAV18, GhRAV20 and GhRAV30) and stem (GhRAV3 and GhRAV18), indicating their potential role in roots and stem development. GhRAV genes can be regulated by phytohormonal stresses (BL, JA and IAA). Our study provides a reference for future studies related to the functional analysis of GhRAVs in cotton.

Systematic Characterization of TCP Gene Family in Four Cotton Species Revealed That GhTCP62 Regulates Branching in Arabidopsis.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play an essential role in regulating various physiological and biochemical functions during plant growth. However, the function of TCP transcription factors in G. hirsutum has not yet been studied. In this study, we performed genome-wide identification and correlation analysis of the TCP transcription factor family in G. hirsutum. We identified 72 non-redundant GhTCP genes and divided them into seven subfamilies, based on phylogenetic analysis. Most GhTCP genes in the same subfamily displayed similar exon and intron structures and featured highly conserved motif structures in their subfamily. Additionally, the pattern of chromosomal distribution demonstrated that GhTCP genes were unevenly distributed on 24 out of 26 chromosomes, and that fragment replication was the main replication event of GhTCP genes. In TB1 sub-family genes, GhTCP62 was highly expressed in the axillary buds, suggesting that GhTCP62 significantly affected cotton branching. Additionally, subcellular localization results indicated that GhTCP62 is located in the nucleus and possesses typical transcription factor characteristics. The overexpression of GhTCP62 in Arabidopsis resulted in fewer rosette-leaf branches and cauline-leaf branches. Furthermore, the increased expression of HB21 and HB40 genes in Arabidopsis plants overexpressing GhTCP62 suggests that GhTCP62 may regulate branching by positively regulating HB21 and HB40.

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.).

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Identification of BR biosynthesis genes in cotton reveals that GhCPD-3 restores BR biosynthesis and mediates plant growth and development.

MAIN CONCLUSION: Brassinosteroid (BR) synthesis genes in different cotton species was comprehensively identified, and the participation of GhCPD-3 in the BR synthesis signaling pathway for regulating plant development was verified. Brassinosteroid is a natural steroidal phytohormone that plays fundamental roles in plant growth and development. In cotton, detailed characterization and functional validation of BR biosynthesis genes remain rare. Here, 16, 8 and 9 BR biosynthesis genes were identified in Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively, and their phylogenetic relationships, gene structures, conserved motifs of the encoded proteins, chromosomal locations were determined and a synteny analysis was performed. Gossypium hirsutum and Arabidopsis BR biosynthesis genes closely clustered in the phylogenetic tree and fragment duplication was likely the primary cause promoting gene family expansion in G. hirsutum. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed their relevance as BR biosynthesis genes. GhCPD-3 was highly expressed in roots and stems and the loci of single nucleotide polymorphisms (SNPs) were significantly associated with these traits.Ectopic overexpression of GhCPD-3 in the cpd91 Arabidopsis mutant rescued the mutant phenotype by increasing plant height and leaf size in comparison to those of cpd91 and WT plants. Moreover, overexpressed GhCPD-3 in cpd91 mutants showed greater hypocotyl and root lengths than those of cpd91 and WT plants under light and dark conditions, respectively, indicating that BR actively promotes hypocotyl and root growth. Similar to CPD (CONSTITUTIVE PHOTOMORPHOGENIC DWARF), GhCPD-3 restores BR biosynthesis thereby mediating plant growth and development.