Studies On Biosyntheses, Regulations, And Gene Isolations Involved In Carbohydrates And Phenols In Underground Tubers Of Different Genotypes Of Yams

Yams (Dioscorea spp.) are twining and herbaceous perennials belonging to Dioscorea genus ofDioscoreaceae family,containing many of both carbohydrates and phenols in underground tubers,and areusually utilized as the materials for the productions of subsistence food,druggery,and biomass energy.Sofar,species of about 600 in genus Dioscorea have been reported,of which about 10 species have beengenerally cultivated in the world and only 3 species as D.alata L.,D.opposite Thunb.,and D.japonicaThunb.in land areas of China.Because phytochemicals in underground yam tubers usually vary in bothconstituents and contents with genotypes and ambient growth conditions,both collections and classificationsof cultivated-mainly yam varieties in China and the elucidations of their biosynthesis mechanisms of maincarbohydrates and phenols in underground tubers will be very important significances in yielding moreproducts with higher quality for mankind's consumptions.In this present study,six genotypic yams namedrespectively'Zixiao','Zida','Baibian','Baizhu','Putong',and'Yesheng'were studied.Firstly,they weresorted and evaluated in the light of their polymorphisms at the levels of morphological characteristics,soluble proteins in tubers,and SRAP molecular markers.Secondly,both contents of major constituents ofcarbohydrates and phenols and the enzyme activities involved in their biosyntheses were measured byphysiological and biochemical methods,meanwhile,the structural gene cDNA sequences coding for the keyenzymes related to the formation of carbohydrates and phenols were isolated from the underground tubers ofsix genotypes,of which full-length cDNA sequences of the related genes in underground tubers of'Zixiao'were emphasizedly cloned and characterized.Lastly,both contents of major constituents of carbohydratesand phenols and the enzyme activities involved in their biosyntheses in underground tubers were assayed byphysiological and biochemical methods after three genotypic yams as'Zixiao','Baibian',and'Putong'werefoliarly sprayed with SA at concentrations of 0.5,1.0,5.0 mmol.L^-1,respectively.Additionally,the putativerelationships on the biosyntheses of carbohydrates and phenols in yams were assayed.The results obtainedthrough the aforementioned studies were as follows.(1) Based on polymorphisms at the levels of morphological characteristics,soluble proteins in tubers,and SRAP molecular markers,six genotypes of yams were not only all completely differentiated into threeclasses at identification percentage of 100%,but also the same sorted results were obtained by threeclassification methods.Four genotypic yams of'Zixiao','Zida','Baibian',and'Baizhu'belong to D.alataL.,and was also divided into two types named respectively as purple type of yams comprising'Zixiao'and'Zida',and white type of yams containing'Baibian'and'Baizhu'.'Putong'and'Yesheng'was attributed to D.opposite Thunb.and D.japonica Thunb.,respectively.By comparisons of three classification methods interms of polymorphisms of morphological characteristics,soluble proteins in tubers,and SRAP molecularmarkers,they had respectively polymorphism ratios of 65.45 %,67.7 %,89.32 % for theabovementioned 6 genotypes of yams,and genetic similarity coefficients of 0.63～0.96,0.54～0.94,0.36～0.73,and genetic distance values of 0.04～0.37,0.06～0.46,0.27～0.64,and average genetic distancevalues of 0.11,0.15,0.39 between 4 genotypes of D.alata L.were obtained,These results indicated thatSRAP molecular marker more contributed to both analysis of genetic diversity and classification for yarngermplasms on account of its more accurate classification to the aforesaid 6 genotypes of yams.(2) Dry matters in underground tubers of six genotypic yams straightly increased during growth anddevelopment,and accumulated earliest in white type ofD.alata L and latest in'Putong'.AM,AP,Cel,andTSS totally accounted for more than 50% of DW in tubers,and AM and AP were 28.3～39.1%,34.7～43.7% of DW at harvesting,respectively.As opposed to Cel,TSS,Sue,TRS,Glc,Frc,TSP,AM and AP hadgenerally the same trends in contents as DW.Although having the smallest contents in DW,each of assayedsoluble sugars,and Cel,in tubers,'Yesheng'contained the highest ones of DC,TS,AM,AP before the partof the rapidly-bulking stage.Contents of TS,AM and AP in'Putong'were the smallest before the part of therapidly-bulking stage,and were the largest at harvesting.Four D.alata L.ranged between'Putong'and'Yesheng'in the contents of other saccharides except Suc.As opposed to AM and soluble sugars,DW,TS,AP,and