Homeostasis of the temperature sensitivity of respiration over a range of growth temperatures indicated by a modified Arrhenius model

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1 Reserch Homeostsis of the temperture sensitivity of respirtion over rnge of growth tempertures indicted y modified Arrhenius model Ko Noguchi 1, Wtru Ymori 2,3, Kouki Hikosk 4,5 nd Ichiro Tershim 1,5 1 Deprtment of Biologicl Sciences, Grdute School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Jpn; 2 Center for Environment, Helth nd Field Sciences, Chi University, Kshiw, Chi, Jpn; 3 PREST, JST, , Jpn; 4 Grdute School of Life Sciences, Tohoku University, Sendi, Miygi, Jpn; 5 CREST, JST, , Jpn Author for correspondence: Ko Noguchi Tel: Emil: knoguchi@s.s.u-tokyo.c.jp Received: 12 Decemer 214 Accepted: 23 Jnury 215 doi: /nph Key words: cclimtion, cold-sensitive species, cold-tolernt species, respirtion, temperture. Summry The temperture dependence of plnt respirtory rte (R) chnges in response to growth temperture. Here, we used modified Arrhenius model incorporting the temperture dependence of ctivtion energy (E o ), nd compred the temperture dependence of R etween cold-sensitive nd cold-tolernt species. We nlyzed the temperture dependences of lef CO 2 efflux rte of plnts cultivted t low () or high temperture (HT). In plnts grown t HT (HT plnts), E o t low mesurement temperture vried mong species, ut E o t growth temperture in HT plnts did not vry nd ws comprle to tht in plnts grown t ( plnts), suggesting tht the limiting process ws similr t the respective growth tempertures. In plnts, the integrted vlue of log e R, mesure of respirtory cpcity, in cold-sensitive species ws lower thn tht in cold-tolernt species. When plnts were trnsferred from HT to, the respirtory cpcity chnged promptly fter the trnsfer compred with the other prmeters. These results suggest tht similr process limits R t different growth tempertures, nd tht the lower cpcity of the respirtory system in cold-sensitive species my explin their low growth rte t. Introduction Plnt respirtory rte (R) dynmiclly increses with temperture in the rnge of low to modertely high temperture (Atkin & Tjoelker, 23; Atkin et l., 25). The temperture dependence of R nd its chnge in different growth conditions nd species hve een investigted using severl models (Lloyd & Tylor, 1994; Atkin & Tjoelker, 23; Kruse et l., 211; O Sullivn et l., 213). Among these models, the Q 1 model hs often een used. In the Q 1 model, R is given y, T T ½ REF 1 Š R ¼ R REF Q1 Eqn 1 (T REF, low reference temperture ( C); R REF, R t T REF.) The Q 1 reltionship descries proportionl chnge of R in response to temperture increse of 1 C. A literture survey indicted tht Q 1 decreses with n increse in mesurement temperture (Tjoelker et l., 21; Atkin & Tjoelker, 23). The temperture dependence of Q 1 is interpreted s follows: t low mesurement temperture, R is thought to e restricted y the mximl ctivities of the respirtory enzymes, wheres R t higher tempertures my e limited y the vilility of ADP or sustrtes (Atkin & Tjoelker, 23). An Arrhenius function hs lso een used to nlyze the temperture dependence of R (Lloyd & Tylor, 1994; Turnull et l., 23; Shpiro et l., 24). In this model, R t given temperture is given y, h i Eo 1 r T 1 R ¼ R REF e REF T Eqn 2 (T REF, low reference temperture (K); E o, overll ctivtion energy of respirtory processes (kj mol 1 ); r, universl gs constnt (8.314 J mol 1 K 1 ).) The Arrhenius model suggests tht clculted E o cn e relted to metolic processes (Kruse et l., 211). A regression line of the Arrhenius plot, where log e R is plotted ginst the reciprocl of temperture, often devites from the dt t low or high mesurement tempertures, nd thus E o is considered to e temperture dependent. Kruse & Adms (28) suggested tht the linerized expression of temperture-dependent respirtion dt is etter fitted y secondorder polynomil including d (1 3 K 2 ) s fctor to ccount 34

2 New Phytologist Reserch 35 for the dynmic response E o (kj mol 1 ) to mesurement temperture. Then, the ove Arrhenius model (Eqn 2) is modified to: log e R ¼ log e R REF þ E oðrefþ r 1 T REF 1 T þ d T REF T Eqn 3 (R REF, R t T REF ; E o (REF) (kj mol 1 ), overll ctivtion energy t T REF.) In the modified Arrhenius model, E o t ny mesurement temperture cn e otined from the first derivtive of the ove eqution. An integrted vlue over the distinct temperture intervl, Cp(R) (reltive unit), is considered to e mesure of the respirtory cpcity (mximl ctivity of the respirtory system). Cp(R) is thought to etter reflect ltertions of respirtory cpcity in the long term thn R REF ecuse R REF is the rte t one low reference temperture (Kruse & Adms, 28; Kruse et