Effects of leaf development and phosphorus supplyon the photosynthetic characteristics of perennial legumespecies with pasture potential: modelling photosynthesiswith leaf development

Lalith D. B. SuriyagodaA,B,C,E, Hans LambersA, Megan H. RyanA,B and Michael RentonA,B,D

ASchool of Plant Biology and Institute of Agriculture, The University of Western Australia, 35 Stirling Highway,Crawley, WA 6009, Australia.

BFuture Farm Industries Cooperative Research Centre, The University of Western Australia, 35 Stirling Highway,Crawley, WA 6009, Australia.

CFaculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka.DCSIRO Sustainable Ecosystems, Floreat, WA 6014, Australia.ECorresponding author. Email: suriyl01@student.uwa.edu.au

Abstract. Age-dependent changes in leaf photosynthetic characteristics (i.e. parameters of the light response curve(maximum photosynthetic rate (Pmax), quantum yield (F) and the convexity parameter (q)), stomatal conductance (gs) anddark respiration rate (Rd)) of an exotic perennial legume,Medicago sativa L. (lucerne), and two potential pasture legumesnative to Australia, Cullen australasicum (Schltdl.) J.W. Grime and Cullen pallidum A. Lee, grown in a glasshouse for5months at twophosphorus (P) levels (3 (P3) and 30 (P30)mg P kg–1 dry soil) were tested. Leaf appearance rate and leaf areawere lower at P3 than at P30 in all species, with M. sativa being the most sensitive to P3. At any leaf age, photosyntheticcharacteristics didnot differ betweenP treatments.However,Pmax andgs for all the species andF forCullen species increaseduntil full leaf expansion and then decreased. The convexity parameter,q, did not changewith leaf age, whereasRd decreased.The estimates of leaf net photosynthetic rate (Pleaf) obtained through simulations at variable Pmax andFwere lower duringearly and late leaf developmental stages and at lower light intensities than those obtainedwhenFwas assumed to be constant(e.g. for a horizontally placed leaf, during the 1500�C days developmental period, 3 and 19% reduction of Pleaf at lightintensities of 1500 and 500mmolm–2 s–1, respectively). Therefore, developmental changes in leaf photosyntheticcharacteristics should be considered when estimating and simulating Pleaf of these pasture species.

Additional keywords: Australian native legumes, leaf age, light response curve, novel crops.

Introduction

Photosynthesis simulation models can be used to explorepotential plant growth. For example, we want to use suchmodels to predict the productivity of exotic and nativeAustralian perennial legume species with pasture potential inthe low phosphorus (P) soils under both current climates andpredicted climate change scenarios. However, the usefulness ofsuch photosynthesis models will depend on accurately predictingleaf area and emergence rate and on accurately incorporating leafphotosynthetic characteristics and the way they change with leafage and with differences in nutrient supply (Pearcy and Sims1994; Stirling et al. 1994; Kitajima et al. 2002).

Leaves in a canopy/sward experience a wide fluctuation inincident PPFD and as leaves get older they are likely to bepositioned lower in the canopy. Most of the publishedmeasurements of the rate of leaf photosynthesis have beenmade only on leaves soon after full leaf expansion and at

light-saturation (Constable and Rawson 1980; Dwyer andStewart 1986; Heschel et al. 2004; Fletcher et al. 2008). Yet,we know that leaf net photosynthetic rate (Pleaf) first increaseswith leaf age and then decreases (Smillie 1962; Pisum sativum L.(pea); Peat 1970; Solanum lycopersicum L. (tomato); Dwyer andStewart 1986; Stirling et al. 1994; Zea mays L. (maize); Suzukiet al. 1987; Triticum aestivum L. (wheat); Kitajima et al. 2002;Cecropia and Urera species; Xie and Luo 2003; Pyrus serotinaRehd. (Asian pear); Niinemets et al. 2004; Quercus spp. (oak)).These age-dependent changes in Pleaf could be due to changesin one or more parameters of the photosynthetic light responsecurve (PLRC).

The PLRC is defined by three parameters: (i) the quantumyield of CO2 assimilation (F), derived from the slope of theinitial linear region of the response of CO2 uptake to PPFD;(ii) the upper asymptote, representing the light-saturated rate ofCO2 assimilation (Pmax) and (iii) the convexity coefficient (q)

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www.publish.csiro.au/journals/fpb Functional Plant Biology, 2010, 37, 713–725

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describing the curvature of the response between (i) and(ii) (Thornley and Johnson 2000). Where detailed studies ofthe PLRC have been undertaken, measurements have notencompassed the early stages of leaf development (Marshalland Biscoe 1980; Kitajima et al. 2002; Fletcher et al. 2008) orreported varying results/trends with leaf age. For example,only Pmax was found to change with leaf age in Z. mays(Boedhram 1998), Lepechinia calycina (Benth.) Epl. (Fieldand Mooney 1983) and Cecropia and Urere spp. (Kitajimaet al. 2002), whereas only Pmax and F changed in Z. mays(Dwyer and Stewart 1986) and only Pmax and q changed inZ. mays (Stirling et al. 1994). It has been suggested that thedifferences reported above might be due to environmental stressduring the growth period, seasonal environmental changes orto errors in measuring these parameters (Sands 1996; Singsaaset al. 2001). However, it seems likely that the photosyntheticcharacteristics (i.e. parameters of the PLRC, stomatalconductance (gs) and dark respiration (Rd)) will change duringleaves aging and that these changes will have importantimplications for estimating Pleaf and, thus, for simulations ofplant growth.

