Effect of Acute Heat Stress on Nutrient Uptake by Plant. Roots. A Thesis. entitled. Anju Giri

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1 A Thesis entitled Effect of Acute Het Stress on Nutrient Uptke y Plnt Roots y Anju Giri Sumitted to the Grdute Fculty s prtil fulfillment of the requirements for the Mster of Science Degree in Biology Scott A. Heckthorn, PhD, Committee Chir John Gry, PhD, Committee Memer Jonthn M. Frntz, PhD, Committee Memer Ptrici R. Komuniecki, PhD, Den College of Grdute Studies The University of Toledo Decemer, 213

2 Copyright 213, Anju Giri This document is copyrighted mteril. Under copyright lw, no prts of this document my e reproduced without the expressed permission of the uthor.

3 An Astrct of Effect of Acute Het Stress on Nutrient Uptke y Plnt Roots y Anju Giri Sumitted to the Grdute Fculty s prtil fulfillment of the requirements for the Mster of Science Degree in Biology The University of Toledo Decemer, 213 The impct of rupt het stress on plnt nutrient uptke is uncler, since most pst studies hve exmined uptke under highly-rtificil conditions (e.g., detched roots), which likely yielded rtifctul results. Het stress often ffects roots, roots re often more het sensitive thn shoots, nd glol wrming nd incresing het wves men more cute het stress for roots in the future. Hence, we re investigting effects of rupt high tempertures (=cute het or het wve), on nutrient uptke y roots, using tomto s model system. In the current study, we grew plnts t 25/2 o C (dy/night) (= control conditions) nd then trnsferred some plnts to 35/3 o C (moderte het) or 42/37 o C (severe het) for 6 dys, fter which, plnts were moved ck to control conditions for 7 dys to monitor recovery. Smples were hrvested fter 1 nd 6 dys of het tretment, nd fter 7 dys of recovery (dy 13), nd then the concentrtion of mjor nutrient uptke proteins in roots ws determined using protein-specific ntiodies nd ELISA. Photosynthesis ws reduced y severe het, compred to controls, nd recovered within 7 dys of return to control tempertures. Roots were negtively impcted y het (severe > moderte), s indicted y decreses in respirtion, protein concentrtion, iii

4 memrne integrity, nd root mss. Root mss decresed more thn shoot mss with het stress. Totl plnt nutrient content, root nutrient uptke rte, nd the level nd, in some cses, ctivity of nutrient uptke proteins were decresed y oth moderte nd severe het. Importntly, for most mesures of function, modertely-stressed plnts were le to recover within 7 d fter het, ut recovery ws incomplete for severely-stressed plnts. Together, our results indicte tht rupt severe het events cn dmge roots nd decrese root nutrient uptke, y decresing root metolic rte, levels or ctivities of nutrient uptke proteins, nd/or root mss. iv

5 To people who never stopped elieving me; my fmily, friends, ABHI, techers nd of ll to the God who is lwys with me in my every steps.

6 Acknowledgements I m highly indeted to my mjor dvisor Professor Dr. Scott A Heckthorn for his continuous nd congenil help during my reserch nd for the opportunity he provided me to work s first couthor in ook chpter. He inspired me to think cretively nd helped me to enhnce my knowledge. My sincere thnks goes to my committee memers Dr. John Gry nd Dr. Jonthn Frntz for their vlule suggestions nd comments. Thnk you Dr. Frntz for giving me chnce to work in side reserch project: Phosphorus deficiency nd recovery. Also, thnks to D. Sturtz nd R. Friedrich (USDA) for their help on nutrient nlysis. I owe specil thnks to Dr. Ssmit Mishr for her help nd direction during my first reserch work. Also thnks to my l mtes, friends nd professors in the deprtment; Dr. Moorhed, for his consent out my grdution requirement nd for helping me to lern out the foundtions of Ecology, Dr. Myer nd Dr. Qin for providing me insight of stt, Dr. Weintru for letting me to prticipte in seminr clsses nd to the friends from Nepl, United Sttes nd ll over the world for their support physiclly or morlly. Specil thnks to Ligi for her nice nd friendly ehvior. My eternl thnk goes to my prents: Mr. Bhrt Giri, Mrs. Renuk Giri nd my ll fmily memers. Without you, I would hve chieved nothing. My life is ll yours. Specil thnks to my der Mr. Ahishes Lmsl; your love, support nd cre is lwys my leding pth. Thnk you so much for eing prt of my life. I love you more! v

7 Tle of Contents Astrct... iii Acknowledgements...v Tle of Contents... vi List of Figures... viii List of Arevitions... ix List of Symols...x 1 Introduction Mterils nd Methods Plnt growth conditions, temperture tretments, nd hrvesting Photosynthesis nd root respirtion Memrne integrity Nutrient reltions Protein extrction nd quntifiction Nutrient nlysis Nutrient metolism protein quntifiction Sttisticl nlysis...13 vi

8 3 Results Plnt growth nd cron reltions Nutrient reltions... 15(Minli, 27) 4 Discussion...3 References...35 vii

9 List of Figures 3-1 Effect of rupt het stress on iomss of tomto Effect of rupt het stress on P n, G s, C i nd Root R Effect of rupt het stress on reltive electrolyte lekge Effect of rupt het stress on protein content per g Effect of rupt het stress on the concentrtion of C nd N in dry tissue Effect of rupt het stress on totl mcronutrient content Effect of rupt het stress on totl micronutrient content Effect of rupt het stress on the uptke rte of specific nutrients Effect of rupt het stress on reltive levels of nutrient uptke proteins Effect of rupt het stress on reltive levels of nutrient metolism proteins Effect of rupt het stress on reltive ctivities of nutrient uptke proteins Effect of rupt het stress on reltive level of H + -ATPse...29 viii

