acclimation of leaf respiratory properties in alocasia odora following reciprocal transfers of...

Plant, Cell and Environment

(2001)

24,

831–839

©

2001 Blackwell Science Ltd

831

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2001248August 2001728Respiratory acclimation after changes of light environmentK. Noguchi

et al.

Original ArticleBEES SGML

Correspondence: Ko Noguchi. Present address: Department ofBiochemistry and Plant Sciences, University of Western Australia, 35Stirling Highway, Crawley, WA 6009, Australia. Fax: + 61 893801148; e-mail: [email protected]

Acclimation of leaf respiratory properties in

Alocasia odora

following reciprocal transfers of plants between high- and low-light environments

K.

NOGUCHI

,

1,2

N.

NAKAJIMA

1,*

& I.

TERASHIMA

1,2

1

Institute of Biological Sciences, Tsukuba University, Tsukuba, Ibaraki, 305-8572, Japan and

2

Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan

ABSTRACT

Acclimation of respiration to the light environments isimportant for a plant’s carbon balance. Respiratory rates ofmature leaves of

Alocasia odora

, a typical shade-tolerantspecies, were measured during the night for 14 d after recip-rocal transfers between high- (330

mmmm

mol m

----

2

s

----

1

) and low-light (20

mmmm

mol m

----

2

s

----

1

) environments. Following the trans-fer, both the rate of CO

2

efflux and that of O

2

uptake of

A.odora

leaves adjusted to the new light environments. TheO

2

-uptake rates changed more slowly than the CO

2

-effluxrates under the new environments. Leaf mass per area alsochanged after the transfer. We analysed whether substrateavailability or ATP-consumption rates influence the respi-ratory acclimation. Since the addition of sucrose to leaf seg-ments did not influence the O

2

-uptake rates, the change ofrespiratory substrate availability was not responsible forthe respiratory acclimation. The addition of an uncouplerinduced increases in the O

2

-uptake rates, and the degree ofenhancement significantly decreased after the transfer fromlow to high irradiance. Thus, the change in ATP-consump-tion rates was responsible for the changes in respiratoryrates in the plants transferred from low to high light. Poten-tial rates of O

2

uptake, as measured in the presence of boththe substrate and the uncoupler, changed after the transfer,and strongly correlated with the O

2

-uptake rates, irrespec-tive of the directions of transfer (

r

= 0·961). There was astrong correlation between maximal activities of NAD-isocitrate dehydrogenase and the potential rates of O

2

uptake (

r

= 0·933), but a weaker correlation between thoseof cytochrome

c

oxidase and the potential rates (

r

= 0·689).These data indicate that the changes of light environmentsaltered the respiratory rates via the change of the respira-tory ATP demand, and that the altered rates of respirationwill induce the changes of the respiratory capacities.

Key-words

: Acclimation to light environments; maximalactivity of respiratory enzyme; respiration; shade species.

INTRODUCTION

Plants often experience sudden changes in light environ-ment. For example, gap formation causes a sudden increasein irradiance. Various leaf properties, such as respiration,photosynthesis, photoprotection, stomatal conductanceand mesophyll anatomy, change responding to the changesin light environment (Bunce

et al

. 1977; Jurik, Chabot &Chabot 1979; Ferrar & Osmond 1986; Bauer & Thöni 1988;Eschrich, Burchardt & Essiamah 1989; Sims & Pearcy 1991,1992; Naidu & DeLucia 1997; Mohammed & Parker 1999).These changes would contribute to optimizing the carbonbalance of the plants in the new light environment. Forexample, when leaves are subjected to low light, adecreased respiratory rate is essential to realise a positivecarbon balance. This is especially relevant to species withlong-lived leaves with low photosynthetic rates, such asshade plants and ever-green trees. The changes in respira-tion are also important because respiration provides inter-mediates and energy for biosynthesis (Lambers 1985).When leaves are suddenly subjected to high irradiancesthey often suffer from photodamage to photosystem II(Ferrar & Osmond 1986; Mulkey & Pearcy 1992; Naidu &DeLucia 1997; Mohammed & Parker 1999). The increasedrespiratory rate will supply intermediates and energy forconstructing chloroplasts having elevated photosyntheticcapacities and sufficient protecting mechanisms.

Sims & Pearcy (1991) studied acclimation of photosyn-thesis and respiration in leaves of

Alocasia macrorrhiza

, atypical shade-tolerant species. They transferred the plantsfrom high to low growth irradiances, and vice versa. Afterthe transfer to the new light environment, CO

2

-efflux rateson a leaf area basis approached the values that were char-acteristic of the plants continuously grown in the ‘new’environment within 1week, but photosynthetic capacitiesdid not change markedly. In the leaves of two deciduoustrees,

Acer saccharum

and

Quercus rubra

, Naidu & DeLu-cia (1997) also observed that acclimation of respiratoryrates on a leaf area basis occurred within 1week and pho-tosynthetic capacities changed more slowly. Although these*Present address: Ina, Kita-Adachi, Saitama, 362-0808, Japan

832

K. Noguchi

et al.

