Diurnal changes of Rubisco in response to elevated CO2, temperature and nitrogen in wheat grown under temperature gradient tunnels

Environmental and Experimental Botany 53 (2005) 13–27

Diurnal changes of Rubisco in response to elevated CO2,temperature and nitrogen in wheat grown under

temperature gradient tunnels

Pilar Pérez, Rosa Morcuende, Isabel Martın del Molino,Rafael Martınez-Carrasco∗

Instituto de Recursos Naturales y Agrobiolog´ıa de Salamanca, CSIC. Apartado 257, 37071 Salamanca, Spain

Accepted 25 February 2004

Abstract

Growth at elevated CO2 and temperature often leads to decreased Rubisco activity. We investigated the effects of increased CO2,temperature and nitrogen on the diurnal changes in the control of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco)activity in wheat (Triticum aestivumL.). Spring wheat was grown at ambient and 700�mol mol−1 CO2, under ambient and 4◦Cwarmer temperatures and with two levels of nitrogen supply in field tunnels in a Mediterranean environment. At ear emergence,elevated CO2 increased Rubisco activation, but decreased Rubisco protein and, with high nitrogen, Rubisco specific activityand had no effect on therbcS transcript. Warmer temperatures tended to decrease therbcS mRNA level and Rubisco protein,although the effect on Rubisco activity was small. High nitrogen decreased Rubisco activation or specific activity, depending onthe CO2 concentration. It increased Rubisco protein at the end of the night, but accelerated its diurnal loss. The main changesafter anthesis were the disappearance of the decrease in Rubisco specific activity caused by elevated CO2, an increase in thisactivity with above-ambient temperatures combined with high nitrogen and that high temperature and nitrogen did not affectRubisco protein contents. This study suggests that decreased leaf protein and increased levels of a Rubisco inhibitor, rather thangene repression by sugars, are involved in acclimation to elevated CO2. High nitrogen increases this down regulation. Changesduring growth in levels of leaf metabolites and protein may alter the relative importance of levels of inhibitors and Rubiscoamounts for Rubisco regulation.© 2004 Elsevier B.V. All rights reserved.

Keywords: Triticum aestivum; Carbohydrates; Diurnal; Elevated CO2; Elevated temperature; Nitrogen; Protein; Rubisco regulation

Abbreviations: rbcS, Rubisco small subunit; Rubisco,ribulose-1,5-bisphosphate carboxylase oxygenase; RuBP, ribulose-1,5-bisphosphate

∗ Corresponding author. Tel.:+34-923272202;fax: +34-923219609.

E-mail address:[email protected] (R. Martınez-Carrasco).

1. Introduction

Many studies on acclimation to elevated CO2 havepointed to a decrease in Rubisco (Drake et al., 1997)which could be accounted for by the acceleration ofdevelopment and an earlier senescence (Sicher andBunce, 1997), but the decrease in Rubisco precedes

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14 P. Perez et al. / Environmental and Experimental Botany 53 (2005) 13–27

any change in senecence or is not affected by it (Nieet al., 1995b). In recent years, much emphasis hasbeen placed on the control of Rubisco gene expres-sion by high atmospheric CO2 levels. The levels oftranscripts for the Rubisco subunits show small di-urnal variations, from a maximum at approximatelydawn to a minimum in the late afternoon (Nie et al.,1995a; Cheng et al., 1998) and are lower at elevatedCO2 (Van Oosten and Besford, 1995), especially be-fore dawn (Nie et al., 1995a) and in the night (Chenget al., 1998). This diurnal oscillation is antiparallel tothe pattern of carbohydrate accumulation (Nie et al.,1995a) and coincides with high levels of hexoses inthe first hours of the night under elevated CO2 (Chenget al., 1998), suggesting a relationship between highlevels of sugars and gene repression. Growth at ele-vated CO2 leads to an accumulation of carbohydrates(Nie et al., 1995a) and there is evidence that theexpression of photosynthetic genes is inhibited bysugars (Sheen, 1990; Krapp et al., 1993). Despite therelationship between diurnal changes in sugar levelsand gene repression, the correspondence of transcriptlevels to the accumulation of soluble carbohydratesat different stages of development is variable (Nieet al., 1995b; Geiger et al., 1999). Moreover, with ahigh nitrogen supply sugars increase at elevated CO2but no acclimation of photosynthesis or decreasedtranscripts for Calvin cycle enzymes is observed(Geiger et al., 1999). The decrease in Rubisco pro-tein at elevated CO2 is accompanied by lowered totalsoluble protein (Sicher and Bunce, 1997) and total ni-trogen in leaves (Nakano et al., 1997), suggesting thatthe effect of elevated CO2 on photosynthesis is dueto the nitrogen status (Riviere-Rolland et al., 1996;Nakano et al., 1997; Farage et al., 1998; Geiger et al.,1999).

The control of Rubisco contents by elevated CO2may involve multiple effects on mRNA translationand/or protein turnover (Webber et al., 1994; Mooreet al., 1999). In addition to this coarse control of Ru-bisco activity, thekcat of Rubisco decreases with in-hibitory phosphorylated compounds that bind tightlyto Rubisco during the photoperiod (Parry et al., 1993,1997; Keys et al., 1995). The catalytic activity of Ru-bisco increases with elevated CO2 by 8% in soybean,although it is not affected by CO2 in rice (Vu et al.,1997). In turn, Rubisco activation decreased at ele-vated CO2 in some studies (Sage et al., 1988; McKee

and Woodward, 1994; Vu et al., 1997), though not inothers (Nakano et al., 1997).