Most models of canopy photosynthesis that are used toestimate the growth of plants under diverse environmentalconditions are based on the assumption that the parametersdefining the PLRC are the same for all leaves and thatvariation in Pleaf with canopy depth arises solely fromdifferences in incident PPFD (Stirling et al. 1994; Kitajimaet al. 2002). This is an over-simplification, since leaf age alsocontributes significantly to variation in Pleaf. However, Charles-Edwards (1981; p. 70, eqns 3.14 and 3.15) assumed that Pmax ofa leaf is proportional to local irradiance and Thornley (2002,2004) used this approach to calculate canopy photosynthesis andalso correlated the ratio of Pmax and local PPFD with canopynitrogen (N) distribution. Apart from this approximation ofPmax, possible changes in, q and F have not been used inmodels for leaves at different depths in the canopy and thesechanges have not been accounted for when modelling Pleaf,for any species. Thus, to date, q and F have been consideredas constants when calculating Pleaf and in simulating canopyphotosynthesis; the estimates ofq andF generally being obtainedfrom leaves immediately after full expansion. This approachsimplifies the model but fails to incorporate importantinformation. Recently, Kitajima et al. (2002) concluded thatleaf age is a more reliable predictor of Pleaf than % PPFD orleaf N concentration. Therefore, there is a need to study thebehaviour of photosynthetic characteristics with leaf age andincorporate those changes into simulation models in order topredict Pleaf more accurately.

Worldwide, P is one of the most limiting mineral nutrientsfor plant productivity; future global food security is questioneddue to the decreasing quality of P fertilisers and increasingproduction costs (Schachtman et al. 1998; Vance et al. 2003;Cordell et al. 2009). P deficiency can reduce sink activity, solarradiation interception (Plénet et al. 2000b; Rodríguez et al. 2000;Pieters et al. 2001; de Groot et al. 2003) and Pleaf (Jacob andLawlor 1992; Chiera et al. 2002; Ghannoum and Conroy 2007),and thereby restrict plant growth. Even though several studieshave demonstrated a reduction inPleaf andPmaxwhen plants wereunder severelyP limiting conditions (Fredeen et al. 1989;Raoand

Terry 1989; de Groot et al. 2003) and when plants were exposedto excess P causing P toxicity (Shane et al. 2004), the influence oflow P supply on F and q has not been reported for any plantspecies.

Medicago sativa L. (lucerne) is one of the most widelycultivated perennial pasture legumes in Mediterranean regionsand has long been grown in fertile and moist environments inAsia (Griffiths 1949;Small 2009).However,Australianperenniallegumes are also considered to have potential for developmentas alternative pasture species (Dear et al. 2007; Robinson et al.2007). In particular, the genus Cullen has been identified ascontaining several potential perennial pasture species (Cocks2001; Dear et al. 2007; Li et al. 2008). One advantage ofnative pasture legumes is their adaptation to local conditions.For example, many of them have evolved in local,P-impoverished environments (Beadle 1966; Handreck 1997).Uptake of P is poorly regulated at elevated P availability in someAustralian species (Shane et al. 2004) and many native speciesare sensitive to P toxicity (Handreck 1997; Shane et al. 2004).Simulation models could be used to predict and comparethe productivity of M. sativa and Cullen in the low P soilscommonly found in Australia, under both current climatesand under predicted climate change scenarios. However, asargued above, the accuracy of such models will depend onaccurate representation of morphological (e.g. leaf growth) andphysiological (e.g. photosynthetic) characteristics of thesespecies under the relevant conditions. As yet, the changes inphotosynthetic characteristics with leaf age and their interactionwith P supply are not available for either M. sativa or Cullenspecies.

Therefore, the objectives of this study were to: (i) comparethe effects of two levels of P supply on leaf area, emergence rate,photosynthetic characteristics and respiration for two perenniallegumes native to Australia, C. australasicum and C. pallidumA. Lee, and an exotic perennial legume, M. sativa; (ii) studyhow the photosynthetic characteristics change with leaf ageand whether this is influenced by [P]; and, (iii) incorporateany significant changes in the PLRC parameters intosimulation models of leaf photosynthesis in order to comparethe resulting estimates of Pleaf with those resulting from thestandard approach of assuming F and q to be constants. Wehypothesised that: (i) reduced P supply would result in a reducedleaf size, leaf appearance rate, leaf photosynthetic rate at1500mmolm–2 s–1 (P1500) and also reduced values of Pmax, F,q, gs and Rd at any leaf age; (ii) for a given P supply, the threeparameters of the PLRCwould all increase over time until the leafwas fully expanded and then decrease with increasing leaf ageand (iii) modelling Pmax,F and q as changing over the course ofleaf agewould greatly reduce the estimates ofPleaf comparedwiththe standard approach of modelling F and q as constants.

Materials and methodsGrowth conditions

Morphological and physiological responses of three legumespecies were assessed by growing plants in pots with differentP supplies. Cullen australasicum (Schltdl.) J.W. Grime,accessions SA4966 (late flowering) and SA42762 (earlyflowering), Cullen pallidum A. Lee accession SA44387 and

714 Functional Plant Biology L. D. B. Suriyagoda et al.

Medicago sativa L. cv. SARDI-10 were grown in 1m tall, 10 cmdiameter pots. Seeds of C. australasicum and C. pallidum werecollected from the seed-multiplication plots established at theShenton Park field station of the University of Western Australiaand M. sativa seeds from the Genetic Resource Centre atthe South Australian Research and Development Institute. Theexperiment included two P treatments (supply): P3 (3mg P kg–1

dry soil) and P30 (30mg P kg–1 dry soil). Recently in a P responsestudy Pang et al. (2010) reported both M. sativa andC. australasicum achieved a maximum growth (dry weight) at24mg P kg–1 dry soil. Therefore, P3 and P30 used in the currentstudy represent a low and an optimum P supply, respectively, forboth species. Three replicate pots of each species�Pcombination were established. Pots were filled with 12 kg pot–1

of thoroughly washed, steam-sterilised river sand. Base soil [P]was 1–2mgP kg–1 sand and pH (CaCl2) was 6.5, as determinedby CSBP FutureFarm analytical laboratories, Bibra Lake, WA,Australia. All essential nutrients other than P were provided byamending the sandwith (inmgkg–1) 126.6Ca(NO3)2.4H2O, 42.8NH4NO3, 178K2SO4, 101 MgSO4.7H2O, 11 CaCl2.2H2O, 12MnSO4.H2O, 8.8ZnSO4.7H2O, 1.96CuSO4.5H2O, 0.68H3BO3,1.01 NaMoO4.2H2O and 32.9 FeNaEDTA. P was supplied asKH2PO4, with different concentrations for different treatments,as given above. Additional K was supplied in the P3 treatment asKCl to balance K. Pots were watered from the top using a wicksystem,whichmaintained the soil profilemoisture throughout theexperiment at 60–75% field capacity. The experiment was set outin a glasshouse at the University of Western Australia, Perth(31�590S, 115�530E) as a complete randomised design at a densityof 12 potsm–2; pots being randomised at weekly intervals. Giventhe wide spacing of the pots, no guard row pots were used. Threeseedlings were planted in each pot and thinned to one plant perpot at 2–3 weeks. After week 6, 300mL of 2mM NH4NO3

was applied weekly to ensure an adequate nitrogen supply(Denton et al. 2006). The glasshouse was unheated and had anaverage daytime temperature of 23�C during the experiment,which was conducted from May to September 2008.

