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.

Genetic dissection and origin of pleiotropic loci underlying multilevel fiber quality traits in upland cotton(Gossypium hirsutum L.)

Cotton fiber quality is a persistent concern that determines planting benefits and the quality of finished textile products.However,the limitations of measurement instruments have hindered the accurate evaluation of some important fiber characteristics such as fiber maturity,fineness,and neps,which in turn has impeded the genetic improvement and industrial utilization of cotton fiber.Here,12 single fiber quality traits were measured using Advanced Fiber Information System(AFIS)equipment among 383 accessions of upland cotton(Gossypium hirsutum L.).In addition,eight conventional fiber quality traits were assessed by the High Volume Instrument(HVI)System.Genome-wide association study(GWAS),linkage disequilibrium(LD)block genotyping and functional identification were conducted sequentially to uncover the associated elite loci and candidate genes of fiber quality traits.As a result,the previously reported pleiotropic locus FL_D11 regulating fiber length-related traits was identified in this study.More importantly,three novel pleiotropic loci(FM_A03,FF_A05,and FN_A07)regulating fiber maturity,fineness and neps,respectively,were detected based on AFIS traits.Numerous highly promising candidate genes were screened out by integrating RNA-seq and qRT-PCR analyses,including the reported GhKRP6 for fiber length,the newly identified GhMAP8 for maturity and GhDFR for fineness.The origin and evolutionary analysis of pleiotropic loci indicated that the selection pressure on FL_D11,FM_A03 and FF_A05 increased as the breeding period approached the present and the origins of FM_A03 and FF_A05 were traced back to cotton landraces.These findings reveal the genetic basis underlying fiber quality and provide insight into the genetic improvement and textile utilization of fiber in G.hirsutum.

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.

Knock-out of GhPDCT with the CRISPR/Cas9 system increases the oleic acid content in cottonseed oil

Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil(Long et al.2023).Cottonseed oil contains approximately14%oleic acid and 59%linoleic acid.An increase in monounsaturated fatty acids,particularly oleic acid,enhances the oxidative stability and nutritional value of edible oil(Chen et al.2021).

EPSPS regulates cell elongation by disrupting the balance of lignin and flavonoid biosynthesis in cotton

EPSPS is a key gene in the shikimic acid synthesis pathway that has been widely used in breeding crops with herbicide resistance.However,its role in regulating cell elongation is poorly understood.Through the overexpression of EPSPS genes,we generated lines resistant to glyphosate that exhibit an unexpected dwarf phenotype.A representative line,DHR1,exhibits a stable dwarf phenotype throughout its entire growth period.Except for plant height,the other agronomic traits of DHR1 are similar to its transgenic explants ZM24.Paraffin section observations showed that DHR1 internodes are shortened due to reduced elongation and division of the internode cells.Exogenous hormones confirmed that DHR1 is not a classical brassinolide(BR)-or gibberellin(GA)-related dwarfing mutant.Hybridization analysis and fine mapping confirmed that the EPSPS gene is the causal gene for dwarfism,and the phenotype can be inherited in different genotypes.Transcriptome and metabolome analyses showed that genes associated with the phenylpropanoid synthesis pathway are enriched in DHR1 compared with ZM24.Flavonoid metabolites are enriched in DHR1,whereas lignin metabolites are reduced.The enhancement of flavonoids likely results in differential expression of auxin signal pathway genes and alters the auxin response,subsequently affecting cell elongation.This study provides a new strategy for generating dwarfs and will accelerate advancements in light simplification in the cultivation and mechanized harvesting of cotton.

GhWDL3 is involved in the formation and development of fiber cell morphology in upland cotton(Gossypium hirsutum L.)

Background Cotton fiber is a model tissue for studying microtubule-associated proteins(MAPs).The Xklp2(TPX2)proteins that belong to the novel MAPs member mainly participate in the formation and development of microtubule(MT).However,there is a lack of studies concerning the systematic characterization of the TPX2 genes family in cotton.Therefore,the identification and portrayal of G.hirsutum TPX2 genes can provide key targets for molecular manipula-tion in the breeding of cotton fiber improvement.Result In this study,TPX2 family genes were classified into two distinct subclasses TPXLs and MAP genes WAVE DAMP-ENED2-LIKE(WDLs)and quite conservative in quantity.GhWDL3 was significantly up-regulated in 15 days post anthe-sis fibers of ZRI-015(an upland cotton with longer and stronger fiber).GhWDL3 promotes all stem hairs to become straight when overexpressed in Arabidopsis,which may indirectly regulate cotton fiber cell morphology during fiber development.Virus induced gene silencing(VIGS)results showed that GhWDL3 inhibited fiber cell elongation at fiber development periods through regulating the expression of cell wall related genes.Conclusion These results reveal that GhWDL3 regulated cotton fiber cell elongation and provide crucial information for the further investigation in the regulatory mechanisms/networks of cotton fiber length.

