| Amomi Fructus ’Sharen’,a commonly used traditional Chinese herb which dedicated to the treatment for stomach diseases and digestive disorders and of high economic value,is derived from the ripe fruit of Amomum villosum.Due to the excessive high fruit dropping rate in the first month after artificial pollination,the yield of A.villosum remains at a low level permanently.Effective measures to reduce fruitlet abscission is urgent for Amomi Fructus industry.Thus,it is necessary to investigate the mechanisms underlying the fruitlet abscission and its regulation in A.villosum.In the present study,abscission zones(AZ)from the pedicel on the 6th,9th,12th,17th,19th,21st and 24th day after artificial pollination(DAP)of non-abscised fruitlet(ZC)and 16th,17th and 19th DAP of abscised fruitlet(TL)were collected for RNA extraction.Transcriptome analysis was performed using RNA-sequencing to compare the expression profiles between the AZs of ZC and TL.Genes related to the biosynthesis and signal transduction of eight phytohormones(auxin,ethylene,abscisic acid,jasmonic acid,cytokinin,salicylic acid,and brassinosteroid),functioning in plant cell wall or cytoskeleton synthesis,participating in calcium signaling and being predicted to encode transcription factors(TFs)were identified to analysis their expression pattern during abscission.Results are as follows:(1)High quality transcriptome data was obtained for further analysis.Transcriptome sequencing yielded 10.67 Gb raw reads and 10.46 Gb clean reads,with Q20 of 98.14%and Q30 of 93.41%.De novo assembly of these high-quality clean reads generated 89,993 Unigenes with a total length of 115,706,046 nt,average length of 1285 bp,and N50 of 2086 bp.Contig sequences were searched against the databases including NR,NT,Swissprot,KEGG,KOG,Pfam,and GO for functional annotation,in result there were 63,713 Unigenes annotated to any database,with an overall annotation rate of 70.80%.(2)Expression patterns of genes that related to the biosynthesis and signal transduction of eight phytohormones in different comparison group(ZC vs ZC,TL vs TL and ZC vs TL)were studied.Auxin biosynthesis related gene amiE was down-regulated by time in both ZC and TL;the expression of YUCCA was highest at ZC6,then decreased,and decreased again after increasing at ZC17 and ZC19,however,YUCCA did not change significantly in TL.The expression of ALDH(CL5948.Contig3)peaked at ZC12 then decreased;in TL vs TL,ALDH(CL5948.Contig3)was first down-regulated then up-regulated.Three ISS1/VAS1 reached the peak of expression level at ZC17,then were down-regulated;in TL vs TL,ISS1/VAS1 was down-regulated by time(Unigene38827),or it showed an expression trend of first increased and then decreased(CL458.Contig2.CL458.Contig3).Compared with ZC on the same DAP,amiE,YUCCA were significantly up-regulated,and ALDH,ISS1/VAS1 were significantly down-regulated in TL.Auxin signal transduction related gene AUX1 reached the peak of expression level at ZC12 and then decreased at ZC9 or ZC19;TIR1 and ARF were down-regulated by time in ZC;the expression of AUX/IAA decreased from ZC6 to ZC12,then increased and peaked at ZC6 or ZC24;GH3 expressed in ZC with a very low level and was indistinguishable.ILL was up-regulated at ZC12 or ZC19;PIN1 was up-regulated at ZC9,meanwhile PIN3 was down-regulated.The above genes did not change significantly in TL vs TL.Compared with ZC on the same DAP.GH3,SAUR32,ILL6 were significantly up-regulated,and AUX1,TIR1,AUX/IAA(IAA1,IAA4,IA49,IAA10,IAA17,IA421,IAA26,IAA30),ARF,SAUR66,ILL1,PIN were significantly down-regulated in TL.The expressions of ACO and ACS which are key enzymes in the ethylene biosynthesis pathway did not change significantly in ZC vs ZC or TL vs TL,but were significantly up-regulated during fruitlet abscission.Ethylene signal transduction related gene ETR2 and EBF were slightly down-regulated by time in ZC;the expression of SIMMK peaked at ZC12 then decreased;expression pattern of EIN2 was irregular,it increased at ZC12 and ZC21.The expression of above genes were up-regulated or down-regulated at TL17.Compared with ZC on the same DAP,ETR2,ERF were significantly up-regulated,and SIMMK,EIN2,EBF were significantly down-regulated in TL.The expression of abscisic acid biosynthesis or metabolism related