J. MER. SOC. HORT. SCI. 130(6):842 847. 2005. Chnges in Protein Content, Protese tivity, nd mino id Content ssoited with Het Injury in Creeping entgrss Yli He College of griulturl nd iologil Siene, Shnghi Jio Tong University, Shnghi, 201101, P.R. Chin Xiozhong Liu nd ingru Hung 1 Plnt iology nd Pthology, Rutgers University, 59 Dudley Rod, New runswik, NJ 08901 DDITIONL INDEX WORDS. grostis stolonifer, high temperture, lef senesene, lipid peroxidtion STRCT. Vrious physiologil proesses my deteriorte in response to inresing tempertures, ontriuting to the deline in turf qulity for ool-seson turfgrsses during het stress. This study ws performed to investigte metoli hnges (memrne lipid peroxidtion, totl protein ontent, mino id ontent, nd protese tivity) ssoited with turf qulity deline for reeping entgrss (grostis stolonifer Huds.) in response to grdully inresing tempertures for short durtion nd prolonged exposure to lethlly high temperture. Plnts were sujeted to inresing tempertures of 20, 25, 30, 35, nd 40 C for 7 dys t eh level of temperture [grdul het stress (GHS)] or exposed to high temperture of 40 C for 28 dys [prolonged het stress (PHS)] in growth hmers. During the GHS tretment, signifint deline in turf qulity ourred when plnts were exposed to 30 C for 7 dys; simultneously, mlondildehyde (MD) ontent inresed nd totl protein ontent in shoots deresed signifintly ompred to those t 20 C. Protese tivity inresed t 25 C nd then deresed s temperture ws elevted from 30 to 40 C during the GHS tretment. mino id ontent deresed under GHS, eginning t 25 C. Under the PHS tretment, turf qulity delined nd MD ontent inresed signifintly, eginning t 14 dys of PHS, while totl protein ontent deresed t 7 dys of PHS. Protese tivity nd mino id ontent inresed t 7 dys of PHS, nd then delined with longer stress durtion. Our results indited tht protese tivity, nd mino id nd totl protein ontent were more responsive to GHS or PHS thn tht of lipid peroxidtion nd turf qulity. Chnges in metoli prmeters of protese tivity, mino id nd totl protein ontent, nd lipid peroxidtion my ontriute to lef senesene nd poor turf performne under severe or prolonged het stress onditions for reeping entgrss. Cool-seson turfgrsses grown in temperte regions often suffer from het stress when temperture exeeds 24 C (DiPol nd erd, 1992). Loss in green olor or lef senesene is typil symptom of het injury in turfgrsses. Therefore, the mintenne of green turf nopy of ool-seson turfgrsses during summer months is mong mjor onerns in turfgrss mngement. Understnding the underlying sis of lef senesene indued y het stress is essentil for mintining high qulity turf of ool-seson turfgrsses in wrm limti res. Oxidtion of lipids (lipid peroxidtion) of ell memrnes is uiquitous feture of lef senesene, whih my our during nturl senesene nd n e indued y environmentl stresses (Thompson et l., 1987). The level of lipid peroxidtion is positively orrelted with memrne dmge nd lef senesene, nd, therefore, is used widely s senesene inditor (Dhinds et l., 1981). Previous studies reported tht het-indued lef senesene nd turf qulity deline ws ssoited with inresed level of lipid peroxidtion in reeping entgrss (Hung et l., 2001; Lrkindle nd Hung, 2004; Liu nd Hung, 2000) nd other ool-seson turfgrss speies (Jing nd Hung, 2001). In ddition to lipid peroxidtion, loss of proteins is often ssoited with het stress injury nd lef senesene (nderson Reeived for pulition 24 Fe. 2005. epted for pulition 25 June 2005. knowledgments: The uthors express thnks for the Center of Turfgrss Siene, Rutgers Univ., for finnil support of this study. Thnks lso go to Dr. Shimon Rhmilevith, Mihelle DCost, Yn Xu, nd Steve MCnn for ritil review of the mnusript. 