Photosynthetic measurements

Leaf photosynthetic measurements were taken from the 8thnodal leaf, counted from the base of the main stem excludingthe cotyledonary node. Pleaf, gs and Rd were measured with aportable gas-exchange system (LI-6400 portable, Li-Cor Inc.,Lincoln, NE, USA) equipped with a light source (6400–02BLED, Li-Cor Inc.) at 7–10 day intervals until harvest.Measurements began 7–8 days after the leaf appeared andwere made at solar noon �2 h. Photosynthetic rates at ninelevels of PPFD were measured on selected leaves using the‘auto light curve’ program with a minimum time of 120 s anda 3% coefficient of variation as the trigger to move to thenext PPFD setting. The light-response curves started at500mmolm–2 s–1, followed by 2000mmolm–2 s–1, then back to500mmolm–2 s–1 and finally 0mmolm–2 s–1. This allowed a rapidstabilisation of photosynthesis at each PPFD level. Standardconditions of 380mmolmol–1 CO2 and 25�C were maintainedwithin the leaf chamber. Ambient RH of the incoming air to theleaf chamber was left at that of the glasshouse environment(30–55%).

Growth measurements and plant analysisLeaf area of the 8th main stem nodal leaf was measured bydrawing the leaf area on a piece ofwhite paper at 1-week intervalsuntil full expansion. Those leaf measurements were used tocorrelate leaf development/expansion with leaf photosyntheticcharacteristics. Plantswere harvested 5months after germination.At the time of final harvest, the number of main stem nodes perplant, excluding the cotyledonary node, was recorded.Main stemnodal counts observed at the time of final harvest were used tocompare species and P treatments and were expressed as the leafappearance rate during the growth period. Leaves were detachedfrom each plant and used tomeasure the leaf area of themain stemleaves and total plant leaf area with a Li-Cor LI-3000 portablearea meter, which was equipped with LI-3050A transparentbelt conveyer accessory (Li-Cor Inc.). The first few leaves thatdropped from the main stem before the final harvest werecollected and used for leaf area measurements. Leaf arearepresents the area of all three leaflets of a single trifoliateleaf. Leaves were dried at 70�C for 1 week and weighed.Measurements were used to estimate the specific leaf area(SLA) of individual plants. Leaf, stem and root [P] of eachplant were measured separately. A subsample of ~100mg wastaken and digested in nitric/perchloric acid and then analysedusing the molybdo-vanado-phosphate method (Kitson andMellon 1944). Leaf [P] is expressed on a leaf area basis([P]area). Leaf, stem and root [P] and DW of each componentwere used to calculate the total P content per plant. Leaf [N] wasdetermined by dry combustion (Nelson and Sommers 1996)using an elemental CN analyser (Elementar AnalysensystemeGmbH, Hanau, Germany).

The concept of thermal time (�C days) was used as ameasure of leaf age. Cumulative thermal time (leaf_age) wascalculated:

leaf�age ¼X

ðT � TbÞ; ð1Þwhere, T and Tb are daily mean and base temperatures,respectively. The base temperature for M. sativa (5�C) (Ficket al. 1988) was used for all species. The day at which the firstphotosynthetic measurements were taken was considered as the0�C day.

Effects of P supply and species on leaf characteristics

The significance of differences in leaf area (per plant and differentnodal positions on the main stem), SLA and leaf [P] due to Psupply and species were tested with Proc GLM for ANOVA inSAS (SAS Institute Inc., Cary, NC, USA). Leaf area measured atthe 8th nodal leaf until full expansion was also tested using ProcGLM. Means were separated with the LSMEANS procedure.All significant differences were expressed at P < 0.05. Leafappearance rates were compared using categorical analysis(Proc CATMOD in SAS).

Estimating the parameters of the PLRCRelationships betweenPleaf and irradiance (Ileaf) for each leaf andfor each sample date, were modelled with the non-rectangularhyperbola (Thornley and Johnson2000) described inEqn2, usingProc NLIN in SAS, with the Gauss–Newton method. Parameter

Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 715

estimates and 95% confidence intervals were obtained for Pmax,F and q.

Pleaf ¼ 12q

ðFI leaf þPmaxÞ�ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðFI leaf þPmaxÞ2�ð4FqI leafPmax

qÞ:

ð2ÞAnalysis of repeatedly measured photosynthetic data

A repeated-measure ANOVA (RM-ANOVA) was used to testwhether the estimated parameters of PLRC, P1500, gs and Rd

varied significantly over the course of leaf development. Each leafage levelwas treated as a time point byusing the ‘repeated’ optionof Proc Mixed, and then the PLRC parameters were modelledusing species, P supply and leaf-age and their interactions asexplanatory variables. As RM-ANOVA can be sensitive to theassumed variance-covariance structure of the data (Potvin et al.1990), we first fitted four types of covariance structures(compound symmetric, first-order autoregressive, spatialpower and unstructured), each based on different assumptionsabout howvariances and covariances change acrossmeasurementlevels (Littell et al. 1996, 1998). We then selected the bestcovariance structure by comparing Akaike information criteria(AIC) scores (Heschel et al. 2004). This approach suggested anunstructured variance-covariance matrix to be the best, so thisassumption was used when testing effects of species, P supply,leaf age and their interactions on photosynthetic characteristics.

Analysis of photosynthetic characteristics at each leaf age

When the interaction between species (or P supply) and leafage was significant, photosynthetic characteristics were testedseparately at each recorded stage of leaf development.Photosynthetic characteristics considered as response variableswere P1500, gs, Rd and the estimates of the three PLRC modelparameters; Pmax, q and F. An ANOVA was performed in SASusing Proc GLM. Means were separated with the LSMEANSprocedure. All significant differences are expressed at P < 0.05.