© 2001 Blackwell Science Ltd,


,

24

, 831–839

studies did not clarify physiological mechanisms that areresponsible for the respiratory acclimation, Sims & Pearcy(1991) suggested that the rapid acclimation of respirationwas due to direct effects of light and/or carbohydrate avail-ability, but not to maintenance costs of photosyntheticapparatus.

Bunce

et al

. (1977) examined CO

2

-efflux rates and max-imal activities of malate dehydrogenase (MDH) in leaves of

Glycine max

, following transfer from high to low growthirradiance, and vice versa. Both the respiratory rates andthe MDH activities changed to the values that were char-acteristic of the plants grown continuously in the ‘new’environments by the fourth day after the transfer. Theirdata indicated that the acclimation of the abundance of res-piratory machinery (enzymes and transporters) occurred aswell as the respiratory rates.

In general, the respiratory rate is limited by a combina-tion of the availability of photosynthates and the rate ofconsumption of respiratory ATP (Lambers 1985; Amthor1995). For example, in a constant-light environment, theATP-consuming processes mainly limit the respiratory ratein the leaves of

Alocasia odora

, another typical shade-tolerant species, whereas the availability of photosynthateslimits the respiratory rates in the leaves of

Spinaciaoleracea

, a sun plant (Noguchi & Terashima 1997). Thus, toknow underlying mechanisms of the respiratory acclima-tion, we should first examine the changes in the above twofactors.

In this study, we analysed mechanisms of respiratoryacclimation in the fully expanded leaves of

A

.

odora

thatwere transferred to a new light environment. We measuredCO

2

-efflux rates of whole leaves and O

2

-uptake rates of leafsegments for 14d after the transfer to the new light envi-ronments. We also examined the effects of sucrose, arespiratory substrate, and carbonyl cyanide

p

-(trifluo-romethoxy)-phenylhydrazone (FCCP), an uncoupler ofoxidative phosphorylation from a respiratory chain activity,on O

2

-uptake rates of the leaf segments to assess whetherthe respiratory rate was limited by the availability of sub-strates or by the ATP-consuming processes. To estimatechanges in the abundance of respiratory machinery, wemeasured potential O

2

-uptake rates in the presence of bothsucrose and FCCP. We also measured maximal activities oftwo respiratory enzymes, NAD-isocitrate dehydrogenaseof tricarbonic acid (TCA) cycle and cytochrome

c

oxidaseof the mitochondrial respiratory chain.

MATERIALS AND METHODS

Plant material and growth

Plant material and growth conditions were similar to thosedetailed previously (Noguchi & Terashima 1997).

Alocasiaodora

(Lodd.) Spach. plants were grown in pots in vermic-ulite (diameter, 10·5cm; depth, 17·5cm; one plant per pot)in a controlled environment (KG-50HLA-S, Koito, Osaka,Japan). Light was supplied by cool-white fluorescence tubes(FPR96EX-N/A, National, Osaka, Japan). Black shade

cloth was used to reduce irradiance levels. Photosyntheticphoton flux densities were 330 and 20

m

mol m

-

2

s

-

1

in thehigh- and low-light environments, respectively. The day/night air temperatures were 27/20

∞

C, relative humidity60% and day-length 8 h (from 0300 to 1100). The plantswere used about 1 month after propagation.

Transfers and sampling schedule

When the respiratory rates of the fully expanded leavesattained constant levels, the plants were transferred fromhigh- to low- and from low- to high-light environments.Only the fully expanded leaves were used for measure-ments. For the sake of simplicity, the plants transferredfrom low to high light are called LH plants and those trans-ferred from high to low light are HL plants. The plants thatwere continuously growing in low-and high-light environ-ments are called LL and HH plants, respectively. Ten plantswere placed in the low- and high-light conditions, respec-tively. Half of them were transferred to the high- or low-light conditions.

Measurements of respiratory rates and sampling weremade during the night on days 2 and 0 before and days 1, 3,5, 8 and 14after the transfer.