1 Corresponding uthor; e-mil ddress: hung@esop.rutgers.edu nd Pdhye, 2004; Cllis, 1995; Hensel et l., 1993; Smrt, 1994). Mny studies reported tht protein degrdtion ws elerted during het stress in vrious plnt speies (Hensel et l., 1993; Smrt, 1994; Cllis, 1995) inluding ool-seson turfgrss speies (He et l., 2005; Jing nd Hung, 2002). Proteins in plnt ells re lssified into different groups sed on their soluility properties nd loliztion in ells (Loponen et l., 2004). Slt- nd wter-solule proteins (slt-solule protein) re usully lolized in ytoplsm nd lled ystoplsmi protein. s SDS gives good soluiliztion of memrne proteins (mes nd Nikido, 1976; Hurkmn nd Tnk, 1986), SDS-solule proteins re minly ell memrne proteins. In this study, we exmined protein ontent of oth groups in reeping entgrss under het stress. Proteins re tolized y proteses into mino ids, nd derived mino ids my e trnsported to growth or storge orgns during lef senesene (Feller nd Fisher, 1994; Peoples nd Dlling, 1988). Protein loss nd inresed protese tivity ould e mrkedly entuted y high temperture stress, whih my elerte lef senesene (l-khti nd Pulsen, 1984). Despite the importne of het-indued lef senesene for ultivtion of ool-seson turfgrsses, the underlying mehnisms re not well understood. How hnges in mino id umultion, protese tivity, nd protein degrdtion re ssoited with het injury in ool-seson turfgrsses, nd the level of high tempertures or durtion of het stress using hnges in these metoli ftors re not ler. Therefore, the ojetives of this study were to 1) exmine protein nd mino id ontent, protese tivity, nd lipid peroxidtion of reeping entgrss in response 842 J. MER. SOC. HORT. SCI. 130(6):842 847. 2005.
to GHS nd PHS; nd 2) determine level of temperture nd durtion of het stress ritil for metoli hnges ssoited with lef senesene nd turf qulity deline. The temperture levels tht ffet eh metoli prmeter were evluted y exposing plnts to GHS (from 20 to 40 C t 5 C intervls). Het stress durtion tht uses hnges in eh prmeter ws determined y exposing plnts to het stress (40 C) for prolonged period of time (28 d) nd monitoring metoli hnges over time. Mterils nd Methods PLNT MTERILS ND GROWTH CONDITIONS. Sod plugs of ʻPennrossʼ reeping entgrss were olleted from 3-yer-old field plots t the Rutgers Univ. Hortiulture Frm II, North runswik, N.J., in My 2002. Sods were trnsferred into polyvinyl hloride (PVC) tues (10-m dimeter nd 60 m long) filled with wshed snd. Plnts were mintined in greenhouse for 1 month nd then moved into four growth hmers set t 20/16 C (dy/night temperture), 75% reltive humidity, 14-h photoperiod, nd 400 μmol photons m 2 s 1 of photosynthetilly tive rdition. Plnts were mintined in growth hmers for 40 d prior to imposing temperture tretments. Plnts were fertilized one per week with 40 ml of full-strength Hoglndʼs solution (Hoglnd nd rnon, 1950) nd ut twie per week to keep the height t out 5 m. TEMPERTURE TRETMENTS. To exmine turf qulity nd metoli hnges with inresing tempertures, four pots of plnts (four replites) were exposed sequentilly for 7 d to eh of the following tempertures: 20, 25, 30, 35, nd 40 C (dy/night temperture) (GHS). Leves were smpled rndomly from eh pot for nlysis t the end of eh 5 C temperture inrement (t 7 d of 20 C, 20 to 25 C, 20 to 25 to 35 C, 20 to 25 to 30 to 35 C, nd 20 to 