Hunhun Wu,Zhongying Ren,Lei Zheng,Mengzhen Guo,Jingyu Yng,Liyong Hou,Ghulm Qnmber,Fugung Li,,*,Zuoren Yng,,*

a State Key Laboratory of Cotton Biology,Cotton Research Institute of Chinese Academy of Agricultural Sciences,Anyang 455000,Henan,China

b National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,Hubei,China

c Zhengzhou Research Base,State Key Laboratory of Cotton Biology,Zhengzhou University,Zhengzhou 450001,Henan,China

Keywords:Cotton fiber Basic helix-loop-helix GhPAS1 Plant architecture Virus induced gene silencing

ABSTRACT 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(bHLH)transcription factor,GhPAS1(PAGODA1 SUPPRESSOR 1)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 bHLH transcription factor,regulates plant development and architecture in cotton.These findings may help breeders and researchers develop cotton cultivars with desirable agronomic characteristics.

1.Introduction

Brassinosteroids (BRs) are a class of plant-specific steroid phytohormones that play essential roles in plant growth and development by regulating cell elongation,cell division,and cell differentiation [1–3].BRs are perceived at membrane-localized receptors,and downstream cytoplasmic regulators transduce BR-mediated signals to the nucleus,thereby activating the transcription of BR-responsive genes that direct cell growth [4,5].InArabidopsis,CPD(CONSTITUTIVE PHOTOMORPHOGENIC DWARF)andDWF4(DWARF4) are rate-limiting enzyme genes in the BR biosynthesis pathway,encoding respectively C-23 hydroxylase(CYP90A1) [6] and C-22 hydroxylase (CYP90B1) [7],and BRs regulate their transcription.Mutations inAtCPDandAtDWF4result in abnormal phenotypes,such as dwarfing,round and dark green leaves,de-etiolation,and lower fertility [8–10].BRI1,a receptor kinase in the BR signal transduction pathway,binds directly to BR ligands and triggers a signal cascade in the cytoplasm that drives cell growth [11,12].bri1mutants showed severe BRdefective phenotypes,characterized by dark green and curled leaves,short petioles,delayed flowering time,and senescence[13],and exogenous application of BL restored the mutant phenotype.Previous studies [14–17] indicated that BRs interact with other phytohormones or environmental signals to coordinate plant growth and development.

Plant architecture refers to the three-dimensional structure of a plant and is determined mainly by factors affecting branches,inflorescence morphology,and plant height[18].Many genes that regulate plant architecture have been identified in various species[18–20].For instance,AtREV(REVOLUTE) initiates lateral meristem development and activates the expression of known meristem regulators inArabidopsis[21].Similarly,OsTB1(TEOSINTE BRANCHED 1)is expressed in axillary buds and negatively regulates lateral branches in rice [22].Down-regulation ofOsMADS18led to delayed seed germination and seedling growth,whereasOsMADS18overexpression produced plants with fewer tillers[23].Plant architecture is determined by the complex interactions of genes,proteins,and metabolite systems[24–26].DELLA proteins such asAtSLR1(SOLITARY ROOT),AtGAI(GIBBERELLIC ACID INSENSITIVE),wheatRHT(REDUCED HEIGHT),and maizeD8(DWARF8)[27–29]are negative regulators of the gibberellin(GA)signal transduction pathway and control plant height.In rice,overexpression of OsMIR396d produced a semi-dwarf phenotype with increased leaf angle.OsMIR396d participates in the interaction network of the BR and GA signal transduction pathways,and its overexpression increases BR signal transduction,however,it damages the biosynthesis and signal transduction pathway of GA[30].Thus,BR and GA may control plant architecture at multiple levels.