l., 211). In ddition to the ove three models, R cn e modeled using polynomil eqution fitted to plots of log R vs mesurement temperture (O Sullivn et l., 213). The temperture dependence of R is often different etween plnts grown t different tempertures. This long-term chnge in the temperture dependence of R (temperture cclimtion of R) hs een seprted into two types, Type I nd Type II. In Type I cclimtion, the temperture sensitivity of R (e.g. Q 1 ) is ltered, nd in Type II cclimtion, n overll shift of the temperture response curve is oserved (Atkin & Tjoelker, 23). Type II cclimtion often results in higher degree of cclimtion thn Type I cclimtion ( higher degree of temperture homeostsis). Some studies reported tht cold-tolernt species showed higher degree of temperture homeostsis of R thn cold-sensitive species (Kurimoto et l., 24; Ymori et l., 29). The temperture cclimtion of R hs lso een descried y some models (Smith & Dukes, 213), ut few studies hve exmined differences in the temperture cclimtion of R etween cold-tolernt nd cold-sensitive species in detil. The degree of temperture cclimtion of R hs een reported to depend strongly on the durtion fter shift to different growth temperture nd on the developmentl stges of plnt orgns (Armstrong et l., 28; Ymori et l., 29). However, how ech prmeter of the respirtory models chnges depending on the durtion fter temperture shift nd/or developmentl stges is still unknown. Here, we focused on how growth temperture influences the prmeters of the temperture dependence of R nd wht differences in prmeters were oserved etween cold-sensitive nd cold-tolernt species. We exmined the following four questions, using dt on lef R from Ymori et l. (25, 29). In these studies, the temperture dependence of CO 2 efflux rtes of mture leves were mesured using plnts grown t high (HT) nd low tempertures (). Ymori et l. (25) lso trnsferred plnts grown t HT to nd compred the degree of cclimtion of CO 2 efflux rtes mong severl durtions fter the trnsfer. The questions we ddressed were: In previous studies using the modified Arrhenius model (Kruse & Adms, 28; Kruse et l., 212), the rtes of CO 2 efflux nd O 2 uptke were mesured using the clorimetry method. Is the model pplicle to CO 2 efflux rte dt mesured using conventionl infrred gs nlyzer (IRGA)? In previous studies, some respirtory prmeters were tightly relted with ech other (e.g. d nd E o (REF)). Are such the reltionships found for dt mesured y IRGA? Wht prmeters of the model chnge depending on long-term cclimtion to growth temperture? In prticulr, is the temperture dependence of E o chnged y growth temperture? Wht prmeters of the model differ etween cold-sensitive nd cold-tolernt species? After trnsfer from HT to, wht prmeters chnge during cclimtion in the trnsferred plnts? Mterils nd Methods We nlyzed dt on drk CO 2 efflux rtes of leves tht were mesured in our previous studies (Ymori et l., 25, 29). The nlyses were conducted with 11 species: Cucumis stivus L. Nnshin (cucumer), Nicotin tcum L. Smsun NN (tocco), Oryz stiv L. Nipponre (rice), Solnum lycopersicum L. House-momotrou (tomto), S. tuerosum L. Dnshku (potto), Vici f L. Nintokuissun (rod en), Secle cerele L. Wrko (winter rye), Triticum estivum L. Hruyutk (spring whet), T. estivum L. Hokushin (winter whet) 9 Triticosecle Wittmck Presto (triticle), nd Spinci olerce L. Tori (spinch). In our previous studies, C. stivus, N. tcum, O. stiv nd S. lycopersicum were considered coldsensitive species, wheres S. cerele, S. olerce, S. tuerosum, T. estivum (spring), T. estivum (winter), Triticosecle nd V. f were considered cold-tolernt species (Ymori et l., 29). However, the rtes of photosynthesis nd respirtion of -grown tomto were comprle to those of HT-grown plnts (Ymori et l., 29); this tomto cultivr showed some cold-tolernt chrcteristics. Plnts were grown t either 15 C: 1 C or 3 C:25 C (dy : night). These re referred to s nd HT conditions nd the plnts grown t nd HT re clled nd HT plnts, respectively. Some spinch plnts grown t HT were trnsferred to conditions when the seventh true leves were t prticulr developmentl stges. The plnts/leves trnsferred from HT to were clled HL-plnts/leves. HL-mture plnts were trnsferred to when their seventh leves were just fully expnded; they were kept t for nother 2 wk. HL-young plnts were trnsferred to when their seventh leves were out one-fifth the re of the fully expnded leves; they were kept for c. 1 month t until full mturtion of their seventh leves. HL-new plnts were trnsferred to when their seventh leves were out to emerge; they were kept t for c. 2 months (Ymori et l., 25). Drk CO 2 efflux rtes in the most recent fully expnded leves were mesured using portle gs exchnge system (LI-64; Li-Cor, Lincoln, NE, USA). The rtes were mesured t tempertures rnging from 9 to 39 C t 3 C intervls for spinch leves nd t tempertures rnging from 1 to 35 C t 5 C intervls, nd t 38 C for ll of the other species except for plnts of