25 to 30 to 35 to 40 C. Therefore, tretments of GHS were expressed s 20 C, 20 to 25 C, 20 to 25 to 30 C; 20 to 25 to 30 to 35 C, nd 20 to 25 to 30 to 35 to 40 C in Figs. 1 5. To determine turf qulity nd metoli hnges with het stress durtion, four pots of plnts were moved diretly from growth hmer t 20 C to four hmers set t 40 C (dy/night temperture), nd mintined t 40 C for 28 d (PHS). During oth temperture tretments, the growth hmers were set t the environmentl onditions desried ove. Plnts were wtered twie per dy, fertilized one per week with 40 ml of full-strength Hoglndʼs solution (Hoglnd nd rnon, 1950) nd ut twie per week to keep the height t 5 m. EVLUTION OF TURF QULITY ND LEF LIPID PEROXIDTION. Turf qulity ws visully rted for four pots of plnts exposed to either GHS or PHS tretment. Turf qulity ws evluted sed on turf olor nd shoot density on sle of 0 (worst, plnts were rown nd ded) to 9 (est, plnts were green nd helthy). The level of lipid peroxidtion in shoots ws evluted in terms of MD ontent s mesure of the severity of lef senesene (Dhinds et l., 1981). Mlondildehyde is finl produt of peroxidtion of unsturted ftty ids in phospholipids of ellulr memrnes (Smirnoff, 1995). MD ontent ws mesured using modified method of Dhinds et l. (1981) nd Zhng nd Kirkhm (1994). From eh of four pots t eh temperture or stress durtion tretment, 0.5 g of fresh shoots ws olleted nd frozen in liquid N 2. Frozen tissues were ground in 4 ml solution ontining 50 mm phosphte uffer (ph 7.0), nd 1% (w/v) polyvinylpolypyrrolidone. The homogente ws entrifuged t 15000 g n for 20 min, nd superntnt (lef extrtion) ws olleted nd used for MD ontent mesurement. 2-mL liquot of lef extrtion solution ws dded to tue ontining 1 ml 20% (v/v) trihloroeti id nd 0.5% (v/v) thiorituri id. The mixture ws heted in wter th t 95 C for 30 min, ooled to room temperture, nd then entrifuged t 10,000 g n for 10 min. The sorne of the superntnt t 532 nm ws determined nd the nonspeifi sorne t 600 nm ws sutrted using spetrophotometer (Spetroni Genesys 2; Spetroni Instruments, Rohester, N.Y.). The MD ontent ws lulted using the extintion oeffiient of 155 mm 1 m 1 (Heth nd Pker, 1968). PROTEIN EXTRCTION ND QUNTIFICTION. Protein ontent ws mesured using the method of rdford (1976). 0.5-g smple of fresh leves ws olleted from eh of four pots in eh tretment. Frozen shoots were ground in liquid N 2 to fine powder. Protein ws extrted in 3 ml of Tris-HCl uffer (0.10 mm This- HCl, nd 0.15 M NCl) (ph 7.6). Smples were then entrifuged twie t 16,000 g n t 4 C for 0.5 h to get ler superntnt for hydrophili protein ontent mesurement. The pellet ws susequently re-suspended with 3 ml SDS-Tris-HCl uffer [0.10 mm Tris-HCl, 0.15 M NCl, nd 2% (w/v) SDS] (ph 7.6) nd spun nd mixed for 1 h. Extrted suspensions were then entrifuged t 16,000 g n t 4 C for 0.5 h nd superntnt ws olleted for hydrophoi protein ontent mesurement. Protein ontent ws determined y the method of rdford (1976). riefly, 100 μl of protein extrtion (diluted five times) ws mixed with 3 ml of Coomssie G-250 regent (1% Coomssie rillint lue G, 4.7% ethnol, nd 8.5% phosphori id), nd the sorne ws mesured t 595 nm fter 5 nd 30 min of retion using spetrophotometer (Spetroni Genesys 2). ovine serum lumin ws used s stndrd. Totl protein ontent ws the sum of sltsolule protein ontent nd SDS-solule protein ontent. MINO CID NLYSIS. mino id ontent ws determined in shoots olleted from eh of four pots t eh temperture tretment using the method of Rosen (1957) with some modifitions. riefly, fresh shoots (1.0 g) were frozen in liquid N 2 nd ground to fine powder. mino id ws extrted in 6 ml of 80% ethnol. Smples were then entrifuged t 16,000 g n t 4 C for 30 min, nd superntnt ws olleted for mino id mesurement. Onehlf milliliter of ynide-ette uffer (0. 