growth responses to elevated co2 in nadp-me, nad-me and pck c4 grasses and a c3 grass from south...

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Volume 28, 2001© CSIRO 2001

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© CSIRO 2001 10.1071/PP99104 0310-7841/01/0100013

Aust. J. Plant Physiol.

, 2001,

28

, 13–25

Growth responses to elevated CO

2

in NADP-ME, NAD-ME and PCK C

4

grasses and a C

3

grass from South Africa

Stephanie J. E. Wand

A

, Guy F. Midgley

B

and William D. Stock

C

A

Department of Horticultural Science, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Corresponding author; email: [email protected]

B

Ecology and Conservation, National Botanical Institute, Private Bag X7, Claremont 7735, South Africa.

C

Botany Department, University of Cape Town, Private Bag, Rondebosch 7700, South Africa.

Abstract.

The potential use of C

4

biochemical subtypes [nicotinamide adenine dinucleotide phosphate-malicenzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) and phospho

enol

pyruvatecarboxykinase (PCK)] as delimiters of plant functional types (PFTs) with distinct responses to rising atmosphericCO

2

concentrations was investigated in South African grass species. Gas exchange and above-ground growth inambient and elevated CO

2

(360 and 660 µmol mol

–1

, respectively) were determined in three NADP-ME species,two NAD-ME species, two PCK species and one C

3

species, all excavated from the same field site. Plants weregrown in open-top chambers in a greenhouse for 178 d. Net CO

2

assimilation rates were only significantlyincreased in one NAD-ME species, but stomatal conductances decreased (in six out of eight species, by a mean of46%) and instantaneous leaf water-use efficiency increased (in all species, by a mean of 89%) in elevated CO

2

.These responses did not differ between photosynthetic pathways. Parameters derived from photosynthetic CO

2

andlight response curves were also not differentially influenced by CO

2

treatment between pathways. Gas exchangeresponses were generally poorly related to CO

2

responsiveness. Significant increases in leaf growth and canopy leafarea in elevated CO

2

were found in two NADP-ME species, whereas increases in non-leaf above-ground growthwere measured in three species representing all three C

4

subtypes. Growth responses in elevated CO

2

wereapparently not simply correlated with biochemical subtype characteristics, although the most significant responses(particularly at the leaf level) were found for the NADP-ME pathway. This result was more likely attributable to thesignificant positive correlation found between CO

2

responsiveness of leaf growth and relative leaf regrowthpotential of individual species, the latter being higher in the two responsive NADP-ME species. Therefore,categorisation of PFTs according to relative growth potential may be more appropriate for predictions of CO

2

responsiveness in C

4

grasses.

Keywords

: C

3

grass, C

4

grass, C

4

photosynthetic subtype, climate change, elevated CO

2

, above-ground growth.

Introduction

The rising concentration of atmospheric CO

2

, predicted todouble from pre-industrial levels (

ca

280 µmol mol

–1

) byabout the middle of the 21st century (Trabalka

et al.

1985;Watson

et al.

1996), could have potentially large impacts onterrestrial ecosystem functioning. Global terrestrial photo-synthesis represents a major sink for CO

2

(Schimel 1995),with some ecosystems capable of significant increases inprimary productivity in high CO

2

. Although the C

4

grami-noid component of tropical and subtropical savanna andgrassland ecosystems accounts for

ca

21% of total global

productivity (Lloyd and Farquhar 1994), empirical studies ofthese species’ responses to elevated CO

2

are few. This ispartially due to the widespread perception that C

4

species donot respond significantly to increased CO

2

(Bowes 1993).However, wild C

4

grasses are not inherently less responsivethan wild C

3

grasses (Wand

et al.

1999). Past studies of C

4

species (mainly from the northern hemisphere) havefocussed mainly on a few economically important ordominant species, and do not shed light on the potentialrange of CO

2

responsiveness within the Poaceae in naturalrangelands. African grasses have been very poorly studied

Abbreviations used:

A

, net CO

2

assimilation rate;

A

max

, light-saturated rate of net CO

2

assimilation;

c

i

, intercellular CO

2

concentration;

g

s

, stomatalconductance; IRGA, infrared gas analyser;

J

max

, CO

2

-saturated rate of net CO

2

assimilation; NAD-ME, nicotinamide adenine dinucleotide-malicenzyme; NADP-ME, nicotinamide adenine dinucleotide phosphate-malic enzyme; PCK, phospho

enol

pyruvate carboxykinase; PFTs, plantfunctional types; PPFD, photosynthetic photon flux density; SLM, specific leaf mass; WUE, instantaneous water-use efficiency.

14 S. J. E. Wand

et al.

and may show unique responses, particularly in regions oflow nutrient availability such as eastern South Africa. It isintractable to test the responses of large numbers ofindividual species. Thus, valid predictions of the responsesof C

4

-dominated ecosystems to rising CO

2

will depend onthe search for alternative methods of assessing the responsesof representative species.

In recent years, the concept of plant functional types(PFT) has developed, particularly with respect to issuessurrounding global change (Box 1996; Poorter

et al.

1996;Smith

et al.

1996; Diaz and Cabido 1997). This approach isbased on the view that species can be grouped variously onthe basis of shared physiological or other functionalresponses to environmental perturbations, e.g. photosyn-thetic pathway, morphogenetic and architectural traits,reproductive and phenological traits (Diaz 1995; Diaz andCabido 1997). This provides a framework for predictingplant and ecosystem responses at larger spatial scales (Box1996), including potential modelling, provided one candefine the vegetational composition on the basis of relativePFT representation.