3 36 Reserch New Phytologist cucumer, tomto, rod en nd tocco, nd HT plnts of tomto, rod en nd tocco. The drk CO 2 efflux rtes on lef re sis were nlyzed using the modified Arrhenius eqution (Kruse & Adms, 28; Kruse et l., 211). A regression curve ws fitted to the dt of ech lef smple, nd the prmeters were clculted. The men nd stndrd error of the men (SEM) of the prmeters were clculted using the dt of three plnts. The R of spinch leves ws mesured t different tempertures from those of the other species, nd thus, in the nlyses in Figs 2 5, we excluded the dt from spinch. Anlysis of covrince (ANCOVA), two-wy nlysis of vrince (ANOVA), Welch s t-test, Dunnett s test nd Tukey s multiple comprison test were conducted using the R sttisticl softwre pckge (R Development Core Tem, 23). Results Dependence of R on mesurement temperture nd model fitting The temperture dependence of R is shown for the dt from spinch leves grown t HT nd (Fig. 1). The curves clculted y the Q 1 model devited from some dt points t high temperture (Fig. 1). An Arrhenius plot nd modified Arrhenius plot re lso shown (Fig. 1,c). On the one hnd, in the stndrd Arrhenius plot of R ginst the reciprocl of temperture, not ll dt conformed to the expected liner reltionship (Fig. 1). On the other, in the modified Arrhenius plot, second polynomil curves fitted well to ll of the dt (Fig. 1c). From the fitted curve, R t the reference low temperture (9 C), R REF, the ctivtion energy t this reference temperture, E o (REF), nd the dynmic response of E o to temperture, d, were clculted. These vlues re shown in Fig. 1(c). The E o vlue t ny mesurement temperture cn e clculted from the first derivtive of the modified Arrhenius eqution. Tht is to sy, the temperture dependence of E o cn e estimted. The temperture dependence of E o ws different etween HT nd plnts, ut the E o vlues t the respective growth tempertures were similr (Fig. 1d). Susequently, we exmined the temperture dependence of E o nd E o vlues t growth temperture using severl cold-sensitive nd cold-tolernt species. Next, we clculted Cp(R), mesure of the respirtory cpcity in spinch leves. For spinch leves, Cp (R) ws clculted using the dt otined from 9 to 39 C. The Cp(R) vlues re lso shown in Fig. 1(c). For the species other thn spinch, the reference low temperture, T REF, ws 1 C, nd Cp(R) ws clculted using the dt otined from 1 to 35 C. Reltionships etween prmeters We nlyzed the reltionships etween prmeters otined from the modified Arrhenius model (Fig. 2). A previous study reported strong negtive reltionship etween E o (REF) nd R REF (Kruse & Adms, 28). We exmined whether such reltionship ws oserved, whether there ws ny difference in the reltionship etween HT nd plnts, nd whether there ws ny difference etween cold-sensitive nd cold-tolernt species. In this study, we found negtive reltionships etween E o (REF) nd R REF, ut the reltionships etween E o (REF) nd R REF were different etween HT nd plnts (Fig. 2). For the HT plnts, we found cler negtive reltionship (r =.957). When compred t the sme R REF, E o (REF) ws higher in plnts thn in HT plnts. Moreover, in the plnts different reltionships were oserved etween cold-sensitive nd cold-tolernt species, wheres in the HT plnts such differences were not found. The cold-sensitive species showed lower R REF t ny given E o (REF) thn the coldtolernt species mong the plnts. Next, we compred R REF with Cp(R), which re considered mesures of the respirtory cpcity. The former hs een frequently used s n indictor of Type II cclimtion (Fig. 2, Atkin & Tjoelker, 23). The two prmeters were well correlted (r =.941). However, when we focused on dt from ech growth temperture seprtely, the reltionships were different etween the two growth tempertures nd the rnge of dt ws wider for R REF thn for Cp(R). In the plnts, oth vlues were lower in cold-sensitive species thn in cold-tolernt ones. We conducted two-wy ANOVAs for the dt of R REF, E o (REF), d nd Cp(R) where fctors were growth temperture (HT vs ) nd cold tolernce (cold-sensitive vs cold-tolernt). In the clcultion, we ssumed tht the tomto cultivr used in our studies ws cold-tolernt, s mentioned ove. In the results of R REF nd Cp(R), ech fctor nd interction were sttisticlly significnt (Supporting Informtion Tle S1). Next, we exmined the reltionship etween d nd E o (REF) s in previous reports (Kruse & Adms, 28; Kruse et l., 212). We lso found negtive reltionship etween d nd E o (REF) (Fig. 3, r =.91). At the intercept