2 mm NCN in 2.65 M ette uffer) (ph 5.4) nd 0.5 ml 3% (w/v) ninhydrin solution in methyl ellosolve (ethylene glyol monomethyl ether) were dded in 1 ml of 20-times-diluted mino id extrtion. The mixed solution ws heted for 15 min in 100 C wter th. Three milliliters of 50% (v/v) isopropyl lohol wter diluents ws then dded in the solution nd shken vigorously. The retion solution ws llowed to ool to room temperture. The sorne of the retion solution ws red t 570 nm using spetrophotometer (Spetroni Genesys 2). Glyine ws used s stndrd to lulte mino id ontent. PROTESE CTIVITY MESUREMENT. Protese ssys were performed using modified method of enell nd Pulsen (1998). Fresh shoots (1.0 g) were olleted from eh of four pots in eh temperture tretment nd were homogenized in 4 ml of old medium ontining 25 mm N-2 hydroxyethylpiperzine-n -2- ethnesulfoni id (Hepes) uffer (ph 7.5), 1 mm ethylenediminetetr eti id (EDT), nd 4 mm dithiothreitol (DTT). The homogenized smples were entrifuged twie t 16000 g n t 4 C for 30 min. Crude protese tivity ws ssyed y dding 0.1-mL liquots of enzyme extrt to 1.5-mL 200 mm sodium itrte-itri id uffer (ph 5.2) nd 0.4 ml of 1% (w/v) zosein in test tues. The referene tue ws prepred with 0.1 ml of medium for protese extrtion to sustitute the enzyme extrt. J. MER. SOC. HORT. SCI. 130(6):842 847. 2005. 843
The tues were inuted for 2 h t 37 C. 1-mL smple of 15% (w/v) trihloroeti id (TC) ws dded to smple nd referene tues to stop the retions. Smple nd referene tues were then inuted t 4 C for 30 min. The resulting preipitte ws removed y entrifugtion t 4000 g n for 5 min twie nd superntnt ws filtered y six lyers of heeseloth in syringe. The sorne of the ler superntnt ws red t 340 nm in spetrophotometer (Spetroni Genesys 2). The sorne of the referene ws sutrted from the smple. One unit of enzyme tivity ws defined s the hnges of the sorne y 0.1. STTISTICL NLYSIS. Tretments were rrnged in rndomized omplete-lok design with four replites. ll mesurements were mde in four replites (pots), nd three su-smples from eh extrtion of smples in eh replite were used for the nlysis of protese tivity, protein ontent, nd mino id ontent. Mens of the three su-smples were used to present single replition in nlysis of vrine (NOV). Men seprtions were performed with the Fisherʼs proteted lest signifine differene test t P = 5 (Steel nd Torrie, 1980). Results CHNGES IN TURF QULITY ND LIPID PEROXIDTION WITH INCRESING TEMPERTURES ND STRESS DURTION. Turf qulity deresed from 8.0 to 7.5 s temperture ws elevted from 25 to 30 C during the GHS tretment, nd further delined to 7.0 or lower when plnts were exposed to higher tempertures from 35 to 40 C (Fig. 1). Turf qulity lso delined with inresing durtion of PHS, eginning t 14 d of tretment (40 C) (Fig. 1). Turf qulity dropped to elow 6.0 y 28 d of PHS (Fig. 1). In ontrst to turf qulity hnge, lef MD ontent inresed signifintly under GHS (Fig. 2). signifint inrese in MD ontent ws first deteted t 30 C nd the inrese ontinued t higher tempertures (35 nd 40 C). MD ontent lso inresed signifintly with the durtion of PHS, eginning t 14 d of tretment (Fig. 2). MD ontent y 28 d of PHS ws lmost two times s high s the pre-stress level (0 d). The inrese in MD ontent ws onomitnt with turf