Zhaoen Yang

PAG1, a cotton brassinosteroid catabolism gene, modulates fiber elongation

Cotton (Gossypium hirsutum) is the major source of natural textile fibers. Brassinosteroids (BRs) play crucial roles in regulating fiber development. The molecular mechanisms of BRs in regulating fiber elongation, however, are poorly understood. pagoda1 (pag1) was identified via an activation tagging genetic screen and characterized by genome walking and brassinolide (BL) supplementation. RNA-Seq analysis was employed to elucidate the mechanisms of PAG1 in regulating fiber development. pag1 exhibited dwarfism and reduced fiber length due to significant inhibition of cell elongation and expansion. BL treatment rescued its growth and fiber elongation. PAG1 encodes a homolog of Arabidopsis CYP734A1 that inactivates BRs via C-26 hydroxylation. RNA-Seq analyses showed that the constitutive expression of PAG1 downregulated the expression of genes involved in very-long-chain fatty acids (VLCFA) biosynthesis, ethylene-mediated signaling, response to cadmium, cell wall development, cytoskeleton organization and cell growth. Our results demonstrate that PAG1 plays crucial roles in regulating fiber development via controlling the level of endogenous bioactive BRs, which may affect ethylene signaling cascade by mediating VLCFA. Therefore, BR may be a critical regulator of fiber elongation, a role which may in turn be linked to effects on VLCFA biosynthesis, ethylene and cadmium signaling, cell wall- and cytoskeleton-related gene expression.

Genome-wide analysis of WOX genes in upland cotton and their expression pattern under different stresses

BackgroundWUSCHEL-related homeobox (WOX) family members play significant roles in plant growth and development, such as in embryo patterning, stem-cell maintenance, and lateral organ formation. The recently published cotton genome sequences allow us to perform comprehensive genome-wide analysis and characterization of WOX genes in cotton.ResultsIn this study, we identified 21, 20, and 38 WOX genes in Gossypium arboreum (2n = 26, A2), G. raimondii (2n = 26, D5), and G. hirsutum (2n = 4x = 52, (AD)t), respectively. Sequence logos showed that homeobox domains were significantly conserved among the WOX genes in cotton, Arabidopsis, and rice. A total of 168 genes from three typical monocots and six dicots were naturally divided into three clades, which were further classified into nine sub-clades. A good collinearity was observed in the synteny analysis of the orthologs from At and Dt (t represents tetraploid) sub-genomes. Whole genome duplication (WGD) and segmental duplication within At and Dt sub-genomes played significant roles in the expansion of WOX genes, and segmental duplication mainly generated the WUS clade. Copia and Gypsy were the two major types of transposable elements distributed upstream or downstream of WOX genes. Furthermore, through comparison, we found that the exon/intron pattern was highly conserved between Arabidopsis and cotton, and the homeobox domain loci were also conserved between them. In addition, the expression pattern in different tissues indicated that the duplicated genes in cotton might have acquired new functions as a result of sub-functionalization or neo-functionalization. The expression pattern of WOX genes under different stress treatments showed that the different genes were induced by different stresses.ConclusionIn present work, WOX genes, classified into three clades, were identified in the upland cotton genome. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. Moreover, the expression patterns suggested that the duplicated genes might have experienced a functional divergence. Together, these results shed light on the evolution of the WOX gene family, and would be helpful in future research.

Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae

BackgroundVerticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized. Identifying an endogenous resistance gene may be useful for controlling this disease.ResultsWe cloned the ribosomal protein L18 (GaRPL18) gene, which mediates resistance to Verticillium wilt, from a wilt-resistant cotton species (Gossypium arboreum). We then characterized the function of this gene in cotton and Arabidopsis thaliana plants. GaRPL18 encodes a 60S ribosomal protein subunit important for intracellular protein biosynthesis. However, previous studies revealed that some ribosomal proteins are also inhibitory toward oncogenesis and congenital diseases in humans and play a role in plant disease defense. Here, we observed that V. dahliae infections induce GaRPL18 expression. Furthermore, we determined that the GaRPL18 expression pattern is consistent with the disease resistance level of different cotton varieties. GaRPL18 expression is upregulated by salicylic acid (SA) treatments, suggesting the involvement of GaRPL18 in the SA signal transduction pathway. Virus-induced gene silencing technology was used to determine whether the GaRPL18 expression level influences cotton disease resistance. Wilt-resistant cotton species in which GaRPL18 was silenced became more susceptible to V. dahliae than the control plants because of a significant decrease in the abundance of immune-related molecules. We also transformed A. thaliana ecotype Columbia (Col-0) plants with GaRPL18 according to the floral dip method. The plants overexpressing GaRPL18 were more resistant to V. dahliae infections than the wild-type Col-0 plants. The enhanced resistance of transgenic A. thaliana plants to V. dahliae is likely mediated by the SA pathway.ConclusionOur findings provide new insights into the role of GaRPL18, indicating that it plays a crucial role in resistance to cotton “cancer”, also known as Verticillium wilt, mainly regulated by an SA-related signaling pathway mechanism.

Resequencing of 243 diploid cotton accessions based on an updated A genome identifies the genetic basis of key agronomic traits

The ancestors of Gossypium arboreum and Gossypium herbaceum provided the A subgenome for the modern cultivated allotetraploid cotton. Here, we upgraded the G. arboreum genome assembly by integrating different technologies. We resequenced 243 G. arboreum and G. herbaceum accessions to generate a map of genome variations and found that they are equally diverged from Gossypium raimondii. Independent analysis suggested that Chinese G. arboreum originated in South China and was subsequently introduced to the Yangtze and Yellow River regions. Most accessions with domestication-related traits experienced geographic isolation. Genome-wide association study (GWAS) identified 98 significant peak associations for 11 agronomically important traits in G. arboreum. A nonsynonymous substitution (cysteine-to-arginine substitution) of GaKASIII seems to confer substantial fatty acid composition (C16:0 and C16:1) changes in cotton seeds. Resistance to fusarium wilt disease is associated with activation of GaGSTF9 expression. Our work represents a major step toward understanding the evolution of the A genome of cotton.The authors report an improved genome assembly of G. arboretum and resequencing of 243 diploid cotton accessions. GWAS and QTL-seq identify a number of candidate loci that associate with seed oil content, disease resistance and yield traits in cotton.

GhCaM7-like, a calcium sensor gene, influences cotton fiber elongation and biomass production.

Calcium signaling regulates many developmental processes in plants. Calmodulin (CaM) is one of the most conserved calcium sensors and has a flexible conformation in eukaryotes. The molecular functions of CaM are unknown in cotton, which is a major source of natural fiber. In this study, a Gossypium hirsutum L.CaM7-like gene was isolated from upland cotton. Bioinformatics analysis indicated that the GhCaM7-like gene was highly conserved as compared with Arabidopsis AtCaM7. The GhCaM7-like gene showed a high expression level in elongating fibers. Expression of β-glucuronidase was observed in trichomes on the stem, leaf and root in transgenic Arabidopsis plants of a PROGhCaM7-like:GUS fusion. Silencing of the GhCaM7-like gene resulted in decreased fiber length, but also caused reduction in stem height, leaf dimensions, seed length and 100-seed weight, in comparison with those of the control. Reduced expression of the GhCaM7-like gene caused decreased Ca2+ influx in cells of the leaf hypodermis and stem apex, and down-regulation of GhIQD1 (IQ67-domain containing protein), GhAnn2 (Annexins) and GhEXP2 (Expansin). These results indicate that the GhCaM7-like gene plays a vital role in calcium signaling pathways, and may regulate cotton fiber elongation and biomass production by affecting Ca2+ signatures and downstream signaling pathways of CaM.

