This document describes a study that examined the effects of leaf age and phosphorus supply on the photosynthetic characteristics of three perennial legume species - Medicago sativa, Cullen australasicum, and Cullen pallidum. The study found that maximum photosynthetic rate and stomatal conductance increased with leaf age until full expansion, then decreased, while dark respiration decreased with leaf age. Phosphorus supply affected leaf area and emergence rate but not photosynthetic parameters. Modelling changing photosynthetic parameters over leaf age reduced estimated leaf photosynthesis compared to assuming constant parameters.

1. CSIRO PUBLISHING
www.publish.csiro.au/journals/fpb Functional Plant Biology, 2010, 37, 713–725
Effects of leaf development and phosphorus supply
on the photosynthetic characteristics of perennial legume
species with pasture potential: modelling photosynthesis
with leaf development
Lalith D. B. Suriyagoda A,B,C,E, Hans Lambers A, Megan H. Ryan A,B and Michael Renton A,B,D
A
School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia.
B
Future Farm Industries Cooperative Research Centre, The University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia.
C
Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka.
D
CSIRO Sustainable Ecosystems, Floreat, WA 6014, Australia.
E
Corresponding 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) and
dark respiration rate (Rd)) of an exotic perennial legume, Medicago sativa L. (lucerne), and two potential pasture legumes
native to Australia, Cullen australasicum (Schltdl.) J.W. Grime and Cullen pallidum A. Lee, grown in a glasshouse for
5 months at two phosphorus (P) levels (3 (P3) and 30 (P30) mg P kg–1 dry soil) were tested. Leaf appearance rate and leaf area
were lower at P3 than at P30 in all species, with M. sativa being the most sensitive to P3. At any leaf age, photosynthetic
characteristics did not differ between P treatments. However, Pmax and gs for all the species and F for Cullen species increased
until full leaf expansion and then decreased. The convexity parameter, q, did not change with leaf age, whereas Rd decreased.
The estimates of leaf net photosynthetic rate (Pleaf) obtained through simulations at variable Pmax and F were lower during
early and late leaf developmental stages and at lower light intensities than those obtained when F was assumed to be constant
(e.g. for a horizontally placed leaf, during the 1500C days developmental period, 3 and 19% reduction of Pleaf at light
intensities of 1500 and 500 mmol m–2 s–1, respectively). Therefore, developmental changes in leaf photosynthetic
characteristics 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 light-saturation (Constable and Rawson 1980; Dwyer and
Photosynthesis simulation models can be used to explore Stewart 1986; Heschel et al. 2004; Fletcher et al. 2008). Yet,
potential plant growth. For example, we want to use such we know that leaf net photosynthetic rate (Pleaf) first increases
models to predict the productivity of exotic and native with leaf age and then decreases (Smillie 1962; Pisum sativum L.
Australian perennial legume species with pasture potential in (pea); Peat 1970; Solanum lycopersicum L. (tomato); Dwyer and
the low phosphorus (P) soils under both current climates and Stewart 1986; Stirling et al. 1994; Zea mays L. (maize); Suzuki
predicted climate change scenarios. However, the usefulness of et al. 1987; Triticum aestivum L. (wheat); Kitajima et al. 2002;
such photosynthesis models will depend on accurately predicting Cecropia and Urera species; Xie and Luo 2003; Pyrus serotina
leaf area and emergence rate and on accurately incorporating leaf Rehd. (Asian pear); Niinemets et al. 2004; Quercus spp. (oak)).
photosynthetic characteristics and the way they change with leaf These age-dependent changes in Pleaf could be due to changes
age and with differences in nutrient supply (Pearcy and Sims in one or more parameters of the photosynthetic light response
1994; Stirling et al. 1994; Kitajima et al. 2002). curve (PLRC).
Leaves in a canopy/sward experience a wide fluctuation in The PLRC is defined by three parameters: (i) the quantum
incident PPFD and as leaves get older they are likely to be yield of CO2 assimilation (F), derived from the slope of the
positioned lower in the canopy. Most of the published initial linear region of the response of CO2 uptake to PPFD;
measurements of the rate of leaf photosynthesis have been (ii) the upper asymptote, representing the light-saturated rate of
made only on leaves soon after full leaf expansion and at CO2 assimilation (Pmax) and (iii) the convexity coefficient (q)
Ó CSIRO 2010 10.1071/FP09284 1445-4408/10/080713

2. 714 Functional Plant Biology L. D. B. Suriyagoda et al.
describing the curvature of the response between (i) and Terry 1989; de Groot et al. 2003) and when plants were exposed
(ii) (Thornley and Johnson 2000). Where detailed studies of to excess P causing P toxicity (Shane et al. 2004), the influence of
the PLRC have been undertaken, measurements have not low P supply on F and q has not been reported for any plant
encompassed the early stages of leaf development (Marshall species.
and Biscoe 1980; Kitajima et al. 2002; Fletcher et al. 2008) or Medicago sativa L. (lucerne) is one of the most widely
reported varying results/trends with leaf age. For example, cultivated perennial pasture legumes in Mediterranean regions
only Pmax was found to change with leaf age in Z. mays and has long been grown in fertile and moist environments in
(Boedhram 1998), Lepechinia calycina (Benth.) Epl. (Field Asia (Griffiths 1949; Small 2009). However, Australian perennial
and Mooney 1983) and Cecropia and Urere spp. (Kitajima legumes are also considered to have potential for development
et al. 2002), whereas only Pmax and F changed in Z. mays as alternative pasture species (Dear et al. 2007; Robinson et al.