qulity deline initited t 30 C during GHS nd t 14 d during PHS tretment. CHNGES IN PROTEIN CONTENT WITH INCRESING TEMPER- TURES ND STRESS DURTION. Chnges in protein ontent under GHS nd PHS followed the sme pttern s tht for turf qulity. Totl protein ontent deresed during GHS from 20 to 40 C (Fig. 3) nd s het stress (40 C) durtion ws prolonged to 28 d (Fig. 3). The initil deline in totl protein ontent ws oserved t 30 C during GHS (Fig. 3) nd t 7 d during PHS (Fig. 3). Totl protein ontent deresed y 75% t the end of GHS nd PHS. Turf qulity 8.5 8.0 7.5 7.0 6.5 MD ontent (nmol.g -1 fresh wt) 6.0 20 20-25 30-40 5 4 3 2 1 20 20-25 30-40 Temperture ( o C ) Temperture ( o C ) Turf qulity 1 8.0 6.0 4.0 2.0 Durtion of het stress (d) Fig. 1. Chnges in turf qulity of reeping entgrss during grdul het stress (GHS) (), nd prolonged het stress (PHS) (). Turf qulity ws evluted sed on turf olor nd shoot density on sle of 0 (worst, plnts were rown nd ded) to 9 (est, plnts were green nd helthy). Different letters indite signifint differene t P = 5 mong temperture tretments for GHS or different tretment durtions for PHS. rs represent SE (n = 4) nd different letters indite signifint differene t P = 5. MD ontent (nmol.g -1 fresh wt) 8 7 6 5 4 3 2 1 Durtion of het stress (d) Fig. 2. Chnges in mlondildehyde (MD) ontent in shoots of reeping entgrss during grdul het stress (GHS) (), nd prolonged het stress (PHS) (). Different letters indite signifint differene t P = 5 mong temperture tretments for GHS or different tretment durtions for PHS. rs represent SE (n = 4) nd different letters indite signifint differene t P = 5. 844 J. MER. SOC. HORT. SCI. 130(6):842 847. 2005.
CHNGES IN PROTESE CTIVITY WITH INCRESING TEMPERTURES ND STRESS DURTION. Protese tivity inresed to the highest level s temperture ws elevted to 25 C (1.4 times tht t 20 C) (Fig. 4). It then deresed when temperture ws further inresed to 30, 35, nd 40 C. In response to PHS t 40 C, protese tivity inresed to ove the pre-stress level t 7 d of tretment, nd then deresed with longer het stress durtion (Fig. 4). CHNGES IN MINO CID CONTENT WITH INCRESING TEMPER- TURES ND STRESS DURTION. mino id ontent deresed stedily s temperture ws inresed from 20 to 35 C during the GHS (Fig. 5). The initil deline ws oserved t 25 C nd signifint inrese when the temperture ws inresed from 35 to 40 C during GHS. When plnts were exposed to 40 C for prolonged durtion, mino id ontent inresed t 7 d of tretment, ut then deresed grdully from 7 to 28 d (Fig. 5). Disussion The results in this study suggested tht GHS to 30 C nd PHS t 40 C for 14 d ws detrimentl for reeping entgrss, s mnifested y the elerted lef senesene (mesured s inresed lipid peroxidtion) nd turf qulity deline. Hung nd Go (2000) lso reported tht 30 C ws the threshold for turf qulity deline in reeping entgrss in seprte study. They ttriuted turf qulity deline ove 30 C to the imlne etween photosynthesis nd respirtion nd limited rohydrte vilility. The present study demonstrted tht het-indued lef senesene nd turf qulity deline ould lso e relted to hnges in protein ontent, protese tivity, nd mino id ontent, s disussed elow. Mintenne of stle proteins in highly orgnized stte is importnt for proper funtions of ells during high temperture stress due to their involvement in metoli proesses nd memrne funtion (Levitt, 1980). Turf qulity deline nd inreses in MD ontent were ompnied y dereses in protein ontent s temperture ws elevted or het stress durtion ws prolonged. The loss of protein ourred