Cel presented more contents in purple varityies ofD.alata L.than in white varityies of D.alata L..In studied yam genotypes,general patterns of SAI,IAI,and NI activities all reduced,while SuSy activitiesdecreased to harvesting after straightly increasing to the high point at rapidly-bulking stage.lnvertase werelower in activities than SuSy,especially at the early stage of tuber bulking.By contrast to SAI,IN and IAIhad higher activities in'Putong'and'Yesheng'than in four D.alata L during the sampling times.Ascompared to'Yesheng'with the lowest SuSy activities,four D.alata L.showed higher activities of SuSythan'Putong'which had the highest activities at harvesting.SAl and NI other than IAI and SuSy were higheractivities in purple varityies ofD.alata L.than in white varityies ofD.alata L..AGPase and SSS activitiesin the same yam had the similar change patterns,namely,both activities of the other yam genotypes except'Putong'with straight increases in their activities fell to the harvesting after increasing to the highest pointsat the early or rapidly-bulking stage of tuber formation.GBSS and SBE increased in general change trendsof all checked genotypes during growth and development,and straightly increased in'Putong'and'Yesheng'Albeit GBSS and SBE in four D.alata L also straightly enhanced before the end of rapidly-bulking stage,thereafter,changes in activities of SBE other than GBSS reduced.'Yesheng'had the lowest activities ofAGPase,SSS,GBSS,and SBE,and'Putong'was lower in activities of these enzymes than four D.alata L atthe early stage and vice versa at the later stage.With the exception of SBE,AGPase,SSS,and GBSS hadhigher activities in purple varityies of D.alata L.than in white varityies of D.alata L..The contents of other saccharide except Cel and the above-mentioned enzyme activities were higher in'Zida'than in'Zixiao',andin'Baibian'than in'Baizhu'.ANOVA results showed differences in the aforesaid physiological andbiochemical parameters were significant between yam species and between types of D.alata L at themajorities of times,but insignificant for intra-types of D.alata L.At the same time,Suc contentsinsignificantly correlated with activities of both SuSy and the enzymes related to starch biosynthesis,butcorrelation coefficients of ratios of both Suc/TSS and Suc/TRS with them were significant at majorities oftimes.The above-mentioned results indicated that,dry matters in underground tubers of six yam genotypeswere mainly composed of AM,AP,Cel,and TSS;invertase and SuSy had activities in underground tubers ofeach of yam genotypes at every stage,and invertase especially SAI had higher activities at the early stage oftuber formations,and thereafter SuSy was promidant enzyme which caused different sink sizes of six yamgenotypes;the differences in starch between yams mainly attributed to differences in activities of bothAGPase and SSS;saccharide contents were closely relate to the related enzyme activities,of which Sucpossibly up-regulated both SuSy and the aforesaid and starch-related enzymes in forms of Suc/TSS andSuc/TRS;as to the maturing stage of underground tubers,the orders of six genotypes were white type of D.alata L.('Zixiao'and'Zida'),purple type of D.alata L.('Baibian'and'Baizhu'),'Yesheng',and'Putong'.(3) Homologous gene cDNA segments coding for SuSy,AGPase,and SSS were respectively isolatedfrom the underground tubers of six genotypic yams with RT-PCR and RACE techniques,and were 830 bp,784 bp,and 1 104 bp in sizes for putative amino acid sequences of 276,261,and 367,respectively.Theresults of both sirnilarities and systemic analyses of these cDNA segment sequences in combinations of theresults of yam classifications showed that SuSy,AGPase,SSS genes could all used to evaluate therelationship among six yam genotypes.At the same time,SuSy gene complete cDNA sequence was clonedfrom the underground tubers of yam'Zixiao'.DaSuSyl full-length cDNA sequence was 2 673 bp in size,which comprised a 2 445-bp largest open reading frame (ORF),a 7-bp 5'noncoding region and a 221-bp 3'noncoding region with a 24-nt poly (A⁺) tailor.A polypeptide of 814 amino acids with a 92.76-kDamolecular weight and a theoretical pI of 6.42 was putatively encoded by the largest ORF,containing bothtwo conserved functional regions as SuSy and glucosyl-transferase and two phosphorylated sites as Ser¹⁰ atN-end of and SNLDRRET⁷⁸¹ RR(Ser⁷⁷⁴～Thr⁷⁸¹) at C-end of amino acid sequence.At the levels of completecDNA sequences,coding regions and its deduced amino acid sequences,DaSuSyl shared the similarities of45.3 %～71.3 %,45.8 %～74.8 %,50.0 %～84.7 %,respectively,with SuSy genes of the selected andknown species in GenBank,and had the