Much of the diversity within the Poaceae can be attrib-uted to evolutionary divergence in photosynthetic pathways(Hattersley and Watson 1992). Diversity with respect togrowth form, morphology, life history and reproductivesystems is particularly low in this family, so functional typecategorisations do not emerge naturally for these types oftraits. However, categorisations based on photosyntheticmechanisms are fairly robust, well researched and docu-mented, and have obvious application in the search forcommon responses to elevated CO

2

. In South Africa,dominance by C

3

and C

4

species is geographically differen-tiated, with C

3

grasses predominating only in the winterrainfall region and the higher altitudes of the easternmountain ranges (Vogel

et al.

1978). Over most of thesummer rainfall region, more than 75% of grass speciesare C

4

, and in large areas more than 95% are C

4

(Vogel

et al.

1978). Therefore, a subdivision within the C

4

group isrequired to achieve a finer resolution and enhanced predic-tive power of potential vegetation change.

Within the C

4

pathway, three photosynthetic subtypeshave been identified, namely NADP-ME (nicotinamideadenine dinucleotide phosphate-malic enzyme), NAD-ME(nicotinamide adenine dinucleotide-malic enzyme) andPCK (phospho

enol

pyruvate carboxykinase) (Hattersley andWatson 1992; Ehleringer

et al.

1997). Ellis

et al.

(1980, forNamibia) and Hattersley (1992, for Australia) showed thatthe abundance of these subtypes is correlated with meanannual rainfall, with NADP-ME species becoming moreabundant with increasing rainfall, whereas NAD-MEspecies dominate in areas of low rainfall and decrease inabundance as rainfall increases. PCK species are mostabundant at intermediate rainfall. Over South Africa,NADP-ME species dominate towards the eastern high-rain-

fall, nutrient-poor regions, whereas NAD-ME species aremore common towards the more fertile western low-rainfallregions. This suggests that C

4

subtypes may be potentialcandidates for PFTs that are likely to respond differentiallyto climate change, and which lend themselves to spatialmodelling. Will they show differential responses to risingCO

2

?LeCain and Morgan (1998) investigated the CO

2

respon-siveness of North American C

4

grasses from the NAD-MEand NADP-ME subtypes. Contrary to predictions of greaterresponsiveness in NAD-ME species, based on assumptionsof differential CO

2

‘leakiness’ from bundle sheath cells(Hattersley 1982; Farquhar 1983), significant growth stimu-lation was found only in two NADP-ME grass species.Because one of the NAD-ME species had previously showngrowth enhancement (Read

et al.

1997), LeCain andMorgan (1998) concluded that no generalisations could bemade about the CO

2

response of C

4

species based on C

4

subtype and CO

2

leakage. Ziska and Bunce (1997) came to asimilar conclusion after finding variable responses to ele-vated CO

2

within NADP-ME and NAD-ME subtypes, andsuggested that a broader range of species and subtypes beexamined. Subsequently, Ziska

et al.

(1999) reported norelationship between relative ‘leakiness’ of the three sub-types and stimulation of leaf photosynthesis in elevatedCO

2

, although only one species representing each categorywas studied. No study has yet investigated all three C

4

subtypes and the C

3

pathway in one experiment, undersimilar growth conditions and with plant material sourcedfrom the same site.

All previous studies in the northern hemisphere usedspecies characterised by relatively high leaf nitrogenconcentrations, high photosynthetic rates (light-saturatedrate of net CO

2

assimilation [

A

max

] > 40 µmol m

–2

s

–1

) andstrong growth potential, originating as they do from regionsof good fertility, and often weedy in nature or bred as crops(Ziska and Bunce 1997; LeCain and Morgan 1998; Ziska

et al.

1999). The C

4

grasses from South Africa exhibitcomparatively lower leaf nitrogen concentrations, lowerphotosynthetic rates (

A

max

< 22 µmol m

–2

s

–1

; Botha andRussell 1988; Wand 1999) and possibly lower growthpotential than their northern counterparts. However,NADP-ME-dominated eastern regions are generally moreproductive than NAD-ME-dominated western regions.Poorter (1993) determined that CO

2

responsiveness of C

3

species is positively correlated with relative growth rate. Is itpossible that the photosynthetic subtypes in African C

4

grasses may show differential responsiveness to elevatedCO

2

related to varying growth potentials? This should betested before final conclusions are drawn as to the relation-ship between subtype physiology and CO

2

responsiveness.The purpose of our study was to test the CO

2

responsive-ness of all three C

4

photosynthetic subtypes and the C

3

pathway in South African grass species from the same field

CO

2

responses of C

3

and C

4

grass subtypes 15

site (this would eliminate potential carry-over environ-mental effects). The aim was to establish whether photo-synthetic pathway is a useful delimiter of PFTs within the C

4

Poaceae, from a species pool characterised by relatively lowphotosynthetic capacity and possibly low growth potentialcompared to previously studied C

4

species.

Materials and methods

Plant establishment

Eight locally dominant grass species representing either the C

3

, C

4

NADP-ME, C

4

NAD-ME, or C

4

PCK photosynthetic pathways wereselected from a field CO

2

experimental site situated in southernKwazulu-Natal, South Africa (30°40

′ S, 30°00′ E). This experimentutilises the natural CO2 springs in the area (Harris et al. 1997). It wasestablished on a grassed hillside and experiences a mild subtropicalclimate, with an average annual rainfall of 800 mm, most of which fallsin summer. The grassland vegetation of the region is dominated by theC4 photosynthetic type, predominantly of the NADP-ME subtype, andonly one C3 grass species occurs at the site. The soil is leached andacidic (pH 4.3) and of relatively low fertility (total N ~0.15% of soildry mass). The selected species and their subtypes are given in Table 1.The identity and photosynthetic pathway of Alloteropsis semialata ssp.eckloniana (C3) were confirmed using 13C isotope determinations,distinguishing it from the closely related Alloteropsis semialata ssp.semialata (C4). Photosynthetic subtypes for C4 species weredetermined from the literature (Ellis et al. 1980; Schulze et al. 1996),and confirmed by R. P. Ellis (pers. comm.).

