Nazi Nadernejad, Ali Ahmadimoghadam, Javad Hosseinifard and Shahram Pourseyedi

Transcription

1 Amerin-Eursin J. Agri. & Environ. Si., 2 (6): 87-84, 22 ISSN IDOSI Pulitions, 22 DOI:.5829/idosi.ejes Phenyllnin Ammoni-Lyse Ativity, Totl Phenoli nd Flvonoid Content in Flowers, Leves, Hulls nd Kernels of Three Pisthio (Pisti ver L.) Cultivrs 2 3 Nzi Ndernejd, Ali Ahmdimoghdm, Jvd Hosseinifrd nd Shhrm Pourseyedi Biology Deprtment, Fulty of siene, Shhid Bhonr University of Kermn, Irn 2 Nutrition nd Irrigtion Deprtment, Irn s Pisthio Reserh Institute, Rfsnjn, Irn 3 Plnt Breeding Deprtment, Fulty of Agriulture, Shhid Bhonr University of Kermn, Irn Astrt: Phenyllnin mmoni-lyse (PAL) plys pivotl role in the prodution of phenoli ompounds, whih re responsile for the suess of the defense strtegies in hrsh environments in response to different stimuli. Mesurements of the PAL tivity, totl phenolis, totl flvonoids nd nthoynin ontents were performed in flowers, leves nd fruits of three pisthio ultivrs "Ahmdghii", "Ohdi" nd "Kllehghuhi". The results showed tht PAL tivity ws different in ultivrs nd in plnts orgns of pisthio trees (flowers, leves nd fruits). The highest tivity rte of their ompounds were oserved in Ahmdghii ultivr. A positive orreltion ws oserved etween PAL tivity, totl phenolis nd totl flvonoids in leves nd negtive orreltion etween PAL tivity nd nthoynin ontents in leves nd flowers of Ahmdghii ultivr. PAL tivity nd totl phenoli in fruits of pisthio suffered deresed when the mturtion proesses egn. It is suggested tht the hulls of the pisthio fruits, ontin high level of phenoli ompounds (espeilly in Ahmdghii ultivr), my funtion s protetive lyer of defense hemils ginst ultrviolet rdition nd pthogens. The finl onentrtion of phenoli ompounds, flvonoids nd ntoynins in the kernel depend on PAL tivity in the kernel s ultivr. The results led to the onlusion tht inrese in PAL tivity, phenoli ompounds nd flvonoids in Ahmdghii n help the plnt to ope with the stresses etter thn the other ultivrs. Sine phenoli ompounds re ntioxidnt nd svenge free oxygen it is postulted tht Ahmdghii is the most resistnt ultivr to the environmentl stresses. Key words: Ahmdghii ultivr Environmentl stress PAL tivity Phenoli ompound Pisthio INTRODUCTION polyphenols. Flvonoids nd their derivtives re the lrgest nd most importnt group of polyphenols [5, 6]. Phenylpropnoids re derived from trns-innmi Flvonoids nd nthoynins re remrkly divers group id whih is formed from L-phenyllnine in retion of seondry produts with vst rry of iologil tlyzed y enzyme L-phenyllnine mmoni-lyse. funtion, inluding pprent roles in stress protetion PAL (EC ) is n extremely sensitive inditor of [7]. Phenoli ompounds one of the most widely stress onditions nd it is ommonly onsidered s ourring groups of phytohemils re of onsiderle iohemil mrker inditing the synthesis of oth physiologil nd morphologil importne in plnts. struturl nd protetive ompounds [-3]. It plys It is thought tht the moleulr sis for the protetive pivotl role in phenoli synthesis nd mny reports tion of phenoli ompounds in plnts is their emphsize on the orreltion etween inrese in the ntioxidnt nd free rdil svenging properties. orresponding PAL gene/protein expression/tivity The umultion of phenoli ompounds vries strongly nd inrese in the phenoli ompounds in response with the growth stte, development nd responses to to different stimuli [4]. The phenylpropnoid pthwy environmentl stresses nd is result of lne etween inludes lrge rnge of low moleulr weight iosynthesis nd further tolism [8, 9]. Corresponding Author: Nzi Ndernejd, Shhid Bhonr University, Post Code: , Kermn, Irn. P.O. Box , Kermn, Irn. Tel/Fx:

2 Am-Eurs. J. Agri. & Environ. Si., 2 (6): 87-84, 22 Pisthio (Pistsi ver L.) tree is n hull (erly-septemer), kernel t hrvesting time eonomilly importnt plnt whih is ultivted in (lte-septemer). The plnt mterils were susequently vst res of rid nd semi rid environments of frozen in liquid nitrogen nd stored t -8 C for the different elevtions nd preipittion rtes in Irn. nlysis. The experiment ws onduted under similr Hene, the plnt is exposed to different environmentl ondition t irrigtion nd soil type, using Rndomized stresses e.g drought nd ioti ftors. The plnt is Complete Blok Design (RCBD) with three loks. reprodued y grfting different ultivrs on Mesurements were rried out on three replite smples rootstoks nd the ssessment of the est ultivrs from eh lok. with respet to their dpttion to the environmentl stresses is likely to e neessry []. The Mesurements presene of phenoli ompounds in different prts of Phenyllnine Ammoni-Lyse Ativity: Phenyllnine pisthio tree hs een reported nd the ntioxidnt mmonium-lyse (PAL) ws extrted from fresh ell mss tivity nd totl phenoli ompounds of pisthio (3 mg fw) with 6.5 ml of 5 mm ph 8.8 Tris-HCl uffer (P. ver) hve een determined in hull extrts [], skin ontining 5mM of ß-merptoethnol in n ie-ooled nd seed [2, 3]. mortr, ground with pestle for out 5 min. The min ojetive of the present study ws to The homogente ws entrifuged for 3 min nd the ssess phenylpropnoid pthwy metolites in superntnt ws olleted for enzyme ssy. PAL tivity flowers, leves nd fruits of three pisthio ws determined sed on the rte innmi id ultivrs tht re grfted on the sme rootstok prodution. Briefly, ml of the extrtion uffer,.5 ml of P. muti. The ssessments were rried out y mm L-phenyllnine,.4 ml of deionized wter nd. mesuring PAL tivity, totl phenolis, flvonoids ml of enzyme extrt were inuted t 37 C for h. nd nthoynins. The ssessments ould led towrd The retion ws terminted y the ddition of.5 ml of 6 the seletion of the most suitle nd omptile ultivr M HCl nd the produt ws extrted with 5 ml ethyl in order to produe the highest yield with est qulity vi ette followed y evportion to remove the extrting its resistne to the environmentl, ioti nd ioti solvent. The solid residue ws suspended in 3 ml of.5 stress ftors. M NOH nd the innmi id onentrtion ws mesured spetrophotometrilly y the sorne t Regents nd Equipments: Folin-Ciolteu s phenol 29 nm. One unit of PAL tivity is equl to µmol of regent ws purhsed from Merk KGA (Drmstdt, innmi id produed per min [4]. Protein ws Germny). Stndred glli id, queretin 3-rutinosid estimted ording to Brdford [5] using BSA s (rutin) were purhsed from Sigm Chemil CO. stndrd. (St. Louis, MO). All hemils were of nlytil grde. Totl Phenoli Compounds: Totl phenoli ontent ws Equipments Were Used: UV-visile spetrophotometer, determined using the Folin-Ciolteu method Singleton Cry 5 on VARIAN, Austrli, Centrifuge Eppendorf nd Rossi [6] s modified y Velioglu et l. [7]. Smples AG 2233 Hmurg Germny. ( mg) were extrted with 8% methnol ontining % hydrohlori (5 ml) t room temperture for 2 hr on MATERIALS AND METHODS shker. The mixture ws entrifuged t 3g for min. The superntnt ws used for to determine totl Smple Colletion nd Preprtion: Three pisthio phenolis. One hundred miroliter of extrt ws mixed ultivrs "Ahmdghii", "Ohdi" nd "Kllehghuhi" with.75 ml of Folin- Ciolteu regent (previously diluted were evluted on rootstok Pisti tlnti su sp. -fold with distilled wter) nd llowed to stnd t 22 C Muti, in Irn s Pisthio Reserh Institute (IPRI). for 5 min;.75 ml of sodium ironte (6 g/l) solution The plnt mterils were olleted in April (flower) ws dded to the mixture. After 9 min t 22 C, nd July (lef) in 2. Pisthio fruits were rndomly sorne ws mesured t 725 nm. Glli id ws used seleted for smpling throughout ripening, from the for onstruting the stndrd urve. Results were young to the hrvest dte, on the following dtes: expressed s mg glli id (GA) per grm of the fresh green hull (July), green-red hull (August), red weight. 88