with the x-xis of the correltion etween d nd E o (REF), tht is t c. 5 kj mol 1, R cn e descried y clssicl Arrhenius kinetics. At this point, the correltion etween log e R nd the reciprocl temperture ecomes strictly liner. When ll dt from ech growth temperture were pooled, the reltionships etween d nd E o (REF) were clerly different etween HT nd plnts (Fig. 3, P <.1, ANCO- VA, Tle S1). At ny given E o (REF), d ws less negtive for HT thn for plnts. As result, the intercept with the x-xis of the correltion etween E o (REF) nd d shifted from 54. kj mol 1 in the HT plnts to 43.6 kj mol 1 in the plnts (Fig. 3). Temperture dependence of E o nd the difference in temperture dependence etween HT nd plnts We exmined whether the temperture dependence of E o ws chnged y growth temperture. Figure S1(,) shows the temperture dependence of E o in HT nd plnts of ech species. The temperture dependence of E o ws vrile depending on the species (P <.1, two-wy ANOVA, Tle S1). However, when we pooled the dt of ll species nd compred the men vlues of E o etween HT nd plnts, the men E o in HT plnts ws significntly higher thn tht in plnts t ny mesurement temperture (Fig. 4, P <.1, two-wy ANOVA, Tle S1). The men E o vlues were lso chnged y the

4 New Phytologist Reserch 37 Mesurement temperture ( C) () () Fig. 1 Temperture dependence of respirtory rte (), the Arrhenius plot () nd the modified Arrhenius plot (c) using dt from spinch leves grown t high temperture (HT, red) nd low temperture (, lue). In (d), the estimted vlues of overll ctivtion energy of respirtory processes (E o ) t given mesurement temperture re shown from the modified Arrhenius model. The determintion coefficients (R 2 ) re.974 (HT) nd.989 () in (),.957 (HT) nd.992 () in (), nd.997 (HT) nd.996 () in (c). Q 1 in the Q 1 model, E o (kj mol 1 ) in the Arrhenius model, the respirtory rte t low reference temperture (R REF, nmol m 2 s 1 ), the overll ctivtion energy t low reference temperture (E o (REF), kj mol 1 ), the dynmic response of E o to temperture (d, 1 3 K 2 ) nd the integrted vlue over the distinct temperture intervl (Cp(R), reltive unit) in the modified Arrhenius model re shown in the respective pnels. Cp(R) is clculted using the dt from 9 to 39 C. Dt re derived from Ymori et l. (25). R (nmol m 2 s 1 ) (c) log e R R REF : E o (REF): 63.8 δ: 3.9 Cp (R): 2.49 Q 1 : TREF 1 T (1 3 K 1 ) Q 1 : Mesurement temperture (K) R REF : E o (REF): δ: 11. Cp (R): log e R 7.5 E o : E o : T (1 3 K 1 ) Mesurement temperture ( C) (d) Eo (kj mol 1 ) HT (3/25 C) (15/1 C) Mesurement temperture (K) mesurement temperture (P <.1, two-wy ANOVA, Tle S1). However, E o t 3 C (E o (3 C)) in HT plnts ws comprle to E o t 15 C (E o (15 C)) in plnts (P >.5, Welch s t-test). This indictes tht the E o vlues t the respective growth tempertures (E o (growth)) were similr etween HT nd plnts, nd tht the limiting process of R my e similr t different growth tempertures. Respirtory cpcity nd its difference etween coldsensitive nd cold-tolernt species Next, we exmined the difference etween cold-sensitive nd cold-tolernt species. Figure 5() shows the reltionship etween E o (growth) nd Cp(R), which is mesure of the respirtory cpcity. E o (growth) ws similr, wheres Cp(R) ws different etween HT nd plnts. Cp(R) in the plnts ws higher thn in the HT plnts, suggesting tht the respirtory cpcity ws higher in the plnts. On the one hnd, in the HT plnts, Cp(R) ws similr etween the cold-sensitive nd cold-tolernt species, ut in the plnts Cp(R) differed etween cold-sensitive nd cold-tolernt species. On the other hnd, E o (growth) ws similr etween cold-sensitive nd cold-tolernt species, irrespective of growth temperture. We clculted the men vlue of Cp(R) in the cold-sensitive nd cold-tolernt species (Fig. 5). The men vlues of Cp(R) in the plnts were significntly different etween the cold-sensitive nd cold-tolernt species. Chnges in respirtory prmeters fter trnsfer from HT to in spinch leves In the ove nlyses, E o (growth) ws similr etween HT nd plnts, ut Cp(R) ws different. We sked how re the prmeters chnged during cclimtion when the plnts re trnsferred from HT to? The degree of temperture cclimtion of R hs een reported to depend strongly on durtion fter the shift to different growth temperture (Armstrong et l., 28; Ymori et l., 29). We lso sked is there ny difference in durtion for cclimtion to mong the prmeters? In Ymori et l. (25), spinch plnts were trnsferred from HT to. Using the dt from these plnts, we ddressed the ove questions. After the trnsfer, E o (3 C) showed similr vlues irrespective of tretment, wheres E o (15 C) in the HT plnts ws lower thn