Clumps of each species were excavated from the field site, wellaway from the CO2 vent, during April 1997, and transferred by road toCape Town within 2 d of excavation. The clumps were then potted into20-cm-diameter plastic pots using a standard Kirstenbosch potting mix(coarse sand, leaf mould and loam, 2:1:1 v/v) packed around theclumps of field soil. Plants were left in the open, but partially shelteredfrom rain, to acclimatise. In early September 1997, clumps wereseparated into smaller tufts of similar size which were individuallypotted into 3.9-dm3 plastic pots (10 cm diameter, 50 cm length) usingstandard Kirstenbosch potting mix with some remaining field soilaround the roots, to give 16 individual plant samples per species. Carewas taken to minimise variation in initial tuft size, since no baselinebiomass data could be obtained due to constraints on availablematerial. Only M. repens yielded fewer than 16 plants (eight plants).

Pots were moved into the greenhouse to acclimatise. All plants werefed with an organic liquid fertiliser (Seagro Pty Ltd, Cape Town, SouthAfrica; with 5 mL diluted to 2 L, and plants fed 120 mL each) on twooccasions: before the start of the experiment, and again 39 d after thestart. Thereafter, no fertiliser was applied. Plants were watered daily.

Design and CO2 treatment

The experiment was performed in a polycarbonate-clad greenhouse.The experimental design was a split-plot, with CO2 treatment as themain plot and photosynthetic pathway as the subplot, with plotsreplicated four times. Each plot comprised two adjacent hexagonalopen-top chambers constructed of polycarbonate, measuring 0.38 malong each side, and 0.50 m in height (Midgley et al. 1995). Chamberswere placed in pairs on four tables, with each chamber covering16 suspended pots. Air was drawn into each chamber from outside thegreenhouse by means of a 12 V DC brushless computer fan andcirculated in a plenum surrounding the base of the chamber. The innerwall of the plenum was perforated by holes that allowed air to enter thechamber at an equal rate from all directions. The air entering one ofeach pair of adjacent chambers was enriched with CO2 gas 70 mmupstream of the fan to give the desired average CO2 concentration ofca 660 µmol mol–1.

The CO2 supply rate was controlled by means of needle valve flowcontrollers (DK800, Krohne Messtechnik, Duisburg, Germany),calibrated at the start of the experiment and re-adjusted to set pointwhen necessary. A float-metering valve with pressure regulator(DK800RA, Krohne Messtechnik) minimised fluctuations in flow ratesdue to diurnal temperature changes. The air in the other chamber ofeach pair remained at ambient CO2 concentration (ca 360 µmol mol–1).The four elevated CO2 chambers were calibrated individually at plantheight at 16 positions throughout the chamber, using a portableinfrared gas analyser (IRGA, LI-6200, Li-Cor, Lincoln, NE, USA).The measured CO2 ranges after calibration in the four amendedchambers were 601–706, 608–737, 611–704 and 624–687 µmol mol–1,with 90–100% of measurements falling within 10% of the target(660 µmol mol–1).

At the start of the experiment, all plants were defoliated to 5 cmheight. Two plants of each species were placed randomly in eachchamber, except M. repens (one plant per chamber). Pots weresuspended beneath the tables through holes cut in the surface, and heldby means of adjustable plastic collars. The experiment was carried outfrom 3 October 1997 to 30 March 1998, for 178 d. Average greenhouseair temperature was recorded every 3 h using a shielded thermistorpositioned at table height, and connected to a data logger (MCSystems, Cape Town, South Africa). Average monthly midday(1200–1500 h) temperatures were 29.7, 28.8, 31.4, 32.3, 34.1 and30.8°C during October, November, December, January, February andMarch, respectively. These values corresponded to daily maximumsummer temperatures experienced in the field in South Africangrasslands (Schulze 1997). Photosynthetic photon flux density (PPFD)was not recorded during this experiment, but ranged between 900 and1300 µmol m–2 s–1 at plant level from October to January (middayaverage) during previous experiments in the same chambers andgreenhouse (Wand 1999).

Photosynthetic gas exchange

Gas exchange measurements (light and A/ci response curves, whereA is the net CO2 assimilation rate and ci is the intercellular CO2concentration) were performed after 6–12 weeks of CO2 fumigation,using a LI-6400 IRGA (Li-Cor). For each species, 4–5 plants fromeach CO2 treatment were selected at random from different chambers.A/ci responses were determined at 6–7 different cuvette CO2concentrations ranging between 50 and 1000 µmol mol–1 (C4 species)or between 100 and 1000 µmol mol–1 (C3 species). Cuvette CO2

Table 1. C4 and C3 grass species used in the experiment, and their subtypes in the case of C4 species

Subtype abbreviations and the photosynthetic enzymes that charac-terise them are: NADP-ME (NADP-malic enzyme), NAD-ME(NAD-malic enzyme) and PCK (phosphoenolpyruvate carboxykinase

SpeciesPhotosynthetic

pathway C4 subtype

Andropogon appendiculatus Nees C4 NADP-MEDigitaria natalensis Stent C4 NADP-METhemeda triandra Forssk. C4 NADP-MEEragrostis curvula (Schrad.) Nees C4 NAD-MEEragrostis racemosa (Thunb.) Stend. C4 NAD-MESporobolus pyramidalis Beauv. C4 PCKMelinis repens (Willd.) Zizka ssp.

repens FP 23:914C4 PCK

Alloteropsis semialata (R.Br.) Hitchc. ssp. eckloniana (Nees) Gibbs-Russell

C3 —

16 S. J. E. Wand et al.

concentrations were regulated using pressurised CO2 canisters (sodachargers). Leaf temperatures were regulated at 30°C, and light levelskept constant at 1500 (C4 species) or 800 µmol m–2 s–1 (C3 species),since C4 photosynthesis can potentially saturate at higher light levelsthan most C3 plants. Data showed that both C3 and C4 grasses werelight-saturated for photosynthesis at about 800–1000 µmol m–2 s–1,with the exception of Andropogon and Sporobolus, which saturated atabout 1500 µmol m–2 s–1 (see Fig. 2). Light was provided by a red/blueLED light source inside the IRGA cuvette.