10 List of Arevitions µg/g...microgrm per Grm AKT1...Potssium Trnsporter AMT1...Ammonium Trnsporter ANOVA...ANALYSIS OF VARIANCE BOR1...Boron Trnsporter C i...internl Cron-Dioxide d...dy Fig...Figure FRO1...Iron Uptke Protein g...grm GDH...Glutmte Dehydrogense GOGAT...Glutmine Oxoglutrte Aminotrnsferse GS...Glutmine Synthetse G s...stomtl Conductnce H + -ATPse...Proton ATPse mg/g...milligrm per Grm NIP5;1...Boron Chnnel Protein NR...Nitrte Reductse NRT1...Low-ffinity Nitrte Trnsporter NRT2...High-ffinity Nitrte Trnsporter PHT1...Phosphorus Trnsporter P n...net Photosynthesis R/S...Root to Shoot Rtio R root...root Respirtion ix

11 List of Symols B...Boron C...Cron C...Clcium Cu...Copper Fe...Iron K...Potssium Mg...Mgnesium Mn...Mngnese Mo...Molydenum N...Nitrogen NH Ammonium NO Nitrte o C...Degree Celsius P...Phosphorus S...Sulphur x

12 Chpter 1 Introduction High-temperture stress, oth from chronic nd rupt heting, is often limiting fctor for plnt growth, development, nd reproduction (Boyer, 1982; Morrison, 1993; Willims et l., 212; Zinn, Tunc-Ozdemir, & Hrper, 21). Both chronic nd rupt het stress re expected to increse s consequence of nthropogeniclly-driven glol wrming (Intergovernmentl Pnel on Climte Chnge, 21, 27). For exmple, in the midcontinentl United Sttes nd Europe, the frequency, severity, nd durtion of het wves is expected to increse in the future (Meehl & Teldi, 24). In mny cses, it is expected tht increses in extreme high-temperture events (i.e., het wves) will ffect plnts more negtively thn increses in verge tempertures (Gutschick & BssiriRd, 21). Het stress decreses plnt function in mny wys, with negtive effects on growth, photosynthesis, respirtion, reproduction, wter reltions, nd hormone production eing especilly well-studied (B. Hung, Rchmilevitch, & Xu, 212; W. Hung, Kn, & Kovts, 21; Rennenerg et l., 26; Whid, Gelni, Ashrf, & Foold, 27; D. Wng, Heckthorn, Wng, & Philpott, 212; Weis & Berry, 1987; Zinn et l., 21), which in turn cn ffect ecosystem productivity nd species distriution nd diversity (Ching, Iverson, Prsd, & Brown, 28; Ciis et l., 25). 1

13 Both shoots nd roots re sensitive to het-relted dmge, nd roots re often s sensitive, or more, s shoots to het stress (Heckthorn, Giri, Mishr, & Bist, 213; B. Hung et l., 212). Further, roots re often sujected to supr-optiml tempertures e.g, when cnopies re not closed nd soil receives direct sunlight (Grves, 1994; Lrcher, 1996) or in cool-seson species during hot summer months (Ferris, Ellis, Wheeler, & Hdley, 1998; H. Wng, Lemke, Goddrd, & Sprout, 27). The tolernce of roots to het stress scles with the men tempertures of the hitts to which the species re dpted such tht optiml tempertures for root growth re lower in cool-seson species, higher in wrm-seson species, nd still higher in wrm-desert plnts (Heckthorn et l., 213). For exmple, soil tempertures cn exceed 25 o C in the top 2cm of the soil profile in cool-seson whet fields in cool-temperte loctions (Ferris et l., 1998; H. Wng et l, 27), 35-4 C to 1cm depth in su-tropicl mize field (Dlmgo et l., 24), nd 7 C to 4 C from the surfce to 15cm depth in deserts (Jordn & Noel, 1984); in ech of these cses, these soil tempertures exceed optiml tempertures for root growth for the respective species. High-temperture stress reduces root growth, numer, nd mss (B. Hung et l., 212), which ffects the growth of oveground tissue y restricting the supply of wter nd minerl nutrients, ffecting production of hormones synthesized in roots nd trnsported to shoots, nd ltering sink-source reltionships etween shoots nd roots (Ho, Jing, Zhng, Tng, & Shi, 212; B. Hung et l., 212; Rennenerg et l., 26; Whid et l., 27). Reltive to shoots, less reserch hs exmined effects of het stress on roots, nd most of this pst reserch hd focused on root growth nd cron reltions (especilly respirtion) (B. Hung et l., 212). Reltively little pst reserch hs 2

14 investigted how het stress ffects plnt nutrient reltions (Bssirird, 2; Heckthorn, Poeller, Colemn, & Hllerg, 1996; B. Hung et l., 212; Rennenerg et l., 26) nd most of this previous work hs mesured only het effects on nutrient content or concentrtion. In ddition, most of the pst reserch on root het stress hs focused on chronic vs. rupt het stress, ut the responses of roots to chronic wrming cn differ from cute het stress (Heckthorn et. l, 213) In tle 1 (modified from Heckthorn et l. (213)), I included pst studies tht I could identify wherein supr-optiml tempertures were imposed on plnts, nd segregted the studies into those tht imposed root nd shoot heting versus root-only heting on nutrient uptke. Of the few studies tht hve exmined effects of het stress on nutrient reltions, most only mesured het effects on nutrient concentrtion or nutrient content (mount); hence, the effects of het stress on root nutrient uptke proteins re poorly understood. Bsed on the few pst studies, is it known tht het stress often decreses the concentrtion of nutrients in plnts tissues or decreses the totl content of nutrients in the plnts, though effects cn vry mong nutrients nd species (tle1, Heckthorn et l. (213)). Het stress cn lso disrupt enzymes involved in nutrient metolism (e.g., nitrte reductse), (Klimenko, Peshkov, & Dorofeev, 26). Decreses in nutrient cquisition with het stress could potentilly e cused y severl fctors, including decrese in root mss or surfce re nd/or decrese in nutrient uptke per unit root (Bssirird, 2; Brvo-F & Urie, 1981). Decreses in nutrient uptke per unit root might e cused y depletion of lile C (totl non-structurl crohydrte), nd hence energy, in roots (e.g., due to decrese in trnsport of shoot C to roots or n increse in root respirtion) or y direct het dmge to roots (B. Hung et l., 212), which might 3

