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Journal of Plant Physiology 164 (2007) 31—38

0176-1617/$ - sdoi:10.1016/j.

Abbreviationoxidase; AR, alCytR, cytochroaltitude; MA, mTCA, tricarbox�Correspond

fax: +91 1894 2E-mail addr

www.elsevier.de/jplph

Plants at high altitude exhibit higher component ofalternative respiration

Narinder Kumar, Dhiraj Vyas, Sanjay Kumar�

Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur 176 061 (HP), India

Received 6 August 2005; accepted 1 November 2005

KEYWORDSAlternativerespiration;Altitude;Cytochromerespiration;Respiration

ee front matter & 2005jplph.2005.11.001

s: AMSL, above mean sternative respiration; Cme respiration; HA, higedium altitude; MHA,

ylic aciding author. Tel.: +91 18930433.ess: sanjayplp@rediffm

SummaryTotal respiration, capacities of cytochrome (CytR) and alternative respiration (AR)were studied in two varieties of barley (Horedum vulgare) and wheat (Triticumaestivum) each and one variety of pea (Pisum sativum) at low (Palampur; 1300m)and high altitudes (Kibber; 4200m). Similar studies were carried out in naturallygrowing Rumex nepalensis and Trifoilum repenses at Palampur, Palchan (2250m) andMarhi (3250m). All the plants species exhibited lower CytR but significantly higherAR capacity at high altitude (HA) (72–1117% higher) as compared to those at lowaltitude (LA). Glycolytic product, pyruvate and tricarboxylic acid cycle intermedi-ate, citrate increased with increase in altitude. While the role of these metabolitesin relation to HA biology is discussed, significantly higher AR at HA is proposed to bean adaptive mechanism against the metabolic perturbations wherein it might act tolower reactive oxygen species and also provides metabolic homeostasis to plantsunder the environment of HA.& 2005 Published by Elsevier GmbH.

Introduction

High altitude environment is characterized byhigher solar radiations, rapid temperature changes

Published by Elsevier GmbH.

ea level; AO, alternativeytO, cytochrome oxidase;h altitude; LA, lowmedium high altitude;

4 233339;

ail.com (S. Kumar).

and lower partial pressure of gases (Streb et al.,1998). Relatively, larger studies are focused onphotosynthetic and related processes and not muchhas been reported on respiratory behavior despitethat respiration oxidizes carbohydrates into CO2

with concomitant synthesis of several compoundsincluding ATP. Alpine populations have highermitochondrial respiration rates compared tothose from the lowlands (Klikoff, 1966). The low-temperature activities of isolated mitochondriawere found to be positively correlated withaltitude. In high altitude (HA) plants, the high rate

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N. Kumar et al.32

of respiration, probably, had an advantage forhigher metabolic activities of these plants (Stewartand Bannister, 1974). It was also observed that therate of dark respiration enhanced when HA plantswere grown at low temperature. Graves and Taylor(1986, 1988) grew Geum rivale (from HA) andG. urbanum (from low altitude – LA) at lowtemperature and observed that the growth rate ofG. urbanum was lower than that of G. rivale.Results suggested that lower growth rate was dueto lower rate of root respiration; hence the plantwas limited to lower, warmer altitudes.

Plant systems have cyanide-sensitive cytochromerespiration (CytR) with terminal cytochrome oxi-dase (CytO) and cyanide-insensitive alternativerespiration (AR) with the terminal alternativeoxidase (AO). Both these pathways participate inoxygen reduction through mitochondrial electrontransport chain with their respective terminaloxidase. CytR plays unequivocally an importantrole in the transfer of electrons from electron richcompounds, such as reduced pyridine nucleotides,malate and succinate to synthesize ATP and water.AR has been implicated in a number of physiologicalsituations such as during energy overflow in themitochondrial electron transport chain, in reducingmitochondrial reactive oxygen species (Maxwellet al., 1999), thermogenesis (Day et al., 1991;Kumar et al., 1990) and so on. Series of papers havediscussed regulation of activities of the two path-ways (Millenaar and Lambers, 2003), therefore, it isimperative to study CytR and AR while studyingrespiratory behavior.