A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum Identified in a Genome-Wide Association Study

Verticillium wilt disease is one of the most destructive biotic stresses faced by cotton plants. Here, we performed a genome-wide association study (GWAS) in 215 Chinese Gossypium arboreum accessions inoculated as seedlings with Verticillium dahliae to identify candidate loci involved in wilt resistance. We identified 309 loci that had a significant association with Verticillium wilt resistance and - log(P) values >5.0; the highest signal appeared on Ca3 in a 74 kb haplotype block. Five genes were also located within this haplotype block. One of these genes, CG05, was positioned close to the most significant SNP Ca3_23037225 (14 kb); expression of the gene was induced by V. dahliae or by treatment with salicylic acid (SA). Therefore, we suggest that CG05 may respond to invasion by V. dahliae via an SA-related signaling pathway, and we designated this gene as GaGSTF9. We showed that GaGSTF9 was a positive regulator of Verticillium wilt through the use of virus-induced gene silencing (VIGS) and overexpression in Arabidopsis. In addition, the glutathione S-transferase (GST) mutant gstf9 of Arabidopsis was found to be more susceptible to Verticillium wilt than wild-type plants. The levels of endogenous SA and hydrogen peroxide had a significant effect on Arabidopsis plants that overexpressed GaGSTF9, indicating that GST may regulate reactive oxygen species content via catalytic reduction of the tripeptide glutathione (GSH), and then affect SA content. Our data demonstrated that GaGSTF9 was a key regulator mediating cotton responses to V. dahliae and a potential candidate gene for cotton genetic improvement.

Genome-Wide Analysis of the NF-YB Gene Family in Gossypium hirsutum L. and Characterization of the Role of GhDNF-YB22 in Embryogenesis

Members of the NF-YB transcription factor gene family play important roles in diverse processes related to plant growth and development, such as seed development, drought tolerance, and flowering time. However, the function of NF-YB genes in cotton remains unclear. A total of 23, 24, and 50 NF-YB genes were identified in Gossypium arboreum (G. arboreum), Gossypium raimondii (G. raimondii), and G. hirsutum, respectively. A systematic phylogenetic analysis was carried out in G. arboretum, G. raimondii, G. hirsutum, Arabidopsis thaliana, cacao, rice and, sorghum, where the 150 NF-YB genes were divided into five groups (α–ε). Of these groups, α is the largest clade, and γ contains the LEC1 type NF-YB proteins. Syntenic analyses revealed that paralogues of NF-YB genes in G. hirsutum exhibited good collinearity. Owing to segmental duplication within the A sub-genome (At) and D sub-genome (Dt), there was an expanded set of NF-YB genes in G. hirsutum. Furthermore, we investigated the structures of exons, introns, and conserved motifs of NF-YB genes in upland cotton. Most of the NF-YB genes had only one exon, and the genes from the same clade exhibited a similar motif pattern. Expression data show that most NF-YB genes were expressed ubiquitously, and only a few genes were highly expressed in specific tissues, as confirmed by quantitative real-time PCR (qRT-PCR) analysis. The overexpression of GhDNF-YB22 gene, predominantly expressed in embryonic tissues, indicates that GhDNF-YB22 may affect embryogenesis in cotton. This study is the first comprehensive characterization of the GhNF-YB gene family in cotton, and showed that NF-YB genes could be divided into five clades. The duplication events that occurred over the course of evolution were the major impetus for NF-YB gene expansion in upland cotton. Collectively, this work provides insight into the evolution of NF-YB in cotton and further our knowledge of this commercially important species.

Genome-wide analysis of the HD-ZIP IV transcription factor family in Gossypium arboreum and GaHDG11 involved in osmotic tolerance in transgenic Arabidopsis