Measurements of CO

2

efflux of intact leaves

Measurements were made according to Noguchi &Terashima (1997) with some modifications. An attachedwhole leaf was enclosed in a chamber and the rate of CO

2

efflux was determined with an open gas-exchange system.Areas of the leaves ranged from 57·0 to 161 cm

2

. Air waspassed through a soda lime column to remove CO

2

andmixed with 5% (v/v) CO

2

using mass-flow controllers(KOFLOC 3910; Kojima, Kyoto, Japan). The humidity ofthe air was adjusted by changing the temperature of the airthat had been passed through water. The rate of air flowthrough the chamber was adjusted to 8·3

¥

10

-

6

m

3

s

-

1

(=500 m L min

-

1

). The concentration of CO

2

and thehumidity of the air were monitored with an infrared gasanalyser (ZRC; Fuji, Tokyo, Japan) and a dew-pointhygrometer (HYGRO M4; General Eastern, Woburn, MA,USA), respectively. A leaf temperature was measured witha copper–constantan thermocouple and adjusted to20

±

1

∞

C. The respiratory rates were measured at an ambi-ent partial pressure of CO

2

of 36Pa. At the end of the lightperiod (1100h), the plants were brought into a dark room.The respiratory rates were measured for more than 3h dur-ing the night-time from 1200 to 2200h. Rates of CO

2

effluxof

A

.

odora

leaves were almost constant during the night inall the light conditions (data not shown). Thus, we used theaverage values of the CO

2

-efflux rates during the night.

Measurement of O

2

uptake of leaf segments

Measurements were made according to Noguchi &Terashima (1997). The rates of O

2

uptake of leaves weremeasured polarographically with a Clark type liquid-phase

©

2001 Blackwell Science Ltd

,


,

24

, 831–839

Respiratory acclimation after changes of light environment

833

O

2

electrode (Rank Brothers, Cambridge, UK) at 20

∞

C in4 mL of the air-saturated solution containing 50 m

M

2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonicacid (HEPES),10 m

M

2-morpholinoethanesulfonic acid (MES) (pH6·6)and 0·2 m

M

CaCl

2

. The leaf segments (

ª

1·5 cm

2

each) werecut paradermally (in parallel with epidermis) into twopieces with a new razor blade. The slicing was essential forthe experiments with exogenous reagents. As the respira-tory rate of these slices was almost fully inhibited by a com-bination of 20 m

M

salicylhydroxamic acid and 1 m

M

KCN,the exogenous application of inhibitors was effective.Because the slicing transiently enhanced the respiratoryrate, the slices were soaked for more than 20 min in thesame buffer before the measurements. After the soaking,the O

2

-uptake rates of the slices were the same as those inthe segments (1 cm

¥

1·5 cm) of leaves that were incubatedfor more than 20 min in the buffer before the measure-ments. A piece of nylon net was used to keep the slicesabove a stirrer bar and an electrode. The rates of O

2

uptakewere calculated assuming that the concentration of oxygenin the air-saturated buffer at 20

∞

C was 276

m

mol L

-

1

. Aftera constant rate of O

2

uptake was attained in the buffer with-out any reagents, sucrose (110 m

M

, final concentration) orFCCP (1

m

M

, final concentration) was added and thechanges in the rate were analysed. Stock solutions were 1

M

sucrose in the above buffer and 1m

M

FCCP in ethanol.

Measurement of dry weight

Segments were cut from a leaf with a fresh razor blade atnight. After measurement of areas of the segments, the seg-ments were dried at 70

∞

C for more than three days andthen weighed.

Determination of maximal enzymatic activities

We measured the maximal activities of two respiratoryenzymes, NAD-isocitrate dehydrogenase, a TCA-cycleenzyme, and cytochrome

c

oxidase of the mitochondrialrespiratory chain. The maximal activities of NAD-isocitratedehydrogenase were determined according to Noguchi,Sonoike & Terashima (1996). Frozen leaf segments wereground to powder in liquid nitrogen and extracted in abuffer containing 100 m

M

HEPES-KOH (pH7·5), 10 m

M

KH

2

PO

4

, 0·5 m

M

EDTA, 10% (v/v) glycerol and 10 m

M

dithiothreitol with a chilled mortar and pestle. The ratio ofleaf to buffer was 1:7 (mg fresh weight

m

L

-

1

volume). Thehomogenate was filtered through 20

m

m mesh to removetissue debris. For the assay of NAD-isocitrate dehydroge-nase, 20

m

L of the filtrate was added to 680

m

L of a reactionmixture containing 50 m

M

HEPES-KOH (pH7·6), 1 m

M

MnSO

4

, 1·33 m

M

NAD

+

, 17·1 m

M

isocitrate and 0·05%(v/v)Triton X-100. The increase in absorbance at 340 nm due tothe reduction of NAD

+

was monitored for 20 min at 20

∞

C(UV-200S; Shimadzu, Kyoto, Japan). A molar extinctioncoefficient of 6·22 m

M

-

1

cm

-

1

was used (Cox 1969).The maximal activities of cytochrome

c

oxidase weremeasured according to Hendry (1993). Frozen leaf seg-

ments were ground to a powder in liquid nitrogen andextracted in a buffer that contained 100 m