With agricultural modernization and cotton industrialization,short-season adaptation with ideal plant architecture suitable for mechanical harvesting are primary research goals[31,32].The main stem of cotton is monopodial and remains indeterminate during growth and development.Lateral branches grow indefinitely from leaf axils,and then develop and differentiate into sympodial fruiting branches or monopodial vegetative branches[33].Previous studies[34,35]have shown thatFT(FLOWERING LOCUS T)and its antagonistTFL1(TERMINAL FLOWER1)function directly in controlling the balance between determinate and indeterminate growth in barley and tomato.GhFTregulates flower transition in upland cotton and overexpression ofGhFTinArabidopsismediates early flowering,which partially rescues the extremely late flowering phenotype of theft-10mutant [36].SilencingGhFTresults in more vegetative branches and delays flowering in cotton [37].GhHB1is highly expressed in the buds of cotton vegetative branches,delaying flowering and promoting bushy architecture.GhHB1also inhibits the expression ofGhFTand thereby changes cotton photoperiod sensitivity and plant architecture[38].Although much progress has been made in understanding cotton development and architecture,the molecular mechanisms of cotton architecture still need further research.

GhPAG1(PAGODA1) is a homolog of the BR biosynthesis geneCYP734A1and regulates endogenous BRs.Thepag1mutant shows dwarfed plant architecture and short fiber length.However,exogenous application of BR partially restores plant height and short fiber phenotype [39].For this reason,thepag1mutant is considered an ideal genetic material for studying cotton growth and development.In a previous study [40],we used a full-length cDNA-overexpressing gene hunting system inArabidopsisas a heterologous host to identify the functions of cotton genes.We found that a new basic helix-loop-helix (bHLH)transcription factor partially restored the phenotype of the BRdeficient mutantbri1-5.Because subsequent experiments (unpublished) showed that it also partially restored the dwarf and short fiber phenotype of thepag1mutant,we named it GhPAS1(PAGODA1 SUPPRESSOR 1).

In the present study,heterologous overexpression of GhPAS1 inArabidopsisand a BRZ treatment experiment indicated that GhPAS1 is a positive regulator that responds to BR signals and promotes growth and development.Slencing GhPAS1 inhibited plant growth and development and altered plant architecture,reducing plant height,fruit branch length,and boll size.GhPAS1-silenced plants exhibited reduced development of cotton fiber and seed.In summary,GhPAS1 regulates plant development and architecture in cotton.These findings will provide a theoretical basis for cotton genetic improvement and breeding.

2.Materials and methods

2.1.Plant materials and growth conditions

Arabidopsis thalianaecotype Col-0 (WT,wild type),tobacco(Nicotiana benthamiana),and upland cotton(ZM24)seedlings were grown in a climate-controlled greenhouse maintained at 22± 2 °C under long-day conditions (16 h light/8 h dark) at Zhengzhou Research Base,Cotton Research Institute of Chinese Academy of Agricultural Sciences (Zhengzhou,China).Arabidopsisseeds were sterilized with 75% ethanol for 15 min,washed five times with sterile water,held at 4 °C for 48 h,and evenly sown on halfstrength Murashige and Skoog (1/2 MS) medium containing 0.8%w/v agar.Two weeks later,the seedlings were transplanted to soil(3:1 compost:vermiculite) in the greenhouse.

2.2.Generation of transgenic plants

To generate p35S::GhPAS1-FLAG overexpression plants,the fulllength coding sequence of GhPAS1 was PCR-amplified from ZM24 plants and subcloned into the target vector pCAMBIA2300.The construct ectopically expressed the fusion protein under the control of the cauliflower mosaic virus (CaMV) 35S promoter.To build the promoter-driven reporter construct of GhPAS1(pGhPAS1::GUS),the upstream 2000 bp of the transcriptional start site was PCR-amplified and subcloned into the β-glucuronidase(GUS)-expressing binary vector pBI121.All binary vector constructs were transformed intoAgrobacteriumtumefaciensGV3101 and then introduced intoArabidopsisusing the floral dip method [41].T1seeds were harvested and screened on 1/2 MS agar medium containing 50 μg mL-1kanamycin to select transgenic progeny.After homozygosity was reached in the T3generation,two independent overexpression lines were selected for further analysis.