5 38 Reserch New Phytologist () 1 () 2.1 Eo (REF) (kj mol 1 ) Cp (R) HT Cucumer Tocco Rice Tomto Potto Brod en Winter rye Spring whet Winter whet Triticle R REF (nmol m 2 s 1 ) Fig. 2 Reltionships etween the overll ctivtion energy t low reference temperture (E o (REF)) nd the respirtory rte t low reference temperture (R REF ) () nd etween the integrted vlue over the distinct temperture intervl (Cp(R)) nd R REF () in the leves of severl cold-sensitive nd coldtolernt species tht were cultivted t high (HT, 3 C:25 C (dy : night)) nd low tempertures (, 15 C: 1 C). Men nd SEM re shown (n = 3). Cp (R) is clculted using the dt from 1 to 35 C. Dt re derived from Ymori et l. (29). () () 1 δ E o(ref) (kj mol 1 ) HT E o(ref) (kj mol 1 ) HT Cucumer Tocco Rice Tomto Potto Brod en Winter rye Spring whet Winter whet Triticle Fig. 3 Reltionship etween the dynmic response of overll ctivtion energy of respirtory processes to temperture (d) nd the overll ctivtion energy t low reference temperture (E o (REF)) () nd scttergrm etween d nd E o (REF) () in the leves of severl cold-sensitive nd cold-tolernt species tht were cultivted t high (HT) nd low tempertures (). Men nd SEM re shown (n = 3). In (), we pooled ll of the dt from ech growth temperture. Red nd lue symols denote the dt of HT nd plnts. Dt re derived from Ymori et l. (29). E o (kj mol 1 ) Mesurement temperture ( C) tht in the plnts (Fig. 6,). E o (15 C) in HL plnts chnged depending on the durtion t, E o (15 C) in the HL-mture plnts ws significntly higher thn tht in the HT plnts, nd E o (15 C) in the HL-new plnts ws similr to tht in the plnts (Fig. 5). These results indicte tht the metolic process limiting R t low mesurement tempertures my e different etween HT nd plnts nd my chnge depending on the period t. On the other hnd, Cp(R) chnged promptly fter the trnsfer compred with E o (15 C) nd R REF (Fig. 5 d). This mens tht the respirtory enzymes my e induced y tretment for only 2 wk even in mture spinch leves. 2 HT Mesurement temperture (K) Fig. 4 Men vlues of overll ctivtion energy of respirtory processes (E o ) in plnts grown t high (HT) nd low tempertures () t given mesurement temperture. Men nd SEM re shown (n = 1). Dt re derived from Ymori et l. (29). Discussion Model fitting, prmeters from the model nd reltionships etween prmeters Figure 1 shows tht of the three models, the modified Arrhenius model gve the est fitted curve to the dt. This model ws pplicle to the CO 2 efflux rte dt mesured using IRGA s

6 New Phytologist Reserch 39 () 1 () Fig. 5 Reltionship etween the overll ctivtion energy of respirtory processes t the respective growth tempertures (E o (growth)) nd the integrted vlue over the distinct temperture intervl (Cp(R)) (), nd the men vlues of Cp(R) in plnts grown t high (HT) nd low tempertures () of cold-sensitive nd cold-tolernt species (). Ech E o (growth) vlue ws estimted t the respective growth temperture. Men nd SEM re shown (n 3). Different letters in () denote significnt difference (P <.1, Tukey s multiple comprison test). Cp(R) is clculted using the dt from 1 to 35 C. Dt re derived from Ymori et l. (29). Eo (growth) (kj mol 1 ) HT Cucumer Tocco Rice Tomto Potto Cp (R) HT Brod en Winter rye Spring whet Winter whet Triticle Cp (R) HT (Cold-sensitive) HT (Cold-tolernt) (Cold-sensitive) (Cold-tolernt) c well s the clorimetry method in previous works (Kruse & Adms, 28; Kruse et l., 212). From the fitted curve generted y the modified Arrhenius model, we clculted four prmeters, R t the reference low temperture, R REF, the ctivtion energy t this reference temperture, E o (REF), the dynmic response of E o to temperture, d, nd the mesure of respirtory cpcity, Cp(R) (Fig. 1). We lso estimted the temperture dependence of E o from the first derivtive of the curve nd the E o vlues t ech respective growth temperture, E o (growth) (Figs 4, 5). In this study, the men E o vlues decresed with mesurement temperture. However, the temperture dependence of E o, which depends on d, ws vrile depending on the species (Fig. S1). Some species showed n increse in E o with temperture increse. We cnnot understnd why the E o vlue increses with mesurement temperture. The temperture dependence of E o vried even in closely relted species such s the two whet cultivrs nd triticle. This corresponds to the results of Tylor et l. (1998), who showed vrious E o vlues in 14 cultivrs of mize. Also, the temperture dependence of E o my not e relted with cold tolernce (Fig. S1; Tle S1). We nlyzed the reltionships etween prmeters from the modified Arrhenius model. A negtive reltionship etween E o (REF) nd R REF ws oserved (Fig. 2), which corresponded to the results in Kruse & Adms (28). Tht is to sy, the species with lower vlue of R t low mesurement temperture (i.e. R REF ) showed higher vlue of