Between one and four enclosed mid-leaf sections (depending onleaf width) were allowed to equilibrate at 500 µmol mol–1 CO2 untilphotosynthetic rate and stomatal conductance were constant (at least15 min). Thereafter, CO2 concentrations were progressively decreasedto the lowest level, then increased to 1000 µmol mol–1, allowed tostabilise, and then decreased to 650 µmol mol–1 for the last measuredpoint. Care was taken that the beginning and end points lay on asmooth curve, signifying sufficient acclimation time and lack ofcuvette-induced changes in photosynthetic capacity. After the A/ciresponse measurement, leaves were allowed to equilibrate at thetreatment CO2 concentration (360 or 650 µmol mol–1) and light levelsof 2000 (C4 species) and 1250 µmol m–2 s–1 (C3 species).Gas-exchange measurements were then taken at six to sevenprogressively lower light levels up to just above light compensationpoint (about 70 µmol m–2 s–1 PPFD). Leaf temperatures were regulatedat 30°C.

After gas exchange, projected leaf areas were calculated using thelengths and breadths of the enclosed leaf sections. Response curveswere fitted individually using non-linear regression (UNISTAT 3.0)and the monomolecular function y = a(1 – eb-cx) given by Causton andDale (1990). For the A/ci response curves, the fitted curve coefficient‘a’ gave the light- and CO2-saturated rate of net CO2 assimilation(Jmax), ‘b/c’ gave the CO2 compensation point, and ‘aceb’ gave theapparent carboxylation efficiency, the slope of the A/ci response atx = 0. The equivalent constants and formulae for the light responsecurves gave Amax, the light compensation point and the apparentquantum efficiency. In addition, the predicted dark respiration rate wascalculated from the light response using ‘a(1 – eb)’ (Causton and Dale1990).

In addition to curve analysis, gas-exchange values were extractedfrom the A/ci responses at cuvette CO2 concentrations of 360 and650 µmol m–2 s–1 for further analysis. The photosynthesis/transpirationratio (A/E) at these CO2 levels was used as a measure of instantaneousleaf water use efficiency (WUE).

Harvest

Plants were defoliated to 5 cm height during early January (14 weeksafter the start of CO2 fumigation) and again at the end of theexperiment (late March). Green leaves were separated from non-leafshoot material (senesced, sheaths, reproductive) and the fresh mass ofthe green leaf component determined. Blade lengths of 10 fullydeveloped leaves were measured. Leaf areas were determined using adigital image analysis system (Delta-T Devices, Cambridge, UK). Forthe first harvest, whole plant leaf area (‘canopy leaf area’) wasmeasured where possible, but in some species the leaf area and drymass of a subsample of 20 leaves were determined. Canopy leaf areawas then calculated using specific leaf mass (SLM, g m–2) of thesubsample and whole plant leaf dry mass. For the second harvest, theSLM of subsamples of 10 leaves was measured for each species, andcanopy leaf area calculated from whole plant leaf dry mass. Leafregrowth potential was approximated for plants grown in ambient CO2,by calculating normalised leaf regrowth as: NLR = LM2/LM1, whereLM2 is leaf biomass at second harvest, and LM1 is leaf biomass at firstharvest (a period of ~11 weeks). All material was oven-dried at 65°C,weighed, and leaf material milled. Leaf total nitrogen concentrations

were determined for each plant on pooled dry leaf material from bothharvests, using the micro-Kjeldahl method (Jones and Case 1990).

Statistical analysis

Gas exchange and harvest data for individual species for each harvestwere subjected to one-way ANOVA. Potential interaction betweenphotosynthetic pathway and CO2 response for gas exchange data wasanalysed by two-way ANOVA. For each harvest, biomass data werecombined according to photosynthetic pathway and subjected to asplit-plot ANOVA, with CO2 treatment as the main plot andphotosynthetic pathway as the sub-plot. In order to test for possiblechanges in the CO2 response of photosynthetic types between theharvests (interaction), and, in the potential absence of such interaction,to provide a more powerful statistical test for CO2 main effects throughincreased replication, a repeated measures two-way ANOVA wasperformed on each species, with CO2 treatment and harvest date asfactors. The significance level for all analyses was set at P ≤ 0.10. Allanalyses were carried out using UNISTAT 3.0. Correlations wereestablished between leaf growth potential, CO2 enhancement of leafgrowth, and leaf nitrogen concentration in C4 species representing allthree subtypes, using linear regression.

Results

Gas exchange

A measured at growth CO2 concentration was not signifi-cantly different between treatments, except in E. curvulawhich showed a significant increase in photosynthetic rate inhigh CO2 (Table 2). Stomatal conductances (gs) weresignificantly decreased in elevated CO2 in all except twospecies. Instantaneous leaf WUE was strongly enhanced inall species, by 68% or more.