15 decrese the production or function of nutrient trnsport proteins. For most minerl nutrients, the ulk of their uptke is medited y the ctivity of specific nutrient uptke proteins, nd uptke protein ctivity depends on oth the concentrtion of uptke proteins per unit root, s well s the rte t which ech protein works. However, lmost nothing is known out the effect of het stress on nutrient trnsporter proteins in roots. To investigte the effects of het stress on nutrient uptke nd nutrient uptke proteins in roots, we determined the effects of n rupt het stress on the uptke of N, P, K, Fe, nd B y tomto roots, s well s het effects on the concentrtion of mjor uptke proteins for nitrte, mmonium, potssium, phosphorus, iron, nd oron. In ddition, we lso determined effects of het on the levels of key N ssimiltion enzymes: nitrte reductse (NR), glutmte dehydrogense (GDH), glutmine synthetse (GS), nd glutmte oxoglutrte mino trnsferse (GOGAT). From rtes of nutrient uptke per unit root mss, nd reltive concentrtion of nutrient uptke per unit root mss, we could then estimte the reltive ctivity of nutrient uptke proteins per unit root mss. Finlly, we lso mesured effects of het on shoot nd root growth, photosynthesis, nd root respirtion, protein content, nd memrne dmge, to determine the extent to which het-relted decreses in root growth lso ffect nutrient uptke, nd if root C vilility vs. het-relted dmge; cn explin effects of het on nutrient uptke. 4

16 Tle 1: Studies of nutrient-relted het-stress effects on plnts. Studies with whole-plnt heting Species Tissue Temperture ( C) 1 Triticum estivum, T. turgidum susp. durum (4 cultivrs) Triticum estivum, T. turgidum susp. durum (4 cultivrs) Triticum estivum, T. turgidum susp. durum (4 cultivrs) Pssiflor edulis (3 cultivrs) Root, shoot, nd spike Root, Shoot, nd spikes Root, shoot, nd spike 25/14,31/2 (dy/night) 25/14,31/2 (dy/night) 25/14,31/2 (dy/night) Plnt 2/15,25/2, 3/25 (dy/night) Nutrient - % & totl Cu % & totl Fe, Mn (2 cultivrs) % & totl C,Mg K (mg plnt -1 ) 5 Nutrient - % & totl Cu,Zn % & totl Fe, Mn (2 cultivrs) % & totl C,Mg N,S,Mg, C,P (mg plnt -1 ) B,Cu,Fe, N,Mn,Z n (µg plnt -1 ) Nutrient -no effect N,Zn Source (Dis & Lidon, 29) (Dis, Lidon, & Rmlh o, 29) (Dis, Lidon, & Rmlh o, 29) (Menze l, Simpso n, & Winks, 1987) Solnum lycopersicu m, Roots nd leves 1,25,35 %Fe (Rivero, Sánche z, Ruiz, & Romero, 23) Agrostis Root 2/2,2/35, % N,P,K (B.

17 plustris (2 cultivrs) 35/2,35/35 (ir/soil) Studies with root-zone heting only Species Tissue Temperture Nutrient ( o C) 1 - Solnum tuerosum Hordeum vulgre, Sorghum icolor Andropogon gerrdii Cucumis stivus L. Shrp I Antirrhinum mjus L. Peori Solnum lycopersicu m 6 Nutrient - % Cu,Zn t 3 Nutrient -no effect Hung & Xu, 2) Source Shoot 16,2,23,27, (Bgho 3 ur et Tuer % Cu l., 22) Xylem 25,35 K, NO₃ PO 4 flux (Bssiri Rd, Rdin, & Mtsud, 1991) Shoot Root 5,1,15,2,25, 3,34,35.5 % N & P % N & P in root, N uptke rte (N g -1 root) Leves 25,32,35,38 % B % N,P,K,C, Mg,Fe,M n Plnt 8,15,22,29,36 N,P,K,C,Mg,Mn, Fe,Zn (mg plnt -1 ) Plnt 24,27,3,33, 36 Cucumis Plnt 24,27,3,33, Mn,P,Zn Mn, P, Zn (mg plnt -1 ) B,Cu, Mo (DeLuc i, Heckt horn, & Dy, 1992) (Du & Tchi n, 1994) (Hood & Mills, 1994) (Klock, Grves, & Ter, 1996)

18 melo 36 (mg plnt -1 ) Gleditsi tricnthos Plnt 24,27,3,33, 36 Mn (mg plnt -1 ) P,Zn Ze mys Root 3,34,35,36, 37,38 % B,N,P % K,Zn (Ll, 1974) Agrostis Shoot 35 N,P, K (Liu & stolonifer Plustris Root (2=control) N content P,K Hung, 25) Cucumis melo L. Gold Str Lctuc stiv Solnum lycopersicu m Shoot Root 25,3,35,4, 45 % P,Zn % Mn,P,Zn Plnt vs. 2 C,Cu,Fe,K, N,Mg,M n,zn (mg plnt -1 ) Plnt 1,15.6,21.1, 26.7,32.2,37.8 N,P,K,M g, Mn,Zn (mg plnt -1 ) B,Cu Mo (mg plnt -1 ) (Stoltzf us, Ter, & Aiello, 1998) (Tn, He, & Lee, 22) (Tindll, Mills, & Rdclif fe, 199) 1 Temperture tretments re continuous unless otherwise noted. 7

19 Chpter 2 Mterils nd Methods 2.1 Plnt growth conditions, temperture tretments, nd hrvesting Solnum esculentum L. (tomto, cultivr "Big oy") ws used s model system ecuse it is wrm-seson modertely-thermotolernt species (Preczewski, Heckthorn, Downs, & Colemn, 2; D. Wng et l., 28), origintes from wrm su-tropicl hitts tht experience vrile tempertures nd rupt het stress (Moyle, 28), nd hs een used s model in mny het stress studies (Heckthorn et l., 213). Seeds were plnted in fom cues nd germinted in controlled-environment chmer t 4-5 µmol m -2 s -1 PAR (photosyntheticlly-ctive rdition), 14h photoperiod, nd 24 C. Plnts were provided with strter nutrient solution [mcronutrients: 3 mm N, 1 mm C, 1 mm K,.5 mm P,.5 mm Mg, using C(NO 3 ) 2, K(NO 3 ), KH 2 PO 4, nd MgSO 4 ; micronutrients: 4 µm Fe, 6 µm Mn, 6 µm Zn, 4 µm B, 4 µm Cu,.1 µm Mo, using FeDTPA, MnCl 2, ZnSO 4, CuSO 4, H 3 BO 3, nd N 2 MoO 4. Plnts were then wtered dily. After producing 3-4 post-emryonic leves, plnts were trnsferred to opque erted 4L tus with lids nd grown hydroponiclly in complete nutrient solution [mcronutrients: 6.2 mm N, 2 mm C, 2 mm K, 2 mm Mg, 1 mm P, using NH 4 NO 3, C(NO 3 ) 2, K(NO 3 ), KH 2 PO 4, nd MgSO 4 ; micronutrients: 71 µm Fe, 1 µm Mn, 1 µm Cl, 6 µm Zn, 6 µm 8