2.3.Plant phenotype and biomass assessment

Roots and hypocotyls of plants were grown on 1/2 MS agar medium in the presence or absence of indicated concentrations of BL(brassinolide)or BRZ.They were photographed using a Canon camera (EW-83M) and their lengths were measured with Image J software(https://imagej.nih.gov/ij/).Hypocotyl lengths were measured from the hypocotyl base to the apical hook and root lengths were measured from the root tip to the hypocotyl base.The lengths of hypocotyl cells were measured with a scanning electron microscope(SU3500,Hitachi,Japan).Each experiment had at least three biological replicates,and more than 20 seedlings were measured for each line.

For phenotypic analysis and whole-plant biomass determination,seedlings of each line were grown in plastic pots containing soil.Four-week-old rosettes and detached leaves were photographed to measure the rosette diameter and the area of the eighth true leaves using Image J software.Plant height was measured from the stem base to the peak of mature plants.Sevenweek-old plants were used to record the number of siliques per plant,silique lengths,and seed number per silique.The fresh weight of seven-week-old plants was determined for each line,and plants were fully dried at 75°C for 24 h to measure dry weight.A total of 1000 mature seeds were counted and weighed to obtain 1000-seed weight.Three biological replicates were used in each experiment,and more than 20 plants were measured in each repeat.

2.4.Subcellular localization and histochemical GUS staining assay

The coding sequence of GhPAS1 was subcloned into the binary vector pPZP111 to obtain p35S::GhPAS1-GFP.The constructed vectors were transformed intoA.tumefaciensGV3101 [42].The GV3101 strains were cultured in Luria–Bertani liquid medium containing 50 mg L-1rifampicin and 50 mg L-1chloramphenicol,and then shaken at 28 °C,200 r min-1for about 16 h.The strains were centrifuged at 5000 r min-1for 10 min,and the supernatant was discarded.The strain sediment was resuspended in MMA solution(10 mmol L-1MgCl2,10 mmol L-12-(4-morpholino) ethanesulfonic acid,and 150 μmol L-1acetosyringone).The OD600was adjusted to 1.5 and the resuspended strain solution was incubated at room temperature in the dark for 3 h.Mixed solution was transiently co-expressed in healthy leaves of tobacco via agroinfiltration [43].After infiltration,infected plants were kept in the dark for 16 h and then grown under light conditions.After 48 h,fluorescence signals were detected using confocal laser scanning microscopy (Olympus,FV 1200,Japan).The experiment had three independent biological replicates.

For the histochemical GUS assay,12-day-old seedlings were stained in GUS staining buffer(pH 7.0,0.4 mmol L-1potassium ferricyanide,80 mmol L-1sodium phosphate,0.4 mmol L-1potassium ferrocyanide,0.01% triton X-100,8 mmol L-1sodium-EDTA,0.8 mg mL-15-bromo-4-chloro-3-indolyl-β-d-glucuronide,and 20% methanol) and held at 37 °C in the dark for 8 h.The samples were then rinsed with 75% ethanol at least five times and photographed using a fluorescence stereomicroscope (Leica,M165FC,Germany).

2.5.Transcriptional activation assay of GhPAS1

The coding sequence of GhPAS1 was subcloned into the pGBKT7 vector to obtain the pGBKT7-GhPAS1 construct.Constructed vectors were transformed into yeast strain Y2H Gold competent cells using the lithium acetate method (Clontech,PT1172-1,America).The transformed yeast cells were grown on selection media containing SD/-Trp and SD/-Trp/-His/-Ade at 30 °C in the dark for five days,and then incubated with 20 μg mL-1X-α-gal to form blue clones.pGBKT7-53 was used as a positive control.The growth status of yeast colonies and β-galactosidase activity was used to identify transcription activity.

