Effect of Transgenic Bt plus CpTI Cotton on Carboxylesterase and Acetylcholinesterase of Cotton Aphid Aphis gossypii

[Objective] The research aimed to assess the effect of transgenic Bt plus CpTI cotton variety SGK321 on carboxylesterase and acetylcholinesterase of cotton aphid Aphis gossypii and provide theoretical basis for studying the biosafety of transgenic cotton.[Method] Cotton aphids were fed with SGK321 and Shiyuan321（normal parental varieties） for over 40 generations.Enzyme activities were compared between cotton aphids feeding on SGK321 for 1,2,3,41,42 and 43 generations with those on Shiyuan321.[Result] The carboxylesterase activity of cotton aphids feeding on SGK321 for 1 generation was significantly higher than those feeding on Shiyuan321.Acetylcholinesterase activity of cotton aphids feeding on SGK321 for 1,2 and 3 generations were significantly higher than those feeding on Shiyuan321 in the same generation.But there was no significant difference of enzyme activity between cotton aphids feeding on SGK321 for a long term and those feeding on parental cotton.[Conclusion] The cotton aphid that feeding on transgenic Bt plus CpTI cotton SGK321 for a long time has adaptivity to SGK321 by regulating the detoxifying enzyme.

The biotypes and host shifts of cotton-melon aphids Aphis gossypii in northern China

Aphis gossypfi is a globally distributed species and therefore has a highly variable life cycle. Populations of A. gossypii in northern China exhibit greater genotypic diversity and a broader host range, yet the details of life cycles of different biotypes is still unclear. In this study, the Cytb and 16S gene regions of A. gossypfi collected from 5 common summer hosts and 4 primary hosts were analyzed. A total of 57 haplotypes were obtained from 1 046 individual A. gossyp# sequences. The sequence included 44 variable sites, 27 of which were parsimony informative sites and 17 of which were singleton variable sites. The most frequent 3 haplotypes were found in 896 individuals, representing a total of 85.7% of all individuals and 36 haplotypes were found in 1 individual. A neighbor-joining tree was constructed using 21 haplotypes that were found in more than 2 individuals. Considering the individual host plant, 5 biotypes were identified. Type 1 corresponded exactly to the cucurbit host-race and the other 4 biotypes were found as cotton host-races. Type 3 was the most abundant biotype in cotton fields in northern China.

Impacts of Climate Change on the Occurrence Degree of Cotton Aphids in Huang-Huai-Hai and Yangtze River Basins

[Objective] The aim was to analyze the impacts of climate change （tem- perature and precipitation） on the occurrence degree of cotton aphids. [Method] Ec- onometric models were established to explore the impacts of climate change on the occurrence degree of cotton aphids based on the observation data from 50 counties in Huang-Huai-Hai and Yangtze River basins in 1991-2010, including variables on the occurrence degree of cotton aphids, temperature and precipitation, and other factors. [Result] There were significant differences in the effects of temperature and precipitation changes on the occurrence degrees of cotton aphid in different growth stages, and even in the same growth stage, the cotton aphids had different responses to the changes of temperature and precipitation in different re- gions. [Conclusion] It should give full consideration to the climatic conditions in dif- ferent periods of different regions, and it should adopt proper measures to control the cotton aphids on the basis of figuring out the occurrence rule of cotton aphid, so as to ensure cotton production.

Occurrence Rule and Forecast Method of Cotton Aphid in Poyang Lake Area in Jiujiang City

[Objective] The aim was to explore occurrence rules and forecast methods of cotton aphid in Poyang Lake Area in Jiujiang City to enrich prediction methods of cotton aphid in the area. [Method] The occurrence rules and influencing factors of cotton aphid in Pengze County in 28 years were analyzed with comparative analysis, correlation analysis and wavelet analysis. Furthermore, a long-term forecast model of occurrence grade of cotton aphid and a short-term forecast model of weather condi- tion suitability were established based on stepwise regression. In addition, a forecast test was conducted in cotton area in the north of Poyang Lake. [Result] The wavelet analysis showed that in recent 28 years, oscillating period at 4 years was significant for the occurrence grade of cotton aphid in the north of Poyang Lake, but insignifi- cant for cotton at seedling stage. The comparative and correlation analyses suggest- ed that occurrence of cotton aphid is of significant correlation with winter climate and weather conditions at middle and short periods. The prediction test indicated that long-term forecast model of occurrence grade of cotton aphid and short-term forecast model of weather condition suitability based on stepwise regression can be made use of in the areas with similar climate. [Conclusion] The research provides theoreti- cal references for prevention against cotton aphid in cotton-planting area.