No interaction was found between photosyntheticpathway and CO2 response for A, gs and WUE, indicatingthat there were no clear patterns of responsiveness along

Table 2. Percentage change in gas-exchange parameters of eightgrass species measured after 6–12 weeks in elevated CO2, relative

to ambient CO2Data taken from A/ci response curves at cuvette CO2 concentrationsequivalent to growth CO2 concentrations. A = net CO2 assimilationrate, gs = stomatal conductance, and WUE = instantaneous leaf wateruse efficiency. n = 7 or 8 except n = 4 for M. repens. Significance levelsas calculated by one-way ANOVA were: + = P ≤ 0.10, * = P ≤ 0.05,

** = P ≤ 0.01, *** = P ≤ 0.001, no symbol = not significant

Percentage changeA gs WUE

A. appendiculatus (NADP-ME) +11 –48 * +95 ***

D. natalensis (NADP-ME) –9 –55 ** +85 ***

T. triandra (NADP-ME) –30 –61 *** +68 ***

E. curvula (NAD-ME) +71 * –10 +86 **

E. racemosa (NAD-ME) +17 –43 + +118 *

S. pyramidalis (PCK) +6 –50 * +88 ***

M. repens (PCK) +26 –41 +107 ***

A. semialata (C3) +6 –55 *** +115 ***

CO2 responses of C3 and C4 grass subtypes 17

C4 subtype lines (Table 3). Strong overall main effects ofelevated CO2 on gs (–46%) and WUE (+89%) weremeasured. Photosynthetic pathways differed with respectto their photosynthetic characteristics: aStudent-Newman-Keuls Multiple Range test (P ≤ 0.05)showed that the C3 pathway had higher gs and lower WUEthan all three C4 pathways.

Analysis of photosynthetic CO2 response curves (Fig. 1;Table 3) revealed no significant interaction between photo-synthetic pathway and CO2 treatment, and no main effects ofCO2 treatment. Differences in curve parameters betweenphotosynthetic pathways were attributable to a higher CO2compensation point in the C3 species compared to all threeC4 subtypes, and lower apparent carboxylation efficienciesin C3 and NAD-ME species than in NADP-ME and PCKspecies.

There was likewise no interaction between photo-synthetic pathway and CO2 treatment for light responsecurve parameters (Fig. 2; Table 3), except for a weakinteraction for dark respiration, which was due to theabsence of a significant CO2 response in PCK species. TheC3, NADP-ME and NAD-ME pathways showed significantdecreases in dark respiration rate in elevated CO2, which ledto decreases in the photosynthetic light compensation point.Photosynthetic pathways differed from one another with

respect to dark respiration rate and apparent quantum useefficiency (Student-Newman-Keuls Multiple Range test(P ≤ 0.05): these values were lower in the C3 and NADP-MEpathways than in the NAD-ME and PCK pathways.

Above-ground growth

In January, A. appendiculatus (NADP-ME) had significantlygreater leaf dry mass, shoot dry mass, canopy leaf area andleaf length in elevated CO2 (Table 4). T. triandra(NADP-ME) also showed significant increases in leaf drymass, non-leaf dry mass (senesced and sheath material, noreproductive tillers), total shoot dry mass and canopy leafarea in elevated CO2. Of the two Eragrostis species(NAD-ME), only E. curvula showed significant increases innon-leaf shoot dry mass (senesced and sheath material, andreproductive tillers) and total shoot biomass in elevatedCO2. S. pyramidalis (PCK) had greater non-leaf dry mass(senesced and sheath material, and reproductive tillers) andtotal shoot dry mass in high CO2. M. repens (PCK) andA. semialata (C3) showed no significant growth responses toelevated CO2.

In March, CO2 responses were relatively smaller than inJanuary and not statistically significant in most cases(Table 4). Only T. triandra showed a significant increase intotal shoot dry mass, due to increases in non-leaf dry mass.E. curvula had slightly shorter leaf blades in elevated CO2,whereas A. semialata had slightly longer leaf blades. Forboth harvests combined, leaf total nitrogen concentrationswere significantly reduced in elevated CO2 inA. appendiculatus, T. triandra and A. semialata. Leaf [N]was similar between the three C4 subtypes, but was twice ashigh in the C3 than the C4 pathway (Fig. 3).

When data from species belonging to the same photo-synthetic pathway were combined, significant CO2-inducedincreases in leaf dry mass, non-leaf dry mass and total shootdry mass, as well as increases in canopy leaf area and leaflength were found in January across all pathways (Fig. 3;Table 5: CO2 main effect). In March, only total shoot drymass and canopy leaf area were significantly increased. Nointeraction was found between photosynthetic pathway andCO2 response, although one-way ANOVA for individualphotosynthetic types showed statistically significant CO2

responses mostly in the NADP-ME pathway (results notshown).

A two-way ANOVA with repeated measures revealed nosignificant interactions between harvest date and CO2

response (Table 6). This allows for valid interpretation basedon the CO2 main effect over both harvests. Statisticallysignificant increases (≥ 34%) were found for total leafbiomass, non-leaf shoot biomass, total shoot biomass andcanopy leaf area in the NADP-ME pathway, whereas theNAD-ME pathway had only increased total shoot biomass

Table 3. Statistical significance of the effects of elevated CO2 ongas exchange measured at growth CO2 concentration, andphotosynthetic CO2 response (A/ci) and light-response curveparameters of grass species of differing photosynthetic

pathway, PP)A = net CO2 assimilation rate, gs = stomatal conductance,WUE = instantaneous leaf water-use efficiency, Jmax = light- andCO2-saturated net CO2 assimilation rate, Amax = light-saturated netCO2 assimilation rate. Significance levels, as calculated by two-wayANOVA, were: + = P ≤ 0.10, * = P ≤ 0.05, ** = P ≤ 0.01,

*** = P ≤ 0.001, NS = not significant

Main effects InteractionParameter PP [CO2] PP × [CO2]