M

KPi (pH7·5),250 m

M

sorbitol, 0·2 mM NaEDTA with a chilled mortarand pestle. The ratio of leaf to buffer was 1:7 (mg freshweight mL-1 volume). Each homogenate was filteredthrough 20 mm mesh to remove tissue debris. The filteredhomogenate was centrifuged at 15000 g for 3 min at 4 ∞C.The pellet was resuspended in 50 mM KPi buffer. For theassay of cytochrome c oxidase, 10 mL of the resuspendedcrude extract was transferred to a cuvette that contained570 mL of 50 mM KPi (pH7·5) and 100mL of reduced cyto-chrome c. The decrease in absorbance at 550nm was mon-itored for 20 min at 20∞C. A molar extinction coefficientof 20 m M-1 cm-1 was used (Hendry 1993). The reducedcytochrome c was prepared by desalting 0·5 mL of 50 mM

KPi buffer that contained 5 mg horse heart cytochrome cand 11mg ascorbic acid with a Sephadex G 50 column(1·3 ¥ 5 cm).

Statistical analyses

All the data were analysed with StatView (ver. 5·0J, SASInstitute Inc., Cary, NC, USA).

RESULTS

On a leaf area basis, both the CO2-efflux and O2-uptakerates of high-light leaves (HH) were higher than those oflow-light leaves (LL) throughout the experimental period(Fig. 1). When A. odora plants were transferred to the high-or low-light environment, the respiratory rates of theirleaves changed. The CO2-efflux rates of the whole leavesshowed quicker responses to the new environments thanthe O2-uptake rates of the leaf segments, both in HL and inLH plants. The O2-uptake rates of HL and LH plantschanged little at day 1 after the transfer. However, the CO2-efflux rates of HL and LH plants changed significantly atday 1 after the transfer.

We also expressed the respiratory rates on a leaf drymass basis (Fig. 2), which reduced the differences in bothCO2-efflux and O2-uptake rates between HH and LLplants, but the difference was still significant. After thetransfer to the low- or high-light environment, both therates changed. Expressed on a dry mass basis, the O2-uptake rates also showed slower responses to the new envi-ronments than the CO2-efflux rates, both in HL and in LHplants. In order to express the respiratory rates on a drymass basis, we used leaf mass per area (LMA). The LMA ofHH plants was almost twice that of LL plants throughoutthe experimental period (Fig. 3). After the transfer to thelow- or high-light conditions, LMA also changed. Althoughthe LMA of HL plants decreased, the difference in LMAbetween HH and HL was small. The LMA of LH plantsincreased at day 3 after the transfer, and was about 1·5times greater than the LMA of LL plants at day 14 after thetransfer. In order to assess what factors influence thechanges of respiration, values on a fixed (i.e. area) basis

834 K. Noguchi et al.

© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 831–839

would be better than those on a variable (i.e. dry mass)basis. Thus, we expressed all the values on a leaf area basisfor further analyses.

In order to assess whether either the rates of ATP con-sumption or the substrate availability limited the respira-tory rate after the transfer, we examined the effect of anuncoupler, FCCP, or a substrate, sucrose, on the O2-uptakerates of leaves. FCCP increased the O2-uptake rates in HHand LL plants to a large extent (Fig. 4a & b), which sug-gests that the low ATP-consumption rate limited the respi-ratory rates in both HH and LL plants. In the presence ofFCCP, the percentage increase in O2 uptake of the leaves ofLL plants was higher than that of HH plants throughout theexperimental period. If the limitation by the rates of ATPconsumption was responsible for the changes of the respi-ratory rate, then the degree of the increase in O2 uptake bythe addition of FCCP is expected to be considerably differ-ent before and after the transfer. However, in HL plants,the effects of FCCP were not significantly greater thanthose in HH plants. On the other hand, in LH plants, the

effect of FCCP was significantly smaller than that in LLplants. The differences of the effects of FCCP between LLand LH plants were, however, much smaller than expected.Assuming that the changes in the respiratory rates of LHplants were explained only by the changes in ATP con-sumption, the O2-uptake rates in the presence of FCCP ofLH plants should be the same as those of LL plantsthroughout the experimental period. It is then possible tocalculate the expected values of the percentage increase inO2 uptake of LH plants after the addition of FCCP from theO2-uptake rates in LL plants in the presence of FCCP andthe O2-uptake rates of LH plants in the absence of FCCP.The expected percentage increases in O2 uptake after theaddition of FCCP were as low as 11% at day 5 and 0% atday 14 in LH plants, whereas the actual values were muchhigher (Fig. 4b). Sucrose hardly influenced the respirationin any of the plants, and thus the substrate level did notlimit the respiratory rates in these plants (Fig. 4c & d).

We tested whether the abundance of the respiratorycomponents changed after the transfer. Figure 5a shows the

Figure 1. Changes in (a) the rate of CO2 efflux and (b) O2 uptake of A. odora leaves, expressed on a leaf area basis. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. Error bars represent standard deviation. Differ-ent character shows significant differences (Tukey–Kramer’s multiple comparison test, P<0·05).

