Wenchao Yin,Nana Dong,Mei Niu,Xiaoxing Zhang,Lulu Li,Jun Liu,Bin Liu,Hongning Tong*

National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:Soybean Brassinosteroid Senescence Gravitropism WRKY

A B S T R A C TBrassinosteroids(BRs)are endogenous phytohorm ones that play im portant roles in regulating plant grow th and developm ent.In this study,w e evaluated the effects of brassinolide(BL,one of the active BRs)on soybean and identified roles of the horm one in regulating m ultiple aspects of plant grow th and developm ent.BL application prom oted hypocotyl and epicotyl elongation in the light but blocked epicotyl elongation in the dark.High levels of castasterone and BL accum ulated in light-grow n plants.BL disrupted shoot negative gravitropism,w hereas gibberellin did not.BL delayed leaf senescence.Transcriptom e analysis showed that BL induced cell w all-m odifying genes and auxin-associated genes but suppressed a class of WRKY genes involved in senescence and stress responses,show ing the com plex roles of BRs in m ultiple biological processes.

1.Introduction

Brassinosteroids(BRs)are a class of steroid horm ones that play im portant roles in regulating plant grow th and developm ent.A near-complete BR m etabolic schem e and the BR prim ary signaling pathw ay have been published[1-3].The BR signal is perceived by the m em brane receptor kinases BRI1 and BAK1[4,5],is then transduced to cytoplasm com ponents such as BSK1,BSU1,and BIN2 by a series of phosphorylation or dephosphorylation events[6-8],and finally regulates the transcriptional factors BZR1 and BES1[9,10]targeting m any BR-responsive genes[11,12].BRs regulate both cell elongation and division and are essential for morphogenesis in darkness[13-16].BRs are involved in m any other developm ent processes,including flow ering,leaf senescence,pollen developm ent and m ale sterility,chloroplast developm ent,and root grow th and developm ent[11,17-20].BRs also regulate biotic and abiotic stress responses[21,22].In rice,BRs regulate plant height,leaf angle and kernel size,w hich are im portant agronom ic traits affecting rice yield[23].Several studies have deployed BR-associated genes for genetic improvement of plant perform ance in the field and show n prom ising results,demonstrating the high potential of the hormone in crop engineering[24-26].

Soybean(Glycine m ax L.Merrill)is a crop plant that is a m ajor source of protein and vegetative oil for hum ans and livestock.How ever,w e currently know little about the detailed functions of BRs in soybean.In this study,w e investigated the effects of brassinolide(BL),one of the active BRs,on soybean grow th and developm ent in different tissues at different development stages under different conditions.We identified some unexpected roles of the hormone that differ from those in Arabidopsis or rice.We show ed that BL inhibits epicotyl elongation in darkness,disrupts shoot gravitropism,and suppresses leaf senescence.These results w ill support the utilization of soybean for genetic screening and for dissecting the m olecular m echanisms underlying these BR functions.Com bined w ith transcriptome analyses,our results provide clues for further exploration of BRfunctions and applications in crop plants.

2.Materials and methods

2.1.Plant material and hormone treatment

Seeds of soybean(cv.Tianlong 1)w ere directly sow n in w elldrained pots filled w ith m oist soil.In a GXZ-800D grow th cham ber(Ningbo Jiangnan,China),soybean seedlings were grow n under 24°C and a photoperiod of 14 h light(nom inal 600μm ol m-2s-1)/10 h dark.To detect the effect of BL on soybean grow th in darkness,the plants w ere grow n under the sam e conditions except for the light illum ination for 7 days after germ ination.

BL(Wako,Japan)w as dissolved in dim ethyl sulfoxide(DMSO)to produce a 20 m m ol L-1stock solution,w hich w as diluted to 0.001,0.01,0.1,1,and 10μm ol L-1w ith w ater to test the effect of concentration gradients of BLon seedling grow th.Unless otherw ise indicated,1μm ol L-1BL w as used for other applications.Brassinazole(BRZ,TCI,Japan)was dissolved in DMSO to produce a 20 m m ol L-1stock solution,and 20μm ol L-1BRZ diluted in w ater w as prepared and used.Gibberellin(GA3,Sigm a,USA)w as dissolved in ethanol to produce a 1 m mol L-1stock solution,which w as diluted to 0.001,0.01,0.1,1,and 10μm ol L-1in w ater for use.Paclobutrazol(PAZ,Sigm a,USA)w as dissolved in ethanol to produce a 100 m m ol L-1stock solution,w hich w as diluted to 10μmol L-1for treatm ent.For the treatments,w ater containing the chem icals w as supplied in a tray w here the pots w ere put(50 m L per pot)for absorption by plant roots.Neat solvent(DMSO or ethanol)w as used for the m ock treatm ent.Depending on the experimental purpose,the treatment was perform ed at different tim es(defined by days after sow ing,DAS)as indicated.For RNA sequencing(RNA-seq),1μm ol L-1BL or mock w as added at 13 DAS,and epicotyl tissues were collected after 6 h of treatm ent.An additional 24 h of treatm ent w as included for q RT-PCRassay.