The increase in atmospheric CO2 concentrationmay be associated with a rise in temperature of1.5–6◦C (Schneider, 1992). Rubisco protein is de-creased by 23% in rice and by 17% in soybean astemperatures increase from 32 to 38◦C and from 28to 40◦C, respectively; in addition, the total activityand activation of Rubisco decreases in rice, while onlyactivation decreases in soybean (Vu et al., 1997). ThatRubisco protein, but not total activity in soybean, de-creases at high temperature indicates that the apparentkcat is up-regulated (Vu et al., 1997). Rubisco catalyticactivity increases with temperature and shows values40–70% lower in C3 species from warm than fromcool habitats (Sage, 2002). It has been observed thatgrowth temperature alters the relative stimulation ofphotosynthesis by elevated CO2 in response to temper-ature, reflecting an acclimation to this environmentalparameter (Hikosaka et al., 1999; Bunce, 2000a; Ziska,2001); the apparent specificity of Rubisco for CO2(Bunce, 2000b) or long-term adjustments in the po-tential rate of electron transport and the maximum ve-locity of RuBP-saturated carboxylation (Ziska, 2001)seem to account for this acclimation to temperature.

Studies of the response of Rubisco activity to risingCO2 and temperature in field crops, with the fluctua-tions in light, temperature and humidity of the naturalenvironment, particularly under Mediterranean condi-tions of limiting water and warm temperatures, aresparse. The aim of this work was to assess whether Ru-bisco activity and its diurnal fluctuation in flag leavesof wheat are affected, at the various control levelsdescribed, by combined increases in CO2 and tem-perature in the air and to ascertain whether the ni-trogen supply modifies these effects. Carbohydrateswere analysed to examine their relationship with Ru-bisco activity. In order to approach the natural envi-ronment of Mediterranean wheat crops, this study wasconducted in the field, without the restrictions to rootgrowth imposed by pots, under temperature gradienttunnels—to track the diurnal and seasonal fluctuationsin temperature—with a water supply equivalent to theaverage rainfall during the growing season. The flagleaf at the key growth stage of ear emergence was se-lected for this study. This was the youngest fully ex-panded leaf at a time when acclimation to elevatedCO2 is more likely than in younger plants and the

P. Perez et al. / Environmental and Experimental Botany 53 (2005) 13–27 15

elongating last internode of the stem and the ear pro-vided an active sink for assimilates. Changes duringgrowth on the control of Rubisco by CO2, tempera-ture and nitrogen were assessed by comparing leavessampled at ear emergence and after anthesis.

2. Materials and methods

Spring wheat (Triticum aestivumL., cv. Areces)was sown in a clay sand soil at a rate of 180 kg ha−1

and 0.13 m row spacing on 13 February. Before sow-ing, N (as NH4NO3), P and K fertilizers (80, 40 and40 kg ha−1, respectively) were applied. The crop waswatered weekly through a drip irrigation system pro-viding amounts of water equivalent to the average rain-fall in this area during the period of the experiment(198 mm between February and June). The experimentwas carried out at the IRNASA farm at Salamanca(41◦N, 800 m above sea level).

Temperature gradient tunnels were mounted on thecrop on 23 March rather than at sowing to select cropareas with a uniform plant cover. Mean crop densitiy atear emergence was 534± 77 shoots per metre square.The tunnels were 9 m long, 2.2 m wide and 1.7 m highat the ridge and followed the design ofRawson et al.(1995). Briefly, each tunnel had transparent walls androof and comprised three modules separated by hori-zontally slotted polycarbonate septa to reduce the mix-ing of air between modules due to convection. Twoinlet fans and an outlet fan continuously circulatedair through the tunnel at the speed required to main-tain a temperature difference between the two extrememodules. Three small fan heaters placed in the out-let plenum were used to help maintain the tempera-ture difference at night and whenever solar radiationwas insufficient to raise the temperature. A two-probePt-100 system measuring the difference between theinlets of the inlet and outlet fans was connected toproportional integrative differential (PID) controllerswith outputs to the fans and the heaters. To raise CO2levels in the air, the signal of an infrared gas analysermonitoring the CO2 concentration at the outlet modulewas fed into another PID controlling a solenoid valvewhich injected CO2 at the two inlet fans. Ventilatedtemperature and humidity sensors and air probes forCO2 analysis were placed at the centre of each module.The data were recorded continuously by a computer.

This study was conducted in two tunnels, one keptat the ambient air CO2 concentration and another at700�mol mol−1 during the light hours. CO2 enrich-ment in the night period may be irrelevant, becausedirect effects of CO2 on leaf respiration in the darkseem not to exist, the reports in the literature appear-ing to be artefactual (Jahnke and Krewitt, 2002). Thetemperature difference between the extreme modulesin a tunnel was set at 4◦C. Additional (40 kg ha−1) ni-trogen was added to one of the longitudinal halves ofeach tunnel 34 days after sowing, such that two levelsof this nutrient (80 and 120 kg ha−1) were compared.The samplings were repeated in four consecutive sec-tions within the two module halves. The experimentaldesign (Fig. 1) and its stastistical analysis are furtherdiscussed below.Fig. 2 shows that the diurnal coursesof air temperature, relative humidity and CO2 concen-tration were controlled satisfactorily. Air humidity inthe high-temperature module was lower with elevatedthan ambient CO2, probably due to decreased tran-spiration at elevated CO2. An increase in temperatureand an associated decrease in air humidity during thelight hours occurred from April to May—early June.No relevant changes in CO2 concentrations were ob-served during this period.