(a)

(b)

Figure 2. Changes in the (a) rate of CO2 efflux and (b) O2 uptake of A. odora leaves, expressed on a leaf dry mass basis. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. Error bars represent standard deviation. Different character shows significant differences (Tukey–Kramer’s multiple comparison test, P<0·05).

(a)

(b)


Respiratory acclimation after changes of light environment 835

O2-uptake rates in the presence of sufficient substrate anduncoupler. These rates represent the respiratory potentialof the leaves. The respiratory potential of HH leaves wassignificantly greater than that of LL leaves throughout theperiod of measurements. The difference in the respiratorypotential between HL and HH, and LH and LL, becamesignificant by day 3 after the transfer. The correlationbetween the O2-uptake rates and the respiratory poten-tial was very strong [Pearson’s correlation coefficient

(r) = 0·961, P < 0·0001] (Fig. 6a). The maximal activities ofNAD-isocitrate dehydrogenase in the HL and LH plantsshowed changes after the transfer to the new light environ-ments (Fig. 5b). The changing patterns of NAD-isocitratedehydrogenase activity in these plants were similar to thoseof the respiratory potential; there was a good correlationbetween the respiratory potential and the maximal activityof NAD-isocitrate dehydrogenase (r = 0·933, P < 0·0001)(Fig. 6b). On the other hand, the maximal activity of cyto-chrome c oxidase in the HL and LH plants did not show thevery clear changes (Fig. 5c); Pearson’s correlation coeffi-cient was lower (r = 0·689, P < 0·001, Fig. 6b).

DISCUSSION

The present data clearly indicate that, in Alocasia leaves,the changes in light environments altered the O2-uptakerates via the changes of ATP-consumption rates. Thealtered rates of respiration will modify the abundance ofrespiratory machinery.

The respiratory rates of the HL and LH plants accli-mated to the new light environments (Fig. 1). Comparingthe CO2-efflux and O2-uptake rates, there was a clear dif-ference in the period that was required to acclimate to thenew light environments. In LH plants, biosynthesis ofphotosynthetic and photoprotective machineries would beinduced (Ferrar & Osmond 1986) and nitrogen assimilationfor biosynthesis would be enhanced. Therefore, in LHplants, NADH from glycolysis and the TCA cycle would beconsumed by biosynthesis and nitrogen assimilation, ratherthan by the respiratory electron chain for ATP production.Nitrate assimilation and biosynthesis of the reduced com-

Figure 3. Changes in leaf mass per area of A. odora leaves. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. Error bars represent standard deviation. Different character shows significant differences (Tukey–Kramer’s multiple comparison test, P < 0·05).

Figure 4. Effects of the addition of (a, b) FCCP or (c, d) sucrose on the rates of O2 uptake of A. odora leaves. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. Error bars represent standard deviation. * represents a significant difference (P < 0·05), ** (P < 0·01), and *** (P < 0·001) according to Student’s t-test.

(a) (b)

(c) (d)



pounds typical of plant biomass often increase the respira-tory quotient (RQ; CO2 efflux/O2 uptake) (Lambers,Chapin & Pons 1998). This may account for the observationthat the CO2-efflux rates showed a faster response to high

irradiances than the O2-uptake rates in LH plants. In HLplants, components that were involved in photosynthesisand photoprotection would become surplus to the demandat the low irradiances, and some of these componentswould be broken down gradually. In HL plants therefore apart of the respiratory substrates would be degradationproducts of cellular constitutions, such as photosyntheticenzymes and transporters. These reduced substrates, com-pared with sucrose, would lower RQ (Lambers et al. 1998).Estimated RQ with the rates of CO2 efflux and O2 uptakewas quite different between HH and LL plants. The differ-ence of RQ may be attributed to the difference of rates ofnitrate assimilation or biosynthesis. As the attached wholeleaf was used for the measurements of the CO2-efflux ratesand the leaf segments for those of the O2-uptake rates, wecould not calculate an exact value for RQ. Accurate andsimultaneous measurements of the rates of CO2 efflux andO2 uptake will be needed to assess the changes of RQ in LH

Figure 5. Changes of O2-uptake rate in the presence of sufficient respiratory substrate and uncoupler (a). Changes of maximal activity of (b) NAD-isocitrate dehydrogenase and (c) cytochrome c oxidase. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. Error bars represent standard deviation. Different character shows significant differences (Tukey–Kramer’s multiple comparison test, P<0·05). There is no significant difference between treatments in maximal activity of cytochrome c oxidase (c).