E o t low mesurement temperture (i.e. E o (REF)). Kruse & Adms (28) oserved negtive correltion etween E o (REF) nd R REF over the sesons, wheres in this study the reltionships were different etween HT nd plnts (Fig. 2). The R REF vlue in plnts ws much higher thn tht in HT plnts when the dt of the sme E o (REF) were compred. Next, we compred R REF with Cp(R) (Fig. 2). R is thought to e restricted y the mximl ctivities of the respirtory enzymes t low mesurement temperture, nd thus R REF hs een frequently used s n indictor of Type II cclimtion (Atkin & Tjoelker, 23). However, in some references, Cp(R) is suggested to etter reflect ltertions of respirtory cpcity in the long term thn R REF ecuse R REF is the rte t one low reference temperture (Kruse & Adms, 28; Kruse et l., 211) nd overll respirtory chnges re often oserved in Type II cclimtion. The two prmeters were well correlted, ut when we used dt from ech growth temperture seprtely, the reltionships were different etween the two growth tempertures. In this study, we cnnot specify which prmeter, R REF or Cp(R), is etter indictor of respirtory cpcity. We need to compre the two prmeters with mximl respirtory enzyme ctivities in the future. A negtive correltion etween d nd E o (REF) ws lso oserved (Fig. 3). The intercept with the x-xis of the correltion etween d nd E o (REF) ws c. 5 kj mol 1. This vlue ws similr to tht in Kruse & Adms (28) nd Kruse et l. (212), which they estimted using O 2 uptke rte dt. However, the vlue in this study ws much different from tht of the CO 2 efflux rte in Kruse et l. (212). Although this discrepncy cnnot e explined, tissue ge my influence the intercept with the x-xis ecuse Kruse et l. (212) used much younger sink leves thn our mture source leves. In the present study, growth temperture cused the x-xis intercept of the correltion etween E o (REF) nd d to shift from 54. kj mol 1 in the HT plnts to 43.6 kj mol 1 in the plnts (Fig. 3). HT plnts showed higher temperture dependence of R thn plnts. Here, the rtes were mesured t 3 or 5 C intervls (Fig. 1). In recent studies, respirtory rtes hve een mesured with higher resolution thn in this study (H uve et l., 212; O Sullivn et l., 213; Heskel et l., 214). The modified Arrhenius model hs not een pplied to this high-resolution dt yet.

7 4 Reserch New Phytologist () 6 () E o ( 3 C) E o (15 C) 6 c * (kj mol 1 ) 4 2 (c) Cp (R) HT HL mture HL young HL new (d) (kj mol 1 ) (nmol m 2 s 1 ) * R REF HT HL mture HL young c c HL new c c Fig. 6 The overll ctivtion energy of respirtory processes (E o )t3 C(E o (3 C)) (), E o t 15 C(E o (15 C)) (), the integrted vlue over the distinct temperture intervl (Cp(R)) (c) nd the respirtory rte t low reference temperture (R REF ) (d) in leves of spinch plnts grown t high (HT) nd low tempertures (), nd plnts trnsferred from HT to (HL-plnts). HL-mture plnts were trnsferred to when their seventh leves were just fully expnded; they were kept t for nother 2 wk. HL-young plnts were trnsferred to when their seventh leves were out one-fifth the re of the fully expnded leves; they were kept for c. 1 month t until full mturtion of their seventh leves. HL-new plnts were trnsferred to when their seventh leves were out to emerge; they were kept t for c. 2 months. Men nd SEM re shown (n = 3). Different letters denote significnt difference (P <.5, Tukey s multiple comprison test). * in () denotes significnt difference etween ech E o (15 C) nd E o (3 C) of the HT plnts (P <.5, Dunnett s test). Cp(R) is clculted using the dt from 9 to 39 C. Dt re derived from Ymori et l. (25). The E o vlue t the growth temperture (E o (growth)) The E o vlues t the growth temperture, E o (growth), were similr etween HT nd plnts (Figs 4, 5). This suggests tht the limiting process of R my e similr t different growth tempertures, ut we cnnot explin why such similr vlues re mintined irrespective of growth temperture. This homeostsis my e influenced y flux of the TCA cycle, sustrte flux to the mitochondril mtrix nd NADH consumption y the respirtory chin. Also, the homeostsis my mintin constnt rtio of NADH/NAD + in the mitochondril mtrix. A constnt NADH/ NAD + rtio would e importnt ecuse n increse in the rtio should reduce the rtio of uiquinol to uiquinone followed y the production of superoxide in the respirtory chin or enggement of lterntive oxidse (AOX). We do not know wht rections limit CO 2 efflux rte in leves, ut Arujo et l. (212) indicted tht the rections of the TCA cycle hve high flux control coefficient (FCC) vlues. The rection with high FCC my influence the homeotic E o (growth) t different growth tempertures. The mechnism of the homeotic E o (growth) needs to e clrified in the future. Using the modified