HD-ZIP IV proteins belong to the homeodomain-leucine zipper (HD-ZIP) transcription factor family and are involved in trichome development and drought stress in plants. Although some functions of the HD-ZIP IV group are well understood in Arabidopsis, little is known about their function in cotton. In this study, HD-ZIP genes were identified from three Gossypium species (G. arboreum, G. raimondii and G. hirsutum) and clustered into four families (HD-ZIP I, II, III and IV) to separate HD-ZIP IV from the other three families. Systematic analyses of phylogeny, gene structure, conserved domains, and expression profiles in different plant tissues and the expression patterns under osmotic stress in leaves were further conducted in G. arboreum. More importantly, ectopic overexpression of GaHDG11, a representative of the HD-ZIP IV family, confers enhanced osmotic tolerance in transgenic Arabidopsis plants, possibly due to elongated primary root length, lower water loss rates, high osmoprotectant proline levels, significant levels of antioxidants CAT, and/or SOD enzyme activity with reduced levels of MDA. Taken together, these observations may lay the foundation for future functional analysis of cotton HD-ZIP IV genes to unravel their biological roles in cotton.

Genome-Wide Study of YABBY Genes in Upland Cotton and Their Expression Patterns under Different Stresses

Members of the YABBY gene family, a small plant-specific family of genes, have been proposed to function in specifying abaxial cell fate. Although to date little has been learned about cotton YABBY genes, completion of the cotton genome enables a comprehensive genome-wide analysis of YABBY genes in cotton. Here, a total of 12, 12, and 23 YABBY genes were identified in Gossypium arboreum (2n = 26, A2), G. raimondii (2n = 26, D5), and G. hirsutum (2n = 4x = 52, [AD]t), respectively. Sequence analysis showed that the N-terminal zinc-finger and C-terminal YABBY domains in YABBY proteins are highly conserved among cotton, Arabidopsis, and rice. Eighty-five genes from eight sequenced species naturally clustered into five groups, and the YAB2-like group could be divided into three sub-groups, indicating that YABBYs are highly conserved among the examined species. Orthologs from the At and Dt sub-genomes (where “t” indicates tetraploid) showed good collinearity, indicating that YABBY loci are highly conserved between these two sub-genomes. Whole-genome duplication was the primary cause of upland cotton YABBY gene expansion, segmental duplication played important roles in YABBY gene expansion within the At and Dt sub-genomes, and the YAB5-like group was mainly generated by segmental duplication. The long-terminal repeat retroelements Copia and Gypsy were identified as major transposable elements accompanying the appearance of duplicated YABBY genes, suggesting that transposable element expansion might be involved in gene duplication. Selection pressure analyses using PAML revealed that relaxed purifying selection might be the main impetus during evolution of YABBY genes in the examined species. Furthermore, exon/intron pattern and motif analyses indicated that genes within the same group were significantly conserved between Arabidopsis and cotton. In addition, the expression patterns in different tissues suggest that YABBY proteins may play roles in ovule development because YABBYs are highly expressed in ovules. The expression pattern of YABBY genes showed that approximately half of the YABBYs were down-regulated under different stress treatments. Collectively, our results represent a comprehensive genome-wide study of the YABBY gene family, which should be helpful in further detailed studies on the gene function and evolution of YABBY genes in cotton.

Genome-Wide Analysis Elucidates the Role of CONSTANS-like Genes in Stress Responses of Cotton

The CONSTANS (CO)-like gene family has been well studied for its role in the regulation of plant flowering time. However, their role remains poorly understood in cotton. To better understand the possible roles of CO-like in cotton, we performed a comprehensive genome-wide analysis of CO-like genes in cotton. Phylogenetic tree analysis showed that CO-like genes naturally clustered into three groups. Segmental duplication and whole genome duplication (WGD), which occurred before polyploidy, were important contributors to its expansion within the At (“t” indicates tetraploid) and Dt subgenomes, particularly in Group III. Long-terminal repeat retroelements were identified as the main transposable elements accompanying 18 genes. The genotype of GhCOL12_Dt displayed low diversity; it was a candidate involved in domestication. Selection pressure analyses indicated that relaxed purifying selection might have provided the main impetus during the evolution of CO-like genes in upland cotton. In addition, the high expression in the torus and calycle indicated that CO-like genes might affect flowering. The genes from Group II, and those from Group III involved in segmental duplication or WGD, might play important roles in response to drought and salt stress. Overall, this comprehensive genome-wide study of the CO-like gene family would facilitate further detailed studies in cotton.