2.2.Hormone quantification

Hypocotyls of eight plants(1 g)w ere collected for hormone measurement following Ding et al.[27].Castasterone(CS),BL,typhasterol (TY), 6-deoxocastasterone (6-deoxoCS), 28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS),homocastasterone(homoCS),28-hom obrassinolide(28-homoBL),teasterone(TE),and norteasterone(norTE)w ere quantified and only those successfully detected are show n.

2.3.RNA-seq and qRT-PCRanalysis

Three biological repeats w ere perform ed for RNA-seq analyses.For each repeat,epicotyls of six plants w ere sampled.RNA w as isolated w ith TRIzol reagent(Invitrogen,USA)and then used for library construction using a NEBNext Ultra RNA Library Prep Kit for Illum ina(NEB,USA)follow ing the m anufacturer's recom m endations.Clustering of index-coded sam ples w as perform ed on a c Bot Cluster Generation System using a Nova-seq Cluster Kit(Illum ina,USA)according to the m anufacturer's instructions.After cluster generation,the library preparations w ere sequenced on an Illum ina Novaseq S6000 platform and 150 bp paired-end reads w ere generated.Differentially expressed genes(DEGs)w ere defined by a tw ofold expression difference w ith false discovery rate(FDR)＜0.05.One m icrogram of RNA w as reverse-transcribed using a q PCR RT Kit(Toyobo,Japan)for q RT-PCR verification,w hich w as perform ed using SYBR-green PCR m ix on a LightCycler 96 machine following the manufacturer's instructions(Roche,USA).ACTIN11 w as used as internal reference for all the analyses.Prim er sequences are described in Table S1.

3.Results

3.1.BL promotes hypocotyl and epicotyl elongation

BL w as added at 5 DASw hen plant hypocotyls em erged from the soil.After an additional four days of grow th,clear differences in both hypocotyl and epicotyl length w ere observed betw een BL-treated and m ock-treated plants(Fig.1A).Particularly in the epicotyl,BL induced an elongation m ore than tw ice that of the m ock treatm ent(Fig.1B).Sim ultaneously,BL inhibited the size of the first pair of true leaves,w ithout affecting leaf vein num ber(Fig.1C).BL w as also added at 3 DASand the effect of different concentrations of BL on epicotyl and hypocotyl elongation(Fig.1D-E)w as tested.After an additional seven days of grow th,application of 0.1μm ol L-1BL significantly prom oted hypocotyl elongation,and the increase becam e more m arked as the concentration of applied BL increased(Fig.1E).BL also suppressed root grow th,particularly w hen applied at high concentrations(Fig.S1).

3.2.BL blocks epicotyl development in darkness and light induces BRaccumulation

Surprisingly,in darkness,BL largely suppressed epicotyl elongation but show ed little effect on hypocotyl elongation(Fig.2A-C).Whether in light or darkness,BRZ,the BR synthesis inhibitor,show ed little effect on soybean grow th and developm ent.We w ere curious w hether the light condition modulated BRcontent to regulate plant growth.We also w anted to determ ine w hether BRZ could inhibit the endogenous BR level.Therefore,w e sim ultaneously quantified BR content in BRZ-treated and mock-treated plants grow n either in light or in dark(Fig.2D-G).Because the epicotyl failed to elongate w hen BLw as applied,w e could collect only hypocotyl tissues for the m easurem ent.Among BR form s quantified,w e successfully detected 28-nor CS,homo CS,CS,and BL.Strikingly,both CS and BL and 28-nor CS and hom oCS accumulated to higher abundance in light-grow n than in dark-grow n plants(Fig.2D-G).In both light and darkness,BRZ show ed little effect on either CSor BL content.We suspected that BRZ w as not an effective BR inhibitor in soybean,although another possibility w as that our culture conditions or treatm ent m ethod(in soil)were not appropriate for BRZ function.

Fig.1-Effects of BL on seedling grow th.(A)Com p arison of m ock-and BL-treated plants in pots.(B)Com parison of rep resentative mock-and BL-treated p lants.(C)Com parison of leaf morphology.(D-E)Statistical com parison of epicotyl and hyp ocotyl lengths of m ock-and BL-treated p lants.Bars ind icate stand ard deviation(SD,n=12).BL at 1μm ol L-1 w as used for the treatm ent in(A-C).**P＜0.01 and***P＜0.001 by t-test.