(Dwyer and Stewart 1986) and only Pmax and q changed in 2007). In particular, the genus Cullen has been identified as
Z. mays (Stirling et al. 1994). It has been suggested that the containing several potential perennial pasture species (Cocks
differences reported above might be due to environmental stress 2001; Dear et al. 2007; Li et al. 2008). One advantage of
during the growth period, seasonal environmental changes or native pasture legumes is their adaptation to local conditions.
to errors in measuring these parameters (Sands 1996; Singsaas For example, many of them have evolved in local,
et al. 2001). However, it seems likely that the photosynthetic P-impoverished environments (Beadle 1966; Handreck 1997).
characteristics (i.e. parameters of the PLRC, stomatal Uptake of P is poorly regulated at elevated P availability in some
conductance (gs) and dark respiration (Rd)) will change during Australian species (Shane et al. 2004) and many native species
leaves aging and that these changes will have important are sensitive to P toxicity (Handreck 1997; Shane et al. 2004).
implications for estimating Pleaf and, thus, for simulations of Simulation models could be used to predict and compare
plant growth. the productivity of M. sativa and Cullen in the low P soils
Most models of canopy photosynthesis that are used to commonly found in Australia, under both current climates
estimate the growth of plants under diverse environmental and under predicted climate change scenarios. However, as
conditions are based on the assumption that the parameters argued above, the accuracy of such models will depend on
defining the PLRC are the same for all leaves and that accurate representation of morphological (e.g. leaf growth) and
variation in Pleaf with canopy depth arises solely from physiological (e.g. photosynthetic) characteristics of these
differences in incident PPFD (Stirling et al. 1994; Kitajima species under the relevant conditions. As yet, the changes in
et al. 2002). This is an over-simplification, since leaf age also photosynthetic characteristics with leaf age and their interaction
contributes significantly to variation in Pleaf. However, Charles- with P supply are not available for either M. sativa or Cullen
Edwards (1981; p. 70, eqns 3.14 and 3.15) assumed that Pmax of species.
a leaf is proportional to local irradiance and Thornley (2002, Therefore, the objectives of this study were to: (i) compare
2004) used this approach to calculate canopy photosynthesis and the effects of two levels of P supply on leaf area, emergence rate,
also correlated the ratio of Pmax and local PPFD with canopy photosynthetic characteristics and respiration for two perennial
nitrogen (N) distribution. Apart from this approximation of legumes native to Australia, C. australasicum and C. pallidum
Pmax, possible changes in, q and F have not been used in A. Lee, and an exotic perennial legume, M. sativa; (ii) study
models for leaves at different depths in the canopy and these how the photosynthetic characteristics change with leaf age
changes have not been accounted for when modelling Pleaf, and whether this is influenced by [P]; and, (iii) incorporate
for any species. Thus, to date, q and F have been considered any significant changes in the PLRC parameters into
as constants when calculating Pleaf and in simulating canopy simulation models of leaf photosynthesis in order to compare
photosynthesis; the estimates of q and F generally being obtained the resulting estimates of Pleaf with those resulting from the
from leaves immediately after full expansion. This approach standard approach of assuming F and q to be constants. We
simplifies the model but fails to incorporate important hypothesised that: (i) reduced P supply would result in a reduced
information. Recently, Kitajima et al. (2002) concluded that leaf size, leaf appearance rate, leaf photosynthetic rate at
leaf age is a more reliable predictor of Pleaf than % PPFD or 1500 mmol m–2 s–1 (P1500) and also reduced values of Pmax, F,
leaf N concentration. Therefore, there is a need to study the q, gs and Rd at any leaf age; (ii) for a given P supply, the three
behaviour of photosynthetic characteristics with leaf age and parameters of the PLRC would all increase over time until the leaf
incorporate those changes into simulation models in order to was fully expanded and then decrease with increasing leaf age
predict Pleaf more accurately. and (iii) modelling Pmax, F and q as changing over the course of
Worldwide, P is one of the most limiting mineral nutrients leaf age would greatly reduce the estimates of Pleaf compared with
for plant productivity; future global food security is questioned the standard approach of modelling F and q as constants.