Cloning of Partial Sodium Channel Gene From Strains of Fenvalerate-Resistant and Susceptible Cotton Aphid(Aphis gossypii Glover)

The strain of fenvalerate-resistant cotton aphids was selected using fenvalerate insecticide in the laboratory, the resistance factor of the strain was 199.54. Three degenerate primers were designed and used to perform PCR amplification. A cDNA encoding partial sodium channel gene was cloned from the fenvalerate-resistant and -susceptible strains. There were two nucleotide acid differences between fenvalerate-resistant strain and -susceptible strain, resulting in an amino acid mutation, Met→Leu. It is predicted that the mutation is related to the cotton aphid resistance to fenvalerate.

Energy Metabolism Pattern in Cotton Aphid, Aphis gossypii Glover （Hem.： Aphididae） Exposure to Neonicotinoids

This study was mainly made on the role of energy revisers in cotton aphid, Aphis gossypii Glover （Hemiptera： Aphididae） countered with imidacloprid and thiametoxam. We measured used energy, available energy and the content of total energy in three strains of cotton aphid which were from very resistant to neonicotinoid, sup to semi-sensitive and sensitive speccies, respectively. It was observed changes in energy resources rate in all of the aphid strains at which each substrate was metabolized under stress. Our findings indicated among energy sources, total lipid in susceptible strain was increased in counter of spraying and it was decreased in resistance and semi sensitive strains. Whereas, total protein was decreased in all of the strains encountering with neonicotiniod stress. Total glycogen was increased significantly in different aphid strains and sugar was decreased in countered of spraying.

Energy Intensity and Fitness Cost of Cotton aphid Exposure to Neonicotinoids

Three populations of Aphis gossypii of distinct susceptibility to neonicotinoids show differences in the accumulation and mobilization of energy reserves, what may allow the production of their defensive tools against two neonicotinoid insecticides without impairing their reproductive performance. Bioassays were also carried out for these aphid populations. According to the results obtained, the Ag-R population showed resistant rate significantly higher than the other two populations. Levels of some biochemical characteristics were determined in the resistant, susceptible and semi susceptible populations of cotton aphid. The results obtained in the assays with biochemical parameters indicated significant differences in activity among the populations, with higher activity in using glycogen in the Ag-R population. The inverse activity trends oft （generation time） and DT （doubling time） in both resistant and susceptible populations, one showing fitness disadvantage without insecticide exposure and the other not showing it, may underlay the mitigation of insecticide resistance physiological costs observed in the Ag-R population.

Strigolactones modulate cotton fiber elongation and secondary cell wall thickening

Cotton is one of the most important economic crops in the world,and it is a major source of fiber in the textile industry.Strigolactones(SLs)are a class of carotenoid-derived plant hormones involved in many processes of plant growth and development,although the functions of SL in fiber development remain largely unknown.Here,we found that the endogenous SLs were significantly higher in fibers at 20 days post-anthesis(DPA).Exogenous SLs significantly increased fiber length and cell wall thickness.Furthermore,we cloned three key SL biosynthetic genes,namely GhD27,GhMAX3,and GhMAX4,which were highly expressed in fibers,and subcellular localization analyses revealed that GhD27,GhMAX3,and GhMAX4 were localized in the chloroplast.The exogenous expression of GhD27,GhMAX3,and GhMAX4 complemented the physiological phenotypes of d27,max3,and max4 mutations in Arabidopsis,respectively.Knockdown of GhD27,GhMAX3,and GhMAX4 in cotton resulted in increased numbers of axillary buds and leaves,reduced fiber length,and significantly reduced fiber thickness.These findings revealed that SLs participate in plant growth,fiber elongation,and secondary cell wall formation in cotton.These results provide new and effective genetic resources for improving cotton fiber yield and plant architecture.

