Journal of Experimental Botany, Vol. 49, No.320, pp. 583-591, March 1998

Daniel A. Sims1,2,3, Jeffrey R. Seemann2 and Viqi Luo1

1 Desert Research Institute, Reno, NV, USA

2 Department of Biochemistry, University of Nevada, Reno, NV 89506, USA'

Received 19 March 1997; Accepted 25 September 1997

Abstract

The rate and extent of leaf thickness and areadevelopment are important determinants of wholeplant photosynthetic capacity. The interactive effectsof photon flux density (PFD), nitrogen supply and CO2concentration on leaf expansion rate were measuredas well as final leaf size and thickness of soybean.Leaf thickness and final area were not correlated withleaf relative expansion rate (RER) suggesting thatthese parameters are controlled by different mechan-isms and that final leaf dimensions are determined bythe duration rather than the rate of leaf expansion.Carbohydrate supply did not explain the variation inleaf RER since RER increased with increasing CO2concentration, but decreased with increasing PFD.Leaf thickness and final area were related to resourcesupply but not in a simple fashion. Both positive andnegative correlations between leaf thickness and car-bohydrate and nitrogen concentrations were obtaineddepending on the environmental variable responsiblefor the variation. In contrast, there was a simple pro-

portional relationship between whole plant relativegrowth rate and a correlate of leaf thickness (leafwater content per unit area), suggesting that leaf thick-ness responds to the balanced supply of all resources,in the same fashion as RGR, rather than to any indi-vidual resource.

Introduction

Leaves which develop at elevated CO2 concentrations areoften thicker than those on ambient CO2 plants. In astudy of four Populus clones, Radoglou and Jarvis (1990)found increases in leaf thickness of 8% to 16% in responseto elevated CO2. Similar results were reported by Leadleyet al. (1987) for soybeans where they found a 10%increase in leaf thickness for elevated CO2-grown plants.Growth at high photon flux density (PFD) also increasesleaf thickness (Boardman, 1977; BjOrkman, 1981). Theincrease in leaf thickness with high PFD is often closelycorrelated with increases in photosynthetic capacity perunit leaf area, suggesting that the high photosyntheticcapacities of sun leaves result from increased volume ofmesophyll tissue, and thus increased quantities of photo-synthetic enzymes, without a change in photosyntheticcapacity per unit volume of tissue (Louwerse andZweerde, 1977; Patterson et al., 1977, 1978; Sims andPearcy, 1992).

Whereas PFD acclimation generally has little effect onphotosynthetic capacity per unit leaf thickness, elevatedCO2 appears to decrease photosynthetic capacity per unitthickness since thickness increases while photosyntheticcapacity per unit area generally decreases (Gundersonand Wullschleger, 1994). However, elevated CO2 doesnot always decrease photosynthetic capacity per unit areaand in some cases even increases it, at least in soybean(Campbell et al., 1988). This could result from thecombination of increased leaf thickness and decreasedphotosynthesis per unit volume if the increase in thickness

Key words: Anatomy, carbohydrates; Glycine max,

microscopy, water content.

3 To whom correspondence should be addressed at the University of Nevada. Fax: +17027841419. E-mail: dsims@maxey.dri.edu

@ Oxford University Press 1998

584 Sims et al.

was greater than the decrease in photosynthesis per unitvolume. Luo et al. (1994) developed a PhotosyntheticAcclimation to CO2 (PAC) model that made use of theopposing changes in leaf thickness and photosyntheticcapacity ~r unit thickness to explain the wide variationin acclimation responses to CO2.

Considerable effort has been expended to understandthe mechanisms controlling the biochemically-basedchanges in photosynthetic capacity per unit volume oftissue in response to CO2 concentration (Stitt, 1991). Incontrast, there have been few studies of the factorscontrolling changes in leaf thickness. The response of leafthickness to elevated CO2 suggests that carbohydratesupply is involved in the regulation mechanism. However,this does not preclude the involvement of other factorssuch as nitrogen supply and direct effects of PFD. Herethe effects of PFD, nitrogen supply and CO2 concentrationon leaf thickness are compared in order to explore themechanisms controlling leaf thickness.