3 Am-Eurs. J. Agri. & Environ. Si., 2 (6): 87-84, 22 Totl Flvonoids: One hundred milligrms of smples were RESULTS extrted with ml 8% queous methnol. The mixture ws entrifuged for min t 2g. Superntnts were Flowers nd Leves: The results showed signifint used for susequent nlysis. The flvonoid ontent ws differenes in PAL tivity, totl flvonoids, totl mesured employing the olorimetri ssy desried y phenoli ompounds nd nthoynin ontents in leves Zhishen et l. [8]..5 ml liquots of extrts were dded nd flowers mong three ultivrs. However, totl to ml volumetri flsk ontining 4.5 distilled wter..3 flvonoid ontents of the ultivrs were not signifintly ml 5% sodium nitrite ws dded to eh liquot fter 5 different in the flowers while they were different mong min, then.6 ml of % luminum hloride ws dded. their leves (Tle -A,B). PAL tivity showed high After 6 min, 2 ml of M sodium hydroxide ws dded to trend in Ahmdghii leves nd flowers ompred to the the mixture following y the ddition of 2. ml distilled other ultivrs. The highest totl phenoli ontent ws wter. Asorne ws reorded t 5 nm nd flvonoid oserved in flowers of Ahmdghii nd the lowest ontent ws expressed s mg of rutin equivlent per mount in Kllehghohi, lso totl phenoli ontents ws g of fresh weight. highest in the leves of Ahmdghii nd the lowest oserved in Ohdi. The highest totl flvonoid ontent Anthoynins: Determintion of nthoynin ontents ws oserved in the leves of Ahmdghii ut no ws rried out using the method of Wgner [9]. Smples signifint differene ws oserved etween the totl (. g) were soked in ml idified methnol [methnol: flvonoid ontents of the flowers in three pisthio HCl 99: v/v]. The tissues were rushed nd kept t 25 C ultivrs. Anthoynin ontents were lower in for 24 h in the drk. The extrts were then entrifuged t Ahmdghii ultivr thn the other s leves nd were 4,g for 5 min t room temperture. The sorption rte lower thn Ohdi nd equl to Kllehghohi in their of the superntnt ws red y spetrophotometer t 55 flowers (Tle ). Correltions were found mong the nm. To lulte the mount of nthoynins, the exmined prmeters. Negtive orreltion existed extintion oeffiient 33, mol m ws used nd etween PAL tivity nd nthoynin ontents of leves nthoynin ontent were expressed s µ mol g fw. nd flowers. There ws high positive orreltion (99%) etween PAL tivity nd totl phenolis nd flvonoids Sttistil Anlysis: Dt of eh prmeter were in the leves (Tle 2). sujeted to two-wy ANOVA. Signifint differenes etween the mens of tretments were determined with Fruits (Hull, Kernel): The results showed 95% onfidene (p.5) limit y Dunn multiple rnge signifint differenes in PAL tivity, totl test (DMRT) using SPSS. Dt re shown s the mens of flvonoids, totl phenoli ompounds nd three replites. Correltions etween the level hrters nthoynin ontents in hulls (stge green, green-red were determined t 95% nd 99% (Tle 2). nd red) nd kernels mong three ultivrs (Tle 3). Tle : PAL tivity, totl phenoli, totl flvonoid nd ntoynin ontent in flowers (A) nd leves (B) of pisthio ultivrs A: flowers PAL tivity Totl phenoli Totl flvonoid Antoynin ontent Cultivr (unit mg protein) (mg GA g fw) (mg Ru g fw) (µ mol g fw) Ohdi.57± ± ± ±.67 Kllehghohi.87± ± ± ±.53 Ahmdghii.5± ±.7 38.± ±.3 B: leves PAL tivity Totl phenoli Totl flvonoid Antoynin ontent Cultivr (unit mg protein) (mg GA g fw) (mg Ru g fw) (µ mol g fw) Ohdi 2.32± ± ± ±.9 Kllehghohi 2.36±. 7.25±.23 3± ±.2 Ahmdghii 4.52± ± ± ±.75 Results re men±se (n = 9), different letters in the sme olumn represent signifint differenes t ( p=.5) ording to Dunn test. GA: glli id, Ru: rutin, unit: (PAL unit = µmol innmi id produed per min 89