erlier thn turf qulity deline nd lipid peroxidtion inrese during PHS, suggesting tht it ould ontriute to long-term het stress injury in shoots for reeping entgrss. The redution in totl protein ontent under het stress hs een previously reported in other speies (Chitny et l., 2001; Myer et l., 1990). The progressive derese in totl protein ontent in shoots of reeping entgrss under het stress ould e due to the impirment of protein synthesis nd/or inresed degrdtion. He et l. (2005) reported tht synthesis of new proteins ws oserved in reeping entgrss under either grdul or sudden het stress, long with deline in protein ontent. Colletively, these results suggested protein degrdtion ould ply lrger Totl protein ontent ( % dry wt) 3 25.0 2 15.0 1 5.0 20 20-25 30 Temperture ( o C) -40 Protese tivity (unit/g dry wt per min) 25.0 2 15.0 1 5.0 20 20-25 30 Temperture ( o C ) d - 40 Totl protein ontent ( % dry wt) 3 25.0 2 15.0 1 5.0 d d Protese tivity (unit/g dry wt per min) 25 20 15 10 5 0 Durtion of het stress (d) Durtion of het stress (d) Fig. 3. Chnges in totl protein ontent in shoots of reeping entgrss during grdul het stress (GHS) (), nd prolonged het stress (PHS) (). Different letters indite signifint differene t P = 5 mong temperture tretments for GHS or different tretment durtions for PHS. rs represent SE (n = 4) nd different letters indite signifint differene t P = 5. Fig. 4. Chnges in protese tivities in shoots of reeping entgrss during grdul het stress (GHS) (), nd prolonged het stress (PHS) (). Different letters indite signifint differene t P = 5 mong temperture tretments for GHS or different tretment durtions for PHS. rs represent SE (n = 4) nd different letters indite signifint differene t P = 5. J. MER. SOC. HORT. SCI. 130(6):842 847. 2005. 845
role in loss of net protein thn the inhiition of protein synthesis in reeping entgrss exposed to GHS or PHS. It hs een reported previously tht inresed proteolyti tivity is orrelted with the loss of totl solule protein in folir senesene (Feller et l., 1977; Mrtin nd Thimnn, 1972; Peterson nd Huffker, 1975). Nonspeifi protese tivity hs een reported to inrese y 4-fold t 25 C nd y 28-fold t 35 C from 0 to 21 d fter the initition of temperture tretments in winter whet (Tritium estivum) during grin development stge (l-khti nd Pulsen, 1984). Our results indited tht enhned proteolyti tivity might ount for protein loss in leves of reeping entgrss exposed to modertely high temperture (25 nd 30 C) or short-term severe het stress (40 C for 7 d). The deline in protein with inresing tempertures to higher levels (35 nd 40 C) or het stress for longer durtion (for 14 to 28 d t 40 C), however, ws not relted to hnge in protese tivities. The protese tivities deresed s the temperture inresed further to 35 nd 40 C nd the het stress prolonged longer thn 7 d. Therefore, the redution in protese tivity under higher tempertures or long-term stress my reflet het injury in protein stility nd thus enzyme funtions (Levitt, 1980). Protein ggregtion ourred in sweet pe (Lthyrus odortus L.) nd vin (Cthrnthus roseus L.) leves t tempertures greter mino id ontent (% dry wt) mino id ontent (% dry wt) 1.0 0.8 0.6 0.4 0.2 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0 20 20-25 30 Temperture ( o C ) d -40 Durtion of het stress (d) Fig. 5. Chnges in mino id ontent in shoots of reeping entgrss during grdul het stress (GHS) (), nd prolonged het stress (PHS) (). Different letters indite signifint differene t P =5 mong temperture tretments for GHS or