The effect of light, nitrogen and CO2 on the rate ofleaf expansion was also measured. Acceleration of leafdevelopment by elevated CO2 may contribute to thevariability in photosynthetic acclimation responses toCO2. Besford et al. ( 1990) found that photosynthesis oftomato leaves exposed to ambient and elevated CO2reached the same maximum value during leaf develop-ment, but that elevated CO2 leaves developed morerapidly and reached the maximum sooner. Consequently,elevated CO2 appeared first to increase and then decreasephotosynthetic capacity depending on the stage of leafdevelopment. Increased rates of leaf expansion are oftenobserved for elevated CO2 plants (Cure et al., 1989;Leadley and Reynolds, 1989; Ferris and Taylor, 1994;Gay and Hauck, 1994; Taylor et al., 1994; Gardner et al.,1995). Leaf expansion rates and final leaf areas andthicknesses were measured to determine whether CO2effects on these parameters are related and thus might becontrolled by the same mechanisms.

to 350 ppm CO2 and the other two at 700 ppm CO2. Carbondioxide concentration in each chamber was measured onceevery 6 min by an infrared gas analyser (model 6262, Li-CorInc, Lincoln, NE, USA). A datalogger (model CRIO, CampbellScientific, Logan, UT, USA) collected the data and controlledthe duration of CO2 injection into the chambers on a 30 s cycleto maintain the CO2 setpoint. Because the ambient CO2concentration within the greenhouse was quite variable, CO2scrubbers were used in the 350 ppm chambers to maintain aconstant CO2 concentration. The scrubber boxes measured14 x 45 x 56 cm, and were constructed from Plexiglas with twofans in the top and an open grill covered by screening in thebottom. CO2 in the air flowing through the boxes was absorbedby soda lime ('Sodasorb', WR Grace & Co. Atlanta, GA,USA). These boxes were placed inside the chambers and thefans were operated continuously. Control to the 350 ppmsetpoint was achieved through additions of CO2.

Within each pair, of chambers four PFD environments wereestablished. The highest and lowest PFD environments were inone chamber and the two intermediate PFD environments werein the other chamber. Each chamber was divided into high andlow PFD halves. The front (south) half received full sun orslightly reduced PFD under neutral density black plastic shadecloth (40% grade). The back (north) half received lower PFDsunder thicker shade cloth (60 and 90"/0). PFD was measuredcontinuously throughout the experiments with one galliumarsenide photodiode (model GII18, Hamamatsu Corp.,Bridgewater, NJ, USA) in each PFD treatment connected to adatalogger (model CRlO, Campbell Scientific, Logan, UT,USA). These sensors were previously calibrated against aquantum sensor (model 190s, Li-Cor Inc., Lincoln, NE, USA).The mean daily PFD over the 4-6 week growing period of theplants was used in the data analysis.

Nutrient treatments were randomly assigned within the lightand CO2 treatments and were begun 3 d following seedlingemergence. All plants were watered to excess daily with eithera 1/2 strength Hoagland solution (7.5 mmol NO3, 0.5 mmolPO4, 3 mmol K, 2.5 mmol Ca, 1 mmol Mg, 1 mmol SO4,0.067 mmol Fe-EDTA, plus micronutrients) or a similar solutionmodified to reduce nitrate concentrations while keeping allother ions constant, except for SO4 and CI which were used inequal portions to maintain charge balance. Five nitrogentreatments (0,0.9, 1.9, 3.8, and 7.5 mM nitrate) were appliedto the plants in the highest PFD treatment whereas the otherPFD treatments received only 0.9 or 7.5 mM nitrate treatments.Pots were flushed with deionized water once per week to avoidsalt build-up.

There were four plants per treatment except for the fivenitrogen levels in the highest PFD where there were onlythree plants.