4 Am-Eurs. J. Agri. & Environ. Si., 2 (6): 87-84, 22 Tle 2: Correltions etween hrters ANf ANl TFl TPHl PALf PALl Anf ANl TFl TPHl.965(**) PALf PALl -.965(**).942(**).976(**) AN: ntohynin, TF: totl flvonoid, TPH: totl phenoli, PAL : PAL tivity, (f: flower, l: lef ) ** Correltion is signifint t. level Tle 3: Totl flvonoid (A) nd Antoynin ontent (B) in green hull, green-red hull, red-hull nd kernel of pisthio ultivrs (during the fruit ripening) A: Totl flvonoid (mg Ru g fw) Cultivr green green red hull red hull kernel Ohdi 38.9±. 3.9± ± ±.4 Kllehghohi 32.87± ± ± ±. Ahmdghii 3.33± ± ± ±.5 B: Antoynin ontent (µ mol g fw) Cultivr green green red hull red hull kernel Ohdi 7.5±.3 7.5± ± ±.8 Kllehghohi 7.27±. 7.72± ± ±.7 Ahmdghii 6.5± ± ± ±.5 Results re men±se (n = 9), different letters in the sme olumn represent signifint differenes t ( p=.5) ording to Dunn test. Ru: rutin, unit: (PAL unit = µmol innmi id produed per min) PAL tivity (unite mg - protein) green hull red-green hull Ohdi ultivr July Aug erly-sep lte-sep PAL Phe red hull PAL tivity ( unit mg - protein) kernel Totl phenoli (mg GA g - fw) - protein) PAL tivity (unite mg Kllehghohi ultivr July Aug erly-sep lte-sep 35 PAL Phe green hullred-green hull Ahmdghii ultivr July Aug erly-sep lte-sep PAL Phe green hullred-green red hull hull Fig. : Chnges in totl phenoli ompounds ( ) nd PAL tivity ( kernel Totl phenoli ( mg GA g - fw) red hull kernel ) in pisthio ultivrs during ripening fruits. (GA: glli id, unit: PAL unit = µmol innmi id produed per min) Totl phenoli (mg GA g - fw) 8

5 Am-Eurs. J. Agri. & Environ. Si., 2 (6): 87-84, 22 Totl phenolis nd PAL tivity of Pisthio fruits from oserved etween the inrese of PAL tivity nd totl three ultivrs studied were different remrkly (Fig ). phenolis in leves. Inrese in PAL tivity nd the The results showed derese in PAL tivity nd totl umultion of high levels of phenoli ompounds in phenolis during the fruit ripening in ll ultivrs nd t ll Ahmdgii ultivr in this study n suggest tht this prts of the fruits, the highest rte in eh stge ws ultivr is more resistnt to the stresses thn the others. oserved in Ahmdgii ultivr. In erly stge of fruit Our results lso onfirm the positive role of phenoli ripening, in green hll, totl phenoli ompounds in ll ompounds in plnt protetion gin stress ondition. ultivrs were high nd grdully derese t the end of It seems the etter resistne of pisthio ultivrs n e ripening. In fruits ripening, in red hull stge the highest hrterized y inrese in PAL tivity nd phenoli nthoynin ontent were oserved in Ohdi nd level tht t s the ntioxidnts of retive oxygen Ahmdghii ultivr deteted the highest totl speies generted under stress influene. flvonoids. The hrvest time, kernels showed the lowest Positive orreltion etween PAL tivity nd totl rte pl tivity nd totl phenoli ompounds. In this flvonoids in this study shows tht inrese in PAL stge, totl phenoli ontents nd PAL tivity of kernel tivity possily indues the prodution of flvonoids. In from three pisthio ultivrs were the lowest thn the greement with the present results, Cheng et l. [24] other stges. The highest PAL tivity, totl phenolis showed tht the tivity of PAL hs diret effet on nd totl flvonoids otined in kernel of Ahmdghii flvonoid formtion nd tht the long wve light nd nthoynin ontents were highest in Ohdi ultivr inresed the tivity of PAL nd CHS (the fist enzyme thn the others. Although ll ultivrs hve strted in the flvonoid s iosynthesis) nd lso enhned umulting nthoynin t the first stge, they showed flvonoids