The bHLH transcription factor GhPAS1 mediates BR signaling to regulate plant development and architecture in cotton

Cotton (Gossypium spp.) is the most important natural textile fiber crop in the world.The ideal plant architecture of cotton is suitable for mechanical harvesting and productivity in modern agricultural production.However,cotton genes regulating plant development and architecture have not been fully identified.We identified a basic helix-loop-helix (b HLH) transcription factor,GhPAS1 (PAGODA1 SUPPRESSOR1) in G.hirsutum (Upland cotton).GhPAS1 was located in the nucleus and showed a strong transcription activation effect.Tissue-specific expression patterns showed that GhPAS1 was highly expressed in floral organs,followed by high expression in early stages of ovule development and rapid fiber elongation.GhPAS1 overexpression in Arabidopsis and BRZ (brassinazole,BR biosynthesis inhibitor) treatment indicated that GhPAS1 positively regulates and responds to the BR (brassinosteroid) signaling pathway and promotes cell elongation.GhPAS1 overexpression in Arabidopsis mediated plant development in addition to increasing plant biomass.Virus-induced gene silencing of GhPAS1 indicated that down-regulation of GhPAS1 inhibited cotton growth and development,as plant height,fruit branch length,and boll size of silenced plants were lower than in control plants.Fiber length and seed yield were also lower in silenced plants.We conclude that GhPAS1,a b HLH transcription factor,regulates plant development and architecture in cotton.These findings may help breeders and researchers develop cotton cultivars with desirable agronomic characteristics.

A comprehensive overview of cotton genomics,biotechnology and molecular biological studies

Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis,which makes it of high research and application value.To date,numerous research on cotton has navigated various aspects,from multi-genome assembly,genome editing,mechanism of fiber development,metabolite biosynthesis,and analysis to genetic breeding.Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers.Mature multiple genome editing systems,such as CRISPR/Cas9,Cas12(Cpf1)and cytidine base editing(CBE),have been widely used in the study of candidate genes affecting fiber development.Based on this,the cotton fiber cell development network has been preliminarily drawn.Among them,the MYB-b HLH-WDR(MBW)transcription factor complex and IAA and BR signaling pathway regulate the initiation;various plant hormones,including ethylene,mediated regulatory network and membrane protein overlap fine-regulate elongation.Multistage transcription factors targeting Ces A 4,7,and 8 specifically dominate the whole process of secondary cell wall thickening.And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development.Furthermore,research on the synthesis of cotton secondary metabolite gossypol,resistance to diseases and insect pests,plant architecture regulation,and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties.This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects,thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.

Genome-wide analysis of BES1 genes in Gossypium revealed their evolutionary conserved roles in brassinosteroid signaling

Brassinosteroids(BRs), which are essential phytohormones for plant growth and development, are important for cotton fiber development. Additionally, BES1 transcription factors are critical for BR signal transduction. However, cotton BES1 family genes have not been comprehensively characterized. In this study, we identified 11 BES1 genes in G. arboreum, 11 in G.raimondii, 16 in G. barbadense, and 22 in G. hirsutum. The BES1 sequences were significantly conserved in the Arabidopsis thaliana, rice, and upland cotton genomes. A total of 94 BES1 genes from 10 different plant species were divided into three clades according to the neighbor-joining and minimum-evolution methods. Moreover, the exon/intron patterns and motif distributions were highly conserved among the A. thaliana and cotton BES1 genes. The collinearity among the orthologs from the At and Dt subgenomes was estimated. Segmental duplications in the At and Dt subgenomes were primarily responsible for the expansion of the cotton BES1 gene family. Of the GhBES1 genes, GhBES1.4_At/Dt exhibited BL-induced expression and was predominantly expressed in fibers. Furthermore, Col-0/mGhBES1.4_At plants produced curled leaves with long and bent petioles. These transgenic plants also exhibited decreased hypocotyl sensitivity to brassinazole and constitutive BR induced/repressed gene expression patterns. The constitutive BR responses of the plants overexpressing mGhBES1.4_At were similar to those of the bes1-D mutant.