different tretment durtions for PHS. rs represent SE (n = 4) nd different letters indite signifint differene t P = 5. thn 40 C (nderson nd Pdhye, 2004). s the loss of protein is dominnt feture of lef senesene during het stress nd muh of the protein is proly tolized y speifi proteses (Mrtin nd Thimnn, 1972), further studies re required to identify nd hrterize speifi senesene-relted proteses in order to understnd proteolyti mehnisms responsile for het-indued protein degrdtion nd lef senesene. The deline in protein ontent during het stress hs een found to e ssoited with n inrese in free mino id umultion in some plnt speies, suh s whet (l-khti nd Pulsen, 1984) nd mulerry (Morus l L.) (Chitny et l., 2001). In our study, within 7 d of het stress t 40 C, inresed totl mino id ontent prlleled the derese in protein ontent nd the inrese in protese tivity. During the initil stge of lef senesene, proteins must e degrded y peptide hydrolses to mino ids prior to the export of the protein nitrogen (Feller, 1990; Peoples nd Dlling, 1988). However, the derese of protein ontent ws not lwys prlleled y the umultion of mino id. s het stress prolonged to 14, 21, nd 28 d, mino id ontent lso deresed. Our results suggest tht the hnges of protein ontent, protese tivity, nd mino id levels nd their reltionship under stress were dependent on the temperture level nd the durtion of het stress. mino id ontent is not only ffeted y proteolysis, ut lso is determined y other proesses, inluding mino id synthesis, omprtmentliztion, trnsport of mino ids, nd metolism to other end-produts (Notor et l., 2002). The deline in mino id ontent in leves my reflet remoiliztion of mino ids from senesent leves into other tissues to ope with the highenergy demnds ugmented during high temperture stress. Levitt (1980) suggested tht evolution of het dpttion ould involve onformtionl flexiility, perhps prtly through rellotion of ertin mino ids. The deline in mino id ontent under severe het stress ould lso e relted to the lekge from ells through dmged memrnes. Inresed mino id lekge hs lso een deteted in reeping entgrss exposed to het stress (35 C), whih hs een ssoited with lipid peroxidtion (Lrkindle nd Hung, 2004). In ft, lipid peroxidtion ws enhned in response to GHS or PHS in this study, lthough diret lekge of mino id ws not exmined. Compring temperture- nd time-responses of different prmeters reveled tht protese tivity nd mino id ontent were more sensitive inditors of het injury thn other prmeters exmined. The hnges in protese nd mino id ourred t 5 C lower or 7 d erlier thn the hnges in protein ontent (t 30 C GHS) nd memrne lipid peroxidtion nd turf qulity deline (t 30 C of GHS nd 14 d of PHS). These results lso suggested tht het injury in reeping entgrss, s mnifested y turf qulity deline, under GHS or PHS, ws ssoited with dereses in protese tivity, mino id ontent, protein ontent, nd inreses in lipid peroxidtion. Literture Cited l-khti, K. nd G.M. Pulsen. 1984. Mode of high temperture injury to whet during grin development. Physiol. Plnt. 61:363 368. mes, G.F.-L. nd K. Nikido. 1976. Two-dimensionl gel eletrophoresis of memrne proteins. iohemistry15:616 623. nderson, J.. nd S.R. Pdhye. 2004. Protein ggregtion, rdil snvenging pity, nd stility of hydrogen peroxide defense systems in het-stressed vin nd sweet pe. J. mer. So. Hort. Si. 129:54 59. enell, M. nd G.M. Pulsen. 1998. Effiy of tretments for delying 846 J. MER. SOC. HORT. SCI. 130(6):842 847. 2005.
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