2.6.Virus-induced gene silencing

Virus-induced gene silencing(VIGS)is a powerful tool for identifying functional genes,especially in plants species that are difficult to transform stably [44,45].In this study,an improved VIGS vector based on cotton leaf crumple virus (CLCrV) [46] was used to silence GhPAS1 continuously and effectively.A silenced fragment of GhPAS1 was PCR-amplified and inserted into pCLCrVA to generate pCLCrV-GhPAS1.Plasmids pCLCrVB,pCLCrVA,and pCLCrV-GhPAS1 were individually introduced intoA.tumefaciensstrain LBA4404 and the culture was pelleted and re-suspended individually in MMA solution as described above.Agrobacteriumharboring pCLCrVA or pCLCrV-GhPAS1 were mixed with an equal volume ofAgrobacteriumharboring pCLCrVB after three hours of incubation at room temperature.Needleless syringes were used to infiltrate mixedAgrobacteriumsolution into the abaxial sides of cotyledons of two-week-old cotton seedlings.Plants injected with empty vector CLCrV were used as control plants (CK).After the plants were cultured continuously for about four weeks,DNA was extracted from young leaves,and positive plants were identified by PCR using the primers VA_F and VA_R of the pCLCrVA vector and VB_F and VB_R of the pCLCrVB vector.Next,total RNA was extracted from young leaves,and the relative expression level of GhPAS1 was detected by RT-qPCR using GhPAS1-qPCR_F and GhPAS1-qPCR_R primers (Table S1) [46].Mature fibers of each silenced plant were harvested and sent to the Center of Cotton Fiber Quality Inspection and Testing,Ministry of Agriculture of China (Anyang,China) for fiber quality identification.Short fibers on the seed surface were removed with sulfuric acid.After drying,a total of 100 cotton seeds were counted and weighed to obtain 100-seed weight.Three biological replicates were used in each experiment,and more than 20 plants were measured in each repeat.

2.7.Total DNA and RNA isolation and RT-qPCR

Total DNA was isolated from approximately 100 mg of young leaves using a plant genomic DNA extraction kit (Bioteke,Beijing,China).Total RNA was extracted from root,stem,leaf,flower,ovule(1,3,5 DPA,days post anthesis),and fiber(7,10,15,20 DPA)tissue from ZM24 using the RNAprep Pure Plant Kit (TianGen,Beijing,China).DNA and RNA were characterized by agarose gel electrophoresis and spectrophotometry.To synthesize the first-strand cDNA,the EasyScript All-in-One First-strand cDNA synthesis SuperMix for RT-qPCR (TransGen,Beijing,China) was used per the manufacturer’s guidelines.cDNA from all samples was adjusted to an equal concentration (0.5–5 ng μL-1).TransStart Top Green qPCR SuperMix kit (TransGen) was used to perform RT-qPCR in a LightCycler 480 (Roche,Switzerland).The RT-qPCR program was 94 °C for 30 s,then 45 cycles of 94 °C for 5 s,and 60 °C for 30 s.Each PCR reaction was performed in triplicate,and three biological replicates were quantified.AtACTIN(AT3G18780.1) andGhHistone3(GenBank accession no.AF024716) were used as internal controls forArabidopsisand cotton,respectively.Primers are listed in Table S1.Relative expression levels were calculated by the 2-ΔΔCTmethod.

2.8.Phylogenetic tree analysis

Protein sequences of GhPAS1,GrPAS1,and GaPAS1 were downloaded from cottonFGD (https://cottonfgd.org/).Protein sequences of GhPAS1 homologs from other species including one monocotyledon (Vitis vinifera) and 11 dicotyledons (A.thaliana,Durio zibethinus,Herrania umbratica,Populus trichocarpa,Carica papaya,Manihot esculenta,Camellia sinensis,Olea europaea,Tarenaya hassleriana,Theobroma cacao,andCoffea arabica) were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/).The DNAMAN program (https://en.freedownloadmanager.org/Windows-PC/DNAMAN.html) was used to align GhPAS1 with homologous proteins from other species.The neighbor-joining method was then applied to generate a phylogenetic tree using MEGA 6.1[47].The expression level of GhPAS1 was extracted from online transcriptomic data to identify its tissue-specific expression pattern [48].

2.9.Statistical analysis

Because the interval of the mean value of each parameter in each group was estimated using SE,all values were expressed as mean ± SE.Comparisons between groups were performed using one-way ANOVA followed by Tukey-Kramer multiple comparisons.Differences were considered significant atP<0.05 and highly significant atP<0.01.