20 Cu, 5 µm B,.1 µm Mo, using Fe DTPA, MnCl 2, ZnSO 4, CuSO 4, H 3 BO 3, nd N 2 MoO 4 ]. Solution ph ws monitored dily nd mintined t ph 5.6 with ddition of 1N KOH, nd solution tempertures were monitored using thermometer. Nutrient solution ws chnged every 3-5 dys. Plnts were moved dily to minimize positions effects inside the chmer. Plnts were grown in hydroponics in order to minimize wter stress during het stress, nd thus e le to scrie tretment responses to het lone, s well s to void confounding plnt responses with rhizosphere influences, s would occur in soil. Plnts were grown under the ove control conditions for five dys to llow for post-trnsfer cclimtiztion. Then plnts (n = 4 per tretment comintion, per hrvest) were rndomly ssigned one of three temperture tretments: control = 2/25 o C night/dy, moderte heting = 3/35 o C night/dy, nd severe heting = 37/42 o C night/dy (light nd photoperiod s ove). After 6 dys of het tretment, ll plnts were then grown under control conditions for 7 dys to follow post-heting recovery. A suset (n = 4) of plnts were hrvested on the dy efore het tretments egn (dy ), to serve s pre-tretment controls for ll temperture tretments; therefter plnts from the three temperture tretments were hrvested, fter 24 hours (1 dy) nd 6 dys of het stress, nd fter 7 dys of post-heting recovery. Plnts were seprted into leves, stems, nd roots (roots fter wshing with DI wter). Biomss ws determined fter oven drying t 7 o C for t lest 48h. Su-smples of fresh root tissue for protein nlysis were immeditely frozen in liquid N 2 fter hrvest nd stored t -8 C Photosynthesis nd Root respirtion 9

21 To monitor the effects of het tretment on C reltions of shoots nd roots, we mesured stedy-stte net photosynthesis (P n ; net CO 2 exchnge) immeditely efore hrvesting, nd root respirtion immeditely fter hrvesting, using n infrred gs nlyzer (IRGA) (Model 64, LiCOR, Lincoln, Nersk, USA) equipped with 6-cm 2 lef-re cuvette which controlled environment conditions (CO 2, light, nd temperture). Photosynthesis ws mesured on recently-expnded ttched leves (with different newly-expnded leves on d 13) receiving direct light prior to mesurement, t 37 ppm CO 2, under sturting light (15 µmol m -2 s -1 PAR), nd t the sme temperture s plnts were experiencing in the growth chmer (25, 35, or 42 o C during heting, 25 o C during recovery). For root respirtion, su-smples of whole roots, detched from plnt were immeditely enclosed in humidified 5-ml plstic tues seled with multiple lyers of prfilm, nd then roots were incuted for 24 h t the sme temperture s the experimentl tretments (25, 35, or 42 o C during heting, 25 o C during recovery). CO 2 concentrtion in the tues ws then determined y injecting su-smple of the ir (25 ml) from the tue into the closed IRGA cuvette (system flow set to slightly > (25 mol s -1 ), which llowed the injected ir to displce existing ir in the cuvette nd IRGA cell in excess for 2-3 seconds required for determintion of cuvette CO 2 level y the immeditely-downstrem IRGA cell). CO 2 emitted per g of root ws clculted from the concentrtion of CO 2 nd ir volume in the incution tue Memrne integrity To monitor het-relted dmge to root memrnes, reltive electrolyte lekge in roots ws mesured y incuting 1 cm of root tip in 2 ml of deionized wter 1

22 overnight t room temperture. Conductivity of the solution ws then mesured nd referred to s initil conductivity (EC initil ). Afterwrds, root tissue ws killed y utoclving t 14 o C, nd the conductivity of ded tissue ws then determined nd referred to s finl conductivity (EC finl ). Reltive electrolyte lekge (REL) ws then used s n index of dmge (I d ), nd ws clculted s the percent conductivity of live roots vs. tht of killed roots (Liu & Hung, 22) Nutrient reltions Protein extrction nd quntifiction: Totl protein ws extrcted from tissue s in Mishr, Heckthorn, nd Frntz (212), y grinding 4mg of frozen root tissue in liquid N 2 in mortr nd pestle nd then in 2 ml of extrction uffer of the following composition:.5 M Tris ph-8,.1 M potssium chloride,.9 M sucrose, 5 mm ethylene dimine tetr-cetic cid, 2% (v:v) β-mercptoethnol, 1 µm leupeptin, nd 1 mm phenyl methyl sulfonyl fluoride. The homogente ws trnsferred to 15-mL tue, to which ws dded n equl volume of phenol, nd then the tues were incuted for 2 min t room temperture nd centrifuged t 56 rcf (reltive centrifugl force) for 15 min t 4 C to seprte queous nd orgnic phses. The upper phenol phse ws recovered, nd fter ddition of n equl volume of extrction uffer, centrifuged s ove. Superntnt otined ws stored overnight t -2 C in five volumes of mmonium cette, to precipitte protein. Precipitted protein ws then wshed two times with mmonium cette nd three times with 8% cetone, followed y finl wsh with 1% cetone. Protein smples were dried nd then re-soluilized in smple uffer contining 1 mm Tris ph 6.8,.5% sodium dodecyl sulphte, nd 1% glycerol. Totl protein concentrtion in the smple ws determined using detergent-solule 11