Pectin methylesterase inhibitors GhPMEI53 and AtPMEI19 improve seed germination by modulating cell wall plasticity in cotton and Arabidopsis

The germination process of seeds is influenced by the interplay between two opposing factors,pectin methylesterase(PME)and pectin methylesterase inhibitor(PMEI),which collectively regulate patterns of pectin methylesterification.Despite the recognized importance of pectin methylesterification in seed germination,the specific mechanisms that govern this process remain unclear.In this study,we demonstrated that the overexpression of GhPMEI53is associated with a decrease in PME activity and an increase in pectin methylesterification.This leads to seed cell wall softening,which positively regulates cotton seed germination.AtPMEI19,the homologue in Arabidopsis thaliana,plays a similar role in seed germination to GhPMEI53,indicating a conserved function and mechanism of PMEI in seed germination regulation.Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.Additionally,the pathways of abscicic acid(ABA)and gibberellin(GA)in the transgenic materials showed significant changes,suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.In summary,GhPMEI53 and its homologs alter the mechanical properties of cell walls,which influence the mechanical resistance of the endosperm or testa.Moreover,they impact cellular phytohormone pathways(e.g.,ABA and GA)to regulate seed germination.These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction,and contribute to a more comprehensive understanding of the PME/PMEI gene superfamily in plants.

GbLMI1 over-expression improves cotton aboveground vegetative growth

Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

Cotton pads-derived carbon materials/reduced graphene oxide modified with polypyrrole for electrode of supercapacitors

This study investigates the influence of electropolymerization conditions on the deposition of polypyrrole(PPy)onto cotton-derived carbon fiber(CF)modified with reduced graphene oxide(rGO)for supercapacitors applications using an experimental/theorical approach.The surface modification of CF by rGO and/or by PPy electrodeposited at 10,25 and 50 mV s^(-1) was thoroughly examined physicochemical and electrochemically.Composite electrodes comprising CF-rGo-PPy,synthesized via electropolymerization at 25 mV s^(-1),demonstrated a remarkable increase in capacitance,showcasing~742 F g^(-1) compared to 153 F g^(-1) for CF.SEM,N_(2)-surface area,XPS,and TD-DFT approach revealed that the higher capacitance observed in CF-rGo-PPy electrodes underscores the influence of morphology and charged nitrogen species on the electrochemical performance of these modified electrodes.Notably,this electrode material achieves a specific capacitance retention of~96%of their initial capacitance after 10000 cycles at 0.5 A g^(-1) measured in a two-electrodes cell configuration.This work also discusses the influence of the scan rate used for pyrrole electropolymerization on the pseudocapacitance contribution of PPy and its possible effect on the porosity of the material.These results highlight the importance of appropriate electropolymerization conditions that allow obtaining the synergistic effect between CF,rGO and PPy.

Exogenous melatonin improves cotton yield under drought stress by enhancing root development and reducing root damage

The exogenous application of melatonin by the root drenching method is an effective way to improve crop drought resistance.However,the optimal concentration of melatonin by root drenching and the physiological mechanisms underlying melatonin-induced drought tolerance in cotton(Gossypium hirsutum L.)roots remain elusive.This study determined the optimal concentration of melatonin by root drenching and explored the protective effects of melatonin on cotton roots.The results showed that 50μmol L-1 melatonin was optimal and significantly mitigated the inhibitory effect of drought on cotton seedling growth.Exogenous melatonin promoted root development in drought-stressed cotton plants by remarkably increasing the root length,projected area,surface area,volume,diameter,and biomass.Melatonin also mitigated the drought-weakened photosynthetic capacity of cotton and regulated the endogenous hormone contents by regulating the relative expression levels of hormone-synthesis genes under drought stress.Melatonin-treated cotton seedlings maintained optimal enzymatic and non-enzymatic antioxidant capacities,and produced relatively lower levels of reactive oxygen species and malondialdehyde,thus reducing the drought stress damage to cotton roots(such as mitochondrial damage).Moreover,melatonin alleviated the yield and fiber length declines caused by drought stress.Taken together,these findings show that root drenching with exogenous melatonin increases the cotton yield by enhancing root development and reducing the root damage induced by drought stress.In summary,these results provide a foundation for the application of melatonin in the field by the root drenching method.