3.3.BL disrupts shoot negative gravitropism and delays leaf senescence

The shoot apical part consistently tended to bend dow n w hen BL w as applied at either the early or late stage of soybean grow th(Fig.S2).This phenom enon w as m ost pronounced in the hypocotyl w hen BL w as applied early in germ ination(Fig.3A).When a series of BL concentrations w ere applied,even 0.01μm ol L-1BL induced the inclination of the hypocotyl,and a high concentration of BL,such as 10μm ol L-1,led to completely downw ard grow th of the plant(Figs.3A,S3-A).GA also strongly prom otes hypocotyl elongation.To exclude the possibility that the hypocotyl bent dow n because of the increase in height,w e also tested the effect of GA on this aspect.Even at the very high concentration of 10μm ol L-1,GA failed to induce clear inclination of plants(Fig.S3-B).These results indicated that BL plays specific and critical roles in m odulating shoot negative gravitropism.

BL significantly suppressed leaf senescence under our grow th conditions(Fig.3B-C).When the first pair of sim ple leaves turned yellow w ithout BL treatm ent,the first pair w ith BL treatm ent rem ained fully green(Fig.3B-C).Application of BL at the flow ering stage(40 DAS)also alleviated the leaf senescence symptom(Fig.S4).BLslightly promoted flow ering,indicating that BL-suppressed senescence w as not due to a delayed developm ent program(Fig.S5).

3.4.BL promotes the expression of auxin-associated and cell wall-modifying genes

Transcriptom e analyses revealed that 151 genes w ere upregulated and 199 dow nregulated by BL treatm ent(Table S2).These genes,based on Gene Ontology analyses(http://w w w.geneontology.org/),w ere enriched m ostly in“response to grow th hormone”and then“auxin-activated signaling pathway”,“response to cyclopentenone”,“cell to cell junction assem bly”, “de-etiolation” and “negative regulation of response to w ater deprivation”(Table S3).Some of these biological pathw ays are highly consistent w ith hormone functions.For example,auxin is well know n to be involved in gravitropic response,and BRs play a role in the de-etiolation process.We selected all 11 genes w ith functional annotation related to auxin(Swiss-Prot annotation,http://www.expasy.ch/sprot)and found that,strikingly,all were BL-upregulated genes(Table 1).We also identified 13 genes encoding extensin,expansin,or xyloglucan endotransglucosylase(XET,encoded by BRU1 or BRU1-like genes)that had potential function in cell wall m odification,and 12 of these genes show ed increased expression in BL-treated samples(Table 1).We selected four auxinassociated genes and tw o XET-encoding genes for q RT-PCR verification.All were highly induced after BLapplication at either 6 or 24 h,indicating that our RNA-seq data w ere reliable(Fig.S6).These results suggested that BL promoted the expression of related genes to regulate cell elongation and gravitropism.

3.5.BL suppresses expression of WRKY and other families of genes

Fig.2-Effects of BLand BRZon seedling grow th in d arkness and BRquantification in dark-and light-grow n p lants.(A)Com parison of m ock-,BRZ-,and BL-treated p lants in the dark.(B-C)Statistical com parison of epicotyl(B)and hypocotyl(C)lengths in(A).(D-G)Quantification of BRs in d ark-and light-grow n p lants.(D)CS;(E)BL;(F)28-norCS;(G)hom o CS.FW,fresh w eight;ND,not d etected.In(B-C),bars indicate SD(n=12).In(D-G),bars ind icate SD(n=3).BLat 1μmol L-1 or BRZ at 20μm ol L-1 w as used for treatment.Chem icals w ere ad ded at 3 DAS,w ith treatm ent for one w eek.*P＜0.05,**P＜0.01,and***P＜0.001 by t-test.

Next,w e focused attention on families of genes w ith consistent regulation,as these genes could play im portant roles in BR responses.All of the eight WRKY family genes,six calciumbinding protein-encoding genes,five cysteine-rich receptor-like protein kinase genes,and four cytochrome P450 genes w ere dow nregulated by BL application(Table 2).These genes m ight m ediate BRregulation of various biological processes,particularly the WRKYs,w hich accounted for the largest number and had the highest fold change.Other unlisted WRKY members,such as

Glyma.04G223300 and Glyma.18G208800,also show ed markedly decreased expression in one or tw o repeats.In Arabidopsis and rice,m any studies demonstrate the critical roles of WRKY members in regulating leaf senescence and stress responses.In soybean,expression of WRKY53a (Glyma.08G021900)and WRKY53b(Glyma.16G026400)are senescence-associated[28].Of five WRKY genes,including WRKY53a and WRKY53b,selected for qRT-PCR verification,all showed decreased expression after BL application for either 6 or 24 h(Fig.S7-A).Thus,BLsuppressed the expression of WRKY genes,some of w hich may be associated w ith leaf senescence and stress responses.