Genome-wide identification and expression profiling of photosystem II(PsbX)gene family in upland cotton(Gossypium hirsutum L.)

Background Photosystem II(PSII)constitutes an intricate assembly of protein pigments,featuring extrinsic and intrinsic polypeptides within the photosynthetic membrane.The low-molecular-weight transmembrane protein PsbX has been identified in PSII,which is associated with the oxygen-evolving complex.The expression of PsbX gene protein is regulated by light.PsbX’s central role involves the regulation of PSII,facilitating the binding of quinone molecules to the Qb(PsbA)site,and it additionally plays a crucial role in optimizing the efficiency of photosynthesis.Despite these insights,a comprehensive understanding of the PsbX gene’s functions has remained elusive.Results In this study,we identified ten PsbX genes in Gossypium hirsutum L.The phylogenetic analysis results showed that 40 genes from nine species were classified into one clade.The resulting sequence logos exhibited substantial conservation across the N and C terminals at multiple sites among all Gossypium species.Furthermore,the ortholo-gous/paralogous,Ka/Ks ratio revealed that cotton PsbX genes subjected to positive as well as purifying selection pressure might lead to limited divergence,which resulted in the whole genome and segmental duplication.The expression patterns of GhPsbX genes exhibited variations across specific tissues,as indicated by the analysis.Moreover,the expression of GhPsbX genes could potentially be regulated in response to salt,intense light,and drought stresses.Therefore,GhPsbX genes may play a significant role in the modulation of photosynthesis under adverse abiotic conditions.Conclusion We examined the structure and function of PsbX gene family very first by using comparative genom-ics and systems biology approaches in cotton.It seems that PsbX gene family plays a vital role during the growth and development of cotton under stress conditions.Collectively,the results of this study provide basic information to unveil the molecular and physiological function of PsbX genes of cotton plants.

Mepiquat chloride priming confers the ability of cotton seed to tolerate salt by promoting ABA-operated GABA signaling control of the ascorbate–glutathione cycle

Background Ensuring that seeds germinate and emerge normally is a prerequisite for cotton production,esp.in areas with salinized soil.Priming with mepiquat chloride(MC)can promote seed germination and root growth under salt stress,but its mechanism has not been fully elucidated.In this study,physiological and biochemical experiments revealed that MC-priming promotes the tolerance of cotton seeds to salt stress by increasing the ability of antioxidant enzymes related to the ascorbate-glutathione(AsA-GSH)cycle to scavenge reactive oxygen species(ROS).Results Results revealed that treatment with inhibitors of abscisic acid(ABA)and γ-aminobutyric acid(GABA)biosynthesis reduced the positive effects of MC-priming.Similarly,MC-priming increased the contents of ABA and GABA under salt stress by stimulating the expression levels of GhNCED2 and GhGAD4 and the activity of calmodulin-binding(CML)glutamate decarboxylase(GAD).Further analysis showed that an inhibitor of ABA synthesis reduced the positive impacts of MC-priming on the content of GABA under salt stress,but the content of ABA was not affected by the GABA synthesis inhibitor.Furthermore,a multi-omics analysis revealed that MC-priming increased the abundance and phosphorylation levels of the proteins related to ABA signaling,CML,and Ca^(2+)channels/transporters in the MC-primed treatments,which resulted in increased oscillations in Ca^(2+)in the MC-primed cotton seeds under salt stress.Conclusion In summary,these results demonstrate that MC-mediated ABA signaling operates upstream of the GABA synthesis generated by GAD by activating the oscillations of Ca^(2+)and then enhancing activity of the AsA-GSH cycle,which ensures that cotton seeds are tolerant to salt stress.

