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.

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.

Significant Improvement of Cotton Verticillium Wilt Resistance by Manipulating the Expression of Gastrodia Anlffungal Proteins

Dear Editor Verticillium wilt caused by Verticillium dahliae Kleb. is the most destructive disease in cotton-growing areas around the world. The infection significantly reduces cotton yield and fiber quality due to leaf chlorosis, necrosis or wilting, leaf or boll abscission, and even plant death. The losses may reach up to 80% of lint cotton yield （Wei et al., 2015）.

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.

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.

Recent progression and future perspectives in cotton genomic breeding

Upland cotton is an important global cash crop for its long seed fibers and high edible oil and protein content.Progress in cotton genomics promotes the advancement of cotton genetics,evolutionary studies,functional genetics,and breeding,and has ushered cotton research and breeding into a new era.Here,we summarize high-impact genomics studies for cotton from the last 10 years.The diploid Gossypium arboreum and allotetraploid Gossypium hirsutum are the main focus of most genetic and genomic studies.We next review recent progress in cotton molecular biology and genetics,which builds on cotton genome sequencing efforts,population studies,and functional genomics,to provide insights into the mechanisms shaping abiotic and biotic stress tolerance,plant architecture,seed oil content,and fiber development.We also suggest the application of novel technologies and strategies to facilitate genome-based crop breeding.Explosive growth in the amount of novel genomic data,identified genes,gene modules,and pathways is now enabling researchers to utilize multidisciplinary genomics-enabled breeding strategies to cultivate"super cotton",synergistically improving multiple traits.These strategies must rise to meet urgent demands for a sustainable cotton industry.

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.)is one of the most important fiber crops worldwide.In the last two decades,transgenesis and genome editing have played important roles in cotton improvement.However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation.Here,we developed a shoot apical meristem(SAM)cell-mediated transformation system(SAMT)that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton(Gossypium hirsutum),Sea island cotton(Gossypium barbadense),and Asiatic cotton(Gossypium arboreum).Through SAMT,we successfully introduced two foreign genes,GFP and RUBY,into SAM cells of some recalcitrant cotton genotypes.Within 2–3 months,transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants.The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP-and RUBY-positive plants,as well as in their progenies,indicating that the transgenes were stably integrated into the genome and transmitted to the next generation.Furthermore,using SAMT,we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing.In summary,the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.

Fighting against fall armyworm by using multiple genes pyramiding and silencing(MGPS) technology

The pest Fall Armyworm(FAW),Spodoptera frugiperda belonging to the order Lepidoptera,is one of the most devastating insect pests resulting in severe economic losses to maize,rice,wheat,cotton and soybean in South America,Africa and Asia.It consists of two morphologically indistinguishable strains:the“corn strain”feeding mainly on maize,cotton and sorghum;the“rice strain”mostly feeding on rice and various grasses(Gouin et al.,2017).Adults of FAW have strong migration ability to fly 100 km per night.In 2018 it was detected in India and then spreads to Bangladesh,Thailand,Myanmar,and China in 2019(http://www.fao.org/fall-armyworm/en/).It is a polyphagous lepidopteran herbivore,attacks more than 350 plant species,and causes destructive damage to many economically important crops.The FAW causes up to 30%-60% yield losses.

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.

Freeze substitution Hi-C,a convenient and cost-effective method for capturing the natural 3D chromatin conformation from frozen samples

Chromatin interactions functionally affect genome architecture and gene regulation,but to date,only fresh samples must be used in High-through chromosome conformation capture(Hi-C)to keep natural chromatin conformation intact.This requirement has impeded the advancement of 3 D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time.Here,we develop a freeze substitution Hi-C(FS-Hi-C)technique that overcomes the need for fresh samples.FS-Hi-C can be used with samples stored in liquid nitrogen(LN2):the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution.After confirming that the FS step preserves the natural chromosome conformation during sample thawing,we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum.Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains,we found that FS-HiC outperforms the in situ Hi-C in terms of library quality,reproducibility,and valid interactions.Thus,FS-HiC will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.

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