On day 3 after the beginning of ear emergence inthe whole experiment (22 May)—ear emergence wasadvanced about 3 days by warm temperatures—flag

Fig. 1. Lay-out of the experiment in temperature gradient tunnelsat ambient (360�mol mol−1) and elevated (700�mol mol−1) CO2,ambient and ambient+4◦C temperatures and 80 (low nitrogen)and 120 (high nitrogen) kg nitrogen ha−1.


Fig. 2. Mean daily courses of air temperature, humidity and CO2

concentration in temperature gradient tunnels set at either ambient(360�mol mol−1, open symbols) or elevated (700�mol mol−1,closed symbols) CO2 and ambient (circles) or ambient+4◦C(squares) temperatures.

leaves (two per replicate) were harvested and im-mediately plunged into liquid nitrogen just beforedawn, 4–6 h later, 1–2 h before dusk and 2–3 h intothe dark period; light intensities were<10, 1700, 100and<10�mol m−2 s−1, respectively. On day 9 after100% anthesis in each temperature regime (3 and 9June for plants under ambient and ambient+4◦C,respectively), another sample of the same size washarvested 4–6 h after the start of the light period. Thesamples were kept in liquid nitrogen until analysis.After determining the fresh weight in subsamples offrozen leaves, leaf area was determined by imageanalysis and chlorophyll was extracted in 80% ace-tone and measured (Arnon, 1949). This allowed theresults to be expressed on a leaf area basis.

For Rubisco activity assays, a procedure based onthat described byLilley and Walker (1974), as mod-

ified by Ward and Keys (1989)and Sharkey et al.(1991), was followed. Aliquots of the frozen leaveswere ground in a mortar with liquid nitrogen and ex-tracted with 100 mM Bicine–NaOH (pH 7.8), 10 mMMgCl2, 10 mM �-mercaptoethanol and 2% PVPP(w/v). An aliquot of the whole extract was used todetermine chlorophyll contents (Arnon, 1949) and theremainder was centrifuged at 13000×g. The total timefrom extraction to the assay of initial Rubisco activitywas less than 2.5 min. Activity was assayed by addingextract to a mixture of 100 mM Bicine (pH 8.2),20 mM MgCl2, 10 mM NaHCO3, 10 mM KCl, 1 mMribulose-1,5-bisphosphate (RuBP), 0.2 mM NADH,5 mM ATP, 5 mM creatine phosphate, 52 units/mlphosphocreatine kinase, 12 units/ml phosphoglycer-ate kinase, 11 units/ml glyceraldehyde 3-phosphatedehydrogenase and recording the decrease in ab-sorbance at 340 nm minus 400 nm for 40–60 s, at astoichiometry of 2:1 between NADH oxidation andRuBP carboxylation. To assay total Rubisco activity,an aliquot of the extract was incubated with NaHCO3and MgCl2 for 10 min at room temperature prior tothe addition of coupling enzymes and NADH, thereaction was started by adding RuBP. The activationstate was estimated as initial activity as a percentageof total activity. Commercial coupling enzymes sus-pended in ammonium sulphate were precipitated bycentrifugation and dissolved in 20% glycerol (Sharkeyet al., 1991). With the assay buffer described, the ini-tial lag in the reaction reported by others (Ward andKeys, 1989; Sharkey et al., 1991) was not observed.The amount of Rubisco in a subsample of frozen leafmaterial was determined by densitometric scanningof SDS-PAGE gels, as described previously (Martındel Molino et al., 1995). Rubisco specific activitywas determined by dividing total activity by Rubiscoactive site contents.

For analysis of RNA, frozen leaf material was ho-mogenised with guanidine buffer. The supernatantwas extracted with phenol/chloroform (1:1, (v:v)) andafter phase separation the aqueous phase was washedwith chloroform. Total nucleic acids were precipi-tated with sodium acetate and ethanol. The pellet waswashed with ethanol, dried and finally resuspendedin DEPC water. Purity was checked by measuring ab-sorption at 230, 260, 280 and 320 nm and the absorp-tion at 260 nm was used for quantification (Logemannet al., 1987). For Northern blots, 10�g of RNA were


separated electrophoretically on 1.2% agarose gelscontaining 7% formaldehyde (Sambrook et al., 1989)and transferred to membranes (Hybond N, Amer-sham Pharmacia Biotec). The membrane was bakedand prehybridization was performed as described byKrapp et al. (1993). The 32P-labelled (radiolabellingwas by random primer using the Boehringer kit) Ru-bisco small subunit (rbcS) cDNA of wheat kindlyprovided by Dr. Christine Raines (Department of Bio-logical Sciences, John Tabor Laboratories, Universityof Essex, UK) was added to the prehybridisation mix-ture and hybridisation was carried out for at least 12 h.The filters were washed and autoradiography was car-ried out at−80◦C with Kodak Xomat films, using asingle intensifying screen. The relative levels ofrbcSexpression were determined by image analysis usingthe Scion ImagePC (Scion, MD, USA) software.