Materials and methods

Plant material and growth conditions

Seeds of a non-nodulating variety of soybean, Glycine max (L.)Merr. (line D68-0099, Group VI maturity in a 'Lee' back-ground, Soybean Production Research, Stoneville, MS, USA,Hartwig, 1994), were planted in March in 81 pots in a 50/50(v/v) mixture of fine sand and sandy loam topsoil. Four seedsper pot were planted initially and then thinned to one per potafter emergence. Sixteen to 23 pots were placed in each of fournaturally lit growth chambers inside a greenhouse at the DesertResearch Institute in Reno, NV , USA. The chambers had glasswalls and tops (lxlm base and 1.75m in height). Thegreenhouse walls and roof were constructed from rigid, double-walled acrylic sheets ('Exolite', Cryo Industries, Orange; CT ,USA). Temperatures were controlled to 28 :t2 °C in the daytimeand 20 :t 1 °C at night. Relative humidity at midday was 66 :t 7%.

Plants were grown in four growth chambers, two controlled

Leaf expansion

After 4 weeks growth, an expanding trifoliate leaf with a centralleaflet measuring between 0.5 and 3 cm in length was markedon each plant with a small piece of wire tied around the petiole.Marked leaves were mostly at trifoliate node 4 for the highlight/high nutrient plants but ranged down to node 2 for thelow light/low nutrient plants since the later plants were smaller .Some studies have reported increases in leaf mass per unit areaand thickness with increasing nodal position in soybean (Luggand Sinclair, 1979, 1980). In measurements of a similar set ofplants (data not shown) increases in leaf dry mass per unit areawere found with increasing leaf nodal position but significantchanges in leaf thickness were not found. Consequently,sampling of leaves at different nodes for this experiment should

GO 2 and leaf development 585

have had little effect on the conclusions regarding leaf thickness.The length of each of these leaflets was measured daily for 1week and then every 2 d for another week until all the leafletshad completed expansion. Since preliminary measurementsshowed that the square of leaf length was directly proportionalto leaflet area, this was used to estimate the rate of leaf area

expansion.

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Fig. I. Trifoliate central leaflet area and leaflet relative expansion rate(RER) for soybean plants growing in a range of photon flux densities(PFD), nitrogen supplies and CQ2 concentrations. Symbols: 700 ppmCQ2 (open symbols), 350 ppm CQ2 (filled symbols), 7.5 mM nitrate(circles) and 0.9 mM nitrate nitrogen (triangles). Arrows indicateresource levels used in the other gradient.

Leaf characteristics

One to 2 weeks following the completion of the central leafletexpansion measurements, the same leaflets were harvested. Theplants were now about 7 weeks old and were starting to fillpods. This work was done at least 1 h after dark to ensure fullhydration of the leaves. After excision of the midrib and asmall section from the middle of the blade to be used for leafanatomy measurements, the remaining leaflet was quicklyweighed to determine fresh mass, leaf area was measured withan area meter (model 3000, Li-Cor Inc., Lincoln, NE, USA)and the samples were dried for 48 h at 60 °C prior todetermination of dry mass. Preliminary measurements showedthat water loss from the leaves between the time of excisionand fresh mass determination was less than 1% of totalfresh mass.

Leaf cross-sections were prepared by hand with a razor bladefrom the small section cut from the middle of the leaf blade.The cross-sections were then photographed with a photomicro-scope (model Nos BX60 and PM-30, Olympus America Inc,Lake Success, NY, USA) and the photographs used todetermine total leaf thickness as well as the thickness of theepidermal, palisade and spongy cell layers.

Total non-structural carbohydrate (TNC) contents of maturecentral leaflets collected from the same plants during the periodof expansion of the leaves used for the anatomy measurementswere measured by an enzyme-coupled colorimetric technique(Hendrix, 1993). These leaves were collected in late afternoon,when TNC contents should be maximal. Half of each leaf wasimmediately frozen in liquid nitrogen, and stored at -80 °Cuntil TNC analysis. The other half was dried for 48 h at 60 °Cprior to measurement of total nitrogen content (model 2400CHN analyser, Perkin Elmer, Norwalk, CT, USA).

could be artefactual. However, since the conclusions are basedon the main effects of light and CO2 rather than the interactionterms this was not felt to be a serious concern.