At growth [CO2]:A NS NS NSgs * –46% *** NSWUE *** +89% *** NS

A/ci response:Jmax NS NS NSCO2 compensation point ** NS NSApparent carboxylation

efficiency*** NS NS

Light response:Amax NS NS NSLight compensation point NS –33% ** NSDark respiration rate *** –34% ** +

Apparent quantum use efficiency

*** NS NS


Andropogonappendiculatus

–5

0

5

10

15

20

25

30

35

40

0 100 200 300 400 500

Ci (µmol mol–1)

A (

µmo

l m–2

s–1

)

Digitaria natalensis

–5

0

5

10

15

20

25

30

35

40

0 100 200 300 400 500

Themeda triandra

–5

0

5

10

15

20

25

30

35

40

0 100 200 300 400 500

Eragrostis curvula

–5

0

5

10

15

20

25

30

35

40

0 100 200 300 400 500

A (

µmo

l m–2

s–1

)A

(µm

ol m

–2 s

–1)

A (

µmo

l m–2

s–1

)

C i (µmol mol–1)

Ci (µmol mol–1)

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Fig. 1. Photosynthetic CO2 response curves (A/ci) of C4 and C3 grass species grown under ambient (squares) or elevated (triangles) CO2concentration. Symbols represent all data points and polynomials represent averages for individually modelled (replicate) curves. Arrows indicateA values at operational ci, as measured at cuvette CO2 concentrations of 360 and 650 µmol m–2 s–1.


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Fig. 2. Photosynthetic light response curves of C4 and C3 grass species grown under ambient (squares) or elevated (triangles) CO2.Measurements were made at growth CO2 concentration. Symbols represent all data points and polynomials represent averages for individuallymodelled (replicate) curves.


(31%), and the PCK and C3 pathways showed no significantresponses.

SLM was reduced between the first and second harvestsin all four pathways, but elevated CO2 did not alter SLM inany photosynthetic type (data not shown).

Correlations

Normalised leaf regrowth after ~11 weeks was weaklypositively correlated (R2 = 0.3463) with leaf nitrogenconcentration in the C4 grasses (Fig. 4a). A good positivecorrelation (R2 = 0.6634) was, however, found between thepercentage CO2 enhancement of leaf growth and thenormalised leaf regrowth rate (Fig. 4b). Leaf nitrogenconcentration was not a good predictor of CO2 enhancementof leaf growth (Fig. 4c, R2 = 0.2664).

Discussion

In this study, two of the three NADP-ME species(A. appendiculatus and T. triandra) showed significantabove-ground growth enhancement in elevated CO2, mainlydue to stimulation of leaf growth. None of the NAD-ME,PCK or C3 species showed significant leaf biomass enhance-ment, but E. curvula (NAD-ME) and S. pyramidalis (PCK)had greater non-leaf shoot biomass in elevated CO2. The C3grass (A. semialata ssp. eckloniana) did not show significantphotosynthetic or growth responses at the time of measure-ment due to photosynthetic downregulation (Fig. 1).

Our findings are similar to those of LeCain and Morgan(1998) for C4 species from the North American short-grassprairie, who reported that two out of three species studiedbelonging to the NADP-ME pathway had significant growthresponses in elevated CO2, compared to none from theNAD-ME pathway. Although Ziska and Bunce (1997) foundgrowth increases in species representing both theNADP-ME and NAD-ME pathways, only the NADP-MEspecies (the least ‘leaky’) used in a subsequent study (Ziskaet al. 1999) showed significant growth increases in elevated

CO2. Unfortunately this result was tenuous since subtypes(level of ‘leakiness’) were not replicated. It is tempting toconclude that NADP-ME species are more responsive thanthe other two C4 subtypes, although LeCain and Morgan(1998), Ziska and Bunce (1997) and Ziska et al. (1999) aremore cautious in their interpretation owing to incon-sistencies. They all concluded that no generalisations couldyet be made about the CO2 response of C4 species based onC4 subtype.

The statistical analysis for the present study does notreveal significant differences in the CO2 response betweendifferent photosynthetic pathways when analysed separatelyfor each harvest (no interaction between pathway and CO2

response). Only when data for both harvests were combineddid significant leaf and total shoot growth enhancementemerge for the NADP-ME pathway, with NAD-ME speciesshowing only a marginal increase in total shoot biomass.

In all C4 species, gas exchange characteristics at the timeof measurement were poorly or not at all related to growthand CO2 responsiveness. Two species showed longer-termphotosynthetic downregulation (D. natalensis andA. semialata ssp. eckloniana; Fig. 1) and one showedphotosynthetic upregulation (E. curvula; Fig. 1). Theremainder were either saturated at ambient CO2 concen-tration, or the increase in A under elevated CO2 was notsignificant. Rates were generally low. On the other hand,Ziska et al. (1999) found stimulation of photosynthetic ratesin all C4 grass species studied (from North America) havinghigh photosynthetic capacity, but growth responses in onlyone. It would appear that differential gas exchangeresponses are not clearly linked with growth responses in thevarious photosynthetic pathways. Lack of a clear concord-ance between gas exchange and growth in elevated CO2 hasalso characterised studies using C3 species.