Subsamples of leaves stored in liquid nitrogen wereextracted three times in 1 ml 80% ethanol—10 mMHepes–KOH (pH 7.5) at 80◦C for 30 min and the ex-tracts were pooled. Then, the residue was extractedthree times in 1 ml water at 80◦C for 30 min andthe extracts were pooled. Chlorophyll in the extractswas analysed as described above. Glucose, fructoseand sucrose in ethanol extracts were determined ac-cording toJones et al. (1977). Since most commer-cial invertases hydrolyse oligofructans (Koroleva et al.,1998), sucrose contents were estimated from glucose

Table 1Example of the analysis of variance used in this study with two CO2 concentrations, two air temperatures, two nitrogen levels, fourreplicate samples and four sampling times in a nested design, with nitrogen and temperature in a stratum within CO2, replicates in astratum within temperature and nitrogen and sampling hour as a further stratum within replicates

Sum of squares Degrees of freedom Mean square Variance ratio

CO2 (C) 1467.9 1 1467.9 27.323SubTN stratum 322.3 6 53.7Temperature (T) 1.4 1 1.4 0.008Nitrogen (N) 0.8 1 0.8 0.005CT 47.8 1 47.8 0.276CN 199.2 1 199.2 1.150TN 22.2 1 22.2 0.128CTN 50.9 1 50.9 0.294Sub replicate stratum 4157.2 24 173.2Sub hour stratum 11606.0 96 120.9Hour 2144.8 3 714.9 19.560Error 328.9 9 36.5

Total 17553.5 127

Details are shown on the distribution of degrees of freedom and error terms used to estimate variance ratios.

rather than from fructose released by invertase. Forfructan analysis, aliquots from both the ethanol andwater extracts were rendered 360 mM HCl, heatedat 60◦C for 30 min and then neutralised with KOHbefore analysing glucose and fructose; fructan (fruc-tose equivalents) in the hydrolysed ethanol extractwas estimated by subtracting the amounts of free- andsucrose–fructose; fructose in excess of glucose in thesucrose analysis was assumed to correspond to fruc-tans and was added to these. Fructose in the hydrolysedwater extract was added to obtain total fructan con-tents. Starch in the residue from ethanol–water extrac-tions was digested with amyloglucosidase and amylaseand determined according toap Rees et al. (1977).

The restriction of the number of available tunnelsto just two, with the consequent lack of replicationfor CO2, was a drawback in the experimental design.In addition, randomisation of treatments was limitedwith temperature gradient tunnels, which unavoidablyplaced in a same tunnel module the two combinationsof nitrogen with a given temperature. With a lay-outof samples within samples, analyses of variance wereperformed as in a nested design according toSnedecorand Cochran (1967), with temperature and nitrogen asa stratum included in CO2 and replicates as a stratumincluded in that for temperature and nitrogen. Detailsof one of such analyses are summarized inTable 1.Thevariance ratio for CO2 was obtained by dividing the


mean square for CO2 (1 degree of freedom) by thenitrogen and temperature within CO2 mean square (6degrees of freedom). Thus, the variability of the CO2levels was compared with the variability of individualnitrogen and temperature treatments after removingany overall similarity between treatments belonging tothe same CO2. Although debatable, this method of ob-taining the variance ratio following theSnedecor andCochran (1967)model seemed more adequate thancomparing the CO2 mean square against a residualmean square representing the random variability be-tween treatments after removing CO2 as well as nitro-gen and temperature effects. Similarly, the nitrogen,temperature and interactive effects (1 degree of free-dom each) were compared against the replicates withinCO2-temperature-nitrogen mean square (24 degrees offreedom). Time effects were evaluated by includingthe hour of day as a further stratum in the analysis (3against 9 degrees of freedom). The standard errors ofdifferences, rather than simply the standard error ofmeans, are shown in the figures, since the former arebetter estimates of the treatment effects.

3. Results

Flag leaf photosynthesis during the morning hours(data not shown) was higher in plants grown at ele-vated than ambient CO2. When measured at the sameCO2 concentration, however, elevated CO2 plantshad decreased photosynthesis rates, except that plantsgrown at above ambient temperatures and abundantnitrogen supply showed smaller or no significantdecreases in photosynthesis.

Fig. 3. Diurnal changes in Rubisco activity, activation state,specific activity and Rubisco protein content of flag leaves ofwheat in response to CO2, temperature and nitrogen 3 days afterear emergence. Plants were grown in the field under tempera-ture gradient tunnels at either ambient (360�mol mol−1) or ele-vated (700�mol mol−1) CO2, ambient (circles) or ambient+ 4◦C(squares) temperatures and 80 (open symbols) or 120 (closed sym-bols) kg nitrogen ha−1. Light intensities before dawn, 4–6 h later,1–2 h before dusk and 2–3 h into the dark period were<10, 1700,100 and<10�mol m−2 s−1, respectively. Each point is the meanof four replicates. Vertical bars represent twice the standard errorof the difference for main effects of CO2 (a), temperature and ni-trogen (b) and time (c). The standard errors of the differences fortreatment interactions are omitted for clarity.