Whole plant relative growth rate

Plants were grown under conditions similar to those describedabove except that there were only two levels of each environ-mental factor (P FD of 25 or 3.7 mol m -2 d -1, nitrogen supply

of 7.5 or 0.75 mM nitrate and CO2 concentration of 350 or 700ppm). The initial harvest was made after 4 weeks growth in thetreatments. The final harvest was made I week later for thehigh PFD plants and 2 weeks later for the low PFD plants.Roots were washed free of soil and the plants were divided intoroot, stem and individual leaf fractions. Leaf area (model 3000,Li-Cor Inc., Lincoln, NE, USA ) and fresh mass weredetermined separately for each leaf. Dry mass (following dryingfor 48 h at 60 °C) was determined for each leaf and for the rootand stem fractions. For the comparison of RGR with leaf watercontent per unit area, values for the first trifoliate leaves, whichwere all fully expanded, were used.

Results

Increasing PFD and nitrogen supply increased fullyexpanded leaflet area, but had surprisingly little effect onrelative expansion rate (RER, calculated for the point atwhich the leaflet was 30% of final area) of the leaflets(Fig. I). RER was actually greatest at the lowest PFDwhere final leaf areas were the smallest. Elevated CO2had no effect on final leaflet area in most treatments,except for the highest and lowest PFDs at high nitrogensupply where an increase in leaf area for elevated CO2plants was observed. Elevated CO2 increased RER inmost of the treatments but there did not appear to beany relationship between changes in RER and final leafarea.

Total leaf thickness increased strongly with increasingPFD whereas increasing nitrogen supply resulted insmaller but also significant increases in leaf thickness(Figs 2, 3). Increased leaf thickness resulted primarilyfrom increases in the mesophyll cell layers with muchsmaller changes in total epidermal thickness (Figs 4, 5).All leaves appeared to have two palisade cell layers, butthe size of the cells in the second layer were greatlyreduced in the low PFD plants (Fig. 2). There was a

Statistical analysis

The results were analysed by ANOVA (SAS, Cary, NC, USA).Since limited growth chamber space made it impossible toreplicate each of the light and CO2 treatments some caution iswarrented in interpreting the interaction terms. In particular,effects observed only for the two middle PFDs but not at thehighest and lowest PFDs (which were in a different chamber)

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586 Sims etal.

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Fig. 2. Representative leaf cross-sections from plants grown at highnitrogen (7.5 mM nitrate), a range of photon flux densities (PFD) andtwo CQ2 concentrations. Black bar at bottom represents 0.2 mm.

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Fig. 3. Representative leaf cross-sections from plants grown at highphoton flux density ( 17.4 mol m -1 d- 1 ), a range of nitrogen supplies

and two COl concentrations. Black bar at bottom represents 0.2 mm.

Since leaf thickness is difficult to measure, several easilymeasured parameters which might serve as a correlate ofthickness were tested. Leaf dry mass per unit area showedresponses to PFD, nitrogen and CO2 which were verysimilar to those seen for leaf thickness (Fig. 7). However,it was not the best correlate for leaf thickness (Fig. 8).Leaf dry mass, fresh mass and water content (fresh massminus dry mass) per unit area were all significantlycorrelated with leaf thickness, but the correlation was

significant interaction between nitrogen supply and CQ2concentration such that elevated CQ2 increased total leafthickness in the high nitrogen treatments, but had nosignificant effect on thickness at low nitrogen ( Fig. 4;Table I). Whereas PFD effects on thickness were similarfor the palisade and spongy mesophylllayers (Fig. 4),CQ2 had a significant effect only on spongy mesophyllthickness (Table I ). CQ2 did not have any significanteffect on total epidermal thickness.