due to the decreasing quality of P fertilisers and increasing
production costs (Schachtman et al. 1998; Vance et al. 2003;
Materials and methods
Cordell et al. 2009). P deficiency can reduce sink activity, solar
radiation interception (Plénet et al. 2000b; Rodríguez et al. 2000; Growth conditions
Pieters et al. 2001; de Groot et al. 2003) and Pleaf (Jacob and Morphological and physiological responses of three legume
Lawlor 1992; Chiera et al. 2002; Ghannoum and Conroy 2007), species were assessed by growing plants in pots with different
and thereby restrict plant growth. Even though several studies P supplies. Cullen australasicum (Schltdl.) J.W. Grime,
have demonstrated a reduction in Pleaf and Pmax when plants were accessions SA4966 (late flowering) and SA42762 (early
under severely P limiting conditions (Fredeen et al. 1989; Rao and flowering), Cullen pallidum A. Lee accession SA44387 and

3. Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 715
Medicago sativa L. cv. SARDI-10 were grown in 1 m tall, 10 cm Growth measurements and plant analysis
diameter pots. Seeds of C. australasicum and C. pallidum were Leaf area of the 8th main stem nodal leaf was measured by
collected from the seed-multiplication plots established at the drawing the leaf area on a piece of white paper at 1-week intervals
Shenton Park field station of the University of Western Australia until full expansion. Those leaf measurements were used to
and M. sativa seeds from the Genetic Resource Centre at correlate leaf development/expansion with leaf photosynthetic
the South Australian Research and Development Institute. The characteristics. Plants were harvested 5 months after germination.
experiment included two P treatments (supply): P3 (3 mg P kg–1 At the time of final harvest, the number of main stem nodes per
dry soil) and P30 (30 mg P kg–1 dry soil). Recently in a P response plant, excluding the cotyledonary node, was recorded. Main stem
study Pang et al. (2010) reported both M. sativa and nodal counts observed at the time of final harvest were used to
C. australasicum achieved a maximum growth (dry weight) at compare species and P treatments and were expressed as the leaf
24 mg P kg–1 dry soil. Therefore, P3 and P30 used in the current appearance rate during the growth period. Leaves were detached
study represent a low and an optimum P supply, respectively, for from each plant and used to measure the leaf area of the main stem
both species. Three replicate pots of each species  P leaves and total plant leaf area with a Li-Cor LI-3000 portable
combination were established. Pots were filled with 12 kg pot–1 area meter, which was equipped with LI-3050A transparent
of thoroughly washed, steam-sterilised river sand. Base soil [P] belt conveyer accessory (Li-Cor Inc.). The first few leaves that
was 1–2 mg P kg–1 sand and pH (CaCl2) was 6.5, as determined dropped from the main stem before the final harvest were
by CSBP FutureFarm analytical laboratories, Bibra Lake, WA, collected and used for leaf area measurements. Leaf area
Australia. All essential nutrients other than P were provided by represents the area of all three leaflets of a single trifoliate
amending the sand with (in mg kg–1) 126.6 Ca(NO3)2.4H2O, 42.8 leaf. Leaves were dried at 70C for 1 week and weighed.
NH4NO3, 178 K2SO4, 101 MgSO4.7H2O, 11 CaCl2.2H2O, 12 Measurements were used to estimate the specific leaf area
MnSO4.H2O, 8.8 ZnSO4.7H2O, 1.96 CuSO4.5H2O, 0.68 H3BO3, (SLA) of individual plants. Leaf, stem and root [P] of each
1.01 NaMoO4.2H2O and 32.9 FeNaEDTA. P was supplied as plant were measured separately. A subsample of ~100 mg was
KH2PO4, with different concentrations for different treatments, taken and digested in nitric/perchloric acid and then analysed
as given above. Additional K was supplied in the P3 treatment as using the molybdo-vanado-phosphate method (Kitson and
KCl to balance K. Pots were watered from the top using a wick Mellon 1944). Leaf [P] is expressed on a leaf area basis
system, which maintained the soil profile moisture throughout the ([P]area). Leaf, stem and root [P] and DW of each component
experiment at 60–75% field capacity. The experiment was set out were used to calculate the total P content per plant. Leaf [N] was
in a glasshouse at the University of Western Australia, Perth determined by dry combustion (Nelson and Sommers 1996)
(31590 S, 115530 E) as a complete randomised design at a density using an elemental CN analyser (Elementar Analysensysteme
of 12 pots m–2; pots being randomised at weekly intervals. Given GmbH, Hanau, Germany).
the wide spacing of the pots, no guard row pots were used. Three The concept of thermal time (C days) was used as a
seedlings were planted in each pot and thinned to one plant per measure of leaf age. Cumulative thermal time (leaf_age) was
pot at 2–3 weeks. After week 6, 300 mL of 2 mM NH4NO3 calculated:
was applied weekly to ensure an adequate nitrogen supply X
(Denton et al. 2006). The glasshouse was unheated and had an leafÀ age ¼ ðT À T b Þ; ð1Þ
average daytime temperature of 23C during the experiment,
which was conducted from May to September 2008. where, T and Tb are daily mean and base temperatures,
respectively. The base temperature for M. sativa (5C) (Fick
et al. 1988) was used for all species. The day at which the first
Photosynthetic measurements photosynthetic measurements were taken was considered as the
Leaf photosynthetic measurements were taken from the 8th 0C day.