Modelling PleafSince the previous analysis revealed the influence of leaf ageon Pmax and F to be significant, the relationships between leafage and Pmax and leaf age and F were then established usingsimple mathematical models. The parameter values of thesemodels were fitted through the maximum likelihood approachusing Proc NLIN in SAS. When species or P supply was notfound to significantly affect Pmax and F at each leaf age, datawere pooled. The changes in gs and Rd over the course of leafdevelopment were modelled in a similar way, following theapproach used by Stirling et al. (1994) for maize.

The way that Pleaf for a horizontally placed leaf (k= 1m2

ground (m2 leaf)–1)) exposed to irradiances of 500, 1000 and1500mmolm–2 s–1 changed over the course of leaf developmentwas estimated using two different methods based on the non-rectangular hyperbola function (Eqn 2) with its three parametersPmax,q andF. In thefirstmethod (the ‘standard’method),F andqwere assumed to be constant (0.035mol CO2 mol–1 quanta and0.91, respectively, calculated after full leaf expansion) and onlyPmax was assumed to change with time. In the second method,both Pmax and F were assumed to change with time, whereas q

was still assumed to be constant (0.91). This was because theprevious analysis had shown thatF and Pmax varied significantlywith leaf development while q did not. We then compared thedifference in estimated Pleaf resulting from the two methods, thatis, from assuming a variable or a constant F.

Simulations were then performed to represent a canopy with aLAI of 4, with angular leaf orientation (k= 0.6). The value of kused here is similar to that derived for broad bean (Vicia faba)(Monteith andUnsworth 1990). The equation proposed byMonsiand Saeki (1953, translated in 2005; Monsi and Saeki 2005) tomeasure the light incident on a leaf (Ileaf) at a depth of L (LAI = L)was used to calculate the light interception by different layers ofthe canopy:

I leaf ðLÞ ¼ I0e�kL; ð3Þ

where I0 is the irradiance on the upper canopy surface, set at1500mmol PPFDm–2 s–1. Once Ileaf (L) was calculated for eachleaf layer, two sets of simulations were performed to estimatePleaf using method 1 (only Pmax varying) andmethod 2 (Pmax andF varying) described above. The percentage difference in Pleaf

resulting from method 1 and method 2 at a given light level for agiven leaf was then calculated. We then compared the estimatedvalues of Pleaf between the two methods for different leaf layersacross all times of leaf development.

Results

Influence of P supply on leaf area and appearance

Leaf area of a single leaf increased up to the 10th nodal positionfrom the first true leaf for Cullen species and up to the 17th nodalposition for M. sativa and then gradually decreased withincreasing node number (Fig. 1). Leaf area of a single leaf wasnot affected by the P supply up to 13th, 17th, 11th and 7th nodalposition for C. australasicum accessions SA4966 and SA42762,C. pallidum and M. sativa, respectively. After these nodalpositions, leaf area of a single leaf decreased at P3 comparedwith P30 for all species. At the time of harvesting, number ofmain-stem nodes per plant (leaf appearance rate) at P3 was lowercompared with that of P30 for all species (Fig. 1).

The expansion of the 8th main stem leaf with age is shown inFig. S1 available as an Accessory Publication to this paper. Leafarea did not differ between P supply for either Cullen species oramongCullen species at any time of leaf development. However,leaf area of the 8th leaf ofM. sativawas smaller than that ofCullenspecies and leaves in the P3 were smaller than those in P30 forM. sativa.

Leaf [P] was lower at P3 than at P30 for all species (seeTable S1 available as an Accessory Publication to this paper).There were no differences in leaf [P] among species at P3.However, at P30, leaf [P] of Cullen species was higher thanthat ofM. sativa. Similarly, total P plant–1 was lower at P3 than atP30 for all species. There were no differences in total P plant–1

among species at either P3 or P30. Plants took up less than 15%ofthe total P present per pot, irrespective of the P treatment andspecies. SLA of M. sativa was almost twice the SLA of Cullenspecies (Table S1). P supply did not change SLA of any species.LAI varied between P treatments and among species. LAI at P3was lower than that at P30 for all the species and LAI ofC. pallidum was lower than that of M. sativa. Leaf [N] of

716 Functional Plant Biology L. D. B. Suriyagoda et al.

Cullen species at the final harvest was lower (0.02 g N g–1 leafDW) than that of M. sativa (0.04 g N g–1 leaf DW), irrespectiveof the P treatment.

Changes in photosynthetic characteristics due to Psupply, species and leaf age

The RM-ANOVA used to test the variability of P1500, gs, Pmax,F and Rd indicated significant effects due to leaf age and

a P supply� species� leaf age interaction. This confirmed theage-dependent changes of P1500, Pmax, F, gs and Rd. Therefore,each response variable was further tested at different leaf agelevels as reported below.

P1500 showed a variable response with leaf age for all speciesat both P3 and P30 (Fig. S2). P1500 increased rapidly withleaf expansion, reached a maximum and remained at a plateauof 22–35mmolCO2m

–2 s–1 at 150�300�C days, followed bya gradual decrease until 0–9mmol CO2m

–2 s–1 at 1518�C daysfor all species. The age at which P1500 reached a peak coincidedwith the time at which a leaf achieved full expansion (Figs S1,S2). The rate of increase of P1500 during early stages of leafdevelopment was higher than the rate of decrease of P1500 at latestages.P1500 did not differ betweenP3 andP30 for a given speciesat any leaf age, except for C. australasicum accession SA42762at 168�C days and beyond 1082�C days. During these periodsC. australasicum accession SA42762 plants maintained a higherP1500 at P3 than at P30. Furthermore, P1500 did not differ amongspecies at a given leaf age.

The pattern of change in gs with leaf age (Fig. 2) was verysimilar to that observed for P1500 (Fig. S2), as gs increasedrapidly, reached a maximum of 0.56–0.82molm–2 s–1 at150–300�C days and then gradually decreased with increasingleaf age up to 0.01–0.14molm–2 s–1 at 1518�C days. There wereno differences in gs between P3 and P30 of a given species oramong species, at a given leaf age, except for C. australasicumaccession SA42762 at 168�C days and beyond 1082�C days,where gs was higher at P3 than at P30.