None of the altitudinal related respiratory studydissected the respiration for CytR and AR. In thepresent study, we studied the capacity of CytR andAR in the plant species grown at contrastingaltitudes and in the plants growing naturally atdifferent altitudinal locations. Also, data wascollected for important respiratory metabolitesnamely pyruvate [product of glycolysis and a sourceof acetyl CoA to the tricarboxylic acid (TCA) cycle]

Table 1. Characteristic features of locations selected in th

Characteristics Locations

Palampur (LA) P

Altitude (m) 1300 2Latitude 321 060 3200 N 3Longitude 761 330 4300 E 7Atmospheric pressure (kPa) 86.8 7Rh (%) 60–80 6Light intensity (mE/m2 s) 1500–1700 2

and citrate (important metabolites of TCA cycle).Data did exhibit a definitive trend with thealtitude, which has been discussed in relation tothe response of plant to altitude.

Materials and methods

A bi-pronged approach was followed in thepresent study. Seeds were sown at two contrastingaltitudes; and the plant species that grow naturallyat different altitudes were targeted.

Location

Studies were carried out at Palampur [LA; 1300mabove mean sea level (AMSL)], Palchan (mediumaltitude, MA; 2250m AMSL), Marhi (medium HA,MHA; 3250m AMSL) and Kibber (high altitude, HA;4200m AMSL). In context to the present study,Palampur represents the LA. The altitude increasesgradually from Palampur to Palchan and Kibber.Important features of these locations are summar-ized in Table 1.

Plants grown at two altitudes

Barley (Horedum vulgare L.; var., Dolma andSonu), pea (Pisum sativum L.; var., Lincoln) andwheat (Triticum aestivum L.; var., HS-240 and HS-295) were sown at LA during the month of October,while at HA the seeds were sown during May. Theseare the conventional sowing seasons for the plantspecies under study. Data was collected 85 daysafter sowing. Mean monthly day temperaturesduring the month of data recording at LA and HAwere 19.272.2 and 18.672.5 1C, respectively.Studies were carried out on flag leaf for barleyand wheat, and on two opposite leaves adjacent tothe stem at 3rd node position in case of pea.

e present study

alchan (MA) Marhi (MHA) Kibber (HA)

250 3250 420021 170 4100 N 321 200 4700 N 321 200 1100 N71 100 7600 E 771 130 1700 E 781 000 5200 E8.48 68.07 61.10–80 50–70 40–60000–2300 2200–2500 2300–2700

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Respiration at different altitudes 33

Plants naturally growing at different altitude

Studies were also carried out on Rumex nepa-lensis Spreng. and Trifolium repens L. naturallygrowing at LA, MA and MHA. These two speciesnaturally grow on wide altitudinal range (Kumaret al., 2005). Measurements were carried outstarting last week of July. Mean monthly daytemperatures during the month of data recordingat LA, MA and MHA were 19.272.2, 17.571.7 and12.273.2 1C, respectively.

Respiratory measurements

Respiratory studies were carried out on sightusing a Clark type oxygen electrode (DW1 electrodeunit and CB1-D box from Hansatech Limited,England) attached with a constant temperaturecirculating water bath maintained at 25 1C (Kumarand Acharya, 1999). Measurements were carriedout by suspending leaf disc (0.625 cm2) in thereaction vessels of the electrode unit containing1.0mL of HEPES buffer (50mM, pH 7.2) in thepresence of KCN (5mM; inhibitor of CytO) tomeasure AR capacity, salicylhydroxamic acid (in-hibitor of AO; dissolved in 0.1 N KOH and pHadjusted to 7.5, 8mM) to measure CytR capacityand in the absence of any inhibitor to measure totalrespiration.