3.Results

3.1.GhPAS1 is a bHLH transcription factor located in the nucleus and has transcription activity

Fig.1.Subcellular localization,transcriptional activity,and tissue-specific expression pattern of GhPAS1.(A)Transient expression of 35S::GhPAS1-GFP construct in tobacco leaves.DAPI was counterstained to indicate the location of the nucleus.Bar,100 μm.(B) Growth and X-α-GAL staining assay of transformant colonies on SD/-Trp and SD/-Trp/-His/-Ade medium.The pGBKT7-53 vector was used as a positive control.(C)GUS staining assay in overexpression plants with GUS gene driven by the GhPAS1 promoter.Bar,2 mm.(D)and(E)The enlarged states of the leaf and upper end of the main root,respectively.(F)Tissue-specific expression pattern of GhPAS1 in upland cotton by RTqPCR.

Phylogenetic analysis showed that GhPAS1,GaPAS1,and GrPAS1 proteins showed the highest similarity.GhPAS1 was orthologous to PAS1 proteins inT.cacao,H.umbratica,andD.zibethinus(Fig.S1A),suggesting that cotton PAS1 proteins may share a common ancestor with PAS1 in these species.Amino acid sequence alignment showed that PAS1 proteins contain one highly conserved domain,whereas other regions were less conserved.This conserved domain is a typical bHLH domain composed of about 60 amino acids (aa) (Fig.S1B).The N-terminus region of about 15 aa is a basic region with the ability to bind DNA sequence.The C-terminus contains an HLH domain and functions by forming homodimeric or heterodimeric complexes with other proteins[49].InArabidopsis,the homolog of GhPAS1 is a positive regulator that responds to BR signals and promotes plant growth and development [50].We accordingly speculated that GhPAS1 has similar physiological functions in growth and development of upland cotton.

The prediction results of subcellular localization using SignalP 4.0 [51] showed that the N-terminus of GhPAS1 protein did not contain a signal peptide and may be located in the nucleus.To verify that GhPAS1 is a nuclear protein,the reporter construct 35S::GhPAS1-GFP was used for transient expression analysis in tobacco leaf cells.The green fluorescence signals of GhPAS1-GFP and DAPI(DNA-binding dye) overlapped perfectly in the nucleus (Fig.1A)clearly indicating that GhPAS1 was located in the nucleus.Yeast cells transformed with pGBKT7-GhPAS1 and pGBKT7-53 grew well on selection medium containing SD/-Trp and SD/-Trp/-His/-Ade with strong β-galactosidase activity (Fig.1B).Thus,GhPAS1 is localized in the nucleus and has strong transcription activity.

3.2.Tissue-specific expression pattern of GhPAS1

To characterize the tissue-specific expression pattern of GhPAS1,we obtained stable genetic lines with the GUS reporter gene driven by the GhPAS1 promoter.A histochemical staining experiment showed GUS staining in root,root hair,hypocotyl,leaf,and epidermal hair of 12-day-old seedlings(Fig.1C–E).Areas with strong cell differentiation ability showed strong GUS staining,suggesting that GhPAS1 may function directly in cell elongation and cell differentiation.GhPAS1 was expressed predominantly in root,floral organs (calycle,torus,stamen,and pistil),and ovules at different developmental stages.The expression of GhPAS1 was higher in 20-DPA fibers than in other fiber tissues (Fig.S2).RT-qPCR results further indicated that GhPAS1 was expressed predominantly in the flower,and the relative expression level of GhPAS1 was highest in initial and elongation stages of fiber development(Fig.1F).These findings suggested that GhPAS1 is essential in cotton growth and development,especially in the development of flower,ovule,and fiber.

3.3.GhPAS1 overexpression promotes plant development and increases biomass in Arabidopsis

To identify the biological function of GhPAS1,we constructed stably inherited overexpression lines of GhPAS1 inArabidopsis.First,we observed and analyzed the lengths of hypocotyls and roots of seven-day-old seedlings.The hypocotyl length of WT seedlings was 1.04 mm,whereas the hypocotyl lengths of two overexpression lines were 1.31 mm and 1.33 mm with significant respective increases of 25.9%and 27.5%compared to WT seedlings(Fig.2A and C).Further analysis showed that the lengths of hypocotyl cells of GhPAS1 overexpression lines were significantly greater by 35.9% (78.36 μm) than those of WT seedlings(57.64 μm) (Fig.S3A and B).In agreement with these findings,roots of two overexpression lines were also significantly longer than those of WT seedlings(Fig.2A and D).Collectively,these findings suggested that GhPAS1 promotes cell elongation,thereby increasing the length of root and hypocotyl.