Morphological characteristics,developmental dynamics,and gene temporal expressions across various development stages of Aphis gossypii sexual female

Background Aphis gossypii(Hemiptera:Aphididae)is a worldwide polyphagous phloem-feeding agricultural pest,and it can produce offspring by sexual or asexual reproduction.Compared with dozens of generations by parthenogenesis,sexual reproduction is performed in only one generation within one year,and little is known about the sexual reproduction of A.gossypii.In this study,sexual females of A.gossypii were successfully obtained through a previously established induction platform,and the morphological characteristics,developmental dynamics,and temporal gene expression were examined.Subsequently,signaling pathways potentially involved in regulating the growth,development,and reproduction of sexual females were investigated.Results The morphological observation showed that from the 1st instar nymph to adult,sexual females exhibited a gradually deepened body color,an enlarged body size,longer antennae with a blackened end,and obviously protruding cauda(in adulthood).The anatomy found that the ovaries of sexual females developed rapidly from the 2^(nd)instar nymph,and the embedded oocytes matured in adulthood.In addition,time-course transcriptome analysis revealed that gene expression profiles across the development of sexual females fell into 9 clusters with distinct patterns,in which gene expression levels in clusters 1,5,and 8 peaked at the 2^(nd)instar nymphal stage with the largest number of up-regulated genes,suggesting that the 2^(nd)instar nymph was an important ovary development period.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis revealed that a large number of genes in the sexual female adult were enriched in the TGF-beta signaling pathway and Forkhead box O(FoxO)signaling pathway,highlighting their important role in sexual female adult development and reproduction.Conclusion The morphological changes of the sexual female at each developmental stage were revealed for the first time.In addition,time-course transcriptomic analyses suggest genes enriched in the TGF-beta signaling pathway and FoxO signaling pathway probably contribute to regulating the development and oocyte maturation of sexual females.Overall,these findings will facilitate the regulating mechanism research in the growth and development of sexual females by providing candidate genes.

Strigolactone-gibberellin crosstalk mediated by a distant silencer fine-tunes plant height in upland cotton

Optimal plant height is crucial in modern agriculture, influencing lodging resistance and facilitating mechanized crop production. Upland cotton (Gossypium hirsutum) is the most important fiber crop globally;however, the genetic basis underlying plant height remains largely unexplored. In this study, we conducted a genome-wide association study to identify a major locus controlling plant height (PH1) in upland cotton. This locus encodes gibberellin 2-oxidase 1A (GhPH1) and features a 1133-bp structural variation (PAVPH1) located approximately 16 kb upstream. The presence or absence of PAVPH1 influences the expression of GhPH1, thereby affecting plant height. Further analysis revealed that a gibberellin-regulating transcription factor (GhGARF) recognizes and binds to a specific CATTTG motif in both the GhPH1 promoter and PAVPH1. This interaction downregulates GhPH1, indicating that PAVPH1 functions as a distant upstream silencer. Intriguingly, we found that DWARF53 (D53), a key repressor of the strigolactone (SL) signaling pathway, directly interacts with GhGARF to inhibit its binding to targets. Moreover, we identified a previously unrecognized gibberellin-SL crosstalk mechanism mediated by the GhD53-GhGARF-GhPH1/PAVPH1 module, which is crucial for regulating plant height in upland cotton. These findings shed light on the genetic basis and gene interaction network underlying plant height, providing valuable insights for the development of semi-dwarf cotton varieties through precise modulation of GhPH1 expression.

CRISPR-Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca2+in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Widespread incomplete lineage sorting and introgression shaped adaptive radiation in the Gossypium genus

Cotton(Gossypium)stands as a crucial economic crop,serving as the primary source of naturalfiber for the textile sector.However,the evolutionary mechanisms driving speciation within the Gossypium genus remain unresolved.In this investigation,we leveraged 25 Gossypium genomes and introduced four novel assem-blies—G.harknessii,G.gossypioides,G.trilobum,and G.klotzschianum(Gklo)—to delve into the speciation history of this genus.Notably,we encountered intricate phylogenies potentially stemming from introgres-sion.These complexities are further compounded by incomplete lineage sorting(ILS),a factor likely to have been instrumental in shaping the swift diversification of cotton.Our focus subsequently shifted to the rapid radiation episode during a concise period in Gossypium evolution.For a recently diverged lineage comprising G.davidsonii,Gklo,and G.raimondii,we constructed afinely detailed ILS map.Intriguingly,this analysis revealed the non-random distribution of ILS regions across the reference Gklo genome.Moreover,we identified signs of robust natural selection influencing specific ILS regions.Noteworthy variations per-taining to speciation emerged between the closely related sister species Gklo and G.davidsonii.Approxi-mately 15.74%of speciation structural variation genes and 12.04%of speciation-associated genes were esti-mated to intersect with ILS signatures.Thesefindings enrich our understanding of the role of ILS in adaptive radiation,shedding fresh light on the intricate speciation history of the Gossypium genus.