nodal leaf, counted from the base of the main stem excluding
the cotyledonary node. Pleaf, gs and Rd were measured with a Effects of P supply and species on leaf characteristics
portable gas-exchange system (LI-6400 portable, Li-Cor Inc., The significance of differences in leaf area (per plant and different
Lincoln, NE, USA) equipped with a light source (6400–02B nodal positions on the main stem), SLA and leaf [P] due to P
LED, Li-Cor Inc.) at 7–10 day intervals until harvest. supply and species were tested with Proc GLM for ANOVA in
Measurements began 7–8 days after the leaf appeared and SAS (SAS Institute Inc., Cary, NC, USA). Leaf area measured at
were made at solar noon Æ2 h. Photosynthetic rates at nine the 8th nodal leaf until full expansion was also tested using Proc
levels of PPFD were measured on selected leaves using the GLM. Means were separated with the LSMEANS procedure.
‘auto light curve’ program with a minimum time of 120 s and All significant differences were expressed at P 0.05. Leaf
a 3% coefficient of variation as the trigger to move to the appearance rates were compared using categorical analysis
next PPFD setting. The light-response curves started at (Proc CATMOD in SAS).
500 mmol m–2 s–1, followed by 2000 mmol m–2 s–1, then back to
500 mmol m–2 s–1 and finally 0 mmol m–2 s–1. This allowed a rapid
stabilisation of photosynthesis at each PPFD level. Standard Estimating the parameters of the PLRC
conditions of 380 mmol mol–1 CO2 and 25C were maintained Relationships between Pleaf and irradiance (Ileaf) for each leaf and
within the leaf chamber. Ambient RH of the incoming air to the for each sample date, were modelled with the non-rectangular
leaf chamber was left at that of the glasshouse environment hyperbola (Thornley and Johnson 2000) described in Eqn 2, using
(30–55%). Proc NLIN in SAS, with the Gauss–Newton method. Parameter

4. 716 Functional Plant Biology L. D. B. Suriyagoda et al.
estimates and 95% confidence intervals were obtained for Pmax, was still assumed to be constant (0.91). This was because the
F and q. previous analysis had shown that F and Pmax varied significantly
with leaf development while q did not. We then compared the
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi difference in estimated Pleaf resulting from the two methods, that
1 is, from assuming a variable or a constant F.
Pleaf ¼ ðFI leaf þPmax ÞÀ ðFI leaf þPmax Þ2 Àð4FqI leaf Pmax Þ:
2q Simulations were then performed to represent a canopy with a
ð2Þ LAI of 4, with angular leaf orientation (k = 0.6). The value of k
used here is similar to that derived for broad bean (Vicia faba)
Analysis of repeatedly measured photosynthetic data
(Monteith and Unsworth 1990). The equation proposed by Monsi
A repeated-measure ANOVA (RM-ANOVA) was used to test and Saeki (1953, translated in 2005; Monsi and Saeki 2005) to
whether the estimated parameters of PLRC, P1500, gs and Rd measure the light incident on a leaf (Ileaf) at a depth of L (LAI = L)
varied significantly over the course of leaf development. Each leaf was used to calculate the light interception by different layers of
age level was treated as a time point by using the ‘repeated’ option the canopy:
of Proc Mixed, and then the PLRC parameters were modelled
using species, P supply and leaf-age and their interactions as I leaf ðLÞ ¼ I 0 eÀkL ; ð3Þ
explanatory variables. As RM-ANOVA can be sensitive to the
assumed variance-covariance structure of the data (Potvin et al. where I0 is the irradiance on the upper canopy surface, set at
1990), we first fitted four types of covariance structures 1500 mmol PPFD m–2 s–1. Once Ileaf (L) was calculated for each
(compound symmetric, first-order autoregressive, spatial leaf layer, two sets of simulations were performed to estimate
power and unstructured), each based on different assumptions Pleaf using method 1 (only Pmax varying) and method 2 (Pmax and
about how variances and covariances change across measurement F varying) described above. The percentage difference in Pleaf
levels (Littell et al. 1996, 1998). We then selected the best resulting from method 1 and method 2 at a given light level for a
covariance structure by comparing Akaike information criteria given leaf was then calculated. We then compared the estimated
(AIC) scores (Heschel et al. 2004). This approach suggested an values of Pleaf between the two methods for different leaf layers
unstructured variance-covariance matrix to be the best, so this across all times of leaf development.
assumption was used when testing effects of species, P supply,
Results
leaf age and their interactions on photosynthetic characteristics.