Identification of candidate genes for aphid resistance in upland cotton by QTL mapping and expression analysis

Lignin is one of the main components of cell walls and is essential for resistance to insect pests in plants.Cotton plants are damaged by aphid(Aphis gossypii) worldwide but resistant breeding is undeveloped due to scarce knowledge on resistance genes and the mechanism. This study reported a lignin biosynthesisrelated gene identified in the F_(2) population derived from the cross between cotton cultivars Xinluzao 61(resistant to aphid) and Xinluzao 50(susceptible to aphid). A quantitative trait locus was mapped on chromosome D04 with a logarithm of odds(LOD) score of 5.99 and phenotypic effect of 27%. RNA-seq analysis of candidate intervals showed that the expression level of GH_D04G1418 was higher in the resistant cultivar than in the susceptible cultivar. This locus is close to AtLAC4 in the phylogenetic tree and contains a conserved laccase domain. Hence, it was designated GhLAC4-3. Silencing of GhLAC4-3 in Xinluzao 61 via virus-induced gene silencing(VIGS) resulted in decreased lignin content and increased susceptibility to aphids. These results suggest that GhLAC4-3 might enhance aphid resistance by regulating lignin biosynthesis in cotton.

Enhancing cotton resilience to challenging climates through genetic modifications

Cotton is one of the most important fiber crops that plays a vital role in the textile industry.Its production has been unstable over the years due to climate change induced biotic stresses such as insects,diseases,and weeds,as well as abiotic stresses including drought,salinity,heat,and cold.Traditional breeding methods have been used to breed climate resilient cotton,but it requires a considerable amount of time to enhance crop tolerance to insect pests and changing climatic conditions.A promising strategy for improving tolerance against these stresses is genetic engineering.This review article discusses the role of genetic engineering in cotton improvement.The essential concepts and techniques include genome editing via clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(CRISPR-Cas9),overexpression of target genes,downregulation using RNA interference(RNAi),and virus-induced gene silencing(VIGS).Notably,the Agrobacterium-mediated transformation has made significant contributions to using these techniques for obtaining stable transgenic plants.

Identification of candidate genes for early-maturity traits by combining BSA-seq and QTL mapping in upland cotton(Gossypium hirsutum L.)

Cotton breeding for the development of early-maturing varieties is an effective way to improve multiple cropping indexes and alleviate the conflict between grains and cotton in the cultivated fields in China.In the present study,we aimed to identify upland cotton quantitative trait loci(QTLs)and candidate genes related to early-maturity traits,including whole growth period(WGP),flowering timing(FT),node of the first fruiting branch(NFFB),height of the node of the first fruiting branch(HNFFB),and plant height(PH).An early-maturing variety,CCRI50,and a latematuring variety,Guoxinmian 11,were crossed to obtain biparental populations.These populations were used to map QTLs for the early-maturity traits for two years(2020 and 2021).With BSA-seq analysis based on the data of population 2020,the candidate regions related to early maturity were found to be located on chromosome D03.We then developed 22 polymorphic insertions or deletions(InDel)markers to further narrow down the candidate regions,resulting in the detection of five and four QTLs in the 2020 and 2021 populations,respectively.According to the results of QTL mapping,two candidate regions(InDel_G286-InDel_G144 and InDel_G24-InDel_G43)were detected.In these regions,three genes(GH_D03G0451,GH_D03G0649,and GH_D03G1180)have nonsynonymous mutations in their exons and one gene(GH_D03G0450)has SNP variations in the upstream sequence between CCRI50 and Guoxinmian 11.These four genes also showed dominant expression in the floral organs.The expression levels of GH_D03G0451,GH_D03G0649 and GH_D03G1180 were significantly higher in CCRI50 than in Guoxinmian 11 during the bud differentiation stages,while GH_D03G0450 showed the opposite trend.Further functional verification of GH_D03G0451 indicated that the GH_D03G0451-silenced plants showed a delay in the flowering time.The results suggest that these are the candidate genes for cotton early maturity,and they may be used for breeding early-maturity cotton varieties.

GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC(C/T)to orchestrate cotton resistance to Verticillium dahliae

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality.Therefore,the mining of V.dahlia-resistance genes is urgently needed.Proteases and protease inhibitors play crucial roles in plant defense responses.However,the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown.This study provides a comprehensive analysis of the PR6 gene family in the cotton genome.We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family,which belongs to the potato protease inhibitor I family of inhibitors.Thirty-nine PR6s were identified in Gossypium arboreum,G.raimondii,G.barbadense,and G.hirsutum,and they were clustered into four groups.Based on the analysis of pathogen-induced and Ghlmm transcriptome data,Gh PR6-5b was identified as the key gene for V.dahliae resistance.Virus-induced gene silencing experiments revealed that cotton was more sensitive to V.dahliae V991after PR6-5b silencing.The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C).Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V.dahliae,and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V.dahliae.

Cotton ethylene response factor Gh ERF91 is involved in the defense against Verticillium dahliae

Verticillium dahliae causes significant losses in cotton production.To reveal the mechanism of the defense response to V.dahliae in cotton,transcriptomic analyses were performed using cotton cultivars M138(V.dahliae-resistant)and P2(V.dahliae-susceptible).The results revealed 11,076 and 6,640 differentially expressed genes(DEGs)in response to V.dahliae,respectively.The weighted gene co-expression network analysis of 4,633 transcription factors(TFs)indicated a“MEblue”module containing 654 TFs that strongly correlate with resistance to V.dahliae.Among these TFs,the ethylene response factor Ghi_A05G10166(GhERF91)was identified as a putative hub gene with a defense response against V.dahliae.A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V.dahliae exposure in cotton.This study provides fundamental transcriptome data and a putative causal gene(GhERF91)associated with resistance to V.dahliae,as well as genetic resources for breeding V.dahliae-resistant cotton.

Mining elite loci and candidate genes for root morphology-related traits at the seedling stage by genome-wide association studies in upland cotton(Gossypium hirsutum L.)

Root system architecture plays an essential role in water and nutrient acquisition in plants,and it is significantly involved in plant adaptations to various environmental stresses.In this study,a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage,including main root length(MRL),root fresh weight(RFW),total root length(TRL),root surface area(RSA),root volume(RV),and root average diameter(AvgD).The correlation analysis of the six root morphological traits revealed strong positive correlations of TRL with RSA,as well as RV with RSA and AvgD,whereas a significant negative correlation was found between TRL and AvgD.Subsequently,a genome-wide association study(GWAS)was performed using the root phenotypic and genotypic data reported previously for the 242 accessions using 56,010 single nucleotide polymorphisms(SNPs)from the CottonSNP80K array.A total of 41 quantitative trait loci(QTLs)were identified,including nine for MRL,six for RFW,nine for TRL,12 for RSA,12 for RV and two for AvgD.Among them,eight QTLs were repeatedly detected in two or more traits.Integrating these results with a transcriptome analysis,we identified 17 candidate genes with high transcript values of transcripts per million(TPM)≥30 in the roots.Furthermore,we functionally verified the candidate gene GH_D05G2106,which encodes a WPP domain protein 2in root development.A virus-induced gene silencing(VIGS)assay showed that knocking down GH_D05G2106significantly inhibited root development in cotton,indicating its positive role in root system architecture formation.Collectively,these results provide a theoretical basis and candidate genes for future studies on cotton root developmental biology and root-related cotton breeding.

Single‑cell RNA sequencing opens a new era for cotton genomic research and gene functional analysis

Single-cell RNA sequencing(scRNA-seq)is one of the most advanced sequencing technologies for studying transcriptome landscape at the single-cell revolution.It provides numerous advantages over traditional RNA-seq.Since it was first used to profile single-cell transcriptome in plants in 2019,it has been extensively employed to perform different research in plants.Recently,scRNA-seq was also quickly adopted by the cotton research community to solve lots of scientific questions which have been never solved.In this comment,we highlighted the significant progress in employing scRNA-seq to cotton genetic and genomic study and its future potential applications.