umultion. Aording to the literture, gret inrese in nthoynin ontent in red hull stge, mutnt plnts (defiient in CHS) re unle to umulte ssoited with the ripening proess, the proess eing flvonoids nd were found to e more sensitive to more ovious minly in Ohdi ultivr. It ws oserved oxidtive stresses [25, 26]. It is hypothesized tht tht the outer prt of Ohdi ultivr fruits is strongly ntioxidnt flvonoids hve protetive funtion during olored. mny stresses nd seems tht flvonoids umultion in the leves nd flowers of Ahmdghii ultivr ould e, DISCUSSION more thn the others, useful for its resistnt to the environmentl stresses. Hene, Ahmdghii ultivr is The umultion of phenoli ompounds is onsidered more resistne thn Ohdi nd kllehghohi refully ontrolled proess with oth the levels of ultivrs. seondry metolites nd the omposition of the In the present study, negtive orreltions were phenoli pool vrying onsiderly etween orgnisms, oserved etween PAL tivity nd nthoynins in tissues, developmentl stges nd, in reltion to leves nd flowers. However, the ontents of oth totl environmentl onditions [2]. The PAL tivity whih is flvonoids nd phenoli ompounds were highest in highly sensitive to environmentl ondition, ply mjor Ahmdghii ultivr. It hs een indited tht lthough role in ontrolling the flux into totl phenolis. The min PAL tivity is required for nthoynin synthesis, it funtion of phenoil is mintin the stle onentrtion does not neessrily gurntee its synthesis sine the of free rdil y produting nd svenging them nd innmi id n e diverted into numerous other their physiologil funtion my e shown y regultion phenoli or flvonoid ompounds [27]. Aording to of ell redox potentil [2]. Ju et l. [28] nd Feng et l. [29] hnges in Hur et l. [22] indited tht determintion of PAL nthoynins umultion n our independently enzyme tivities should e explined during the stress from hnges in PAL tivity in pple nd per. period to otin etter understnding of how resistnt nd sensitive vrieties differ in their response. In nother experiment, Hur et l. [23] lso reported orreltion etween PAL tivity nd phenoli ompounds in leves of hyrid mize in drought stress nd onsidered the umultion of phenoli ompounds s the indition of tivted defense retion in the drought resistne of tht genotype. In our study, positive orreltion ws Hmouz et l. [3] lso in greement to our results showed tht the ontents of phenolis nd nthoynins were dependent on the plnt genotype. It lso ws oserved in this study tht different ultivrs responded differently in se of the nthoynin ontents. So it n e onluded tht PAL tivity lone did not regulte nthoynin synthesis nd the other enzyme re involved in their synthesis. hlon synthse 8

6 Am-Eurs. J. Agri. & Environ. Si., 2 (6): 87-84, 22 Fruits (Hull- Kernel): Regrding to results oserved in the study, we hve to onlude tht the ripening hrteristis of the fruits in pisthio ultivrs ffet oth on PAL tivity nd totl phenoli ontents (Fig.). Both suffered derese when the mturtion proess egn during pisthio development. Comprtive hnges in PAL tivity nd totl phenolis of three ultivrs show orreltion etween the ompounds where highest vlue oserved in Ahmdghii ultivr. Our results indited tht PAL tivity, whih is highly sensitive to environmentl ondition, ply mjor role in ontrolling the flux into totl phenolis. These dt greed with those of Montero et l. [3], who oserved tht PAL enzyme is involved in iosynthesis of phenoli ompounds, whih re known to umulte during erly stge of development. During orgn growth the phenoli profiles often undergo remrkle hnges inditing tht their metolism is integrted into progrms of growth nd development [32]. Our results indited tht PAL tivity nd totl phenoli ompounds present in