Photosynthetic water use efficiency (PWUE) of leaveswas constant with leaf age (data not shown). PWUE did notdiffer between P3 and P30 for any species at any leaf age,except for C. australasicum accession SA42762, wherePWUE of P3 plants was higher than that of P30 beyond1082�C days.

The pattern of change in estimated Pmax with leaf age (Fig. 3)was also very similar to that observed for P1500 (Fig. S2), asPmax increased with leaf expansion, reached a maximumof 25–36mmolCO2m

–2 s–1 at 150�300�C days, and thengradually decreased to 0–10mmolCO2m

–2 s–1 at 1518�C days.As was found for P1500 and gs, there was no difference in Pmax

between P3 and P30 for any species or among species, at a givenleaf age, except for C. australasicum accession SA42762 at age0�C days and beyond 1255�C days, where Pmax was higher at P3that at P30. Therefore, the patterns of change for P1500, Pmax andgs with leaf age were very similar.

The estimated values ofFwere used to establish relationshipsbetweenF and leaf age for each species (Fig. 4). The response ofF to leaf age differed between Cullen species and M. sativa.Similar to P1500, Pmax and gs,F of Cullen species increased withleaf age, reached a maximum of 0.029–0.042mol CO2mol–1

quanta at 150�300�C days and gradually decreased up to0.0–0.018mol CO2mol–1 quanta at 1518�C days. However,for M. sativa, such a change of F during the development of aleaf was not observed and F was constant at 0.012–0.036mol CO2mol–1 quanta. There was no difference in Fbetween P3 and P30 for any species, at any leaf age.

The convexity of the PLRC did not change with leafdevelopment and no differences were observed among speciesor between P3 and P30 (data not shown). Therefore, q was

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Fig. 1. Leaf area of all leaves on the main stem after full expansion asdependent on the number of nodes present for (a) Cullen australasicumaccession SA4966, (b) C. australasicum accession SA42762, (c) Cullenpallidum accession SA44387 and (d) Medicago sativa cv. SARDI-10,treated with either 3mg P kg–1 dry soil (*) or 30mgP kg–1 dry soil (*).Bars represent s.e. of the means, n= 3. Note that the scale of the x-axis differsamong species.

Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 717

averaged across all species and P treatments and a value of 0.91was obtained.

Dark respiration (Rd) changed significantly with leaf age(Fig. 5). However, the relationship was different to thatobserved for P1500, Pmax, gs or F. The rate of Rd was higher(4.1–6.9mmolCO2m

–2 s–1) at early developmental stages andgradually decreased to 0.01–1.9mmolCO2m

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Modelling Pleaf considering temporal changesin Pmax and F

Since Pmax, F, gs and Rd did not differ between P3 and P30 andamong species data were pooled in most instances and usedto establish relationships between Pmax, F, gs and Rd with leafdevelopment through simple mathematical models (Fig. 6). Theequations and coefficient values defining the relationship

between Pmax, F, gs, Rd and accumulated degree days aresummarised in Fig. 6.

The sensitivity of simulated Pleaf to varying Pmax andF in thelight response curve with leaf development was calculated usinga non-rectangular hyperbola of CO2-assimilation rate (Eqn 2)(Thornley and Johnson 2000). Figure 7a describes therelationship between simulated Pleaf and leaf age based onvariable Pmax (equation in Fig. 6a) and fixed convexity (0.91)together with either fixed (0.035mol CO2 mol–1 quanta) orvariable F (equation in Fig. 6b) at an incident radiation of1500, 1000 or 500mmolm–2 s–1 and when k= 1. Comparingthe three light levels, simulated Pleaf at 1500mmolm–2 s–1 wasthe highest and Pleaf at 500mmolm–2 s–1 was the lowest, for both

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Fig. 2. Effect of leaf age on stomatal conductance (gs) atPAR= 1500mmolm–2 s–1 for (a) Cullen australasicum accession SA4966,(b) C. australasicum accession SA42762, (c) Cullen pallidum accessionSA44387 and (d) Medicago sativa cv. SARDI-10, treated with either3mgP kg–1 dry soil (*) or 30mgP kg–1 dry soil (*). Bars represent s.e.of the means, n= 3.

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Fig. 3. Effect of leaf age on estimated maximum leaf photosynthesis (Pmax)for (a) Cullen australasicum accession SA4966, (b) C. australasicumaccession SA42762, (c) Cullen pallidum accession SA44387 and(d) Medicago sativa cv. SARDI-10, treated with either 3mg P kg–1 dry soil(*) or 30mgP kg–1 dry soil (*). Bars represent s.e. of the means, n= 3.

718 Functional Plant Biology L. D. B. Suriyagoda et al.

constant and variableF (Fig. 7a), as expected. For any given lightlevel, Pleaf simulated using variable F was lower compared withPleaf simulated using a fixed F across all stages of leafdevelopment. Also, the % reduction in Pleaf was higher atlower PPFD. During the time course of leaf development, this% reduction was very high during early leaf expansion and withincreasing leaf age after full expansion (Fig. 7b). When averagedacross the whole period of leaf development, the values of Pleaf

estimated using variable F were 3, 7 and 19% lower than thevalues of Pleaf estimated using fixed F, at 1500, 1000 and500mmol PPFDm–2 s–1, respectively.