Biochemical analyses

Leaves were harvested on sight and storedimmediately in liquid nitrogen. Later, these werestored at �80 1C till further use. Pyruvate andcitrate contents were estimated with minor mod-ifications as described previously by enzymaticmethods (Delhaize et al., 1993; Korff, 1969;Williamson and Corkey, 1969). Samples wereground in liquid nitrogen, followed by extractionin 0.6 N perchloric acid. The extract was centri-fuged at 15,000g for 10min. While the residue wasused to estimate protein content following Folinphenol method (Lowry et al., 1951), supernatantwas used for metabolite estimations. Supernatantwas collected and neutralized to pH 6.0 withK2CO3. The neutralized solution was centrifugedat 15,000g for 10min, and the supernatant solutionwas collected. For pyruvate, extract was incubatedwith phosphate buffer (50mM, pH 7.6) and 15 mL ofNADH of 4mg/mL. The reaction mixture was pre-incubated for 10min to obtain a stable A340 readingbefore the addition of 5 mL of lactate dehydrogen-ase (5 units, Sigma). The decrease in A340 due to

production of NAD was monitored to calculate theamount of pyruvate in the sample.

Citrate was determined enzymatically with ci-trate lyase and malate dehydrogenase (Williamsonand Corkey, 1969). The reaction mixture contained25 mL of the extract, 464 mL of assay mixturecontaining 50mM Tris–HCl buffer pH 7.4, 5mMEDTA, 10mM MgSO4, 10 mL of NADH (2mg/mL) and0.5 mL of MDH (2 units, Sigma). The reactionmixture was pre-incubated for 10min to obtain astable A340 reading. When the reading was stabi-lized, 0.5 mL of citrate lyase (5 units, Sigma) wasadded to start the reaction. The decrease in A340

due to oxidation of NADH was monitored tocalculate the amount of citrate in the sample.Assays were performed with known amount ofcitrate (0–100 mM) using distilled water as blank tocalculate the linearity of assay and the citrateconcentration per se in the extract.

Measurement of chlorophyll fluorescenceand /PS II activity

Chlorophyll-a fluorescence of photosystem II(PS II) and quantum efficiency of PS II (fPS II) weremeasured with a portable pulse-modulated fluor-escence monitoring system (model FMS2, Hansa-tech Instruments Ltd., UK) on intact leaves (Pandeyet al., 2003). After 30min of dark adaptation F0, Fvand Fm were determined with 100% of the availableactinic photosynthetic photon flux density (ap-proximately 3000 mmol/m2 s).

Measurement of water potential (ww)

The water potential of leaf sample was measuredusing a Wescor HR-33 T dew point psychrometerwith a C52 sample holder (Wescor Inc., Logan, UT,USA) as described earlier (Sharma and Kumar,2005). The sampling of leaf sample for waterpotential measurement was done between 9 and10 AM under full sunlight condition. A uniform leafdisc was kept inside the C52 sample holder,incubated for half an hour before the measure-ments were made.

Statistical analysis

Values were analyzed using Complete Rando-mized Block Design according to Gomez and Gomez(1984) and differences between the means weretested against critical difference Po0.01.

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N. Kumar et al.34

Results

Fv/Fm ratio, /PSII activity and ww

No significant change in Fv/Fm, fPSII and cw wasobserved between the plants growing at LA or HA(Table 2). Similar results were observed inthe plants growing along the altitudinal gradient(Table 3).

Respiratory dynamics

Total respiration, capacities of CytR and AR werestudied in plants at all altitudes. Total respirationwas found to lower by 32%, 22%, 14% and 17% inbarley Dolma, barley Sonu, pea Lincoln and wheatHS-295, respectively, at HA as compared to LA(Fig. 1A). Wheat HS-240, however, did not show anysignificant change in total respiration at twoaltitudes (Fig. 1A). Compared to LA, respirationrate of T. repens also decreased by 9% and 52% atMHA and HA, respectively. Respiratory rate inR. nepalensis, however, increased by 47% and114% at MHA and HA, respectively, as comparedto the LA (Fig. 1B).