23 colorimetric ssy (DC Protein Assy, BioRd), using Bovine Serum Alumin s stndrd Nutrient nlysis: The concentrtion of ech mcro- nd micro- minerl nutrients were determined from powdered dry shoots nd roots y the comustion-ms technique for C nd N nd y ICP-OES (Inductively Coupled Plsm Opticl Emission Spectroscopy; model IRIS Intrepid II; Thermo Corp, Wlthm, MA) for remining nutrients s in Mishr, Heckthorn, Frntz, Yu, nd Gry (29). Totl nutrient content in the entire plnt ws clculted from concentrtion of ech nutrient multiplied y the iomss of plnt. Then the root specific uptke rte of ech nutrient (x) (totl g plnt nutrient x per g dry root per d) ws clculted from the totl mount of nutrient x tken up during dy 1-6 (het stress) or from dy 7-13 (recovery) (uptke during d 1-6 = totl plnt nutrient x t d 6 minus totl nutrient x t d 1, uptke for d 7-13 = totl nutrient x t d 13 minus tht t d 6) Nutrient metolism protein quntifiction: The reltive mount of nutrient uptke proteins per unit totl root protein ws determined y quntittive ELISA (Enzyme-Linked immunosorent Assy) using protein-specific ntiodies generted y the Heckthorn l (see Mishr et l. (212) for n exmple protocol). We quntified the following key nutrient-uptke proteins: NRT1 nd NRT2 (the min low- nd highffinity nitrte trnsporters; (Forde, 22; Glss et l., 22)), AMT1 (the primry mmonium trnsporter; (Glss et l., 22)), PHT1 (the primry root phosphorus trnsporter; (Nussume et l., 211)), AKT1 (the min potssium trnsporter; (Gierth & Mäser, 27)), FRO1 (iron reductse, one of the two min Fe uptke proteins in dicots, (Jeong & Connolly, 29; Koyshi & Nishizw, 212)), nd BOR1 nd NIP5;1 (the 12

24 two min B trnsporters; (Miw & Fujiwr, 21; Tkno, Miw, Yun, von Wirén, & Fujiwr, 25)). Then, the reltive mount of ech uptke protein per g fresh root ws clculted from totl root protein per g fresh root. The reltive ctivity per uptke protein ws then determined from specific uptke rte per g of root nd the reltive mount of trnsporter per g of root. Using commercilly-ville ntiodies (Agriser, Sweden) nd ELISA, we lso determined the reltive level per unit root protein of the following key nutrient ssimiltory proteins (Lm, Coschigno, Oliveir, Melo-Oliveir, & Coruzzi, 1996); NR (Nitrte Reductse), GOGAT (Glutmine OxoGlutrte Amino Trnsferse), GS (Glutmine Synthetse), GDH1 (Glutmte DeHydrogense), nd plsmlemm H + - ATPse Sttisticl nlysis Experimentl results were nlyzed sttisticlly using two-wy (temperture x dy) nlysis-of-vrince (ANOVA), with temperture nd dy s fixed fctors, using Sigm-plot softwre (version 12). Following significnt ANOVA results, the Holm- Sidk test ws used to identify significnt differences mong the three temperture tretments. Tretment effects were considered significnt if P<.5. Dt were trnsformed (log-trnsformed for concentrtion nd content dt) if they did not meet ssumptions of ANOVA (normlity nd equl vrince), though this ws rre, nd results presented re untrnsformed mens nd error rs. 13

25 Chpter 3 Results 3.1. Plnt growth nd cron reltions: As in other studies wherein tomto ws grown t < 3 o C(Preczewski et l., 2; D. Wng et l., 28), the growth nd function of tomto plnts in this study ws decresed y rupt heting t 35 C nd 42 C. As symptoms of cute het stress were oserved more rpidly or were greter in plnts heted t 42 C thn t 35 C, herefter, heting t 35 C will e referred s moderte, nd 42 C s severe, het stress. Both root nd shoot iomss ws decresed y het tretments (especilly y 42 o C), nd root growth ws more sensitive thn shoot growth under severe het stress, s indicted y the decline in root:shoot mss t 42 o C (Fig. 1). Biomss of modertely-stressed plnts ws only slightly (nd non-significntly) decresed with 6 dys of heting, nd the iomss of these plnts ws slightly (nd non-significntly) ove unheted controls fter the 7d recovery period under control conditions (i,e y d 13). In contrst, severely-stressed plnts did not recover from het within 7 dys, nd their iomss ws 73% less on controls on dy 13. Surprisingly, we sw increse in net photosynthesis (P n ) with moderte het (35 o C), reltive to control plnts, ut decrese in P n with severe het (d 6 only, with full 14

26 recovery y d 13) (Fig. 2). Severely-stressed plnts opened their stomtes (i.e., incresed stomtl conductnce, G s ), on dy 1 of het stress, likely to increse evportive cooling, ut hd lower G s on dy6 vs. controls; in modertely-stressed plnts, G s ws incresed on dy 6, ut similr to controls on dy 1 (ll tretments were similr on d 13). Except for dy 1 (where 35 o C < controls or 42 o C), lef internl cron dioxide concentrtion (C i ) ws similr mong the tretments, indicting tht decreses in P n were not cused y stomtl closure in heted plnts. Root respirtion (R root ) decresed significntly with heting (oth 35 nd 42 o C), nd recovered y dy 13 only in plnts heted to 35 o C. In contrst to R root, root protein content ws decresed only in plnts heted to 42 o C, with full recovery y dy 13 (Fig. 4). The decline in R root ws not likely cused y decreses in plnt C uptke or protein synthesis, s P n nd root protein content decresed only in plnts heted t 42 o C, ut ws likely direct relted, t lest in prt, to het-relted dmge to roots, since electrolyte lekge (n index of memrne dmge) significntly incresed from roots of ll heted plnts (Fig. 3, d 6 only, with recovery y d 13). The C concentrtion of roots ws incresed following 6 dys of het (especilly t 42 o C), nd this increse persisted fter 7 dys of post-het recovery, ut there ws no significnt effect of heting on shoot %C, excluding on dy 1, where het decresed %C t 35 o C (Fig. 5). Hence, decreses in root mss nd respirtion were not correlted with decreses in root C content Nutrient reltions: In contrst to %C, %N in the roots tended to decline with het stress during the entire experiment (especilly for 42 o C t d 1 nd 6), while in shoots, hets effects on %N were significnt only for 42 o C on dy 6 nd 13, wherein %N decresed. Totl nutrient 15