outer lyer of pisthio fruits tht highest vlue ws oserved in Ahmdghii ultivr. High onentrtion of phenoli ompounds in fruits often go prllel with low inident y pthogen [33]. The lowest fltoxin ontent of pisthio kernels with hulls ompred with hulled kernels is lso proly the result of the fltoxin inhiitory effet of the hulls [34]. There is lso evidene tht flvonoids, in some plnt orgns is restrited to epiderml ells, my funtion in plnts to sreen hrmful rdition [35]. Goli et l. [] lso in greement to our results showed tht pisthio hull is nturl soure of phenoli ompounds nd Tomino et l. [3] indited tht these ompounds re present higher in skin thn in seed. In this study, the outermost lyer of the pisthio fruits nd nut kernels, the hulls, were showed to ontin high level of totl flvonoid nd phenoli ompounds. It seems the hulls of pisthio fruits (espeilly Ahmdghii ultivr) eing exposed to the environment my funtion s protetive lyer, thus phenoli ompounds tend to umultion in derml tissue of plnt odies euse of their potentil roles of protetion ginst ultrviolet rdition nd s defense hemil ginst pthogens nd predtors. Anthoynin ontents in erly stges of pisthio fruits development re low nd inrese during the progress of ripening. The highest rte oserved in Ohdi ultivr in the hulls of mture fruits. It seems nthoynin ontents inrese in the hulls of mture fruits depend on the vriety nd the stge of mturity. These findings re onsistent with previous studies [36]. It ws oserved tht the outer prt of Ohdi ultivr fruits is strongly olored. Although ll ultivrs hve strted umulting nthoynin t the first stge, minly in Ohdi ultivr, they showed gret inrese in nthoynin ontent in red hull stge tht ssoited with the ripening proess. There is no study out the PAL tivity in kernel pisthio in the literture. The present results indite tht PAL tivity nd totl phenolis in kernels of pisthio derese signifintly during fruit ripening in hrvest time nd signifint differene etween pisthio ultivrs exist minly in Ahmdghii ultivr oiniding with higher vlue on PAL tivity. These finding suggested tht PAL my e involved in growth nd development during seed mturtion, lso in the finl onentrtion of phenoli ompounds nd flvonoids nd nthoynins in the kernels. Bllistreri et l. [37] reported pisthio kernels ontin remrkle mount of phenoli ompounds suh nthoynins nd flvonoids. Silv et l. [38] determined high orreltion etween polyphenoli ontent nd the ntioxidnt tivity. The ntioxidnt tivity of pisthio seed ws reported y Tomino et l. [3]. Our results either indited the presene of numer of iotive ompounds in kernel of pisthio, the highest vlue oserved in Ahmdghii ultivr. CONCLUSION An inrese in the tivity of PAL n e onsidered s iohemil mrker for the resistne of the plnts to the environmentl stresses, given tht this enzyme is the key for neessry synthesis of phenoli ompounds ssoited with resistne. Ahmdghii ultivr grfted on muti exmined in this reserh hs high ontent of PAL tivity, phenolis nd flvonoids nd hene hs high ntioxidnt ility in the flowers, leves, hulls nd kernels. The ultivr n e introdued s resistnt genotype to the environmentl stresses nd totl phenolis nd flvonoids nd nthoynins from kernels of this ultivr n ply n importnt role in humn helth. However, to selet the most resistnt ultivrs to the environmentl stresses, further investigtions re required to e rried out on the other ntioxidnts nd in mny other ultivrs grown in pisthio orhrds espeilly when the ultivrs grfted on other rootstoks. ACKNOWLEDGMENTS The uthors knowledge Shhid Bhonr University for providing reserh funds nd Irn s Pisthio Reserh Institute (IPRI) for providing the reserh mteril. 82

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