Total leaflet thickness was not simply a function ofincreasing leaf size. Although there was a significantpositive correlation between leaflet area and thickness forplants grown in the highest PFD, there was no correlationfor the lower PFDs (Fig. 6). Leaves of the same totalarea in high and low PFD were about twice as thick inthe high PFD treatment.

GO 2 and leaf development 587

Low nitrogen High nitrogen

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Fig. 4. Total leaf thickness and the thickness of the palisade, spongyand epidermal (upper and lower combined) cell layers for plants grownin a range of photon flux densities (PFD), two nitrogen supplies (7.5or 0.9 mM nitrate) and two CQ2 concentrations (350 ppm (filledsymbols) or 700 ppm (open symbols».

Fig. s. Total leaf thickness and the thickness of the palisade, spongyand epidennal (upper and lower combined) cell layers for plants grownin a range of nitrogen supplies and two CO2 concentrations (350 ppm(filled symbols) or 700 ppm (open symbols».

whereas variation due to nitrogen supply resulted in apositive correlation (Fig. 9).

In a separate experiment (unpublished data) usingsimilar plants and treatments, leaf water content per unitarea (shown here to be a good correlate of leaf thickness)was well correlated with whole plant relative growth rate(Fig. 10). This relationship was not significantly affectedby any of the PFD, nitrogen or CO2 treatments.

best for leaf water content. Elevated CO2 significantlyreduced the ratio of leaf thickness to leaf dry mass perunit area (Table 1 ). This ratio was also significantlyaffected by PFD and nitrogen supply. The relationshipbetween leaf thickness and fresh mass per unit area wasbetter than that for dry mass, but PFD still had asignificant effect. None of the treatments had a significanteffect on the ratio of leaf thickness to water content,suggesting that leaf water content is the most robustpredictor of leaf thickness.

Leaf thickness was not a simple function of either totalnon-structural carbohydrate content (TNC) or nitrogencontent as percentage of dry mass (Fig. 9). TNC as apercentage of leaf dry mass in mature leaves was positivelycorrelated with leaf thickness when variation was inducedby variation in PFD, but was negatively correlated whenthe variation was induced by differeI)ces in nitrogensupply (Fig. 9). These relationships were exactly theopposite for nitrogen concentration. Variation due tovariation in PFD resulted in a negative correlation

Discussion

The results suggest that elevated CO2 has at least twoindependent effects on soybean leaf development. Growthof soybeans at elevated CO2 concentrations acceleratedleaf relative expansion rate (RER) in most of the PFDand nitrogen treatments. Elevated CO2 also increased leafthickness in the high PFD and high nitrogen treatments.

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Fig. 6. Trifoliate central leaflet thickness as a function of total centralleaflet area for soybeans grown at three photon flux densities (2.3(circles), 4.2 (triangles) and 17.4 mol m-2 d-l (squares), one inter-mediate PFD was left out for clarity) and two CQ2 concentrations (350ppm (filled symbols) or 700 ppm (open symbols».

Increased leaf thickness (Thomas and Harvey, 1983;Leadley et al., 1987; Vu et al., 1989) and increased leafexpansion rates (Leadley and Reynolds, 1989; Cure et al.,1989) have previously been reported for elevated CO2grown soybeans. However, we are not aware of anystudies where the CO2 effects on both these parameterswere measured in the same experiment. It was found thatthe extent of the increase in leaf thickness was notcorrelated with the stimulation of leaf RER, suggestingthat these CO2 effects result from independentmechanisms.

Comparison of the light, nitrogen and CO2 effects mayprovide some insight into these mechanisms. Both elev-ated CO2 and high PFD increase photosynthesis and thuscarbohydrate production. Comparison of the PFD and