Table 2. Fv/Fm ratio, fPSII activity and leaf water potenti

Plant species Fv/Fm ratio fPSII ac

LA HA LA

Barley Dolma 0.80770.049 0.84770.067 0.7117Barley Sonu 0.77170.038 0.82470.062 0.6877Wheat HS-240 0.72670.077 0.79870.087 0.6277Wheat HS-295 0.70870.066 0.68770.073 0.6087Wheat VL-116 0.74870.025 0.71970.018 0.6237

Values (n ¼ 5, 7SE) for LA and HA were analyzed using CRBD and diffdifference between the means at the two altitudes was obtained in

Table 3. Fv/Fm ratio, fPSII activity and leaf water potentigradient at LA (1,300m), MA (2, 250m) and MHA (3, 250m)

Plant Species Parameters Location

LA

Rumex nepalensis Fv/Fm 0.8127fPSII 0.6687cw (MPa) �2.677

Trifolium repens Fv/Fm 0.7367fPSII 0.647cw (MPa) �2.457

Value (n ¼ 5, 7SE) for all altitudes were analyzed using CRBD and difdifference between the means at different altitudes was obtained in

CytR capacity was found to be lower by 73%, 66%,60%, 45% and 56% in barley Dolma, barley Sonu, peaLincoln, wheat HS-240 and wheat HS-295, respec-tively, at HA compared to those grown at LA(Fig. 1C). Whereas, AR capacity was higher by150%, 87%, 72%, 150% and 78% in barley Dolma,barley Sonu, pea Lincoln, wheat HS-240 and wheatHS-295, respectively, grown at HA compared tothose grown at LA (Fig. 1E).

CytR capacity was found to be lower by 50% and52% in R. nepalensis at MA and MHA, respectively,compared to the plants growing at LA. These valueswere 36% and 93% in T. repens (Fig. 1D). On thecontrary, AR capacity was higher by 573% and 1117%in R. nepalensis at MA and MHA, respectively. Thisincrease for T. repens was 280% and 262% (Fig. 1F).

Organic acid level

Respiration is influenced by the respiratorymetabolites, principally pyruvate and citrate (Mill-ar et al., 1993; Vanlerberghe and McIntosh, 1996).Pyruvate increased by 133%, 37%, 104%, 102% and85% in barley Dolma, barley Sonu, pea Lincoln,wheat HS-240 and wheat HS-295, respectively, at

al (cw) of the plant species grown at the LA and HA

tivity cw (MPa)

HA LA HA

0.022 0.68870.058 �2.3470.15 �2.370.210.084 0.70870.031 �1.8870.14 �1.8670.200.011 0.65970.068 �1.9670.12 �1.7370.130.029 0.64870.049 �2.3170.21 �1.9370.220.023 0.58970.067 �1.7870.56 �2.0070.46

erences among mean were tested against PX0.01. No significantthe table.

al (cw) in the plant species growing along the altitudinal

s

MA MHA

0.049 0.8370.038 0.82470.0560.037 0.72770.078 0.64670.0450.78 �3.1270.48 �2.8770.34

0.064 0.7870.084 0.7770.0270.037 0.68370.061 0.6270.0580.56 �2.1870.39 �2.4770.62

ferences among mean were tested against PX0.01. No significantthe table.

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Figure 1. Total respiration, capacities of cytochrome and alternative respiration in plant species grown at low (&) andhigh altitudes (’); and in the plants growing naturally at low (&), medium (IIII) and medium high altitudes (’). Errorbars denotes 7SE (n ¼ 4). Different letters above the bar, show significant difference at Po0.01.

Respiration at different altitudes 35

HA compared to the plants growing at LA (Fig. 2A).These values were 81%, 485%, 112%, 45%, 45% and137% for citrate (Fig. 2C).

No significant change in pyruvate content wasobserved at MA compared to LA in R. nepalensis,but it was higher by 481% at MHA as compared to LA(Fig. 2B). Pyruvate content in T. repens at MA andMHA was higher by 85% and 121%, respectively,as compared to LA (Fig. 2B). Citrate content inR. nepalensis at MA and MHA increased by 68% and496%, respectively, as compared to LA. Thesevalues in T. repens were 433% and 662% (Fig. 2D).