3.1. Ear emergence

3.1.1. Rubisco3.1.1.1. Rubisco activity. In general, initial Rubiscoactivity (Fig. 3A and B) showed a decrease from the


end of the night to the end of the light period and thenincreased during the two first hours of the night. El-evated CO2 decreased initial Rubisco activity at theend of the night and mid-morning and then the effectdisappeared due to a faster decline with ambient thanelevated CO2. With a high nitrogen supply, the ini-tial activity of the enzyme was higher at the end ofthe night, as compared to low nitrogen; at other timesof the day, nitrogen had no significant effect on ini-tial activity. Growth temperatures did not affect initialRubisco activity.

Compared to the values obtained at the end of thenight, the activation state of Rubisco (Fig. 3C and D)decreased to a greater extent at ambient than elevatedCO2 in the light period. Two hours into the dark pe-riod, Rubisco activation displayed an increase in plantsgrown at ambient, but not at elevated, CO2. A similarincrease in Rubisco activation after dusk was observedby Parry et al. (1993)and no pre-dawn decrease in ac-tivity was observed in a number of species (Vu et al.,1984; Servaites et al., 1986). The activation state ofRubisco was similar at ambient and elevated CO2 bythe end of the night, while it was higher at elevatedCO2 during the day. A high nitrogen supply decreasedRubisco activation, although in the morning this de-crease occurred only at ambient CO2, as an exception,nitrogen had no effect on the degree of activation ofRubisco by the end of the night. Temperature had noeffects on Rubisco activation.

In plants grown at ambient CO2, total Rubisco ac-tivity (Fig. 3E and F) showed an increase from the endof the night to mid-morning; from there to the end ofthe day, it changed little in plants at the current tem-peratures and increased with 4◦C higher temperatures.Two hours after dusk, in contrast, total Rubisco ac-tivity increased somewhat at the current temperatures,but decreased to values close to those at the end ofthe night at 4◦C above ambient temperatures. By con-trast, at elevated CO2 total Rubisco activity generallydecreased from the end of the night to the first part ofthe light period and then increased until 2 h after dusk.Compensating in part the increased activation of Ru-bisco, total activity was decreased by elevated CO2 atthe end of the night and the decrease was even greaterduring the light hours. Abundant nitrogen did not pre-vent the decrease in total Rubisco activity caused byelevated CO2; rather, high nitrogen increased total Ru-bisco activity in plants grown under ambient CO2, but

not under elevated CO2. The increase in temperaturehad no significant effect on total Rubisco activity.

3.1.1.2. Rubisco specific activity.At ambient CO2,the specific activity of Rubisco (Fig. 3G and H), astotal Rubisco activity, generally increased from theend of the night to the end of the day. Two hoursinto the dark period, Rubisco specific activity changedrelatively little at the current temperatures, but un-derwent a marked decrease at 4◦C higher tempera-tures. In plants grown at elevated CO2, Rubisco spe-cific activity also increased from the end of the nightto mid-morning in plants with a low nitrogen supply,but, in contrast, it decreased in this interval in plantswith an ample nitrogen supply. At elevated CO2 therewas no homogeneous pattern of change in specific ac-tivity from the morning to the end of the day and thiswas generally followed by an increase 2 h into the darkperiod.

During the light hours, elevated CO2 decreased Ru-bisco specific activity in plants with abundant nitro-gen and did not affect it with low nitrogen; 2 h afterthe start of the night, elevated CO2 continued to de-crease the specific activity of the enzyme in plantswith high nitrogen and ambient temperatures, but notwith above ambient temperatures. High temperaturesincreased the specific activity at the end of the day inplants at ambient CO2 and decreased it at the begin-ning of the dark period in plants at ambient CO2 andhigh nitrogen. Nitrogen showed the described interac-tion with CO2 on Rubisco specific activity.

3.1.1.3. Rubisco protein content.The diurnalchange in Rubisco protein (Fig. 3I and J) depended onthe supply of nitrogen. Thus, with abundant nitrogenRubisco protein generally decreased from the end ofthe night to the end of the day. With low nitrogen,Rubisco protein showed little change from end of thenight to the final light hours at ambient CO2, whileat elevated CO2 it decreased at mid-morning andchanged little thereafter at the current temperatures,but increased throughout the light hours with highertemperatures. With both nitrogen levels, Rubisco pro-tein increased in the first hours of the night. ElevatedCO2 decreased the amount of Rubisco protein. Hightemperatures decreased the amount of Rubisco inplants with high nitrogen during the light hours, butthe effect disappeared or was reversed 2 h into the


Fig. 4. (A) Diurnal changes inrbcSmRNA levels 3 days after ear emergence in flag leaves of wheat grown in the field under temperaturegradient tunnels either at ambient (360�mol mol−1) or elevated (700�mol mol−1) CO2, ambient or ambient+4◦C temperatures and 80(low N) or 120 (high N) kg nitrogen ha−1. Numbers (1 and 2) represent replicate samples. Equal amounts of RNA were applied to eachlane. (B) Quantified signals (arbitrary units) of diurnal changes inrbcS transcripts. The signal intensity indicates the transcript abundancerelative to total RNA. Symbols as inFig. 2.

dark period. With low nitrogen, however, this effectof high temperatures only occurred under elevatedCO2 during the night hours. A high nitrogen supplyincreased the amount of Rubisco in the night hoursat high temperature and at the end of the night andmid-morning at the current temperatures, but did notmodify Rubisco contents at the end of the day.