closest genetic relationship with some members of SuSy genefamilies of gramineous plants.DaSuSyl expressed only in non-photosynthetic organs of yam was not onlystrongly expressed in the underground tubers,but also its expression abundances progressively increasedfrom the initiation of early stage to the part of middle stage of the underground tuber bulking,and thereaftergradually decreased.Expressions of DaSuSyl,DaAGPasel,and DaSSS1 by RT-PCR and Northern hybridization showed that,three genes did not expressed in leaves of'Zixiao',but expressed more highly inunderground tubers;although DaSuSy1 expressed a little in stems and roots,DaAGPasel and DaSSS1 didnot completely;DaAGPase1 and DaAGPasel were possibly regulated after transcription and DaSSS1 atlevels of transcription.(4) After'Zixiao','Baibian',and'Putong'were foliarly sprayed with salicylic acid (SA) at the levels of0,0.5,1.0,5.0 mmol.L^-1 at the earlier stage and the termination of rapidly-bulking stage of the formation ofunderground yam tubers,respectively,both contents ofTSS,Suc,Glc,Frc,TS,AM,AP,Cel and activities ofSuSy,AGPase,GBSS were assayed.The results showed that,as opposed to5.0 mmol.L^-1 SA,0.5mmol.L^-1could enhance amounts of both Suc synthesized in leaves and its mobilization through stem base ofthe studied yam genotypes,and further increased contents of the physiological and biochemical parametersassayed,especially,these effects showed evidently on spraying at the earlier stage of tuber formations,while1.0 mmol.L^-1 had negative effects on two cultivars of D.alata L.and positive actions on'Putong';differences in the majorities of the assayed parameters in the same yam genotypes were insignificantbetween years;although the aforesaid parameters also bulkly had insignificant differences between 1.0mmol.L^-1 and the control,but were usually significant between 0.5 mmol.L^-1 or 5.0 mmol.L^-1 and the control.These results indicated that,0.5 mmol.L^-1SA contributed to Suc biosynthesis in leaves and its transport andits convesion into starch in underground tubers as opposed to 5.0 mmol·L^-1,wherea effects of 1.0mmol.L^-1varied with genotypes;SA at three levels had paralelled and consistent positive or negative actionson the assayed parameters;the effects of SA on accumulations of dry matters and starch were realized by theregulations of SA to Suc biosynthesis in leaves and transport in stem,and Suc as signal molecularconsistently up-regulated activities of SuSy,AGPase,and GBSS in the forms of Suc/TSS;because SA ofeach concentrations sprayed at different times produced slight and different influences,SA of proper levelsshould be continuously and foliarly sprayed once at every about 30 days from the early stage of tuberformations in order to facilitate accumulations of dry matters and starch in underground tubers,and also D.opposite Thunb.could endure higher levels of SA than D.alata L.(5) Partial cDNA of PAL homologous genes were amplified from underground tubers of six genotypicyams using traditional PCR,RACE-PCR and TAIL-PCR with both a pairs of degenerate primers and somespecific primers.The results of both similarities and systemic analyses of these cDNA segment sequences incombinations of the results of yam classifications showed that,although'Putong','Yesheng',and D.alata L.were differentiated in items of PAL homologous genes partial sequences,four cultivars ofD.alata L did not,so PAL gene was not suitable to evaluate phylogeny of yam genotypes.Meantime,A 2 376-bp full-lengthcDNA of PAL gene were isolated from the tubers of'zixiao',which contains a 1 986-bp ORF,a 28-bp5'UTR and a 362-bp 3'UTR with a 25-bp ploy (A⁺) tailor.The ORF encodes a 661 AA putative peptide withthe MW of 71.974 kDa and a pl of 6.310.Sequence characters for enzyme active site (GTITASGDLVPLSYIA) was observed in the amino acid sequence.DaPAL1 shared over 50 % similaritywith its homologous genes in other species at the levels of complete cDNA sequences,coding regions and itsdeduced amino acid sequences.DaPAL1 were closer to dicot PAL at the nucleotide sequences,and equallycloser to dicot or monocot PAL at amino acid sequences.RT-PCR and Northern blot analysis showed thatDaPAL1 was expressed only in the tubers and its abundance fluctuates in different stages of the life span.Amaximum expression of DaPAL1 was observed at the initiation of the early stage of tuber formation,thereafter,DaPAL1 expression decreased before the harvesting with slight increase.DaPAL1 expression andits enzyme activity and TPC had the same change patterns,which indicated that DaPAL1 was possiblyregulated at transcription level.