Overexpressed BRH1, a RING finger gene, alters rosette leaf shape in Arabidopsis thaliana

Leaves are the most important plant parts for photosynthesis and respiration. Many genes are involved in determining leaf shape;however, little is known about the effects of brassinosteroid (BR) signaling-pathway genes on the development of leaf shape. Here, the brassinosteroid-responsive RING-H2 (BRH1) gene, which is suppressed by 24-epi-brassinolide treatment, was isolated from Arabidopsis thaliana. The amino acid sequence contained a highly conserved RING finger domain. In a phylogenetic analysis,BRH1 clustered closely with GLYMA11G02470.1. The leaves of brh1 mutant plants were not much different to those of the wild-type, while transgenic plants with high BRH1 expression levels had rounder rosette leaves. Mutants of the BR synthesis pathway also had a similar round leaf phenotype, and greater BRH1 expression levels. Moreover, the related marker genes KNAT1,AtHB13 and ROT4, which are known to control leaf shape, altered transcriptional levels in both transgenic BRH1 and BR-synthesis mutant lines. Thus, BRH1 may be involved in the BR signaling pathway and regulate the growth and development of rosette leaves. Research on BRH1 may prove valuable for understanding the regulatory mechanism of leaf shape and improving the leaf shapes of ornamental plants.

Transcriptome Profiling Reveals Auxin and Cytokinin Regulating Somatic Embryogenesis in Different Sister Lines of Cotton Cultivar CCRI24

To get a broader view on the molecular mechanisms underlying somatic embryogenesis （SE） in cotton （Gossypium hirsutum L.）, global analysis of cotton transcriptome dynamics during SE in different sister lines was performed using RNA-Seq. A total of 204 349 unigenes were detected by de novo assembly of the 214 977 462 Illumina reads. The quantitative reverse transcription-polymerase chain reaction （qRT-PCR） measurements were positively correlated with the RNA-Seq results for almost all the tested genes （R2 -- 0.841, correlation was significant at the 0.01 level）. Different phytohormone （auxin and cytokinin） concentration ratios in medium and the endogenous content changes of these two phytohormones at two stages in different sister lines suggested the roles of auxin and cytokinin during cotton SE. On the basis of global gene regulation of phytohormone-related genes, numerous genes from all the differentially expressed transcripts were involved in auxin and cytokinin biosynthesis and signal transduction pathways. Analyses of differentially expressed genes that were involved in these pathways revealed the substantial changes in gene type and abundance between two sister lines. Isolation, cloning and silencing/ overexpressing the genes that revealed remarkable up- or down-expression during cotton SE were important. Furthermore, auxin and cytokinin play a primary role in SE, but potential cross-talk with each other or other factors remains unclear.

Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum

Genetic transformation in some plant species,including cotton(Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing(VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different c DNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA(si RNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of si RNA in target sequence: the disruption energy of the target(DGdisruption), the differential stability of si RNA duplex ends(DSSE), and the internal stability at positions 9–14 of the si RNA antisense strand(AIS), which are calculated by Sfold program(http://sfold.wadsworth.org). We find that the si RNAs with low DGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, Gh PDS,Gh CLA1, Gh AOS1, and Gh CXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.

Comparative phosphoproteomic analysis of BR-defective mutant reveals a key role of GhSK13 in regulating cotton fiber development

Brassinosteroid(BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3 s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1(pag1), and its corresponding wild-type(ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage(10 days after pollination(DPA)) and secondary cell wall synthesis stage(20DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs(GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor GhDNFYB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.

GhKLCR1, a kinesin light chain-related gene, induces drought-stress sensitivity in Arabidopsis

Drought stress results in significant losses in agricultural production, and especially that of cotton. The molecular mechanisms that coordinate drought tolerance remain elusive in cotton. Here, we isolated a drought-response gene GhKLCR1, which is a close homolog of AtKLCR1, which encodes a kinesin light chain-related protein enriched with a tetratrico peptide-repeat region.A subcellular localization assay showed that GhKLCR1 is associated with the cell membrane. A tissue-specific expression profile analysis demonstrated that GhKLCR1 is a cotton root-specific gene. Further abiotic and hormonal stress treatments showed that GhKLCR1 was upregulated during abiotic stresses, especially after polyethylene glycol treatments. In addition, the glucuronidase(GUS) staining activity increased as the increment of mannitol concentration in transgenic Arabidopsis plants harboring the fusion construct PGhKLCR1::GUS. The root lengths of 35 S::GhKLCR1 lines were significantly reduced compared with that of wild type. Additionally, seed germination was strongly inhibited in 35 S::GhKLCR1 lines after 300-mmol L^(-1) mannitol treatments as compared with Columbia-0, indicating the sensitivity of GhKLCR1 to drought. These findings provide a better understanding of the structural, physiological and functional mechanisms of kinesin light chain-related proteins.

Genome sequencing brought Gossypium biology research into a new era

The first sequenced diploid cotton genome was published in2012 by the group led by the Institute of Cotton Research,Chinese Academy of Agricultural Sciences.Cotton genomics research subsequently entered a period of rapid development.The accumulating data have provided new insights into the evolution and domestication of cotton,the development of important agronomic traits,and strategies