The relationship between Pleaf and leaf age in different leaflayers of a closedcanopywas also compared forfixedandvariable

F. According toEqn3, radiation intercepted bydifferent layers ofa canopy of LAI = 4, when Io = 1500mmolm–2 s–1 and k= 0.6were 677, 371, 204 and 112mmolm–2 s–1 for the four layers, fromtop to bottom, respectively. Those intercepted light intensitieswere used for the simulation of Pleaf with leaf development atdifferent leaf layers with a variable Pmax (equation in Fig. 6a)together with either fixed (0.035mol CO2mol–1 quanta) orvariable F (equation in Fig. 6b). Convexity was set to 0.91.As expected, Pleaf was highest for the uppermost leaf layer andgradually decreasedwith increasing depth in the canopy (Fig. 8a).For a given leaf layer, Pleaf at variable F was lower than that ata fixed F at any given time of the leaf development and thisreduction was higher at a very early period of leaf expansion andwith increasing leaf age after full leaf expansion. Furthermore,% reduction of Pleaf estimated using variable F compared with

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Fig. 4. Effect of leaf age on estimated quantum yield (F) for (a) Cullenaustralasicum accession SA4966, (b) C. australasicum accession SA42762,(c) Cullen pallidum accession SA44387 and (d) Medicago sativa cv.SARDI-10, treated with either 3mg P kg–1 dry soil (*) or 30mgP kg–1

dry soil (*). Bars represent s.e. of the means, n= 3.

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Fig. 5. Effect of leaf age on dark respiration (Rd) for (a) Cullenaustralasicum accession SA4966, (b) C. australasicum accessionSA42762, (c) Cullen pallidum accession SA44387 and (d) Medicagosativa cv. SARDI-10, treated with either 3mgP kg–1 dry soil (*) or30mg P kg–1 dry soil (*). Bars represent s.e. of the means, n= 3.

Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 719

using fixedFwas lower for upper leaf layers than for deeper leaflayers (Fig. 8b). When averaged across the period of leafdevelopment, these % reductions were 13, 24, 31 and 32%, forthe four layers, from top to bottom, respectively.

Discussion

Even though numerous studies have reported the age dependentchanges of leaf photosynthetic characteristics, most studies havelooked at only one or two parameters at a time (mostly P1500 andPmax) or were limited to a narrow leaf developmental period(e.g. only after full leaf expansion or only during initialexpansion). In addition, the influence of P on Pmax, F and q isnot known. Therefore, we studied the age dependent changes ofthe photosynthetic characteristics P1500, Pmax,F, q, gs and Rd forthree pasture species during the whole leaf development periodunder two levels of P supply (low and optimum).We now discussour results and the implications of age dependent changes of leafphotosynthetic characteristics for simulating Pleaf.

Low P did not reduce photosynthetic characteristicsat any leaf age

P1500, Pmax, F, q, gs and Rd were not lower at P3 than at P30except for C. australasicum accession SA42762, and thus thehypothesis that low P supply would reduce the rate ofphotosynthetic characteristics of Cullen species and M. sativaat any given leaf age was not supported. The reduction in P1500,Pmax, and gs at P30 for C. australasicum accession SA42762might be an adaptive response to conserve moisture or due toP toxicity. We believe that, since plants at P30 had a higher LAIthan at P3 (Table S1), they experienced higher water loss throughtranspiration, and therefore partial closure of stomata might havereduced the photosynthetic rate. This conclusion is supportedby the higher PWUE of C. australasicum accession SA42762 atP30 compared with that at P3 (data not shown). Reduction ofphotosynthetic rate during P toxicity is highly correlated with areduction in plant growth (Shane et al. 2004). Even though thephotosynthetic rate was reduced at P30 for C. australasicumaccession SA42762, dry weight was higher at P30 than at P3 andleaf [P] at P30 was similar to that of other species in theexperiment. Indeed, leaf [P] for Cullen species and M. sativaat P30 was much lower than reported for many native Australianspecies and crop plants during P toxicity (e.g. Shane et al. 2004;Pang et al. 2010). Therefore, it seems the reduction inphotosynthetic rates soon after full leaf expansion and duringthe late leaf developmental stages of C. australasicum accessionSA42762 treated with P30 was due to limitation in CO2 fluxcaused by partial closure of stomata (Jacob and Lawlor 1991) not

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Fig. 6. Fitted (*) and observed (*) values of (a) maximumphotosynthesis (Pmax), (b) quantum yield (F), (c) stomatal conductance(gs) and (d) dark respiration (Rd) with leaf age. Equations and coefficientsfor the fitted relationships between parameters of the photosynthetic lightresponse curve with leaf age (X – �C days) are also shown. Observed valuesrepresent measured values from all the species.

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Fig. 7. (a) Simulated leaf photosynthesis (Pleaf) with leaf age for an incidentradiation of 1500mmol PPFDm–2 s–1 (triangles), 1000mmol PPFDm–2 s–1

(squares) and 500mmol PPFDm–2 s–1 (circles) with either fixed F(closed symbols) or variable F (open symbols). Simulated maximum leafphotosynthesis (Pmax) is shown as (+) symbols. (b) Estimated % reduction ofPleaf at variableF compared with that of fixedFwith leaf age for an incidentradiation of 1500mmol PPFDm–2 s–1 (triangles), 1000mmol PPFDm–2 s–1

(squares) and 500mmol PPFDm–2 s–1 (circles).

720 Functional Plant Biology L. D. B. Suriyagoda et al.

P toxicity. Thus, overall, our hypothesis that Cullen species andM. sativa would show reduced P1500, Pmax, F, q, gs and Rd atlow P supply at a given leaf age was not supported. This mightbe due to several reasons.

One possible reason is that the lowest [P] of 3mg P kg–1 drysoil used in this study might not have been low enough tosignificantly reduce photosynthetic rate (Khamis et al. 1990;Jacob and Lawlor 1991). However, in a P response study Panget al. (2010) found that both C. australasicum and M. sativaincreased growth until soil [P] reached to 24mg P kg–1 dry soil.Therefore, P3 and P30 in the present study represent low andoptimum supply of P for the growth of C. australasicum andM. sativa, respectively. It is also known that leaf growth under Pdeficiency is reduced before any reduction inPleaf (Rao and Terry1989; Jacob and Lawlor 1991; Plénet et al. 2000b). During theinitial growth stages of the present experiment, leaf size did notdecrease for any species at P3, suggesting that P was not limitingfor the initial growth, even though the growthwas reduced later atP3 (Fig. 1). Thismeans that the photosynthetic rates of the 8th leafmight not have been affected by P3. Even though the canopy [P]wasmeasured, itwas not possible tomeasure the [P] of the 8th leafdue to its small size. Therefore, [P] of the 8th leaf was not knownand it may not have differed between P3 and P30. A secondpossible reason is that low light intensity might have limited Pleaf

more than the low P treatment. As a leaf develops, its position inthe canopy changes and it often becomes shaded by the upper

leaves (e.g. estimated intercepted radiation was 112mmolm–2 s–1

at the 4th layer at the bottom of the canopy, while that at the topleaf layer was at 677mmolm–2 s–1). de Groot et al. (2003) alsofound that difference in Pleaf of low- and high P treatedS. lycopersicum plants reduced more for shaded leaves thanfor unshaded leaves. A third possible reason is that total LAI,leaf size and leaf number at harvest was lower for P3 than for P30.Therefore, the8th leaf ofP3 treatedplantsmight haveexperienceda higher light intensity than that at P30 and thereby acclimated toa higher Pleaf than we expected (Lambers et al. 2008). Furtherresearch may identify upper and lower threshold [P] at whichphotosynthesis is affected in these species and, thus, determine ifthere are important differences in response between native andexotic species, and among native species.