Influence of P supply on leaf area and appearance
Analysis of photosynthetic characteristics at each leaf age Leaf area of a single leaf increased up to the 10th nodal position
When the interaction between species (or P supply) and leaf from the first true leaf for Cullen species and up to the 17th nodal
age was significant, photosynthetic characteristics were tested position for M. sativa and then gradually decreased with
separately at each recorded stage of leaf development. increasing node number (Fig. 1). Leaf area of a single leaf was
Photosynthetic characteristics considered as response variables not affected by the P supply up to 13th, 17th, 11th and 7th nodal
were P1500, gs, Rd and the estimates of the three PLRC model position for C. australasicum accessions SA4966 and SA42762,
parameters; Pmax, q and F. An ANOVA was performed in SAS C. pallidum and M. sativa, respectively. After these nodal
using Proc GLM. Means were separated with the LSMEANS positions, leaf area of a single leaf decreased at P3 compared
procedure. All significant differences are expressed at P 0.05. with P30 for all species. At the time of harvesting, number of
main-stem nodes per plant (leaf appearance rate) at P3 was lower
Modelling Pleaf compared with that of P30 for all species (Fig. 1).
Since the previous analysis revealed the influence of leaf age The expansion of the 8th main stem leaf with age is shown in
on Pmax and F to be significant, the relationships between leaf Fig. S1 available as an Accessory Publication to this paper. Leaf
age and Pmax and leaf age and F were then established using area did not differ between P supply for either Cullen species or
simple mathematical models. The parameter values of these among Cullen species at any time of leaf development. However,
models were fitted through the maximum likelihood approach leaf area of the 8th leaf of M. sativa was smaller than that of Cullen
using Proc NLIN in SAS. When species or P supply was not species and leaves in the P3 were smaller than those in P30 for
found to significantly affect Pmax and F at each leaf age, data M. sativa.
were pooled. The changes in gs and Rd over the course of leaf Leaf [P] was lower at P3 than at P30 for all species (see
development were modelled in a similar way, following the Table S1 available as an Accessory Publication to this paper).
approach used by Stirling et al. (1994) for maize. There were no differences in leaf [P] among species at P3.
The way that Pleaf for a horizontally placed leaf (k = 1 m2 However, at P30, leaf [P] of Cullen species was higher than
ground (m2 leaf)–1)) exposed to irradiances of 500, 1000 and that of M. sativa. Similarly, total P plant–1 was lower at P3 than at
1500 mmol m–2 s–1 changed over the course of leaf development P30 for all species. There were no differences in total P plant–1
was estimated using two different methods based on the non- among species at either P3 or P30. Plants took up less than 15% of
rectangular hyperbola function (Eqn 2) with its three parameters the total P present per pot, irrespective of the P treatment and
Pmax, q and F. In the first method (the ‘standard’ method), F and q species. SLA of M. sativa was almost twice the SLA of Cullen
were assumed to be constant (0.035 mol CO2 mol–1 quanta and species (Table S1). P supply did not change SLA of any species.
0.91, respectively, calculated after full leaf expansion) and only LAI varied between P treatments and among species. LAI at P3
Pmax was assumed to change with time. In the second method, was lower than that at P30 for all the species and LAI of
both Pmax and F were assumed to change with time, whereas q C. pallidum was lower than that of M. sativa. Leaf [N] of

5. Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 717
20 a P supply  species  leaf age interaction. This confirmed the
(a)
age-dependent changes of P1500, Pmax, F, gs and Rd. Therefore,
16 each response variable was further tested at different leaf age
levels as reported below.
12
P1500 showed a variable response with leaf age for all species
8 at both P3 and P30 (Fig. S2). P1500 increased rapidly with
leaf expansion, reached a maximum and remained at a plateau
4 of 22–35 mmol CO2 m–2 s–1 at 150À300C days, followed by
a gradual decrease until 0–9 mmol CO2 m–2 s–1 at 1518C days
0
1 3 5 7 9 11 13 15 17 19 21 23 for all species. The age at which P1500 reached a peak coincided
with the time at which a leaf achieved full expansion (Figs S1,
20 (b) S2). The rate of increase of P1500 during early stages of leaf
16
development was higher than the rate of decrease of P1500 at late
stages. P1500 did not differ between P3 and P30 for a given species
12 at any leaf age, except for C. australasicum accession SA42762
at 168C days and beyond 1082C days. During these periods
8 C. australasicum accession SA42762 plants maintained a higher
4
P1500 at P3 than at P30. Furthermore, P1500 did not differ among
Leaf area (cm2)
species at a given leaf age.
0 The pattern of change in gs with leaf age (Fig. 2) was very
1 3 5 7 9 11 13 15 17 19 21 23 25 27 similar to that observed for P1500 (Fig. S2), as gs increased
20
(c)
rapidly, reached a maximum of 0.56–0.82 mol m–2 s–1 at
150–300C days and then gradually decreased with increasing
16
leaf age up to 0.01–0.14 mol m–2 s–1 at 1518C days. There were
12
no differences in gs between P3 and P30 of a given species or
among species, at a given leaf age, except for C. australasicum
8 accession SA42762 at 168C days and beyond 1082C days,
where gs was higher at P3 than at P30.