Scrutiny of the literature on grass C4 subtype growthresponses to elevated CO2 yields mixed results. Only a few

Table 4. Percentage changes in above-ground biomass, morphology and leaf nitrogen concentration of eight grass species grown underelevated CO2 relative to ambient CO2

Plants were grown from 3 October 1997 and clipped to 5-cm height in January and March 1998. January and March leaf material were pooled forleaf [N] determinations. n = 7 or 8 except n = 4 for M. repens. Significance levels, as calculated individually for species and harvest date by

one-way ANOVA, were: + = P ≤ 0.10, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, no symbol = not significant

Total leaf dry mass Non-leaf dry mass Total shoot dry mass Canopy leaf area Leaf blade lengthJanuary March January March January March January March January March Leaf [N]

A. appendiculatus +74* +36 +52 +34 +72* +35 +77* +32 +35** +8 –10+

D. natalensis +16 +18 –3 +19 +9 +19 +19 +16 +17 +9 +0.8T. triandra +119** +21 +118** +51* +118*** +43* +118*** +29 +9 –4 –9***E. curvula +32 +25 +82+ +22 +69+ +23 +25 — +4 –12* –5E. racemosa +18 +10 +11 +5 +16 +10 +22 +9 –3 +3 –9S. pyramidalis +19 +9 +143+ –15 +50+ –5 +26 +0.3 +10 +3 –4M. repens +3 +4 +47 +43 +29 +29 +5 +2 +14 +10 –3A. semialata +35 +26 +51 +52 +42 +41 +34 +21 +25 +17+ –10*


species belonging to the NAD-ME or PCK pathways havebeen studied, with the majority having the NADP-MEpathway. Significant shoot biomass responses have beenreported in some NAD-ME species e.g. Bouteloua gracilis(Hunt et al. 1996; Read and Morgan 1996; but no responsein LeCain and Morgan 1998), Eragrostis orcuttiana(a noxious weed, Smith et al. 1987), Eleusine indica

(a weedy annual, Sionit and Patterson 1984; Patterson1986), Panicum dichotomiflorum (a weed, Ziska and Bunce1997) and Panicum coloratum (Ghannoum and Conroy1998). Similarly, many weedy NADP-ME species showsignificant growth responses in high CO2. Ziska and Bunce(1997) reported inconsistent responses to high CO2 inNAD-ME and NADP-ME subtypes, represented by a range

Fig. 3. Shoot biomass and leaf chemistry in elevated CO2 in grass species representing four photosynthetic pathways. For each harvest (clippingto 5 cm height in January and March), n = 24 for C4 NADP-ME, n = 13–16 for C4 NAD-ME, n = 12 for C4 PCK, and n = 7 or 8 for C3. For leaf[N], n = 11 or 12 (NADP-ME), n = 8 (NAD-ME), n = 8 (PCK) and n = 4 (C3). Values are means ± s.e. bars.


of weedy and crop species. Weediness was a far strongerpredictor of CO2 responsiveness than subtype. Most wildnon-weedy C4 grass species which respond favourably toelevated CO2 belong to the NADP-ME subtype, notablyAndropogon gerardii (Knapp et al. 1993; Owensby et al.1993; LeCain and Morgan 1998), Themeda triandra (Wandet al. 1996; but response not significant in Wilsey et al.1997), Panicum antidotale (Ghannoum et al. 1997),Sorghastrum nutans (LeCain and Morgan 1998) and Schiza-chyrium scoparium (Polley et al. 1996; but response notsignificant in LeCain and Morgan 1998). However, possiblebias may be introduced by the skewed representation of

subtypes in past studies. In addition, by no means allNADP-ME species respond favourably to elevated CO2,suggesting a genetic (species-specific) limitation.

An interesting link between responsive species of theNADP-ME subtype appears to be their taxonomic relation-ship. As the NADP-ME subtype probably arose three timesin different ancestral lines, there may be considerablegenetic diversity even within this subtype (Hattersley andWatson 1992), which may account for differential respon-siveness to environmental factors such as CO2 concentra-tion. Most of the CO2-responsive genera (e.g. Andropogon,Themeda, Sorghastrum and Schizachyrium) belong to the

Table 5. Statistical significance of the effects of elevated CO2 on shoot growth and leaf nitrogen con-centration of grass species of differing photosynthetic pathway (PP)

Data for species belonging to the same photosynthetic pathway are pooled, with n = 24 (NADP-ME), n =13–16 (NAD-ME), n = 12 (PCK) and n = 7 or 8 (C3) for each harvest. Significance levels, as calculated bysplit-plot ANOVA with CO2 treatment as main plot and PP as subplot, were: + = P ≤ 0.10,

* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, NS = not significant

Main effects InteractionPP × [CO2]PP [CO2]

Growth parameter January March January March January March

Leaf dry mass *** *** +29% * NS NS NSNon-leaf shoot dry mass ** ** +82% ** NS NS NSTotal shoot dry mass *** *** +51% *** +18% * NS NSCanopy leaf area *** *** +40% *** +13% + NS NSLeaf length *** *** +13% + NS NS NSLeaf [N] *** NS NS

Table 6. Statistical significance of the effects of elevated CO2 on above-ground biomass and canopy leaf area of differing photosynthetic pathways, over two harvest times

Data for species belonging to the same photosynthetic pathway are pooled, with n = 24(NADP-ME), n = 13–16 (NAD-ME), n = 12 (PCK) and n = 7 or 8 (C3) for each harvest. Percentagechanges in elevated CO2 are given where effects are significant. Significance levels, as calculated bytwo-way ANOVA with repeated measures, were: + = P ≤ 0.10, * = P ≤ 0.05, ** = P ≤ 0.01,

*** = P ≤ 0.001, NS = not significant

Main effects Interaction

Species Growth parameter [CO2] Harvest date [CO2] × date

C4 NADP-ME Total leaf dry mass +34% * *** NSNon-leaf dry mass +37% + *** NSTotal shoot dry mass +36% * *** NSCanopy leaf area +39% *** *** NS

C4 NAD-ME Total leaf dry mass NS * NSNon-leaf dry mass NS ** NSTotal shoot dry mass +31% + *** NSCanopy leaf area NS *** NS

C4 PCK Total leaf dry mass NS *** NSNon-leaf dry mass NS *** NSTotal shoot dry mass NS *** NSSLM NS *** NS