gene NCED and CYP707A decreased by time in ZC,while A OX and ABA2 showed the opposite trend.In TL vs TL,NCED and CYP707A decreased by time,while AOX and ABA2 were first down-regulated then up-regulated.Compared with ZC on the same DAP,NCED(Unigene 26445).ABA2 were significantly up-regulated,CYP707A was significantly down-regulated in TL.Abscisic acid signal transduction related gene PYR/PYL(CL3012.Contig3,Unigenel1974)reached the peak of expression level at ZC9 then sharply decreased;PP2C was down-regulated by time in ZC;the change of SnRK and ABF in ZC was indistinguishable.The expression of above genes were up-regulated or down-regulated at TL17.Compared with ZC.on the same DAP,PYR/PYL(PYL8,PYL4),PP2C30,PP2C51,PP2C53,PP2C49,PP2C68,SnRK(SPAK1)were significantly up-regulated,ABF(ABI5)was significantly down-regulated in TL.The change of the gibberellin biosynthesis related gene KAO in ZC was indistinguishable,while GA20ox was down-regulated by time,GA13ox and GA2ox reached the peak of expression level at ZC12 then decreased.In TL vs TL,KAO was first up-regulated then down-regulated while GA13ox GA20ox and GA2ox changed gently.Compared with ZC on the same DAP,KAO,GA2ox were significantly up-regulated,GA13ox,GA20ox were significantly down-regulated in TL.Gibberellin signal transduction related gene DELLA,PIF3 and PIF4 were down-regulated by time in ZC,while GID1 was first up-regulated then down-regulated.The above genes did not change significantly in TL vs TL.Compared with ZC on the same DAP,DELLA(SLR1,SLN1),PIF3,PIF4 were significantly down-regulated in TL.The expression pattern of genes that related to jasmonic acid biosynthesis distinguished from each other in ZC;LOX(CL5556.Contig2)was down-regulated by time;AOS change gently;AOC(CL11085.Contig2)was up-regulated at ZC17 and ZC24.In TL/TL,LOX change gently;AOS(Unigene2857)was down-regulated by time;AOC was down-regulated by time(CL5435.Contig3)or was first up-regulated then down-regulated(CL11085.Contig2).Compared with ZC on the same DAP,AOS was significantly up-regulated,and LOX was significantly down-regulated in TL.Jasmonic acid signal transduction related genes JAR1 and MYC2 were down-regulated at ZC9 or ZC12,then decreased;the expression of JAZ peaked at ZC6 or ZC17,lowest at ZC9.The expression of above genes were up-regulated or down-regulated at TL17.Compared with ZC on the same DAP,JAR1 was significantly up-regulated at TL17 while MYC2 was significantly up-regulated in TL19.The expression of cytokinin biosynthesis or metabolism related genes LOG and CKX in ZC had a similar pattern in ZC,they increased at ZC12 and subsequently decreased.In TL vs TL,LOG and CKX5 were first up-regulated then down-regulated while CKX6 was first down-regulated then up-regulated.Compared with ZC on the same DAP,LOG and CKX6 were significantly up-regulated in TL.The expression pattern of cytokinin signal transduction related genes HK3 and HK5 was similar in ZC,both of them were up-regulated at ZC9 or ZC12,but decreased at ZC17;on the contrary,HK4 was down-regulated at ZC9 then to be up-regulated.HK4 expressed in TL with a very low level and was indistinguishable;HK3 and HK5 were first down-regulated then up-regulated in TL.Compared with ZC on the same DAP,HK5 was significantly up-regulated,and HK4 was significantly down-regulated in TL.The expression of salicylic acid signal transduction genes NPR and TGA peaked at ZC12.NPR1 and TGA1 were up-regulated by time in TL,while NPR5 was down-regulated and NPR2,TGA2.2 and TGA4 were first down-regulated then up-regulated;The expression of PR-1 in ZC was much lower than in TL.Compared with ZC on the same DAP,both NPR1,TGA1 and PR-1 were significantly up-regulated in TL.Brassinolide biosynthesis related genes DET2 and DWF4 were generally down-regulated by time in ZC,the expression of CPD increased sharply at ZC12.DET2 and DWF4 were first down-regulated then up-regulated in TL vs TL,while CPD was up-regulated by time.Compared with ZC on the same DAP,DWF4 and CPD were significantly up-regulated,DET2 was significantly down-regulated in TL.Despite increasing at ZC 12,the expression levels of brassinosteroid signal transduction genes BRI1,BIN2,BKI1 and BZR1 in ZC were relatively stable.BSK1 was down-regulated by time(CL904.Contig4)or was first up-regulated then down-regulated(CL9938.Contig1)in ZC;CDL1 was down-regulated at ZC12 then up-regulated at ZC17 or ZC19.In TL vs TL,the change of CDL1,BIN2 and BZR1 was indistinguishable;BRI1 was first up-regulated then down-regulated;BKI1 was down-regulated by time while BSK1 was the opposite.Compared with ZC on the same DAP,BRI1(Unigene32328),BKI1,BIN2(Unigene4539)and BZR1(CL8264.Contig3,CL9802.Contig2)were significantly up-regulated,BRI1(CL2479,Contig1,CL2479.Contig4),CDL1,BIN2(CL469.Contig3,CL5344.Contig2,CL5575.Contigl)and BZR1(Unigene29418)were significantly down-regulated in TL.