4 Photosynthetic water use efficiency (PWUE) of leaves
was constant with leaf age (data not shown). PWUE did not
0
1 3 5 7 9 11 13 15 17 19 differ between P3 and P30 for any species at any leaf age,
except for C. australasicum accession SA42762, where
20 (d ) PWUE of P3 plants was higher than that of P30 beyond
16 1082C days.
The pattern of change in estimated Pmax with leaf age (Fig. 3)
12 was also very similar to that observed for P1500 (Fig. S2), as
Pmax increased with leaf expansion, reached a maximum
8
of 25–36 mmol CO2 m–2 s–1 at 150À300C days, and then
4 gradually decreased to 0–10 mmol CO2 m–2 s–1 at 1518C days.
As was found for P1500 and gs, there was no difference in Pmax
0 between P3 and P30 for any species or among species, at a given
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 leaf age, except for C. australasicum accession SA42762 at age
Main stem node number 0C days and beyond 1255C days, where Pmax was higher at P3
that at P30. Therefore, the patterns of change for P1500, Pmax and
Fig. 1. Leaf area of all leaves on the main stem after full expansion as
dependent on the number of nodes present for (a) Cullen australasicum gs with leaf age were very similar.
accession SA4966, (b) C. australasicum accession SA42762, (c) Cullen The estimated values of F were used to establish relationships
pallidum accession SA44387 and (d) Medicago sativa cv. SARDI-10, between F and leaf age for each species (Fig. 4). The response of
treated with either 3 mg P kg–1 dry soil (*) or 30 mg P kg–1 dry soil (*). F to leaf age differed between Cullen species and M. sativa.
Bars represent s.e. of the means, n = 3. Note that the scale of the x-axis differs Similar to P1500, Pmax and gs, F of Cullen species increased with
among species. leaf age, reached a maximum of 0.029–0.042 mol CO2 mol–1
quanta at 150À300C days and gradually decreased up to
Cullen species at the final harvest was lower (0.02 g N g–1 leaf 0.0–0.018 mol CO2 mol–1 quanta at 1518C days. However,
DW) than that of M. sativa (0.04 g N g–1 leaf DW), irrespective for M. sativa, such a change of F during the development of a
of the P treatment. leaf was not observed and F was constant at 0.012–
0.036 mol CO2 mol–1 quanta. There was no difference in F
Changes in photosynthetic characteristics due to P between P3 and P30 for any species, at any leaf age.
supply, species and leaf age The convexity of the PLRC did not change with leaf
The RM-ANOVA used to test the variability of P1500, gs, Pmax, development and no differences were observed among species
F and Rd indicated significant effects due to leaf age and or between P3 and P30 (data not shown). Therefore, q was

6. 718 Functional Plant Biology L. D. B. Suriyagoda et al.
1.0 40
(a) (a)
0.8
30
0.6
0.4 20
0.2
10
0.0
0
1.0
(b)
0.8 40
(b)
0.6
30
0.4
20
gs (mol m–2 s–1)
0.2
0.0
Pmax (μmol m–2 s–1)
10
1.0
(c) 0
0.8
0.6 40
(c)
0.4
30
0.2
20
0.0
1.0 10
(d )
0.8 0
0.6
0.4 40
(d )
0.2 30
0.0
0 168 288 446 633 778 952 1082 1255 1386 1518 20
Leaf age (°C days)
10
Fig. 2. Effect of leaf age on stomatal conductance (gs) at
PAR = 1500 mmol m–2 s–1 for (a) Cullen australasicum accession SA4966, 0
(b) C. australasicum accession SA42762, (c) Cullen pallidum accession 0 168 288 446 633 778 952 1082 1255 1386 1518
SA44387 and (d) Medicago sativa cv. SARDI-10, treated with either Leaf age (°C days)
3 mg P kg–1 dry soil (*) or 30 mg P kg–1 dry soil (*). Bars represent s.e.
of the means, n = 3. Fig. 3. Effect of leaf age on estimated maximum leaf photosynthesis (Pmax)
for (a) Cullen australasicum accession SA4966, (b) C. australasicum
averaged across all species and P treatments and a value of 0.91 accession SA42762, (c) Cullen pallidum accession SA44387 and
(d) Medicago sativa cv. SARDI-10, treated with either 3 mg P kg–1 dry soil
was obtained.
(*) or 30 mg P kg–1 dry soil (*). Bars represent s.e. of the means, n = 3.