27 content per plnt showed similr responses for ll the mcro- (nitrogen, phosphorus, potssium, sulphur, mgnesium nd clcium) (Fig. 6) nd micro-nutrients (oron, copper, iron, mngnese, molydenum) (Fig. 7). In generl, y dy 6, plnt nutrient content ws slightly nd non-significntly lower in modertely-het-stressed plnts vs. controls, ut much lower in severely-stressed plnts. At 35 o C, nutrient content recovered (often slightly exceeding controls) y dy 13, ut t 42 o C, the effect of het ws long-lsting nd persistent, with no recovery y dy 13. Like totl nutrient uptke, we sw decreses in the uptke rte of mcronutrients (NPK) nd micronutrients (Fe) y roots with het stress, ut only t 42 o C ( nonsignificnt decrese in K nd Fe ws oserved t 35 o C) (Fig. 8). Interestingly, B uptke rte did not decrese with het, ut rther incresed t 35 o C reltive to other tretments. Notly, N nd P uptke rte ws still lower in severely-het-stressed plnts during recovery, wheres K nd Fe uptke lrgely recovered y dy 13. Het stress (oth 35 nd 42 o C) initilly decresed the concentrtion (per g root) of ll the nutrient uptke proteins we mesured (NRT1, NRT2, AMT1, AKT1, PHT1, FRO1, BOR1, nd NIP5;1) (Fig. 9, dy 1). After 6 dys of het stress, the levels of nutrient uptke proteins in modertely-stressed plnts were similr to unheted controls, ut in severely-stressed plnts, levels of these proteins remined elow controls in ll except for NIP5;1 nd AMT1. After 7 dys of post-het recovery, levels of these nutrient uptke proteins hd recovered to control levels for ll ut FRO1 (with smller nonsignificnt decreses for NRT1 nd AKT1). Similr ptterns were oserved in the reltive level of the nutrient ssimiltion proteins exmined (NR, GOGAT, GDH, GS), with decreses in their levels following 1 dy of het stress, recovery of their levels in 16

28 modertely-stressed, ut not in severely-stressed, plnts y dy 6, nd tendency for ner-complete recovery in oth het tretments y dy 13 (Fig. 1). The estimted reltive ctivities of the nutrient uptke proteins during the 6-d het stress decresed significntly only in NRT1 t 42 o C (with non-significnt decreses t 42 o C for AMT1 nd FRO1); no effects of het were oserved for BOR1, NIP5;1, nd NRT2, nd het incresed ctivity for PHT1 (42 o C) (with trend for increses in AKT1) (Fig. 11). During the 7d recovery, only NRT2 ctivity in severely-stressed plnts ws significntly decresed y het (though there were non-significnt trends towrds hetrelted decreses for PHT1, AMT1, nd NIP5;1). Since the uptke of mny nutrients y roots is dependent on proton grdient generted y the plsm memrne H + -ATPse, we lso mesured reltive levels of this protein in roots (Fig. 12). As with the nutrient uptke proteins, het decresed levels of the H + -ATPse, ut decresed levels of this protein during the entire 6d het tretment t oth 35 nd 42 o C. H + -ATPse levels recovered to control levels y the end of the 7d post-het recovery period. 17

29 Shoot Dry Mss (g) o C 35 o C 42 o C (***) (***) (***) (***) (***) (***) 1..5 Root Dry Mss (g) Totl Dry Mss (g) (***) (***) (***) (*) (***) (NS)..2.1 R/S rtio I I I I I I I I Stress Recovery Dys Stress Recovery. Fig. 1: Effect of rupt het stress on iomss of tomto. Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery. Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). 18

30 2 (***) (***) (***) (**) (***) (***) 2. P n ( mol CO 2 m -2 s -1 ) c c Gs (mol H 2 O m-1 s -1 ) Root R ( mol CO 2 g -1 min -1 ) (***) (***) (**) (***) (***) (***) c 25 o C 35 o C 42 o C I I I I I I I I Stress Recovery Dys Stress Recovery Ci ( mol mol-1 ) Fig. 2: Effect of rupt het stress on net photosynthesis (Pn), stomtl conductnce (Gs), lef internl CO2 concentrtion (Ci), nd root respirtion (R) of tomto. Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). 19

31 7 (**) (**) NS) 25 o C Electrolyte Lekge (%) o C 42 o C 6 13 Stress Recovery Dys Fig. 3: Effect of rupt het stress on reltive electrolyte lekge (EL). Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). 2

32 12 (NS) (**) (NS) Root Protein Content (mg g -1 ) o C 35 o C 42 o C I I I I Stress Dys Recovery Fig. 4: Effect of rupt het stress on protein content per grm (fresh weight) of roots. Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). 21

33 45 (***) (NS) (***) (NS) (*) (*) 45 %C in roots %C in shoots %N in roots o C 35 o C 42 o C (***) (NS) (**) (***) (***) (***) %N in shoots I I I I I I I I Stress Recovery Dys Stress Recovery Fig. 5: Effect of rupt het stress on the concentrtion of C nd N in dry tissues of tomto. Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). 22

34 N (mg plnt -1 ) o C 35 o C 42 o C (***) (***) (***) (***) (***) (***) K (mg plnt -1 ) (***) (***) (***) (***) (***) (***) Mg (mg plnt -1 ) P (mg plnt -1 ) (***) (***) (***) (***) (***) (***) c I I I I I I I I Stress Recovery Dys Stress Recovery c S (mg plnt -1 ) C (mg plnt -1 ) Fig. 6: Effect of rupt het stress on the totl content per plnt of ech mcronutrient in tomto (dry mss sis). Plnts were grown t 25/2oC dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37oC dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). 23