Leaf photosynthetic characteristics changed with leaf age

Our second hypothesis stated that a substantial change in P1500,Pmax, F, gs and Rd would occur in Cullen species and M. sativaduring leaf development. P1500 and Pmax showed a tri-phasicresponse to leaf age, with an initial rapid increase in values toreach maxima across a short plateau, followed by a near-lineardecrease with leaf age (Fig. 3, S2), which supported ourhypothesis. The leaf age at which these rates achieved amaximum coincided with the time at which a leaf achievedfull expansion. Similar results have been obtained by Stirlinget al. (1994) and Boedhram (1998) for other crop species. Thisreduction in Pmax after full leaf expansion (down the canopy)is functional if resources such as photosynthetic leaf N areinvested at the best light environment for photosynthesis(Charles-Edwards 1981; Evans and Seemann 1989; Sands1995), thereby achieving the highest Pmax per plant. Thetemporal change in Pmax observed in the present studysupports our second hypothesis and is in agreement withfindings for several other crop species (Marshall and Biscoe1980; Leech and Baker 1983; Dwyer and Stewart 1986;Stirling et al. 1994).

Even though the temporal change inPmax has been extensivelystudied formanycrops, there is a lackof literature on the changeofF and q with the complete leaf developmental period. In thecurrent study the response of F to increasing leaf age for Cullenspecies was similar to that observed forPmax (r= 0.46,P< 0.001)and supported our second hypothesis. The peak estimates of Fobtained at full leaf expansion in the current study were similar toobservations by Field and Mooney (1983), Dwyer and Stewart(1986), Stirling et al. (1994) and Singsaas et al. (2001) for severalother crop species. However, in contrast with these responses,a constant value of F with leaf age has also been observed(Field and Mooney 1983; Stirling et al. 1994). This constant Fresponsewas the same as that observed forM. sativa in the presentstudy, which did not support our second hypothesis. Reportssuggest thatF can vary due to environmental factors such as lightintensity and temperature (Groom et al. 1991), season (Field andMooney 1983; Niinemets et al. 2004), and leaf traits such as leaftransmittance (Ehleringer and Björkman 1977; Osborne andGarrett 1983). Stirling et al. (1994) suggested that limitation instomatal conductance, biophysical processes of energy transferand electron transport can also change F. However, the patternsof change of gs with leaf development for Cullen species and

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Fig. 8. (a) Simulated leaf photosynthesis (Pleaf) with leaf age in a closedcanopy with LAI = 4, for an intercepted radiation of 677mmol PPFDm–2 s–1

at the upper layer (circles), 371mmol PPFDm–2 s–1 at the second layer(squares), 204mmol PPFDm–2 s–1 at the third layer (diamonds) and112mmol PPFDm–2 s–1 at the fourth layer (triangles) with either fixed F(closed symbols) or variableF (open symbols). (b) Estimated% reduction ofPleaf at variable F compared with that of fixed F with leaf age for Fig. 8aat the upper layer (circles), second layer (squares), third layer (diamonds) andthe fourth layer (triangles).

Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 721

M. sativawere similar (Fig. 2). The relationship between gs andFwas significant for Cullen species (r= 0.41, P < 0.05), but notfor M. sativa (r= 0.18, P > 0.05). Therefore, our observation forM. sativa was not in agreement with the suggestion by Stirlinget al. (1994) that stomatal limitation affects the change inFwithleaf age. We believe that the different response of F of Cullenspecies and M. sativa to leaf age might be due to limitations inbiophysical processes of energy transfer and electron transportcaused byN nutrition. Leaf [N] reflects the amount of the primarycarboxylating enzyme in C3 plants (Friedrich andHuffaker 1980;Kitajima et al. 2002). In the present study, leaf [N] of Cullenspecies at the final harvest was lower than that of M. sativa andthis was partly associated with a lower SLA of Cullen speciesthan that of M. sativa. This lower leaf [N] might have beenassociated with a reduced carboxylating capacity in Cullenspecies with leaf age, resulting in a lower value of F.However, further investigation is required to establish such arelationship.

Even thoughPmax andF changedwith leaf age supporting oursecond hypothesis, qwas unchanged. The range ofq observed forCullen species andM. sativa in this study (0.75–1.0) was similarto that observed by Stirling et al. (1994) and Lizaso et al. (2005)for other crop species. The fact that we found q unchanging withleaf development during the current study might be due to thevery large variability of q observed among leaves for a givenspecies� P treatment combination and the ability of the other twoparameters to account for much of the change in the shape of thePLRC.

A decline in Pmax after full leaf expansion was highlycorrelated with the decline in Rd (r= 0.73, P < 0.001). LowerRd at lowerPmax is expected to be duemainly to lower respiratorycosts involved in sugar export (Bouma et al. 1995). After fullexpansion of a leaf, the ratio ofRd toPmaxwas~0.10, in agreementother published results of 0.05–0.12 (Angus and Wilson 1976,Hordeum vulgare L. (barley); Marshall and Biscoe (1980);Stirling et al. (1994), T. aestivum; Noguchi et al. 1996;Z. mays, Spinacia oleracea L. (spinach) and Colocasiaesculenta L. (Asian taro)). However, as leaf age increased, theratio tended to increase, due to a greater reduction of thephotosynthetic capacity of leaves as a result of leaf senescence.