To characterize the effect of GhPAS1 on plant growth and development,we further observed and compared the phenotypic changes of GhPAS1 overexpression plants.In four-week-old plants,the rosette diameter of GhPAS1 overexpression lines increased significantly by 33.3% to 34.5% compared to WT plants(Fig.3A and F).GhPAS1 overexpression significantly increased the area of rosette leaves.For example,the area of the eighth leaf of overexpression lines increased by 70.1% to 73.0% compared to WT plants (Fig.2B and E).Consistently,plant height(Fig.3B and G),inflorescence organs (Fig.3C),stigma height(Fig.3D),and silique length (Fig.3E and K) of GhPAS1 overexpression lines were significantly larger than their counterparts in WT plants.These findings suggested that GhPAS1 promoted plant growth and development.

Given that GhPAS1 promotes plant growth and development,we speculated that GhPAS1 increases plant biomass.Plant biomass measurement showed significant increases of 10.6% to 14.9% in fresh weight (Fig.3H),and 40.8% to 43.9% in dry weight (Fig.3I)of GhPAS1 overexpression lines compared to WT plants.Similarly,21.0%to 29.9%increases in silique number per plant(Fig.3J),15.1%to 17.1% increases in silique length (Fig.3K),13.3% to 16.6%increases in seed number per silique (Fig.3L),and 24.6% to 31.0%increases in 1000-seed weight(Fig.3M)were measured in GhPAS1 overexpression lines compared to WT plants,all of which were significant differences.Thus,GhPAS1 overexpression increased plant biomass inArabidopsis.

3.4.GhPAS1 is a positive regulator of the BR signal pathway

Phytohormones are essential to plant growth and development[52–55].For example,BRs regulate the division,expansion,and differentiation of various cell types throughout the life cycle in plants[5].To determine whether GhPAS1 responds to BR signals,we used BR biosynthesis inhibitor BRZ to treat young seedlings.For seven-day-old seedlings cultured in the dark,hypocotyl lengths of GhPAS1 overexpression seedlings were significantly longer than those of WT seedlings in the presence or absence of BRZ (Fig.4A and D).For seven-day-old seedlings grown under light,root lengths of GhPAS1 overexpression seedlings were longer than those of WT seedlings in the presence or absence of BRZ (Fig.4B and E).Thus,GhPAS1 overexpression reduced the sensitivity of root and hypocotyl to BRZ.Phenotypic observation of two-weekold seedlings showed that the growth potential of GhPAS1 overexpression seedlings was greater than that of WT seedlings in the presence or absence of BRZ,based on leaf area,petiole length,and plant volume(Fig.4C),indicating that GhPAS1 overexpression reduced the sensitivity of seedlings to BRZ.RT-qPCR results showed that the relative expression levels of the key geneBR6OX1in the BR biosynthesis pathway and the key transcription factorsBES1,BZR1,andBEH3in the BR signaling transduction pathway were higher in GhPAS1 overexpression plants than WT plants in the presence or absence of BRZ (Fig.4F and G).Similarly,the relative expression level of BR response geneEXP8was higher in GhPAS1 overexpression plants than in WT plants in the presence or absence of BRZ (Fig.4F and G).We accordingly speculated that GhPAS1 positively responds to BR signals and promotes plant growth and development.

Fig.2.Overexpression of GhPAS1 increases hypocotyl elongation,root length,and leaf area in Arabidopsis.(A)Phenotype of seven-day-old seedlings grown under light.Bar,2 mm.(B) Phenotype of rosette leaves of four-week-old plants.Bar,1 cm.(C) and (D) Lengths of hypocotyls and roots of seven-day-old seedlings grown under light,respectively. n=20.(E) Areas of eighth leaf of four-week-old seedlings. n=20.