C3 Total leaf dry mass NS * NSNon-leaf dry mass NS *** NSTotal shoot dry mass NS *** NSCanopy leaf area NS *** NS


supertribe Andropogonidae (Gibbs-Russell et al. 1990;Hattersley and Watson 1992), or to a branch of the Paniceae(e.g. Panicum antidotale), which, together with theArundinelleae, share the common and exclusive feature of

having lost their outer bundle sheaths as they evolved(Hattersley and Watson 1992). In contrast, other NADP-MEgenera such as Digitaria, Paspalum, Echinochloa andSetaria seem to be responsive to elevated CO2 only if theyare early successional, weedy species growing under favour-able conditions e.g. Echinochloa crus-galli (Sionit andPatterson 1985) and Digitaria sanguinalis (Sionit andPatterson 1984). However, the potential evolutionary link toCO2 responsiveness was recently investigated by Kellogget al. (1999), who found variable and inconsistent responsesin nine grass species representing three independent originsof the C4 photosynthetic pathway. They suggested thatspecies-specific diversity in internal growth regulation wasmore important than phylogenetic origin. Similarly, in adetailed study of 11 C3 grass species, Roumet and Roy(1996) concluded that physiological criteria, life form andselective pressure were far more important in determiningCO2 responses that generic affiliation.

Where does this leave us? It would appear that intrinsic,species-specific controls over growth are more important indetermining the growth response in elevated CO2 than any ofthe options offered above. Our study demonstrates for thefirst time (Fig. 4) the potential relationship between growthpotential and CO2 responsiveness in C4 grasses, similar tothat demonstrated for C3 species (Poorter 1993; Poorter et al.1996; Atkin et al. 1999). Potentially fast-growing wildC3 species have greater biomass responses to elevated CO2(54%) than slow-growing wild C3 species (35%), possiblyowing to stronger sink strength (Poorter 1993). Similarresults have been obtained for a range of fast- andslow-growing Acacia species (Atkin et al. 1999). Respon-siveness based on relative growth potential may offer anunderlying explanation for the apparently greater effect ofhigh CO2 on some NADP-ME species and on weedyC4 species. NADP-ME species tend to dominate in the mesiceastern part of South Africa (the ‘sourveld’, Ellery et al.1995). This vegetation type responds well to high rainfallduring the summer growing season, has a long growingseason (> 120 d), and is favoured by conditions conducive tocarbon assimilation and maximal growth during favourableconditions (Ellery et al. 1995). On the other hand, NAD-MEspecies dominate the semi-arid ‘sweetveld’ towards the westof the country. This vegetation type has a shorter growthseason and is less productive. If NADP-ME grasses haveinherently higher growth potential than NAD-ME species,one would expect a greater growth response to elevated CO2.Nevertheless, even within subtype (e.g. NADP-ME), therewere differences in relative growth potential that gave rise tovarying CO2 responses. D. natalensis had a low relativeregrowth rate and CO2 response of leaf growth (1.11 g g–1

leaf and 16%, respectively) whereas T. triandra was on thetop end of the scale (3.44 g g–1 leaf and 119%, respectively).This would possibly explain the variability found withinsubtypes by various researchers in previous studies.

R 2 = 0.3463

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Fig. 4. Relationships between leaf growth potential, CO2 enhance-ment of leaf growth, and leaf nitrogen concentration in C4 speciesrepresenting all three subtypes. (a) Leaf regrowth rate (over ~11weeks from first to second harvest, ambient-CO2-grown plants)normalised against leaf biomass at first harvest, plotted against leafnitrogen concentration; (b) percentage stimulation of leaf biomass inelevated CO2 plotted against normalised leaf regrowth rate;(c) percentage stimulation of leaf biomass in elevated CO2 plottedagainst leaf nitrogen concentration


The CO2 responsiveness of 11 C3 grass species studiedby Roumet and Roy (1996) was more strongly positivelycorrelated with specific leaf area (SLA, the inverse ofspecific leaf mass, SLM) than with relative growth potential.By contrast, our data show only a weak negative relationship(R2 = 0.2217, regression not shown) between percentageCO2 enhancement of leaf mass and SLM, although SLMwas negatively correlated with leaf nitrogen concentration,[N] (R2 = 0.6467, not shown). CO2 responsiveness was,therefore, also only weakly linked to [N] (Fig. 4c). Thecorrelations suggest that growth potential was, for the sevenspecies studied, only partially controlled by leaf chemistry,and responses to elevated CO2 depended more on thepotential relative growth and sink strength. Thus, theinherent ability to utilise assimilates for cell division andexpansion in rapidly growing organs would determinewhether and what fraction of the assimilates produced inelevated CO2 can be used for extra growth. We suggest thatfurther analysis of published data and/or further experimentswith a larger sample of species are required to establish sucha potential relationship more firmly, and to investigate themechanism involved.

In conclusion, C4 photosynthetic subtypes are most likelynot entirely reliable ‘functional type’ predictors of growthresponsiveness of C4 grass species to elevated CO2. Instead,this study suggests that categorisation of PFTs according torelative growth potential may be more appropriate forpredictions of CO2 responsiveness in C4 grasses. This resultdeserves further investigation and provides a promising leadfor future larger-scale modelling of the responses ofC4-dominated grasslands and savannas to elevated CO2.

Acknowledgments

This study was conducted while S. J. E. Wand was in theemploy of the National Botanical Institute, South Africa,who funded the project. We thank Mr Guy Clegg of PleasantView Farm, for permission to work on his land and excavateplants. We gratefully acknowledge the support of the Rolandand Leta Hill Trust, administered by the World WildlifeFund (Southern Africa), who funded the greenhouse facili-ties. An anonymous reviewer made suggestions that greatlyimproved the original manuscript.

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Manuscript received 13 July 1999, received in revised form 2 August2000, accepted 19 October 2000

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