Dark respiration (Rd) changed significantly with leaf age
(Fig. 5). However, the relationship was different to that
observed for P1500, Pmax, gs or F. The rate of Rd was higher between Pmax, F, gs, Rd and accumulated degree days are
(4.1–6.9 mmol CO2 m–2 s–1) at early developmental stages and summarised in Fig. 6.
gradually decreased to 0.01–1.9 mmol CO2 m–2 s–1 as leaves The sensitivity of simulated Pleaf to varying Pmax and F in the
aged. There were no differences in Rd between P3 and P30 for light response curve with leaf development was calculated using
any species or among species, at any leaf age. a non-rectangular hyperbola of CO2-assimilation rate (Eqn 2)
(Thornley and Johnson 2000). Figure 7a describes the
Modelling Pleaf considering temporal changes relationship between simulated Pleaf and leaf age based on
in Pmax and F variable Pmax (equation in Fig. 6a) and fixed convexity (0.91)
Since Pmax, F, gs and Rd did not differ between P3 and P30 and together with either fixed (0.035 mol CO2 mol–1 quanta) or
among species data were pooled in most instances and used variable F (equation in Fig. 6b) at an incident radiation of
to establish relationships between Pmax, F, gs and Rd with leaf 1500, 1000 or 500 mmol m–2 s–1 and when k = 1. Comparing
development through simple mathematical models (Fig. 6). The the three light levels, simulated Pleaf at 1500 mmol m–2 s–1 was
equations and coefficient values defining the relationship the highest and Pleaf at 500 mmol m–2 s–1 was the lowest, for both

7. Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 719
0.05 8
(a) (a)
0.04 6
0.03
4
0.02
2
0.01
0
0.00
8
0.05 (b) (b)
0.04 6
0.03 4
0.02
Φ (mol CO2 mol–1 quanta)
2
Rd (µmol m–2 s–1)
0.01
0
0.00
8
(c)
0.05
(c)
6
0.04
4
0.03
0.02 2
0.01 0
0.00
8 (d)
0.05
(d) 6
0.04
4
0.03
2
0.02
0
0.01 0 168 288 446 633 778 952 1082 1255 1386 1518
0.00 Leaf age (°C days)
0 168 288 446 633 778 952 1082 1255 1386 1518
Fig. 5. Effect of leaf age on dark respiration (Rd) for (a) Cullen
Leaf age (°C days)
australasicum accession SA4966, (b) C. australasicum accession
Fig. 4. Effect of leaf age on estimated quantum yield (F) for (a) Cullen SA42762, (c) Cullen pallidum accession SA44387 and (d) Medicago
australasicum accession SA4966, (b) C. australasicum accession SA42762, sativa cv. SARDI-10, treated with either 3 mg P kg–1 dry soil (*) or
(c) Cullen pallidum accession SA44387 and (d) Medicago sativa cv. 30 mg P kg–1 dry soil (*). Bars represent s.e. of the means, n = 3.
SARDI-10, treated with either 3 mg P kg–1 dry soil (*) or 30 mg P kg–1
dry soil (*). Bars represent s.e. of the means, n = 3.
F. According to Eqn 3, radiation intercepted by different layers of
a canopy of LAI = 4, when Io = 1500 mmol m–2 s–1 and k = 0.6
constant and variable F (Fig. 7a), as expected. For any given light were 677, 371, 204 and 112 mmol m–2 s–1 for the four layers, from
level, Pleaf simulated using variable F was lower compared with top to bottom, respectively. Those intercepted light intensities
Pleaf simulated using a fixed F across all stages of leaf were used for the simulation of Pleaf with leaf development at
development. Also, the % reduction in Pleaf was higher at different leaf layers with a variable Pmax (equation in Fig. 6a)
lower PPFD. During the time course of leaf development, this together with either fixed (0.035 mol CO2 mol–1 quanta) or
% reduction was very high during early leaf expansion and with variable F (equation in Fig. 6b). Convexity was set to 0.91.
increasing leaf age after full expansion (Fig. 7b). When averaged As expected, Pleaf was highest for the uppermost leaf layer and
across the whole period of leaf development, the values of Pleaf gradually decreased with increasing depth in the canopy (Fig. 8a).
estimated using variable F were 3, 7 and 19% lower than the For a given leaf layer, Pleaf at variable F was lower than that at
values of Pleaf estimated using fixed F, at 1500, 1000 and a fixed F at any given time of the leaf development and this
500 mmol PPFD m–2 s–1, respectively. reduction was higher at a very early period of leaf expansion and
The relationship between Pleaf and leaf age in different leaf with increasing leaf age after full leaf expansion. Furthermore,
layers of a closed canopy was also compared for fixed and variable % reduction of Pleaf estimated using variable F compared with

8. 720 Functional Plant Biology L. D. B. Suriyagoda et al.