3.5.GhPAS1 controls plant development and architecture in cotton

We used VIGS to silence GhPAS1 in upland cotton to further verify the role of GhPAS1 on plant growth and development.The growth and development of silenced plants was significantly inhibited compared to CK plants (Fig.5A),owing to low expression levels of GhPAS1 in silenced plants compared to CK plants(Fig.5B).The plant architecture of GhPAS1 silenced plants was very different from that of CK plants.Specifically,the height of GhPAS1 silenced plants was reduced by 26.0% in comparison with CK plants (Fig.5C).Smaller root architectures (Fig.5D),shorter internodes (especially the seventh and eighth internodes)of the main stem (Fig.5E),shorter fruit branches (sympodial branches) (Fig.5A),and smaller bolls (Fig.5A) were observed in silenced plants than in CK plants.Thus,virus-induced GhPAS1 silencing in upland cotton inhibited plant growth and development and altered cotton plant architecture.The phenotypes of GhPAS1-silenced plants were consistent with the phenotype of GhPAS1 heterologous expression inArabidopsis.These results indicated that GhPAS1 regulates cotton plant development and architecture.

3.6.Silencing GhPAS1 reduces fiber length and seed yield

Cotton fibers and seeds are important indicators for cotton breeding and production [56].Cotton fibers are extremely elongated single cells[57,58].Given that GhPAS1 is a positive regulator promoting cell elongation,we speculated that GhPAS1 is involved in regulating cotton fiber development.Fiber quality measurements showed 1.62 mm shorter fiber(27.82 mm in silenced plants and 29.44 mm in CK plants)(Fig.6A and C),lower fiber uniformity index and fiber strength (Table 1),smaller seed length and width(14.5% to 14.9% reduction in silenced plants) (Fig.6B and D),and 18.6% reduction in 100-seed weight (Fig.6E) in silenced plants compared to CK plants.These results indicated that besides regulating cotton growth and development,GhPAS1 also regulates the development of fibers and ovules.

Table 1 Comparison of fiber quality parameters between GhPAS1-silenced and control plants.

4.Discussion

4.1.Diverse expression of GhPAS1

We identified a new typical bHLH transcription factor GhPAS1 in upland cotton,and its conserved domain showed a basic region and an HLH domain.GhPAS1 is located in the nucleus and has strong transcriptional activity.GhPAS1 was expressed in various tissues,suggesting that GhPAS1 may regulate in plant growth and development.GhPAS1 showed high expression levels in early stages of ovule development and during the phase of rapid fiber elongation,suggesting its potential roles in development of fiber and ovule.Histochemical GUS assay revealed the strong GUS promoter activity of the GhPAS1 gene in root,root hair,hypocotyl,leaf,and epidermal hair of 12-day-old seedlings.The GhPAS1 promoter showed stronger GUS activity in areas with strong cell differentiation ability,suggesting that GhPAS1 may have vital functions in cell differentiation and elongation.Therefore,GhPAS1 may play multiple roles in plant growth and development.

4.2.GhPAS1 promotes cell elongation through the BR signal pathway

bHLH transcription factors are essential in plant growth and development [59–61].Previous studies have identified the functional roles of cotton bHLH transcription factors,focusing mainly on the regulation of fiber cell development [62–64] and trichome development [65].In the present study,GhPAS1 overexpression inArabidopsispromoted cell elongation,mainly of root and hypocotyl,and leaf area of GhPAS1 overexpression plants was greater than that of WT plants.BRZ treatment showed that GhPAS1 overexpression increased the relative expression levels of key genes in the BR signal transduction pathway and reduced seedling sensitivity to BRZ.These results suggested that GhPAS1 promotes cell elongation by positively regulating the BR signal transduction pathway,thereby promoting plant growth and development.