3.1.1.4. rbcS transcript levels.Northern-blots werecarried out with two of the four replicates of all treat-ments and sampling times (Fig 4A). The mRNA levelfor the Rubisco small subunit did not change sig-nificantly during the day (Fig. 4B). High nitrogen

increased therbcS mRNA level. However, this in-crease was not judged as reliable because the highandlow-nitrogen samples were arranged on separate mem-branes. There were no significant effects of CO2 onrbcS transcript levels. In contrast, high temperaturestended to decreaserbcS expression.

3.1.2. Leaf carbohydratesFructose (Fig. 5C and D) increased during the first

part of the light period and decreased thereafter; al-though glucose and fructans (Fig. 5A–B, G–H) fol-lowed the same pattern, it did not reach significance.Sucrose (Fig. 5E and F) increased from the end of the


Fig. 5. Diurnal changes in carbohydrate contents of flag leaves ofwheat in response to CO2, temperature and nitrogen 3 days afterear emergence. Plants were grown in the field under temperaturegradient tunnels. The levels of fructan and starch are in hexoseequivalents. Each point is the mean of four replicates. Symbols asin Fig. 2.

dark period to the first part of the day, remained at thislevel until the end of the light period and then declinedduring the first hours of the night. Starch (Fig. 5I andJ) increased throughout the light period and was mo-bilised at night. Therefore, total non-structural carbo-hydrate contents increased during the day comparedto the end of the night and decreased 2 h into thenight.

Elevated CO2 significantly increased glucose andfructans during the dark period; in the day time thisincrease did not reach significance. Elevated CO2 alsoincreased fructose, sucrose and starch at all day andnight samplings. Thus, total non-structural carbohy-drates always increased at elevated CO2. The highercarbohydrate levels at the end of the night under ele-vated CO2 indicate that the extra carbohydrate accu-mulated during the day was not fully mobilised duringthe dark period. High temperatures decreased the con-tents of fructose, except in plants at elevated CO2 andlow nitrogen and, in the light hours, they decreasedsucrose contents. High temperatures also decreasedstarch contents in plants with ample nitrogen in thelight hours and, in plants with ample nitrogen and el-evated CO2, also 2 h after the start of the night. Ni-trogen deficiency did not affect glucose, fructose andsucrose contents; it increased fructan contents onlyin the light hours and starch contents throughout theday in elevated CO2 plants and, at mid-morning andthe beginning of the night, also in ambient CO2 andhigh-temperature plants.

3.2. After anthesis

3.2.1. Rubisco3.2.1.1. Rubisco activity.At mid-morning 9 days af-ter anthesis, elevated CO2 decreased initial Rubiscoactivity (Fig. 6A) as was observed at ear emergence,except in plants under current temperatures and lownitrogen, which also showed high total and specificRubisco activity and protein compared to other com-binations of factors. Initial Rubisco activity showed apositive response to increased temperatures after an-thesis , except for elevated CO2 and low nitrogen, incontrast with the temperature insensitivity displayedat ear emergence. Nitrogen increased initial Rubiscoactivity at ambient CO2, significantly so at above am-bient temperatures; at elevated CO2 and current tem-peratures, nitrogen decreased initial Rubisco activity


due to the high value with the elevated CO2, currenttemperature and low nitrogen combination.

While elevated CO2 increased Rubisco activationstate at ear emergence, it decreased activation afteranthesis (Fig 6B), except at warm temperature andlow nitrogen. A temperature increase, which had nosignificant effect on the activation of Rubisco at earemergence, caused an increase in activation after an-thesis which was small in all treatment combinationsexcept in elevated CO2 and low nitrogen. At the cur-rent temperatures, high nitrogen did not affect the ac-tivation state of Rubisco, while at high temperatures itdecreased this activation, especially at elevated CO2,an effect which was observed at both temperatures,but only at ambient CO2, at ear emergence.

At above ambient temperatures, elevated CO2 de-creased total Rubisco activity (Fig. 6C), an effectobserved at both temperatures at ear emergence; atambient temperature and low nitrogen, total activitywas high at elevated CO2, as already indicated. Ex-cept for this treatment, a temperature rise increasedtotal Rubisco activity after anthesis, especially atambient CO2 and high nitrogen, in contrast withits lack of effect at ear emergence. Apart from thehigh activity in plants at elevated CO2, ambient tem-perature and low nitrogen, a high nitrogen supplyincreased total Rubisco activity and the interaction ofnitrogen and CO2 on this activity at ear emergencedisappeared after anthesis.

3.2.1.2. Rubisco specific activity.Elevated CO2 in-creased the specific activity of Rubisco (Fig. 6D);this effect of CO2 tended to be higher at current thanwarmer temperatures, which accounted for the un-altered total Rubisco activity under elevated CO2 atthe current temperatures. Therefore, the decrease in

Fig. 6. Effects of CO2, temperature and nitrogen on Rubisco ac-tivity, activation state, specific activity and Rubisco protein con-tent of flag leaves of wheat at mid-morning 9 days after anthesis.Plants were grown in the field under temperature gradient tunnelsat either ambient (360�mol mol−1) or elevated (700�mol mol−1)CO2, ambient (white, dark grey) or ambient+4◦C (pale grey,black) temperatures and 80 (white, pale grey) or 120 (dark grey,black) kg nitrogen ha−1. Each point is the mean of four replicates.Vertical bars represent twice the standard error of the differencefor main effects of CO2 (a) and temperature and nitrogen (b).The standard errors of the differences for treatment interactionsare omitted for clarity.


specific activity caused by elevated CO2 with a highnitrogen supply at ear emergence disappeared afteranthesis. Above ambient temperatures increased Ru-bisco specific activity with high nitrogen, while underelevated CO2 and low nitrogen this activity was highat ambient temperature.