CO2 responses suggest that leaflet RER is not a directfunction of carbohydrate supply since elevated CO2 stimu-lated RER but increasing PFD had a generally negativeeffect. Gay and Hauck ( 1994) reported a similar resultfor Lolium temulentum where leaf growth rate was greatestat low PFD and high CO2. Whereas elevated CO2 gener-ally seems to stimulate leaf expansion (Cure et al., 1989;Leadley and Reynolds, 1989; Ferris and Taylor, 1994;Gay and Hauck, 1994; Taylor et al., 1994; Gardner et al.,1995), the effect of PFD is less consistent. Taylor andDavies ( 1986) found no effect of PFD on early expansionrates of Acer pseudoplatanis and Betula pendula leaves,but increases in leaf expansion rates with increasing PFDhave been reported in other studies (Taylor and Davies,1988; Sims and Pearcy, 1992). It may be that this responsevaries between species, but it is also possible that differ-ences in expansion rates resulted from variation in factorsother than PFD and carbohydrate supply.

Temperature has a much stronger effect on leaf

GO 2 and leaf development 589

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Fig. 8. Leaf thickness as a function of leaf dry mass, fresh mass and water content (fresh mass minus dry mass) per unit area for soybean plantsgrown at a range of photon flux densities, nitrogen supplies and two CQ1 concentrations (350 ppm (filled symbols) or 700 ppm (open symbols».

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0Fig. 9. Leaflet thickness as a function of total non-structural carbohyd-rates (TNC) and total leaf nitrogen per unit leaf dry mass for soybeanplants grown at a range of photon flux densities (PFD, circles) andnitrogen supplies (triangles) and two CQl concentrations (350 ppm(filled symbols) or 700 ppm (open symbols». The correlations werepositive when the driving variable responsible for the variation was thesame as the independant variable but were negative when the oppositeresource provided the variation. For example, when PFD, whichprimarily affects carbohydrate uptake, varied there was a positivecorrelation between leaf thickness and TNC. However, when thevariation resulted from changes in nitrogen supply at a constant PFDthere was a negative correlation between leaf thickness and TNC.

Fig. 10. Leaf water content as a function of whole plant relative growthrate (RGR) for soybeans grown at CQ2 concentrations of 350 ppm(filled symbols) or 700 ppm (open symbols). photon flux densities of25 mol m-2 d-l (circles and up triangles) or 4 mol m-2 d-l (squaresand down triangles) and nitrogen supplies of 7.5 mM nitrate (circlesand squares) or 0.75 mM nitrate (up and down triangles).

expansion rate than either PFD (Milthorpe, 1959) orCO2 (Ackerly et al., 1992). Even when air temperaturesare carefully controlled, leaf temperatures may vary sub-stantially because of differences in radiant heat load andstomatal conductance, which affects transpirational cool-ing. Leaf temperatures of plants in high PFD can beeither higher or lower than those of plants in low PFDdepending on the balance between increased radiant loadand the stomatal response. Elevated CO2 generallyincreases leaf temperature (Campbell et al., 1990), sinceit reduces stomatal conductance and thu~ transpirationalcooling. Since photosynthesis of species with the C4photosynthetic pathway is unaffected by elevated CO2,but stomatal conductance is reduced in the same fashion

as CJ species (Samarakoon and Gifford, 1996), compar-ison of the responses of CJ and C4 plant species can beused to test the importance of CO2 effects on stomata asopposed to photosynthesis. Ackerly et al. (1992) foundthat elevated CO2 stimulated leaf expansion rate in bothAbutilon theophrasti (CJ) and Amaranthus retroftexus(C4), suggesting that changes in expansion rate mighthave been related to stomatal effects.

Reduced stomatal conductance and thus transpirationrates in elevated CO2 might also increase leaf waterpotential and turgor pressure which could stimulate leafexpansion (Morison, 1993; Tyree and Alexander, 1993).However, Gardner et al. (1995) found that leaf turgorpressure was actually reduced for several poplar clones

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supply of all resources, in the same fashion as RGR,rather than to any individual resource. The relationshipbetween RGR and leaf thickness did not simply resultfrom the increased leaf size of more rapidly growingplants since only a weak relationship was found betweenleaf size and thickness. Regardless of whether leaf thick-ness is causally linked to RGR or whether they simplyrespond to the same signals, their close correlation sug-gests that the factors ultimately determining leaf thicknessare complex. Given that leaf area development and leafthickness are themselves important determinants of wholeplant carbon gain and growth (Lambers and Poorter,1992), it is clear that many feedback responses arepossible which may result in quite complex responses atthe whole plant level.