Discussion

Adaptive biology at HA has been a subject ofintense research mainly due to the prevalence ofextremes of environmental conditions. A vastamount of literature exists in the area of photo-synthesis (Korner and Diemer, 1987, 1994; Korner etal., 1988; Kumar et al., 2004, 2005), but much lessis known on respiratory metabolism, which other-wise plays an un-disputated role in energy con-servation and is influenced by environment (Kumarand Sinha, 1994; Millenaar and Lambers, 2003).Therefore, to study the respiratory behavior at

varying altitudes, it becomes crucial to study if theplants experienced any stress. Parameters such asFv/Fm, fPSII activity and cw, which measure thestress of the plants (Tanaka et al., 1999; Tezaraet al., 1999), revealed no significant change at allthe altitudes (Tables 2 and 3) suggesting that theplants were apparently free from those stressesthat affect the above said parameters.

Our results showed that the respiration rateincreased, decreased or remained un-altered withincrease in altitude (Figs. 1A and B). However, aremarkable feature was that CytR was lower and ARcapacity was always higher at HAs (Figs. 1E and F).Secondly, the plants which are normally grown atLA, exhibited lesser increase in AR capacity(72–150% increase) with increase in altitude com-pared to the plants that grow naturally along awider altitudinal range (increase in AR capacity was262–1117% at higher altitudes as compared to LA).The data suggested the possible importance of ARin particular. AR is expected to play an importantrole in plant adjustment under unfavorable condi-tions (Rychter et al., 1988). AR allows stabilizationof redox state of the mitochondrial ubiquinone poolunder the situations of limited CytO activity inrelation to NADH produced through TCA cycle. Alower ratio of reduced to total quinone pool

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BarleyDolma

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Figure 2. Pyruvate and citrate content in plant species grown at low (&) and high altitudes (’); and in the plantsgrowing naturally at low (&), medium (IIII) and medium high altitudes (’). Error bars denotes 7SE (n ¼ 4). Differentletters above the bar, show significant difference at Po0.01.

N. Kumar et al.36

prevents the formation of oxygen fee radicals, thusminimizes damage to plants (Millenaar and Lam-bers, 2003). Role of AR to lower the mitochondrialreactive oxygen production has been shown intransgenic tobacco cells with anti-sense suppres-sion of AO, which resulted in increased productionof reactive oxygen species compared with wild-type cells. Also, the over-expression of AO lowereddown the level of reactive oxygen species (Maxwellet al., 1999). Further, AR is expected to work as ananti-oxidative defense system also when the photo-respiratory cycle is highly active (Pastore et al.,2001), which is indeed very active in the plants atHA (Streb et al., 1998). Photo-respiratory productglyoxylate and hydroxypyruvate could play a role inthe activation of AO (Millar et al., 1996; Pastoreet al., 2001).

There is a lot of debate on the role organic acidsin the regulation of AO activation (Millar et al.,1996). Our results that pyruvate and citratecontent increased with increase in altitude andthat the species that grow naturally along thealtitude gradient had higher content compared tothe one which are grown at HA suggest that thesecould play a role in regulating AR (Popova et al.,1998; Vanlerberghe and McIntosh, 1996). Pyruvatecould pass through inner membrane of mitochon-dria, where it could modulate the activity of AOactivation (Millar et al., 1993). However, this needsto be proved as the role of pyruvate under in vivostimulation of AO is yet to be understood (Millenaarand Lambers, 2003).

High levels of pyruvate and citrate suggestshigher carbon flux through glycolysis and TCA cycle

that will lead to greater production of NADH. Thisshould have been reflected in terms of increasedrate of oxygen uptake, but this was evident only inthe case of R. nepalensis. This suggests re-routingof some these compounds towards other pathwaysfor example, in amino acid biosynthesis (Vanler-berghe and McIntosh, 1997), as acetyl donors and inglyoxylate cycle (Popova et al., 1998).