Arrhenius model, we lso nlyzed dt of the temperture dependence of R in Quercus crispul leves, which were mesured over severl sesons (Hikosk et l., 27). We estimted E o t the men dily temperture during the mesurements (Fig. S2). The E o vlue t the men dily temperture ws unchnged over the sesons lthough E o clculted from the Arrhenius plot incresed sesonlly (Fig. S2). This result supports the possiility tht E o (growth) is mintined over the sesons. In our nlyses using cold-sensitive nd cold-tolernt species, E o (growth) ws similr, ut Cp(R) in the plnts ws significntly different etween the cold-sensitive nd cold-tolernt species (Fig. 5). This result suggests tht the lower respirtory cpcity in the cold-sensitive species my e relted to their lower growth t. A reltionship etween growth rte nd respirtory rte ws oserved in other studies. Kurimoto et l. (24) showed tht cold-tolernt species hd higher degree of temperture homeostsis of R thn cold-sensitive species nd tht reltive growth rtes were positively correlted with root respirtory rtes. Ymori et l. (29) lso showed tht high rte of photosynthesis t low mesurement temperture ws ccompnied y high rte of respirtion t low mesurement temperture. Such reltionship ws found in the cclimtion to. Kruse et l. (212) clculted the instntneous enthlpic growth cpcity, Cp(G), in leves of Euclyptus species grown t four different ltitudes. Cp(G) ws determined from the integrl of the enthlpic growth rte, R SG DH B. The Cp(G) vlue incresed with n increse in ltitude nd ws strongly correlted with Cp(R CO2 ), which corresponds to Cp(R) in this study. Higher growth cpcity nd Cp(R) t my e mechnism to compenste for reduced rection rtes t, in order to mintin similr rtes of growth nd respirtion t contrsting temperture.

8 New Phytologist Reserch 41 Chnges in respirtory prmeters fter trnsfer from HT to E o (15 C) in HL plnts chnged depending on the durtion t (Fig. 6). Such chnges in the temperture dependence of R hve een reported in previous studies. Covey-Crump et l. (22) reported tht the temperture dependence of root respirtion in Plntgo lnceolt chnged fter 7 d of tretment. Azcon-Bieto & Osmond (1983) mesured the CO 2 efflux rte of whet leves nd showed tht E o mesured t the end of night ws different from tht mesured t the end of the light period. These studies suggest tht the temperture dependence of R (e.g. E o ) cn chnge depending on environmentl/endogeneous chnges. Cp(R) ws promptly chnged fter the trnsfer compred with E o (15 C) nd R REF (Fig. 6 d). This mens tht the respirtory enzymes my e induced y tretment for only 2 wk even in mture leves. In previous study, the respirtory enzymes were induced for short period fter environmentl chnges (Noguchi et l., 21). Although we did not mesure the ctivities of the respirtory enzymes in this study, n increse in the respirtory cpcity fter tretment my e importnt for growth t. Conclusion In this study, we successfully pplied the modified Arrhenius model to dt otined using the IRGA system. In previous studies estimting E o, field dt hve often een used. Here, we nlyzed dt from cultivted plnts nd clerly showed differences in the prmeters of this model. The men vlue of E o in HT plnts ws higher thn tht in plnts t ny mesurement temperture, ut E o (growth) ws similr etween HT nd plnts. The underlying mechnism of this constnt E o (growth) hs yet to e clrified, ut it suggests tht the limiting process of R is similr t different growth tempertures. In cold-tolernt species, the increse in Cp (R), the respirtory cpcity, t my e importnt for Type II cclimtion. Also, Cp(R) ws promptly chnged only 2 wk fter trnsfer to. This increse my e importnt for low temperture cclimtion in spinch, which is cold-tolernt species. Acknowledgements We thnk the memers of the Plnt Ecologicl Lortory in the Deprtment of Biologicl Sciences, Grdute School of Science, The University of Tokyo, for dvice on our dt. References Arujo WL, Nunes-Nesi A, Nikoloski Z, Sweetlove LJ, Fernie AR Metolic control nd regultion of the tricroxylic cid cycle in photosynthetic nd heterotrophic plnt tissues. Plnt, Cell & Environment 35:1 21. Armstrong AF, Bdger MR, Dy DA, Brthet MM, Smith PMC, Millr AH, Wheln J, Atkin OK. 28. Dynmic chnges in the mitochondril electron trnsport chin underpinning cold cclimtion of lef respirtion. Plnt, Cell & Environment 31: Atkin OK, Bruhn D, Hurry VM, Tjoelker MG. 25. The hot nd the cold: unrvelling the vrile response of plnt respirtion to temperture. Functionl Plnt Biology 32: Atkin OK, Tjoelker MG. 23. Therml cclimtion nd the dynmic response of plnt respirtion to temperture. Trends in Plnt Science 8: Azcon-Bieto J, Osmond CB Reltionship etween photosynthesis nd respirtion. Plnt Physiology 71: Covey-Crump EM, Attwood RG, Atkin OK. 22. Regultion of root respirtion in two species of Plntgo tht differ in reltive growth rte: the effect of short- nd long-term chnges in temperture. Plnt, Cell & Environment 25: Heskel MA, Greves HE, Turnull MH, O Sullivn OS, Shver GR, Griffin KL, Atkin OK Therml cclimtion of shoot respirtion in n Arctic woody plnt species sujected to 22 yers of wrming nd ltered nutrient supply. Glol Chnge Biology 2: Hikosk K, Neshim E, Hiur T. 27. Sesonl chnges in the temperture response of photosynthesis in cnopy leves of Quercus crispul in cooltemperte forest. Tree Physiology 27: H uve K, Bichele I, Ivnov H, Keererg O, P rnik T, Rsulov B, Tois M, Niinemets U Temperture responses of drk respirtion in reltion to lef sugr concentrtion. Physiologi Plntrum 144: Kruse J, Adms MA. 28. Three prmeters comprehensively descrie the temperture response of respirtory oxygen reduction. Plnt, Cell & Environment 31: Kruse J, Rennenerg H, Adms MA Steps towrds mechnistic understnding of respirtory temperture responses. New Phytologist 189: Kruse J, Turnull TL, Adms MA Disentngling respirtory cclimtion nd dpttion to growth temperture y Euclyptus. New Phytologist 195: Kurimoto K, Dy DA, Lmers H, Noguchi K. 24. Effect of respirtory homeostsis on plnt growth in cultivrs of whet nd rice. Plnt, Cell & Environment 27: Lloyd J, Tylor JA On the temperture dependence of soil respirtion. Functionl Ecology 8: Noguchi K, Nkjim N, Tershim I. 21. Acclimtion of lef respirtory properties in Alocsi odor following reciprocl trnsfers of plnts etween high- nd low-light environments. Plnt, Cell & Environment 24: O Sullivn OS, Lsnth KW, Weersinghe K, Evns JR, Egerton JJG, Tjoelker MG, Atkin OK High-resolution temperture responses of lef respirtion in snow gum (Euclyptus puciflor) revel hightemperture limits to respirtory function. Plnt, Cell & Environment 36: R Development Core Tem. 23. R: lnguge nd environment for sttisticl computing. Vienn, Austri: R Foundtion for Sttisticl Computing. Shpiro JB, Griffin KL, Lewis JD, Tissue DT. 24. Response of Xnthium strumrium lef respirtion in the light to elevted CO 2 concentrtion, nitrogen vilility nd temperture. New Phytologist 162: Smith NG, Dukes JS Plnt respirtion nd photosynthesis in glol-scle models: incorporting cclimtion to temperture nd CO 2. Glol Chnge Biology 19: Tylor DK, Rnk DR, Keiser DR, Smith BN, Criddle RS, Hnsen LD Modelling temperture effects on growth-respirtion reltions of mize. Plnt, Cell & Environment 21: Tjoelker MG, Oleksyn J, Reich PB. 21. Modelling respirtion of vegettion: evidence for generl temperture-dependent Q 1. Glol Chnge Biology 7: Turnull MH, Whitehed D, Tissue DT, Schuster WSF, Brown KJ, Griffin KL. 23. Scling folir respirtion in two contrsting forest cnopies. Functionl Ecology 17: Ymori W, Noguchi K, Hikosk K, Tershim I. 29. Cold tolernt crop species hve greter temperture homeostsis of lef respirtion nd photosynthesis thn cold sensitive species. Plnt nd Cell Physiology 5: Ymori W, Noguchi K, Tershim I. 25. Temperture cclimtion of photosynthesis in spinch leves: nlysis of photosynthetic components nd

9 42 Reserch New Phytologist temperture dependencies of photosynthetic prtil rections. Plnt, Cell & Environment 28: Supporting Informtion Additionl supporting informtion my e found in the online version of this rticle. Fig. S1 Temperture dependences of the overll ctivtion energy of respirtory processes in plnts grown t high nd low tempertures. Fig. S2 Temperture dependence of respirtory rte in Quercus crispul leves mesured sesonlly. Tle S1 Summry of sttisticl results Plese note: Wiley Blckwell re not responsile for the content or functionlity of ny supporting informtion supplied y the uthors. Any queries (other thn missing mteril) should e directed to the New Phytologist Centrl Office. New Phytologist is n electronic (online-only) journl owned y the New Phytologist Trust, not-for-profit orgniztion dedicted to the promotion of plnt science, fcilitting projects from symposi to free ccess for our Tnsley reviews. Regulr ppers, Letters, Reserch reviews, Rpid reports nd oth Modelling/Theory nd Methods ppers re encourged. We re committed to rpid processing, from online sumission through to puliction s redy vi Erly View our verge time to decision is <26 dys. There re no pge or colour chrges nd PDF version will e provided for ech rticle. The journl is ville online t Wiley Online Lirry. Visit to serch the rticles nd register for tle of contents emil lerts. If you hve ny questions, do get in touch with Centrl Office (np-centrloffice@lncster.c.uk) or, if it is more convenient, our USA Office (np-usoffice@lncster.c.uk) For sumission instructions, suscription nd ll the ltest informtion visit