4.3.GhPAS1 overexpression enhances plant growth and biomass in Arabidopsis

Fig.5.Virus-induced GhPAS1 silencing in upland cotton inhibits plant development and alters plant architecture.(A) Phenotypic observation of GhPAS1 silenced plants at maturity.Bar,10 cm.(B) Relative expression level of GhPAS1 in silenced plants.(C) Statistical analysis of plant height at maturity. n=20.(D) Observations of internode of main stem and root architecture.Bar=10 cm.(E) Statistical analysis of internode length of main stem. n=20.

The cell is the basic unit of life in all living organisms.Complex organisms contain differentiated cells and tissues as well as organs composed of multiple tissues with special functions [66].Cell elongation is the main mechanism of plant growth and morphogenesis,and is controlled by various phytohormones and environmental signals [67].BRs are a class of phytohormone promoting growth and developmental processes,including cell elongation,cell division,reproductive development,vascular tissue differentiation,and responses to various stresses [2,68].Because GhPAS1 is a positive regulator promoting cell elongation that responds to BR signals,we speculated that GhPAS1 promotes plant growth and development.Our experimental findings verified this hypothesis.In comparison with WT plants,GhPAS1 overexpression promoted elongation of hypocotyl and root and increased leaf area,as well as increasing rosette diameter and plant height.During reproductive growth stages,inflorescence size,stigma height,and silique size of GhPAS1 overexpression plants were significantly larger than those of WT plants.Measurements of plant biomass indicated that fresh weight,dry weight,and 1000-seed weight of GhPAS1 overexpression lines were larger than those of WT plants.Thus,GhPAS1 overexpression inArabidopsispromotes plant growth and development and increases plant biomass.

4.4.GhPAS1 regulates plant development and architecture in cotton

Plant architecture usually refers to morphological characteristics and spatial arrangements of aerial plant parts,which determine various crop agronomic traits and affect yield [18].Plant architecture is important for adapting to mechanical harvesting and increasing cotton productivity.Previous studies in cotton have shown thatGhAAI66(ALPHA AMYLASE INHIBITOR 66) mediates flowering [69],GhFT1(FLOWERING LOSUC T) promotes flowering and lateral shoot outgrowth [70],whileGhCEN(CENTRORADIALIS),GhTFL1(TERMINAL FLOWER 1),andGhSP(SELF-PRUNING) inhibit flowering[71],thereby altering the development of plant architecture.In this study,virus-induced GhPAS1 silencing in upland cotton inhibited plant growth and development and altered cotton plant architecture,as manifested mainly in plant height,fruit branch length,and boll size of GhPAS1-silenced plants.Fiber length,seed size,and 100-seed weight of GhPAS1-silenced plants were significantly lower than those of CK plants.We concluded that GhPAS1 regulates plant development and architecture and may function directly in fiber and ovule development.Given the role of GhPAS1in regulating cotton plant development and architecture,its physiological functions and regulatory mechanisms invite further investigation.

Fig.6.Silencing of GhPAS1 inhibited fiber and seed development.(A)Phenotype of mature fibers.Bar,1 cm.(B)Phenotype of mature seeds.Bar,1 cm.(C)Statistical analysis of fiber length. n=20.(D) Statistical analysis of seed length and seed width.(E) Statistical analysis of 100 seed weight.

5.Conclusions

GhPAS1 is a newly identified bHLH transcription factor located in the nucleus.GhPAS1 participates in the BR signal pathway and promotes cell elongation.Overexpression of GhPAS1 inArabidopsisand virus-induced GhPAS1 silencing in cotton showed that GhPAS1 regulates plant architecture and promotes plant growth and development.

CRediT authorship contribution statement

Zuoren Yang,Fuguang Li,and Huanhuan Wu:conceived and designed the study.Huanhuan Wu and Zhongying Ren:assisted in the acquisition of experimental materials,agronomic survey,and data analysis.Mengzhen Guo,Jingyu Yang,and Liyong Hou:conducted of primary mapping and agronomic survey.Huanhuan Wu,Ghulam Qanmber,and Zuoren Yang:wrote and improved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank the Center of Cotton Fiber Quality Inspection and Testing,Ministry of Agriculture of China for fiber quality identification.This work was supported by the Funds for Creative Research Groups of China(31621005)and the National Transgenic Major Project of China (2018ZX0800921B).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.10.014.