35 Fig. 7: Effect of rupt het stress on the totl content per plnt of ech micronutrient in tomto (dry mss sis). Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). B (mg plnt -1 ) Mn (mg plnt -1 ) Fe (mg plnt -1 ) Cu (mg plnt -1 ) Mo (mg plnt -1 ) o C 35 o C 42 o C (***) (***) (**) (***) (***) (***) (***) (***) (***) 1 (***) (***) 6 (***) 13 Dys 1 (**) (***) 6 (**) 13 Dys I I I I Stress Recovery Dys 24

36 Fig. 8: Effect of rupt het stress (dy 1-6) nd recovery (7-13) on the uptke rte of specific nutrients y roots of tomto (totl mg for mcronutrients nd µg for micronutrients in the plnt per g root per d). Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). Fe ( g g -1 dy -1 ) K (mg g -1 dy -1 ) P (mg g -1 dy -1 ) N (mg g -1 dy -1 ) o C 35 o C 42 o C (**) (***) (NS) (**) (**) (NS) (**) (**) (NS) (**) (*) (*) (*) (*) (NS) B ( g g -1 dy -1 ) STRESS (dy 1-6) RECOVERY (dy 7-13) 25

37 5 (***) (***) (***) (***) (***) (***) NRT NRT2 AMT (NS) (**) (NS) (***) (***) (NS) AKT1 PHT (*) (***) (*) (***) (*) (NS) FRO1 BOR (**) (***) (*) (NS) (**) (NS) 25 C 35 C 42 C I I I I I I I I Stress Recovery Dys Stress Recovery NIP5;1 Fig. 9: Effect of rupt het stress on reltive levels of specific nutrient trnsport proteins (per g root) in roots of tomto: low-ffinity NO 3, NRT1; high-ffinity NO 3, NRT2; NH 4, AMT1; K, ATK1; P, PHT1; Fe, FRO1; B, BOR1 nd NIP5. Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). 26

38 25 (***) (*) (NS) (***) (**) (**) 3 NR o C 35 o C GOGAT (***) (NS) (**) 42 o C (***) (***) (***) 3 GDH GS I I I I I I I I Stress Recovery Stress Recovery Dys Fig. 1: Effect of rupt het stress on reltive levels of specific nutrient metolism proteins (per g root) in roots of tomto: NR, nitrte reductse; GOGAT, glutmine oxoglutrte mino trnsferese; GDH1, glutmte dehydrogense; GS, glutmine synthetse. Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm-Sidk test; letters presented only for differences). 27

39 Activity of NRT o C 35 o C 42 o C (***) (NS) (NS) (***) (**) (NS) Activity of NRT2 Activity of PHT (***) (NS) (*) (***) (NS) (NS) Activity of AMT1 Activity of FRO (***) (NS) (NS) (***) (*) (NS) Activity of AKT1 4 (***) (NS) (NS) (***) (*) (NS) 8 Activity of BOR Activity of NIP5;1 STRESS (dy 1-6) RECOVERY (dy 7-13) STRESS (dy 1-6) RECOVERY (dy 7-13) Fig. 11: Effect of rupt het stress (dy 1-6) nd recovery (dy 7-13) on the reltive ctivities of specific nutrient uptke proteins in roots of tomto: low-ffinity NO 3, NRT1; high-ffinity NO 3, NRT2; NH 4, AMT1; K, ATK1; P, PHT1; Fe, FRO1; B, BOR1 nd NIP5. Reltive ctivity ws estimted y dividing nutrient uptke per grm of root y the reltive content of ech nutrient uptke protein per g root, during tht period. Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). 28

40 5 (***) (***) (*) 25 o C H + ATPse g -1 roots c 35 o C 42 o C I I I I Stress Dys Recovery Fig. 12: Effect of rupt het stress on reltive level of H + ATPse (per g root). Plnts were grown t 25/2 o C dy/night (control), nd then susets of plnts were het stressed t 35/3 or 42/37 o C dy/night for 1 or 6 d, nd then returned to control conditions for 7 d of recovery (= d 13). Vlues re mens±1 SE for four independent replictes from ech hrvest (dys 1, 6, 13). Within ech vrile, the significnce of min tretment effects (temperture, dy, temperture*dy) is indicted in prentheses (ANOVA results: *P<.5, **P<.1, ***P<.1, NS=not significnt). Different lowercse letters indicte significnt difference mong temperture tretments within ech dy (Holm- Sidk test; letters presented only for differences). 29

41 Chpter 4 Discussion With increses in temperture due to glol wrming, plnts re likely to experience incresingly frequent, hotter, nd longer episodes of rupt het stress (i.e., het wves) in the future, nd this will negtively impct plnt function. Bsed on the limited pst studies, we know tht het stress cn negtively ffect plnt nutrient reltions (Tle 1, nd Heckthorn et l. (213)), ut the effect of het stress, chronic or rupt, on root nutrient uptke rte hs een little studied, nd we re wre of no previous reserch on effects of het stress on nutrient uptke proteins (Bssirird, 2; B. Hung et l., 212). Our results show tht het stress decresed totl nutrient content in tomto, y decresing plnt growth (root growth more thn shoot growth) nd decresing uptke rte of nutrients per g of root. Decreses in root growth nd nutrient uptke rte were likely cused y dmge, rther thn y lower ville C for metolism. The uptke rte of nutrients y roots decresed for most nutrients during severe, ut not moderte het stress, s consequence of decreses in the concentrtion of nutrient uptke proteins, nd, for some nutrients, the ctivity of the uptke proteins. Het effects on roots nd nutrient reltions were often long lsting, with incomplete recovery in severely-stressed plnts even fter 7 dys of post-het recovery. 3