In the present study, gs also showed a tri-phasic response withleaf age, supporting our second hypothesis and was highlycorrelated with P1500 (r= 0.92, P < 0.001) and Pmax (r = 0.89,P < 0.001). Similar responses of gs with leaf age have beenobserved for maize (Dwyer and Stewart 1986). However,PWUE was unchanged with leaf age (data not shown), due toproportional reduction of P1500 and gs as also found by Field andMooney (1983) and Shirke (2001). Lower gs and PWUE areoften, but not always, observedwith leaf aging (Field andMooney1983; Witkowski et al. 1992; Kitajima et al. 2002).

Estimates of Pleaf were affected by changesof F with leaf age

The results of the present study indicate that the parameters ofthe light response curve are subject to change with leaf age.Therefore, photosynthetic capacity of a leaf varies with leafage, or for leaves at different depths of a canopy, at a giventime. Since peak values of P1500, Pmax, gs andF were associated

with full leaf expansion, it is reasonable to use leaves at fullexpansion for comparison purposes. However, extrapolationof leaf photosynthetic rates at full expansion to different leaflayers of a plant requires consideration of light microclimate(proportional Pmax to local irradiance), leaf characteristics(leaf age and N status) and plant age (Charles-Edwards 1981;Sands 1995, 1996; Hollinger 1996). This is why it was importantto test our third hypothesis: that taking into consideration theway that estimated changes of Pmax, F and q with leaf age willreduce estimates of Pleaf. This has direct relevance to modellingthe growth of these species as pastures. It may be particularlyimportant when modelling the effects of grazing, where youngerleaves higher in the canopy will be removed first and the leavesmost likely to remain will be the older and less efficient ones.

In the present study, lower estimates of Pleaf at variable Fcompared with a fixed F, for a horizontally placed leaf(k= 1), under different light intensities (1500, 1000 and500mmolm–2 s–1, Fig. 7a, b) showed that using a fixed Fresults in overestimates of Pleaf at any given time and thatgreater over-estimation of Pleaf occurs at lower light intensities(500mmolm–2 s–1 compared with 1500mmolm–2 s–1). Similarly,when considering a canopy with an angular leaf orientation(k= 0.6) the % reduction of Pleaf at variable F compared witha fixedF was greater for deeper leaf layers and further increasedduring very early expansion andwith leaf age after full expansion(Fig. 8a, b). Therefore, when Pmax and F were considered asfunctions of leaf age and treated as variables in the PLRC,different estimates of Pleaf resulted. Our results show thatwhen using a fixed-parameters approach in actual stand-levelmodelling, overestimation of Pleaf could occur when the LAI isvery high, if the stand is mature (during later growth stages of thepasture stand or at dormancy), soon after grazing (most of themature leaves are still retained but younger more efficient leavesremoved), during cloudy days comparedwith sunny days, duringwinter compared with summer, for shade-grown stands(understory crops or pastures), at higher plant densities and onfertile soils (higher growth rate).

The q parameter did not change with leaf age in the presentstudy. Therefore, the impact of a variable q on Pleaf could not bestudied to test our hypothesis. In cases where q is found to changewith leaf development, the impact of a variable q on Pleaf shouldbe investigated and accounted for in simulation models in orderto estimate Pleaf more accurately.

P affected leaf area and leaf appearance in M. sativacompared with Cullen species

Leaf size andappearance rate ofCullen species andM.sativa at P3were reduced compared with P30 with the response forM. sativabeing much quicker than for the Cullen species, which supportsour hypothesis. Reduced leaf size at low P supply has beenobtained in previous studies for other crop and tree species(Kirschbaum et al. 1992; Plénet et al. 2000a). The lower leaf[P] at harvest for P3 compared with P30 for all the speciesindicates that decreased leaf area and leaf appearance at P3were due to lower availability of P. Furthermore, the decline inthe leaf area was earlier forM. sativa (at 7th nodal position) thanforCullen species (after 11th nodal position). Possible reasons forthe earlier response ofM. sativa could include a higher sensitivity

722 Functional Plant Biology L. D. B. Suriyagoda et al.

to low P supply or a faster decline of P reserves due to a highergrowth rate. However, in this study all species took up less than15%of the total P available in thepot.Also, sinceM.sativadidnothave ahigher growth rate than theCullen species at P3, thedeclinein leaf area and leaf appearance ofM. sativa at P3 compared withthe Cullen species was not due to the lower availability of P, butwas due to a higher sensitivity ofM. sativa to P supply. Leaf sizeand appearance rate are important drivers in models predictingphotosynthesis of whole plants and canopies and it would,therefore, be important to include the observed differences dueto P supply in such models when predicting and comparingthe productivity of Cullen species and M. sativa in low Penvironments.

Conclusions

P3 did not reduce the photosynthetic rate for any of the species ata given leaf age compared with P30.Pmax and gs of all the speciesandF of Cullen species increased until full leaf expansion, weresteady for ~150�C days, and then gradually decreased withincreasing leaf age. Rd decreased with leaf age for all specieswhileF ofM. sativa and q of all species did not change with leafage. When Pmax and F were considered as functions of leaf ageand treated as variables when estimating the temporal changes ofPleaf, estimates of Pleaf obtained using variable F were lowerthan those obtained using a fixed F at any given leaf age. The% reduction ofPleaf was higher at lower light intensities aswell asduringvery early andvery late stages of leaf growth.Both leaf sizeand rate of leaf appearance were reduced at P3 for all the species,while the response for M. sativa was considerably faster.Therefore, it is important to incorporate the effects of leaf ageand light intensity on photosynthetic parameters into carbon-assimilation models for these particular species. More generally,these results indicate that it is important to consider such effectswhen modelling the performance of any plant species.

Acknowledgements

We thank two anonymous referees for their valuable suggestions, whichgreatly improved the original manuscript. This study was supported bythe School of Plant Biology and the Future Farm Industries CooperativeResearch Centre, The University of Western Australia. LDB Suriyagodaalso appreciates the SIRF/UIS Scholarship awarded by the University ofWestern Australia and further scholarship support from the late Frank Ford.

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Manuscript received 21 November 2009, accepted 1 April 2010

Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 725

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