4.Discussion

BRs can induce XET enzyme activity in addition to epicotyl elongation in soybean[29].We show ed that the increase in protein activity w as caused by increased transcription of the encoding genes,further supporting the critical role of XETencoding genes in mediating BR-stim ulated cell elongation.BRU1 or BRU1-like genes m ay be the prim ary targeted genes of the BR signaling pathw ay and could be used as effective marker genes for evaluating BR response in soybean.BL prom oted epicotyl elongation in light but suppressed its elongation in the dark.This tendency is sim ilar to that in Arabidopsis.How ever,unlike in soybean,w here BL m arkedly inhibited epicotyl elongation in darkness,BRinhibition of the hypocotyl in Arabidopsis was relatively weak[30].In rice,BRs strongly prom ote m esocotyl and even internode elongation in young seedlings in darkness.Thus,in the dark,BRs appeared to have opposite functions in soybean and rice.

Fig.3-Effects of BL on shoot gravitropism and leaf senescence.(A)Effects of BL on shoot gravitropism.Chem icals w ere add ed at sow ing,and plants w ere grow n for one w eek.(B-C)Effects of BL on leaf senescence.BL w as add ed at 5 DAS,and plants w ere grow n for an add itional 17 days.

BL disrupted shoot negative gravitropism in soybean.This effect is also observed in Arabidopsis,but only in the dark[31].Tropism responses including gravitropism and phototropism are generally thought to be associated w ith auxin transport and distribution[32-34].Considering that several auxinassociated genes,including an auxin efflux carrier gene,have consistently increased expression w ith application of the hormone,BL might also disrupt shoot negative gravitropism via regulating auxin response.Given that BR functions are closely associated w ith light regulation,w hether BRs also m odulate phototropism rem ains to be investigated.Based on the observation that a BR-deficient mutant usually has dark green leaves,BRs are suggested to prom ote senescence.How ever,the suppression of BAK7,a putative BR signaling component,led to an early senescence phenotype in Arabidopsis[18].A similar phenotype w as not observed in even severe BR-deficient m utants such as bri1 and bin2-D.The finding that BL delayed soybean leaf senescence suggests novel roles of BRs in this process.

WRKY fam ily genes play im portant roles in diverse aspects of plant grow th and developm ent,w ith prom inent roles in senescence and stress response[35].Recently,tw o studies[36,37]revealed the roles of WRKY genes in BR responses in both Arabidopsis and rice.Another study[29]in soybean suggested the involvem ent of WRKY m em bers in lightregulated senescence.Am ong the DEGs identified in our transcriptome analyses,the expression of all WRKY genes decreased in BL-treated sam ples,indicating the pivotal roles of WKRYs in m ediating BR functions in soybean.Com bined with the finding that light regulates BR levels,these results indicate the connection am ong light,BRs,WRKYs,and senescence.BRs as internal developm ent signals likely integrate environm ental cues to m odulate developm ent processes via regulation of the expression of associated genes.Given that these WRKYs w ere detected in epicotyls,it rem ains unclear w hether they play roles in cell grow th.It w ill be interesting to investigate how WRKYs are regulated by BRs during senescence.

Collectively,the results of our study provide clues for further study of BRfunctions at the m olecular level in soybean(Fig.S7-B).How ever,given that only epicotyls w ere used for the RNA-seq analysis,the connection betw een BR function and gene expression requires further investigation.In addition,considering that exogenously applied BL could exert som e artificial effects on plants,genetic evidence is im portant for verification of these BR functions.Whether BRs exert sim ilar effects under different grow th conditions (for exam ple,in the field)rem ains to be tested.Nevertheless,given that som e of the effects have not been reported or not found in other w ell-studied species,w e can uncover BR functional mechanisms in these aspects in soybean.These BR responses provide feasible approaches for screening of related m utants.Such screening w ill prom ote the study of horm one genetics in soybean,further favoring genetic improvement of the crop.

Table 1-BL p rom otes ex pression of auxin-associated and cell w all-m odifying genes.

Acknow ledgm ents

This w ork w as supported by the National Natural Science Foundation of China(91735302,31722037,91435106)and Fundam ental Research Funds for Central Non-Profit of Institute of Crop Sciences,Chinese Academ y of Agricultural Sciences(2060302).

App end ix A.Sup plem entary data

Supplementary figures and tables for this article can be found online at https://doi.org/10.1016/j.cj.2018.10.003.