3.2.1.3. Rubisco protein contents and rbcS transcriptlevels. Except for the high Rubisco protein underambient CO2 and temperature with low nitrogen, el-evated CO2 decreased the amount of Rubisco protein(Fig. 6E) 9 days after anthesis, as it did at ear emer-gence. Warmer temperatures had no effect on Rubiscoprotein, in contrast with the decrease induced in itscontents in plants with high nitrogen at ear emergence.Abundant nitrogen did not affect the contents of Ru-bisco protein after anthesis and thus the positive effectof high nitrogen at ambient temperatures observed atmid-morning at ear emergence, disappeared after an-thesis.

The mRNA level for the Rubisco small subunit wasnot affected by the treatments after ear emergence(data not shown).

3.2.2. Leaf carbohydratesNine days after anthesis elevated CO2 had no

significant effect on the contents of glucose and fruc-tose (Fig. 7A and B) and increased starch contents(Fig. 7E), except at current temperatures and highnitrogen. With low nitrogen, elevated CO2 increasedsucrose contents (Fig. 7C), but with high nitrogenit decreased these contents. Under ambient temper-atures, elevated CO2 decreased the levels of fructan(Fig. 7D), while at warmer temperatures it increasedthese levels. Thus, the overall increase in nonstruc-tural carbohydrate contents caused by elevated CO2 atear emergence had disappeared after anthesis. Aboveambient temperatures decreased fructan contents and,with elevated CO2 and low nitrogen, as well as withambient CO2 and high nitrogen, also the starch con-tents. Consequently, a rise in temperature tended todecrease the contents of non-structural carbohydrates,as was generally observed at ear emergence. Nitrogendeficiency did not affect the contents of glucose, fruc-tose and fructan. Starch contents were higher withlow nitrogen, elevated CO2 and current temperaturesthan with other combination of factors.

Fig. 7. Effects of CO2, temperature and nitrogen on carbohydratecontents of flag leaves of wheat at mid-morning 9 days afteranthesis. Plants were grown in the field under temperature gradienttunnels. The levels of fructan and starch are in hexose equivalents.Each point is the mean of four replicates. Symbols as inFig. 6.


4. Discussion

With the modifications discussed below, initialRubisco activity at ear emergence showed a declineduring the day which was due to a deactivation of Ru-bisco, since total Rubisco activity generally showedan increase from before dawn or mid-morning, de-pending on growth CO2 level. Although Rubisco ac-tivation state often increases with light (Portis, 1992),the higher activation of Rubisco at night hours in ourexperiment is consistent with previous results showingan increase in Rubisco activation in the dark (Parryet al., 1993; Anwaruzzaman, 1999) and no pre-dawndecrease in Rubisco activity in some species, in-cluding Gramineae (Vu et al., 1984; Servaites et al.,1986). The increase during the day in total Rubiscoactivity contrasts with the afternoon decrease reportedin previous studies (Kobza and Seemann, 1989) andwas due—with some exceptions, discussed later—toincreasing specific activities of the enzyme, since Ru-bisco protein contents displayed a decreasing pattern.This inhibition of catalysis, which did not disappearin vitro (He et al., 1997; Sharkey et al., 2001), pointedto the in vivo presence of tight-binding Rubisco in-hibitors (Kobza and Seemann, 1988; Parry et al.,1997). A day-time inhibitor of Rubisco with prop-erties similar to those of pentodiulose-bisphosphate(Kane et al., 1998) has been found in wheat (Keyset al., 1995; Parry et al., 1997); the inhibitor contentscould increase in response to an accumulation of itsprecursors, as has been confirmed for the nocturnalinhibitor 2-carboxyarabinitol 1-phosphate (Andralojcet al., 2002) and can play a major role in regulatingRubisco activity in the light (Parry et al., 1993, 1997;Medrano et al., 1997). The increase up to the end ofthe day in Rubisco specific activity in most plantsgrown at ambient CO2 revealed that the putative in-hibitor was released only slowly from Rubisco activesites. In turn, Rubisco protein displayed diurnal de-creases - similar in magnitude to those observed forNitrate Reductase (Scheible et al., 1997) and solubleprotein (Häder et al., 1997)—which showed a lowcorrespondence with the diurnal changes in RubiscomRNA levels, suggesting that gene transcription wasnot the only regulator of the abundance of this enzyme(Webber et al., 1994; Moore et al., 1999).