(a)

(b)

(c)

Figure 6. (a) Correlations between the O2-uptake rate and the potential rate of O2 uptake in the presence of sufficient substrate and uncoupler. Plants originally grown in high light (circles) or low light (triangles) were transferred to high light (open symbols) or low light (closed symbols) on day 0. (b) Correlations between the maximal enzymatic activities and the O2-uptake rate in the presence of sufficient substrate and uncoupler. Open symbols denote NAD-isocitrate dehydrogenase (NAD-IDH) and closed ones denote cytochrome c oxidase (COX). A line represents y=x.

(a)

(b)



and HL plants and the difference between HH and LLplants. However, our data clearly suggest the changes inRQ after the transfer to the new irradiances.

After the transfer to the low- or high-light condition,LMA changed (Fig. 3). The leaves of two deciduous treespecies showed similar changes of LMA after the transferfrom low- to high-light environment (Naidu & DeLucia1997). In the leaves of HL plants, decreases in the amountsof photosynthates and degradations of cellular componentsmay account for the decrease in LMA, whereas the oppo-site is expected to occur in the leaves of LH plants.

In the constant-light environments, the respiratory ratesof A. odora leaves were not influenced by the abundance ofcarbohydrates (Fig.3 in Noguchi & Terashima 1997; Fig. 4c& d in this study). Exogenous carbohydrates also did notinfluence the O2-uptake rates in HL and LH plants. It isconcluded that the respiration of A. odora leaves waslimited by the rates of ATP-consuming processes underthe constant-light environments (Fig. 4 in Noguchi &Terashima 1997; Fig. 4a & b in this study). Therefore, weexpected that the effects of FCCP would largely changeafter the transfer in HL and LH plants. However, the dif-ferences of the percentage increase of O2 uptake were small(Fig. 4a & b). If the respiratory capacities change in paral-lel with the respiratory rates in HL and LH plants, the largechange of ATP-consumption rates will not result in thelarge change of FCCP effects. Thus, these small changes ofFCCP effects will be ascribed to the changes of the amountsof the respiratory components (Figs 5 & 6). It is, however,worth noting that the present data with FCCP may not rep-resent the true degree of adenylate control in vivo, becauseFCCP also affects pH in cellular components.

Raven (1989) estimated a cost for repairing photodam-aged photosystem II (PSII), which was much lower than adaily carbon gain. As his estimation was based on the spe-cific energy costs of Penning de Vries, Brunsting & van Laar(1974), the cost of protein synthesis was underestimated(de Visser, Spitters & Bouma 1992; Zerihun, McKenzie &Morton 1998). We estimated the cost of PSII repair and itscontribution to the increment of respiratory ATP produc-tion in LH plants, based on the following assumptions:

1 PSII content per leaf area is 1·2mmolm-2 in LH plants(Chow et al. 1988);

2 Half of PSII are photodamaged and resynthesizedduring 5d;

3 In the damaged PSII, only D1 protein is removed andsynthesized (Raven 1989);

4 All the costs of PSII repair are covered by increments ofthe respiratory ATP production;

5 The ATP/O2 ratio is 5;6 The specific cost of protein synthesis is 8·0 mol ATP

mol-1 peptide bond (Noguchi et al. 2001b);7 The number of amino acid residues is D1 protein is 345

(Andersson et al. 1994), and8 PSII repair ends within 5 d of the transfer.

The cost of PSII repair during 5 d and its contribution toincrements of respiratory ATP production for these periods

were 1·7 m mol ATP m-2 and 1%, respectively. Thus, therecalculated repair cost of PSII would still be too low toaccount for the increase in ATP consumption and to influ-ence the respiratory acclimation in LH plants. Therefore,ATP in LH plants was probably consumed for the biosyn-thesis of new cellular components. The transfer of peaplants from low- to high-light environment increased theamount of components of photosynthesis in the leaves, suchas Rubisco, cytochrome f, chloroplast ATPase (Chow &Anderson 1987a, 1987b).

The respiratory potential in HL and LH plants changedafter the transfer to the new light environments (Fig. 5a).The very strong correlation between the O2-uptake ratesand the respiratory potential indicates that the abundanceof the respiratory components (enzymes and transporters)changed after the transfer. In fact, the maximal activity ofNAD-isocitrate dehydrogenase changed after the transferin HL and LH plants (Fig. 5b) and the correlation betweenthe respiratory potential and the maximal activity of NAD-isocitrate dehydrogenase was very strong (Fig. 6b). Thus,we concluded that the changes of ATP-consumption ratescaused the changes of the O2-uptake rates, and that thechanged rates of respiration will alter the abundance of res-piratory components. These results agree with the data ofBunce et al. (1977), which showed the good correlationbetween the respiratory rates and the MDH activities ofsoybean leaves.