Anchorene,a carotenoid-derived growth regulator,modulates auxin homeostasis by suppressing GH3-mediated auxin conjugation

Anchorene,identified as an endogenous bioactive carotenoid-derived dialdehyde and diapocarotenoid,affects root development by modulating auxin homeostasis.However,the precise interaction between anchorene and auxin,as well as the mechanisms by which anchorene modulates auxin levels,remain largely elusive.In this study,we conducted a comparative analysis of anchorene's bioactivities alongside auxin and observed that anchorene induces multifaceted auxin-like effects.Through genetic and pharmacological examinations,we revealed that anchorene's auxin-like activities depend on the indole-3-pyruvate-dependent auxin biosynthesis pathway,as well as the auxin inactivation pathway mediated by Group II Gretchen Hagen 3(GH3)proteins that mainly facilitate the conjugation of indole-3-acetic acid(IAA)to amino acids,leading to the formation of inactivated storage forms.Our measurements indicated that anchorene treatment elevates IAA levels while reducing the quantities of inactivated IAA–amino acid conjugates and ox IAA.RNA sequencing further revealed that anchorene triggers the expression of numerous auxin-responsive genes in a manner reliant on Group II GH3s.Additionally,our in vitro enzymatic assays and biolayer interferometry(BLI)assay demonstrated anchorene's robust suppression of GH3.17-mediated IAA conjugation with glutamate.Collectively,our findings highlight the significant role of carotenoid-derived metabolite anchorene in modulating auxin homeostasis,primarily through the repression of GH3-mediated IAA conjugation and inactivation pathways,offering novel insights into the regulatory mechanisms of plant bioactive apocarotenoids.

Single-cell RNA sequencing reveals a hierarchical transcriptional regulatory network of terpenoid biosynthesis in cotton secretory glandular cells

Plants can synthesize a wide range of terpenoids in response to various environmental cues.However,the specific regulatory mechanisms governing terpenoid biosynthesis at the cellular level remain largely elusive.In this study,we employed single-cell RNA sequencing to comprehensively characterize the transcriptome profile of cotton leaves and established a hierarchical transcriptional network regulating cellspecific terpenoid production.We observed substantial expression levels of genes associated with the biosynthesis of both volatile terpenes(such asβ-caryophyllene andβ-myrcene)and non-volatile gossypol-type terpenoids in secretory glandular cells.Moreover,two novel transcription factors,namely GoHSFA4a and GoNAC42,are identified to function downstream of the Gossypium PIGMENT GLAND FORMATION genes.Both transcription factors could directly regulate the expression of terpenoid biosynthetic genes in secretory glandular cells in response to developmental and environmental stimuli.For convenient retrieval of the single-cell RNA sequencing data generated in this study,we developed a user-friendly web server.Our findings not only offer valuable insights into the precise regulation of terpenoid biosynthesis genes in cotton leaves but also provide potential targets for cotton breeding endeavors.

Hydrogen peroxide mediates high-intensity blue light-induced hypocotyl phototropism of cotton seedlings

Phototropism is a classic adaptive growth response that helps plants to enhance light capture for photosynthesis.It was shown that hydrogen peroxide(H_(2)O_(2))participates in the regulation of blue light-induced hypocotyl phototropism;however,the underlying mechanism is unclear.In this study,we demonstrate that the unilateral high-intensity blue light(HBL)could induce asymmetric distribution of H_(2)O_(2) in cotton hypocotyls.Disruption of the HBL-induced asymmetric distribution of H_(2)O_(2) by applying either H_(2)O_(2) itself evenly on the hypocotyls or H_(2)O_(2) scavengers on the lit side of hypocotyls could efficiently inhibit hypocotyl phototropic growth.Consistently,application of H_(2)O_(2) on the shaded and lit sides of the hypocotyls led to reduced and enhanced hypocotyl phototropism,respectively.Further,we show that H_(2)O_(2) inhibits hypocotyl elongation of cotton seedlings,thus supporting the repressive role of H_(2)O_(2) in HBL-induced hypocotyl phototropism.Moreover,our results show that H_(2)O_(2) interferes with HBL-induced asymmetric distribution of auxin in the cotton hypocotyls.Taken together,our study uncovers that H_(2)O_(2) changes the asymmetric accumulation of auxin and inhibits hypocotyl cell elongation,thus mediating HBL-induced hypocotyl phototropism.