The activation state of Rubisco was higher at ele-vated CO2, especially with an abundant nitrogen sup-

ply, due to a slower deactivation of Rubisco duringthe day. Therefore, the decrease in initial activity ofRubisco at elevated CO2 must be a consequence of alowered total activity, which was not prevented, butwas exacerbated by a high nitrogen supply, in con-trast to previous reports (Riviere-Rolland et al., 1996;Geiger et al., 1999; Stitt and Krapp, 1999). This oc-curred, firstly, because when combined with abundantnitrogen, elevated CO2 reversed or prevented the in-crease in specific activity observed at ambient CO2.This change points to an increase in the level of an in-hibitor of Rubisco parallel to the greater accumulationof carbohydrates particularly of hexoses and sucrosewith high nitrogen and probably also phosphorylatedintermediates caused by elevated CO2. Secondly, el-evated CO2 reduced the amount of Rubisco protein,in agreement with earlier studies reporting an inhibi-tion of gene expression for this enzyme (Van Oostenand Besford, 1995; Nie et al., 1995a; Cheng et al.,1998). However, we observed no significant changesin rbcS expression associated with the accumulationof carbohydrates at elevated CO2. The decrease in Ru-bisco protein was associated, in agreement with oth-ers (Nakano et al., 1997; Geiger et al., 1999), witha decrease in leaf nitrogen, attributable in part to adilution on a leaf area basis and to a change in al-location within the plant rather than to decreased ni-trogen uptake (Martın del Molino et al., unpublisheddata), which is consistent with the results reported byMakino et al. (1997). In contrast with other studies(Geiger et al., 1999), however, decreased leaf nitrogenat elevated CO2 was not relieved by a high nitrogensupply, because the latter did not alter the allocationof nitrogen to the flag leaf (unpublished data). Themain changes in effects of elevated CO2 on Rubiscoafter anthesis were a decrease in the activation stateand no decrease in Rubisco specific activity. The lattermay be due to the fact that levels of carbohydrates andpossibly also of photosynthetic intermediates, were nolonger increased by elevated CO2, probably due to en-hanced carbohydrate export, as the increased ear dryweight suggests (data not shown).

Higher temperatures did not affect the activation orthe total activity of Rubisco at ear emergence as ele-vated CO2 did. In contrast, high temperatures alteredthe specific activity of Rubisco at certain hours of theday. The paralellism of this effect with carbohydratelevels was poor. In addition, high temperatures with


abundant nitrogen, decreased the amount of Rubiscoprotein, in agreement with previous studies (Vu et al.,1997). Here we show that a trend towards decreasedrbcS transcript is involved in the loss of Rubisco athigh temperature. After anthesis, the activation stateof Rubisco and its specific activity with high nitro-gen were stimulated by above ambient temperatures,while Rubisco protein andrbcSlevels were unaffected.Again, the change in specific activity with tempera-ture was not closely related to carbohydrate levels.With abundant nitrogen, a slower loss of Rubisco pro-tein from ear emergence to 9 days after anthesis withwarmer temperatures accounted for the disappearanceof the negative effect of rising temperatures on Ru-bisco protein and associated levels of mRNA (Geigeret al., 1999).

The greater decrease in the morning of initial Ru-bisco activity with a high than with a low nitrogensupply at ambient CO2 was due to a faster decreasein Rubisco activation state, in agreement with otherstudies (Mächler et al., 1988; Cheng and Fuchigami,2000), possibly because the ATP/ADP ratio decreaseswith high nitrogen (Mächler et al., 1988). At vari-ance with this, at elevated CO2 a high nitrogen sup-ply did not decrease Rubisco activation, but tendedto decrease total Rubisco activity at mid-morning asa consequence of decreased specific activity, as al-ready discussed. By contrast, abundant nitrogen gen-erally increased the amount of Rubisco protein at theend of the night and/or mid-morning at both ambi-ent and elevated CO2, as previously reported (Chengand Fuchigami, 2000; Nakano et al., 1997), althoughit accelerated the loss of protein during the light pe-riod. This increase in Rubisco protein could be as-sociated with higher levels ofrbcS transcripts. Afteranthesis, abundant nitrogen increased the specific ac-tivity of Rubisco at high temperatures, a change thatwas not associated with lower carbohydrate contents.More Rubisco protein was lost between the two sam-plings with high than with low nitrogen, probably as aconsequence of increased export to sinks and thus thepositive effect of nitrogen on Rubisco contents and onthe nitrogen-dependent amount of RNA (Geiger et al.,1999) disappeared after anthesis.

In conclusion, this study suggests that decreasedleaf protein and increased levels of a Rubisco inhibitor,rather than gene repression by sugars, are involvedin acclimation to elevated CO2. Warmer temperatures

may decrease Rubisco gene expression with little con-sequence for Rubisco activity. High nitrogen does notprevent, but rather increases down regulation of Ru-bisco under elevated CO2, probably due to increasedlevels of Rubisco inhibitors. Changes during growthin levels of leaf metabolites and protein may alter therelative importance of Rubisco amounts and levels ofinhibitors for Rubisco regulation under a changing cli-mate.

Acknowledgements

The technical co-operation of Libia Hernandez andAngel Verdejo is acknowledged. The staff of the IR-NASA experimental farm helped with crop sowingand fertilizer application. The Northern blots anal-yses were made at the laboratory of Prof. M. Stitt(Botanical Institute, University of Heidelberg, Ger-many) with Marina Bueno CSIC-DFG funds grantedto R. Morcuende. This work was funded by the Span-ish Plan Nacional de Investigación y Desarrollo (grantno. CLI96-0396). We thank Dr. Christine Raines (De-partment of Biological Sciences, John Tabor Labora-tories, University of Essex, UK) for the generous giftof the Rubisco cDNA.

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Documents

Diurnal changes of Rubisco in response to elevated CO2, temperature and nitrogen in wheat grown under temperature gradient tunnels