The maximal activity of cytochrome c oxidase did notshow distinct changes after the transfer to the new lightenvironments (Fig. 5c). In the respiratory electron trans-port chain, there are two pathways from ubiquinol tooxygen, the alternative cyanide-resistant pathway and thecytochrome pathway. The distinctly different changes ofthe maximal activities of cytochrome c oxidase and those ofthe respiratory potential may be caused by the alternativepathway, although A. odora plants show little in vivo activ-ity of the alternative pathway under constant-light environ-ments (Noguchi et al. 2001a). Millenaar et al. (2000) showedthat O2-uptake rates and maximal activity of cytochrome coxidase decreased in parallel after transfer from high-tolow-light environments in roots of Poa annua. They alsoshowed that the alternative pathway activity did not changeafter the transfer, whereas the cytochrome pathway activitydecreased. Our results need to be clarified by analysingthe in vivo activities of cytochrome and alternative path-ways after the transfer of A. odora plants.

The respiratory potential of LL plant was higher thanthe O2-uptake rate of LH plant even 14d after the transfer(Figs 1b & 5a). Why did the abundance of respiratory com-ponents increase in LH plants? This may be because thecosts of the respiratory components would be much lowerthan those of photosynthetic components (Makino &Osmond 1991) and because the sufficient respiratory com-ponents would allow respiratory rates to increase rapidlyunder a stress environment. For example, the leaves of A.odora show a rapid and transient increase of the CO2-effluxrates upon injury (Noguchi, unpublished results). Althoughphysiological mechanisms of this phenomenon are still



unknown, this response may be important to protect a leaffrom herbivores.

We examined the respiratory acclimation of A. odoraleaves to the changes of light environments. The changes inthe light environments caused the changes in the resp-iratory demands and rates. The altered rates of respirationwill induce the changes in respiratory capacities. Theseexcess capacities will permit transient demands for rapidrespiration.

ACKNOWLEDGMENTS

We thank Dr Sachiko Funayama-Noguchi and Dr Shin-IchiMiyazawa for the helpful advice and the members of theTerrestrial Ecological Laboratory, Institute of BiologicalSciences, University of Tsukuba and the members of theEcophysiological Group, Department of Biology, GraduateSchool of Science, Osaka University for kind advice andhelp. This study was supported by a Fellowship from theJapan Society for the Promotion of Science for JapaneseJunior Scientists, the Sasakawa Scientific Research Grantfrom the Japan Science Society, Grant-in-aid for BasicStudies (No. 10440235) and Grant-in-aid under CreativeBasic Research Program (09NP1501), both from theMinistry of Education, Science, Sports and Culture, Japan,and a Grant from the Ministry of Agriculture, Forestoryand Fishery, Japan (Bio-Design Program).

REFERENCES

Amthor J.S. (1995) Higher plant respiration and its relationshipsto photosynthesis. In Ecophysiology of Photosynthesis (eds E.-D. Schulze & M.M. Caldwell), pp. 71–102. Springer-Verlag,Berlin.

Andersson B., Ponticos M., Barber J., Koivuniemi A., Aro E.-M., Hagman A., Salter A.H., Dan-Hui Y. & Lindahl M.(1994) Light-induced proteolysis of photosystem II reactioncentre and light-harvesting complex II proteins in isolatedpreparations. In Photoinhibition of Photosynthesis (eds N.R.Baker & J.R. Bowyer), pp. 143–159. BIOS Scientific Publishers,Oxford.

Bauer H. & Thöni W. (1988) Photosynthetic light acclimation infully developed leaves of the juvenile and adult life phases ofHedera helix. Physiologia Plantarum 73, 31–37.

Bunce J.A., Patterson D.T., Peet M.M. & Alberte R.S. (1977)Light acclimation during and after leaf expansion in soybean.Plant Physiology 60, 255–258.

Chow W.S. & Anderson J.M. (1987a) Photosynthetic responsesof Pisum sativum to an increase in irradiance during growth. I.Photosynthetic activities. Australian Journal of Plant Physiology14, 1–8.

Chow W.S. & Anderson J.M. (1987b) Photosynthetic responsesof Pisum sativum to an increase in irradiance during growth. II.Thylakoid membrane components. Australian Journal of PlantPhysiology 14, 9–19.

Chow W.S., Qian L., Goodchild D.J. & Anderson J.M. (1988)Photosynthetic acclimation of Alocasia macrorrhiza (L.) G.Don. to growth irradiance: structure, function and compositionof chloroplasts. Australian Journal of Plant Physiology 15, 107–122.

Cox G.F. (1969) Isocitrate dehydrogenase (NAD-specific) frompea mitochondria. Method in Enzymology 13, 47–51.

Eschrich W., Burchardt R. & Essiamah S. (1989) The inductionof sun and shade leaves of the European beech (Fagus sylvaticaL.): anatomical studies. Trees 3, 1–10.