(3)The expression differences of genes which relate to cell wall and cytoskeleton synthesis and calcium signaling during fruitlet abscission were studied.Except for genes encoding β-1,3-glucanase,chitinase,β-glucosidase,glycosyltransferase IRX15,fucosyltransferase and three putative polygalacturonase,most genes involved in cell wall and cytoskeleton synthesis or remodeling in the AZ were significantly down-regulated in TL compared to ZC.Such as endo-1,4-D-glucanase EG10,EG24;pectin methylesterase/pectin methylesterase inhibitor PME-like,PME31,PPE8B-like,PPE12,PPE16,PPE34,PPE51;expansin EXP-A2,EXP-A4,EXP-A6,EXP-A7,EXP-A10,EXP-A15,EXP-B16,EXP-B18;galacturonosyltransferase GAUT,GAUT-like1,GAUT-like2,GAUTlike-3,GAUT4,GAUT7,GAUT8-like,GAUT12,GAUT13,GAUT15;cellulose synthase catalytic subunit CESA1,CESA5,CESA-like E6,CESA9;COBRA-like protein COBRA-like1,COBRA-like4;xyloglucan endotransglucosylase/hydrolase XTH5,XTH7,XTH8,XTH9,XTH28,XTH31-like,XTH32;β-galactosidase βGal2,βGal3,βGal5,βGal-like;xyloglucan 6-xylosyltransferase XXT1;glycosyltransferase IRX9;endo-1,4-β-mannanase MAN2-like;UDP-arabinopyranose mutase UMA1;Formin-like protein Formin-likel,Formin-like8.Ca2+-or calmodulin-binding proteins CaM(CL9295.Contig1)CML14,CML18 were up-regulated in TL compared to ZC,while the expression of CaM(CL10915.Contig2),CML27,CML 7,CBL,CRK and CNX was decreased by the time of abscising.(4)Transcription factors which expressed dramatically during abscission in abscised fruitlet pedicel were screened out.To identify transcriptional dynamics associated with fruitlet abscission,TFs that were differentially expressed more than 8-fold in TL relative to ZC were selected for further analysis.Within these 268 TFs,it was found that TFs containing ABI3/VP1,ARF,bZIP,C2C2-GATA,GRAS,GRF,HB,LIM,MADS,mTERF,RWP-RK,TAZ,TCP,Tify and zf-HD domains were down-regulated,while TFs classed in the Alfin-like and SRS families were up-regulated during fruitlet abscising.Furthermore,within the TFs that both significantly higher and lower expression in the AZ,most NAC and WRKY TFs were up-regulated in TL;while most AP2-EREBP,bHLH,C2C2-Dof,C3H,MYB and SBP TFs were down-regulated;These TFs were found to play an important role in hormone signal and stress response,or regulating senescence.Taking together,the concentration of IAA,GA,CTK and other growth-promoting hormones were higher in AZ of non-abscised fruitlet pedicel,which guarantees the normal growth and development of the cells.ETH was accumulated in the AZ while abscising,it accelerated the cell senescence and apoptosis.Down-regulation of genes related to cell wall and cytoskeleton synthesis and remodeling reduced cell wall viscosity and increased tissue fragility and eventually leading to the breakdown of AZ.The biosynthesis or signal transduction activities of ABA,SA,and BRs were enhanced when abscising,they may cooperate with ETH to promote fruitlet abscission,or may play a role in induced resistance to possible pathogen attack after a plant part is shed.Since Ca2+ signal transduction was not active in TL,we speculated that it did not mediate the abscising process directly.Transcription factors may mediate fruit drop process mainly by synergizing hormone biosynthesis or signal transduction,promoting stress response,and regulating senescence.In present study,RNA-seq was performed for the first time to study the fruitlet abscission in A.villosum.It is a whole new approach to understand the molecular regulation of fruitlet abscission in this specie.A large number of genes that are possible related to fruitlet abscission were screened,which may shed light on further research. |
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