(6) Homologous genes cDNA segments coding for CHS and ANS were isolated from the undergroundtubers of six genotypic yams with RT-PCR and RACE techniques,which were 800 bp,280 bp in sizes forputative amino acid sequences of 273,93,respectively.The results of both similarities and systemic analysesof these cDNA segment sequences in combinations of the results of yam classifications showed that,although CHS genes could used to evaluate the relationship among six yam genotypes,ANS genes could not.At the same time,ANS gene complete cDNA sequence was cloned from the underground tubers of yam'Zixiao',which wasl 387 bp containing a 1 077-bp largest ORF,a 9-bp 5' noncoding region and a 301-bp 3'noncoding region.DaANS1 could encode a polypeptide of 358 amino acids with a 40.4-kDa molecularweight and a theoretical pI of 5.26,in which had the 2-oxoglutarate- and Fe²⁺-dependent conservedoxidation regions containing two of conserved Arginine (Arg^295,304) related to the combination of2-oxoglutarate and the Fe²⁺ combination-related amino acid residues as both five of conserved Histidine(His^{238,243,249,276,294}) and three of Aspartate (Asp^240,260,279).DaANS1 shared the higher similarities with ANSgenes of the selected angiosperm species than gymnosperm ANS genes at the levels of complete cDNAsequences,coding regions and its deduced amino acid sequences,and had the closest genetic relationshipwith ANS genes of convolvulaceae plants in dicots of angiosperms,but was used to reasonably sort onlybetween genus or species plants.Expressions of DaCHS1 and DaANS1 by RT-PCR and Northernhybridization showed that,DaCHS1 expressed only in both underground tubers and roots,and had highexpression abundance at every stage,of which most strongly expressed at the former part of rapidly-bulkingstage;DaANS1 expressed in all organs,but more strongly expressed in tubers than in the other organs.DaCHS1 and DaANS1 were possibly regulated at levels of transcription.(7) During growth and development,TPC in underground yam tubers was composed of TFD,TPA,andTTN,which were respectively 0.64～0.93,0.10～0.25,0.06～0.22 of TPC.TAN and TFL mainlyconstituting TFD had respectively 0.53～0.75,0.13～0.34 of TFD in underground tubers of purple type of D.alata L.and 0.49～0.68,0.11～0.29 of TPC,while having respectively 0.49～0.68,0.11～0.29 of TFD inthe other yam tubers and 0.02～0.12,0.38～0.62 of TPC.General change trends of these phenol contents reduced with growth and development of tubers as opposed to ones of starch,and closely cooperated withTAC.Purple type of D.alata L.had higher TPC and TFD contents than the other genotypes,followed byones of'Putong'and white type of D.alata L.,and'Yesheng'was the lowest contents of TPC and TFD.Albeit having the highest content of TAN,purple type of D.alata L.was only higher contents of TFL andTPA than'Yesheng',and lower than'Putong'and white type of D.alata L.,which indicated that there wascompetitive relationship between TPA and TFD and between TAN and TFL.Significant differences incontents of these phenols between yam species and between types of D.alata L were observed at themajorities of stage,but did not for intra-types.The change trends of PAL and ANS activities were not onlythe same as ones of TPC and TAN,but also the results of comparisons in PAL and ANS activities betweengenotypes were similar with those of TPC and TAN.As compared with the control,general change trends ofphenol contents and the related enzyme activities did not have markedly differences after foliar spray of SAat different levels,their values would have somewhat changes about 30 days after spray,furthermore,SAshowed the synergistic actions on all kinds of phenols and activities of PAL and ANS,namely,0.5 mmol.L^-1SA had positive effects by contrast to 5.0 mmol.L^-1,and 1.0 mmol.L^-1 was positive to D.alata L andnegative to'Putong'.The aforesaid parameters of'Zixiao','Baibian,and'Putong'at the majorities of stageshad significant differences between years,between genotypes,and between treatments.Starch content inunderground tubers had strikingly significant and negative correlations to each of phenol contents,activitiesof both PAL and ANS in the same yam genotype,which phenol biosynthesis competed with starch formationfor requirements of sucrose.Phenol formations,activities of PAL and ANS were regulated by sucrose,andalso sucrose as signal molecular affected them in the forms of Suc/TSS,Suc/TRS.As opposed to SuSy,invertase especially SAI seemed to facilitate phenol biosynthesis,After SA treatment,the interrelationshipsof carbohydrates with phenols in underground yam tubers further indicated that differences in phenolcontents between yam species were caused by the effects of foliar-spray SA on biosynthesis and transport ofsucrose in leaves.