Ferrar P.J. & Osmond C.B. (1986) Nitrogen supply as a factorinfluencing photoinhibition and photosynthetic acclimationafter transfer of shade-grown Solanum dulcamara to bright light.Planta 168, 563–570.

Jurik T.W., Chabot J.F. & Chabot B.F. (1979) Ontogeny ofphotosynthetic performance in Fragaria virginiana underchanging light regimes. Plant Physiology 63, 542–547.

Hendry G.A.F. (1993) Respiration: cytochrome c oxidase activity.In Methods in Comparative Plant Ecology (eds G.A.F. Hendry& J.P. Grime), pp. 144–146. Chapman & Hall, London.

Lambers H. (1985) Respiration in intact plants and tissues: Itsregulation and dependence on environmental factors,metabolism and invaded organisms. In Higher Plant CellRespiration (eds R. Douce & D.A. Day), pp. 418–473. Springer-Verlag, Berlin.

Lambers H., Chapin F.S. III & Pons T.J. (1998) PlantPhysiological Ecology, pp. 412–413. Springer-Verlag, Berlin.

Makino A. & Osmond C.B. (1991) Effects of nitrogen nutritionon nitrogen partitioning between chloroplasts and mitochondriain pea and wheat. Plant Physiology 96, 355–362.

Millenaar F.F., Roelofs R., Gonzàlez-Meler M.A., Siedow J.N.,Wagner A.M. & Lambers H. (2000) The alternative oxidase inroots of Poa annua after transfer from high-light to low-lightconditions. Plant Journal 23, 623–632.

Mohammed G.H. & Parker W.C. (1999) Photosyntheticacclimation in eastern hemlock (Tsuga canadensis (L.) Carr.)seedlings following transfer of shade-grown seedlings to highlight. Trees 13, 117–124.

Mulkey S.S. & Pearcy R.W. (1992) Interactions betweenacclimation and photoinhibition of photosynthesis of a tropicalforest understorey herb, Alocasia macrorrhiza, during simulatedcanopy gap formation. Functional Ecology 6, 719–729.

Naidu S.L. & DeLucia E.H. (1997) Acclimation of shade-developed leaves on saplings exposed to late-season canopygaps. Tree Physiology 17, 367–376.

Noguchi K. & Terashima I. (1997) Different regulation of leafrespiration between Spinacia oleracea, a sun species, and Aloca-sia odora, a shade species. Physiologia Plantarum 101, 1–7.

Noguchi K., Sonoike K. & Terashima I. (1996) Acclimation ofrespiratory properties of leaves of Spinacia oleracea L., a sunspecies, and of Alocasia macrorrhiza (L.) G. Don., a shadespecies, to changes in growth irradiance. Plant and CellPhysiology 37, 377–384.

Noguchi K., Go C.-S., Terashima I., Ueda S. & Yoshinari Y.(2001a) Activities of the cyanide-resistant respiratory pathwayin leaves of sun and shade species. Australian Journal of PlantPhysiology 28, 27–35.

Noguchi K., Go C.-S., Miyazawa S.-I., Terashima I., Ueda S. &Yoshinari Y. (2001b) Costs of protein turnover andcarbohydrate export in leaves of sun and shade species.Australian Journal of Plant Physiology 28, 37–47.

Penning de Vries F.W.T., Brunsting A.H.M. & van Laar H.H.(1974) Products, requirements and efficiency of biosynthesis: aquantitative approach. Journal of Theoretical Biology 45, 339–377.

Raven J.A. (1989) Fight or flight: the economics of repair andavoidance of photoinhibition of photosynthesis. FunctionalEcology 3, 5–19.

Sims D.A. & Pearcy R.W. (1991) Photosynthesis and respirationin Alocasia macrorrhiza following transfers to high and lowlight. Oecologia 86, 447–453.



Sims D.A. & Pearcy R.W. (1992) Response of leaf anatomy andphotosynthetic capacity in Alocasia macrorrhiza (Araceae) to atransfer from low to high light. American Journal of Botany 79,449–455.

de Visser R., Spitters C.J.T. & Bouma T.J. (1992) Energy costof protein turnover: theoretical calculation and experimentalestimation from regression of respiration on protein con-centration of full-grown leaves. In Molecular, Biochemical andPhysiological Aspects of Plant Respiration (eds H. Lambers &

L.H.W. van der Plas), pp. 493–508. SPB Academic Publishing,The Hague, The Netherlands.

Zerihun A., McKenzie B.A. & Morton J.D. (1998)Photosynthate costs associated with the utilization of differentnitrogen-forms: influence on the carbon balance of plants andshoot-root biomass partitioning. New Phytologist 138, 1–11.

Received 30 January 2001; received in revised form 20 April 2001; accepted for publication 20 April 2001

acclimation of leaf respiratory properties in alocasia odora following reciprocal transfers of...

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