It appears that at HA, CytR is relatively lesseractive compared to AR (Figs. 1C and D). Underthese situations, role of AR as an energy overflowroute cannot be ruled out (Maxwell et al., 1999).Our study opens path for future research to lookinto some of the interesting observations. Forexample, it is well established that HA locationsare characterized by smaller sized plants (Purohit,2003). Is it that AR plays an important role to thisattribute?

It is quite likely that high AR at HA is an adaptivemechanism against the metabolic perturbationswherein it might act to lower reactive oxygenspecies (Laties, 1982; Maxwell et al., 1999) and alsoprovides metabolic homeostasis to plants (McCaigand Hill, 1977; Rychter et al., 1988) under theenvironment of HA.

Acknowledgments

We thank Dr. P.S. Ahuja, Director, Institute ofHimalayan Bioresource Technology, Palampur forencouragement and providing necessary facilitiesfor conducting the experiments. This research is

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Respiration at different altitudes 37

supported by the Department of Biotechnology(DBT), Government of India vide Grant NumberBT/PR/502/AGR/08/39/966-VI under the Biopros-pection Programme. NK and DV acknowledge DBTfor financial assistance. The manuscript representsIHBT communication number 0449. Authors thankanonymous reviewers for helpful suggestions.

References

Day DA, Dry IB, Soole KL, Wiskich JT, Moore AL.Regulation of alternative pathway activity in plantmitochondria. Deviations from Q-pool behavior duringoxidation of NADH and quinols. Plant Physiol 1991;95:948–53.

Delhaize E, Ryan PR, Randall PJ. Aluminum tolerance inwheat (Triticum aestivum L.) II. Aluminum-stimulatedexcretion of malic acid from root apices. Plant Physiol1993;103:695–702.

Graves JD, Taylor K. A comparative study of Geum rivaleL. and G. urbanum L. to determine factors controllingtheir altitudinal distribution. I. Growth in controlledand natural environments. New Phytol 1986;104:681–91.

Graves JD, Taylor K. A comparative study of Geum rivaleL. and G. urbanum L. to determine factors controllingtheir altitudinal distribution. II. Photosynthesis andrespiration. New Phytol 1988;108:297–304.

Gomez KA, Gomez AA. Statistical procedures for agricul-tural research. New York: Wiley; 1984.

Klikoff LG. Temperature dependence of the oxidativerates of mitochondria in Danthonia intermedia,Penstemon davidsonii and Sitanion hystrix. Nature1966;213:529–30.

Korff RWV. Purity and stability of pyruvate and a-keto-glutarate. Method Enzymol 1969;13:519–23.

Korner C, Diemer M. In situ photosynthesis responses tolight, temperature and carbon dioxide in herbaceousplants from low and high altitude. Funct Ecol1987;1:179–94.

Korner C, Diemer M. Evidence that plants from highaltitudes retain their greater photosynthetic effi-ciency under elevated CO2. Funct Ecol 1994;8:58–68.

Korner C, Farquhar GD, Roksandic Z. A global survey ofcarbon isotope discrimination in plants from highaltitude. Oecologia 1988;74:623–32.

Kumar S, Acharya SK. 2,6-Dichloro-phenol indophenolprevents switch-over of electrons between the cya-nide-sensitive and -insensitive pathway of the mito-chondrial electron transport chain in the presence ofinhibitors. Anal Biochem 1999;268:89–93.

Kumar S, Sinha SK. Possible role of alternative respirationin temperature rise of water stressed plants. J Biosci1994;19:331–8.

Kumar S, Patil BC, Sinha SK. Cyanide respiration isinvolved in temperature rise in ripening mangoes.Biochem Biophys Res Commun 1990;168:818–22.

Kumar N, Kumar S, Ahuja PS. Differences in the activationstate of ribulose-1,5-bisphosphate carboxylase/oxy-

genase in some plant species at two altitudes.Photosynthetica 2004;42:303–5.