The capacity of plants to utilize increased carbohydratesupply under elevated CO2 is frequently hypothesized tocontrol plant responses. Decreased photosynthetic capa-city and photosynthetic enzyme concentrations of leavesgrown at elevated CO2 are hypothesized to result from afeedback process when plants are unable to utilize theincreased carbohydrate production (Stitt, 1991 ). Theseresults suggest that the ability to utilize increased carbo-hydrate supply may also influence leaf thickness. ElevatedCO2 increased leaf thickness to the greatest extent in highnutrient conditions where there was also the greatestgrowth stimulation. This might increase photosyntheticcapacity, because of increased photosynthetic tissuevolume per unit area, under conditions where increasedcarbohydrate production could be used for increasedgrowth. Further work should focus on the factors limitinggrowth and the signals linking growth potential to photo-synthetic and leaf thickness responses.

Acknowledgements

We thank Carol Johnson for nitrogen analyses and GavinChandler for carbohydrate analyses. This work was supportedby USDA NRICGP grant No.940136 to YL and NSF grantNo. IBN 9420054 to JRS.

590 S;ms et al.

grown in elevated CQ2° They concluded that increasedexpansion rates of elevated CQ2 leaves resulted fromincreased cell wall extensibility rather than turgorpressure. Elevated CQ2 also increased the activity ofxyloglucan endotransyglycosylase (Taylor et al., 1994),an enzyme correlated with changes in growth rate andcell wall extension (Fry et al., 1992; Heatherington andFry, 1993). Changes in cell wall properties also appearto account for increased leaf expansion rate in otherspecies in response to increased nitrogen (Taylor et al.,1993) or PFD (Taylor and Davies, 1985, 1988), however,the signals controlling these changes in cell wall propertiesremain to be elucidated.

Cell divisions are largely complete prior to the rapidexpansion phase of leaf development (Maksymowych,1973 ), and there is some evidence that final cell size isalso determined by the length of the cell cycle in meristems(Francis and Halford, 1995). Consequently, final leaf sizeand thickness may be determined quite early in leafdevelopment by processes distinct from those controllingthe rate of expansion during later stages of development.Two studies have recently demonstrated that elevatedCQ2 has effects on development at very early stages.Kinsman et al. (1996) found that elevated CQ2 couldincrease the rates of cell division at the tip of the apicalmeristem, but this effect varied dramatically between thetwo genotypes of Dactylis glomerata that were measured.Robertson et al. ( 1995) found almost 3-fold increases inmitochondrial numbers in very young leaf cells of wheat.

In this study it was found that all the treatments hadgreater effects on mesophyll thickness than on epidermalthickness. In addition, the CQ2 treatments, but not PFDor nitrogen, had a greater effect on spongy mesophyllthickness than on palisade mesophyll thickness. A similarresult for CQ2 treatment was reported for Phaseolusvulgaris by Radoglou and Jarvis ( 1992), but Thomas andHarvey (1983) found the opposite response, a greaterincrease in palisade than in spongy thickness with increas-ing CQ2, for soybean and sweet gum. All that can beconcluded with certainty is that elevated CQ2 increasesmesophyll thickness more than epidermal thickness. Thissuggests a well controlled response targeting specific cell

types.Whereas RER of leaves appeared to be largely inde-

pendent of resource supply, final leaf size and thicknessboth increased with increasing PFD and nitrogen supply.However, the relationships between leaf thickness andcarbohydrate and nitrogen concentrations in matureleaves were complex and dependent on whether the sourceof variation was PFD or nitrogen supply. It may be thatresource concentrations in mature leaves do not accur-ately reflect the concentrations in young developingleaves. However, the strong correlation between leafthickness and whole plant relative growth rate (RGR)suggests that leaf thickness responds to the balanced

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GO 2 and leaf development 591

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