42 As in other previous studies (Heckthorn et l., 213), in roots nd shoots sujected to the sme high tempertures, roots were more sensitive to het stress thn shoots. In this study, oth root nd shoot growth decresed with het stress, with greter decreses t 42 thn 35 vs. 25 o C dytime tempertures, ut the effect ws lrger for roots s indicted y decrese in root-to-shoot rtio (especilly for severely-stressed plnts). Also, photosynthesis (P n ) incresed t 35 o C, nd ws lower thn controls only on dy 6 t 42 o C, yet root respirtion (R root ) decresed t oth 35 nd 42 o C. Similrly, there ws little effect of het on shoot %C, ut root %C incresed with het stress, nd shoot %N decresed only t 42 o C, ut decresed t oth 35 nd 42 o C in roots. The decrese in %N nd other nutrients (dt not shown) could hve contriuted to the increse in %C in the roots. Root respirtion ws strongly decresed y rupt het stress (oth 35 nd 42 o C), nd this would decrese oth growth nd nutrient uptke, s oth re dependent on energy production from respirtion (Atkin & Tjoelker, 23). Though respirtion typiclly increses with temperture, up to point, especilly with chronic wrming t tempertures constituting moderte stress or with short-term tretments t higher tempertures in detched roots (Atkin & Tjoelker, 23; Brvo-F & Urie, 1981; Burton, Melillo, & Frey, 28), decreses in root respirtion re typiclly oserved during cute het stress with intct plnts or during chronic heting t tempertures tht constitute more-severe stress (Heckthorn et l., 213; B. Hung et l., 212). In this study, decreses in root respirtion with heting were not pprently due to decreses in ville sustrte for respirtion, since root %C incresed with het stress, nd this increse in %C ws not likely driven y incresed structurl C, given tht het stress 31

43 decresed root growth reltive to controls. However, to confirm this one would need mesures of root, totl non-structurl crohydrte (TNC) content. Further, photosynthesis decresed only in the severe het tretment, yet R root decresed with oth moderte nd severe heting. These results lso indicte tht het did not impir C trnsloction from shoots to roots, otherwise, root %C would not increse, which is consistent with results of study on rupt het stress in het-tolernt grss (Minli, 27). So, the prole reson for lower R root during het tretment in this study is tht roots sustined direct dmge from the high tempertures, s indicted y het-relted increses in root electrolyte lekge (n indiction of memrne dmge), s oserved in other studies (Liu & Hung, 2, 22). In ddition, root protein content decresed with het stress, which would result from n increse in protein degrdtion nd/or decrese in protein synthesis, nd either cuse would constitute het dmge nd oth re known to occur during het stress in roots (B. Hung et l., 212). Lstly, the decrese in shoot %N t 42 o C likely occurred ecuse het ffected shoot growth less thn increses in totl shoot N content, indicting tht either trnsloction of N from roots to shoots nd/or voltiliztion of shoot N incresed during severe het stress. Consistent with the effects of het stress on totl plnt N content nd %N, moderte het stress did not ffect the rte of nutrient uptke per g of root, ut severe het stress did decrese the uptke rte of 4 of 5 nutrients exmined (N, P, K, Fe vs. B). Hetrelted decreses in nutrient uptke rtes y roots cn e cused y decreses in the concentrtion of nutrient uptke proteins nd/or y decreses in the ctivity (or trnsport or rection rte) of individul uptke proteins. In this study, most of the eight nutrient uptke proteins exmined showed similr responses to het stress; i.e., levels of the 32

44 proteins per g root decresed compred to controls fter 24 h of het tretment t 35 nd 42 o C (excluding NIP5;1), ut fter 6 dys of het tretment, only plnts heted t 42 o C exhiited decresed levels of uptke proteins (excluding NIP5;1 nd AMT1), nd fter 7 dys of post-het recovery, levels of most uptke proteins hd recovered to control levels (excluding FRO1, nd non-significntly, AKT1 nd NRT1). Perhps the het tolernce of NIP5;1 vs. BOR1 nd the other proteins is relted to the fct tht NIP5;1 is chnnel protein, while BOR1 is not, nd B is typiclly n unchrged molecule t physiologicl ph, ut the other nutrients exmined here re chrged (Dords, Chrispeels, & Brown, 2; Noguchi et l., 1997). The effects of het stress on the levels of the four N ssimiltory proteins exmined were very similr to effects on nutrient uptke proteins, though levels of ll four proteins tended to remin slightly lower thn controls fter 7 dys of post-het recovery. For the H + -ATPse, levels decresed during moderte nd severe het stress, ut recovered within 7 dys. In contrst to levels of nutrient uptke proteins, the estimted reltive ctivity of these proteins during nd fter het stress ws vrile. During het stress, decreses in ctivity were oserved only t 42 o C for NRT1 (nd non-significntly in AMT1 nd FRO1), nd ctivity even tended to increse for some proteins. During the recovery period, ctivity of some uptke proteins tended to e lower thn controls, ut similr to, or slightly greter thn, controls for the remining proteins. Importntly, in this study, plnts were grown t high level of ville NO 3 (6.1 mm vs..1 mm NH 4 ); hence, it ws likely tht most of the N uptke ws vi the ctivity of NRT1, the low-ffinity N trnsporter (Forde, 22). Since the true contriution of the three min inorgnic N trnsporters (NRT1, NRT2, AMT1) to N uptke could not e determined in the study, 33

45 ccurte estimtes of the reltive ctivity of the uptke proteins is not possile. Nevertheless, it is likely tht the verge reltive ctivity of these N uptke proteins ws decresed y het stress. In generl, het stress did not decrese the reltive ctivities of the nutrient uptke proteins, consistent with results in detched roots (Brvo-F & Urie, 1981). Similrly, (Chopr, 1983) found tht optiml in vivo NR ctivity in eight different crop species to e >45 o C. In summry, this study showed tht cute het stress cn hve lrge negtive effects on plnt nutrient uptke, nd decreses in nutrient uptke were due to effects on root growth, s well s on the levels nd/or ctivities of nutrient uptke proteins, for some nutrients. Importntly, the totl plnt content of ech of the mcro- nd micro-nutrients exmined showed similr ptterns: often smll decreses t 35 o C vs. lrger decreses t 42 o C t the end of the 6 dy het tretment, nd increses t 35 o C reltive vs. lrge decreses t 42 o C. These effects of moderte vs. severe het stress on totl plnt nutrient were correlted with effects on root-to-shoot mss, nutrient uptke rte per g of root, nd levels of nutrient uptke nd N ssimiltory proteins (excluding NIP5;1); in contrst, reltive ctivities of nutrient trnsport proteins were not well correlted with other nutrient responses to het. So, increses in het stress with glol wrming in the future will likely hve overll negtive effects on plnt nutrient reltions tht will ecome more severe s tempertures rise. 34