Kumar N, Kumar S, Ahuja PS. Photosynthetic character-istics of Hordeum, Triticum, Rumex, and Trifoliumspecies at contrasting altitudes. Photosynthetica2005;43:195–201.

Laties GG. The cyanide-resistant, alternative path inhigher plant respiration. Ann Rev Plant Physiol PlantMol Biol 1982;33:519–55.

Lowry OH, Rosebrough JH, Farr AL, Randall RJ. Proteinmeasurement with Folin phenol reagent. J Biol Chem1951;193:265–75.

Maxwell DP, Wang Y, McIntosh L. The alternative oxidaselowers mitochondrial reactive oxygen productionin plant cells. Proc Natl Acad Sci USA 1999;96:8271–6.

McCaig TN, Hill RD. Cyanide-insensitive respiration inwheat: cultivar differences and affects of tempera-ture, carbon dioxide and oxygen. Can J Bot 1977;55:549–55.

Millar AH, Wistich JT, Wheian J, Day DA. Organic acidactivation of the alternative oxidase of plant mito-chondria. FEBS Lett 1993;329:259–62.

Millar AH, Hoefnagel MHN, Day DA, Wiskich JT. Specificityof the organic acid activation of alternative oxidase inplant mitochondria. Plant Physiol 1996;111:613–8.

Millenaar FF, Lambers H. The alternative oxidase: in vivoregulation and function. Plant Biol 2003;5:2–15.

Pandey S, Kumar S, Nagar PK. Photosynthetic perfor-mance of Ginkgo biloba L. grown under high and lowirradiance. Photosynthetica 2003;41:505–11.

Pastore D, Trono D, Laus MN, Fonzo ND, Passarella S.Alternative oxidase in durum wheat mitochondria.Activation by pyruvate, hydroxypyruvate and glyox-ylate and physiological role. Plant Cell Physiol 2001;42:1373–82.

Popova TN, Pinheiro MAA, de Carvalho P. Citrate andisocitrate in plant mitochondria. Biochem BiophysActa 1998;1364:307–25.

Purohit AN. Plant form and functional behaviour alongthe altitudinal gradient in mountains. J Plant Biol2003;30:199–209.

Rychter AM, Ciesla E, Kacperska A. Participation of thecyanide-resistant pathway in respiration of winterrape leaves as affected by plant cold acclimation.Physiol Plant 1988;73:299–304.

Sharma P, Kumar S. Differential display mediatedidentification of three drought-responsive expressedsequence tags in tea (Camellia sinensis (L.) O. Kuntze).J Biosci 2005;30:231–5.

Stewart WS, Bannister P. Dark respiration rates inVaccinium spp. relation to altitude. Flora 1974;163:S415–21.

Streb P, Shang W, Feieraband J, Bligny R. Divergentstrategies of photoprotection in high-mountain plants.Plant Physiol 1998;207:313–24.

Tanaka Y, Hibino T, Hayashi Y, Tanaka A, Kishitani S,Takabe T, et al. Salt tolerance of transgenic riceoverexpressing yeast mitochondrial Mn-SOD in chlor-oplasts. Plant Sci 1999;148:131–8.

ARTICLE IN PRESS

N. Kumar et al.38

Tezara W, Mitchell VJ, Driscoll SD, Lawlor DW.Water stress inhibits plant photosynthesis by decreas-ing coupling factor and ATP. Nature 1999;401:914–7.

Vanlerberghe GC, McIntosh L. Alternative oxidase: fromgene to function. Annu Rev Plant Physiol Plant Mol Biol1997;48:703–34.

Vanlerberghe GC, McIntosh L. Signals regulating theexpression of the nuclear gene encoding alternativeoxidase of plant mitochondria. Plant Physiol 1996;111:589–95.

Williamson JR, Corkey BR. Assays of intermediates of thecitric acid cycle and related compounds by fluorometricenzyme methods. Method Enzymol 1969;13:434–513.