40 40
Φ (mol CO2 mol–1 quanta) Pmax (µmol m–2 s–1)
(a) (a)
Pmax = 7.5 + 0.22 × e–0.004X R 2 = 57
Pleaf (µmol m–2 s–1)
30
30
20
10 20
0
10
0.05
(b) Φ = 0.011 + 0.0001 × e–0.0026X R 2 = 63 0
0.04
100
0.03 (b)
% reduction in Pleaf
0.02 80
0.01
60
0.00
40
1.0
(c) 20
gs = 0.138 + 0.0068 × e–0.0055X R 2 = 58
gs (mol m–2 s–2)
0.8
0
0.6 0 400 800 1200 1600
0.4 Leaf age (°C days)
0.2
Fig. 7. (a) Simulated leaf photosynthesis (Pleaf) with leaf age for an incident
0.0 radiation of 1500 mmol PPFD m–2 s–1 (triangles), 1000 mmol PPFD m–2 s–1
(squares) and 500 mmol PPFD m–2 s–1 (circles) with either fixed F
8 (closed symbols) or variable F (open symbols). Simulated maximum leaf
Rd (µmol m–2 s–1)
(d) photosynthesis (Pmax) is shown as (+) symbols. (b) Estimated % reduction of
Rd = –0.963 ln(X) + 7.9 R 2 = 77
6 Pleaf at variable F compared with that of fixed F with leaf age for an incident
4 radiation of 1500 mmol PPFD m–2 s–1 (triangles), 1000 mmol PPFD m–2 s–1
(squares) and 500 mmol PPFD m–2 s–1 (circles).
2
0
0 400 800 1200 1600 Low P did not reduce photosynthetic characteristics
Leaf age (°C days) at any leaf age
Fig. 6. Fitted (*) and observed (*) values of (a) maximum P1500, Pmax, F, q, gs and Rd were not lower at P3 than at P30
photosynthesis (Pmax), (b) quantum yield (F), (c) stomatal conductance except for C. australasicum accession SA42762, and thus the
(gs) and (d) dark respiration (Rd) with leaf age. Equations and coefficients hypothesis that low P supply would reduce the rate of
for the fitted relationships between parameters of the photosynthetic light photosynthetic characteristics of Cullen species and M. sativa
response curve with leaf age (X – C days) are also shown. Observed values at any given leaf age was not supported. The reduction in P1500,
represent measured values from all the species. Pmax, and gs at P30 for C. australasicum accession SA42762
might be an adaptive response to conserve moisture or due to
P toxicity. We believe that, since plants at P30 had a higher LAI
using fixed F was lower for upper leaf layers than for deeper leaf
than at P3 (Table S1), they experienced higher water loss through
layers (Fig. 8b). When averaged across the period of leaf
transpiration, and therefore partial closure of stomata might have
development, these % reductions were 13, 24, 31 and 32%, for
reduced the photosynthetic rate. This conclusion is supported
the four layers, from top to bottom, respectively.
by the higher PWUE of C. australasicum accession SA42762 at
P30 compared with that at P3 (data not shown). Reduction of
Discussion photosynthetic rate during P toxicity is highly correlated with a
Even though numerous studies have reported the age dependent reduction in plant growth (Shane et al. 2004). Even though the
changes of leaf photosynthetic characteristics, most studies have photosynthetic rate was reduced at P30 for C. australasicum
looked at only one or two parameters at a time (mostly P1500 and accession SA42762, dry weight was higher at P30 than at P3 and
Pmax) or were limited to a narrow leaf developmental period leaf [P] at P30 was similar to that of other species in the
(e.g. only after full leaf expansion or only during initial experiment. Indeed, leaf [P] for Cullen species and M. sativa
expansion). In addition, the influence of P on Pmax, F and q is at P30 was much lower than reported for many native Australian
not known. Therefore, we studied the age dependent changes of species and crop plants during P toxicity (e.g. Shane et al. 2004;
the photosynthetic characteristics P1500, Pmax, F, q, gs and Rd for Pang et al. 2010). Therefore, it seems the reduction in
three pasture species during the whole leaf development period photosynthetic rates soon after full leaf expansion and during
under two levels of P supply (low and optimum). We now discuss the late leaf developmental stages of C. australasicum accession
our results and the implications of age dependent changes of leaf SA42762 treated with P30 was due to limitation in CO2 flux
photosynthetic characteristics for simulating Pleaf. caused by partial closure of stomata (Jacob and Lawlor 1991) not

9. Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 721
20 (a)
leaves (e.g. estimated intercepted radiation was 112 mmol m–2 s–1
at the 4th layer at the bottom of the canopy, while that at the top
leaf layer was at 677 mmol m–2 s–1). de Groot et al. (2003) also
Pleaf (µmol m–2 s–1)
15
found that difference in Pleaf of low- and high P treated
S. lycopersicum plants reduced more for shaded leaves than
10 for unshaded leaves. A third possible reason is that total LAI,
leaf size and leaf number at harvest was lower for P3 than for P30.
5 Therefore, the 8th leaf of P3 treated plants might have experienced
a higher light intensity than that at P30 and thereby acclimated to
0
a higher Pleaf than we expected (Lambers et al. 2008). Further
research may identify upper and lower threshold [P] at which
100
(b) photosynthesis is affected in these species and, thus, determine if
there are important differences in response between native and
% reduction in Pleaf
80 exotic species, and among native species.
60
Leaf photosynthetic characteristics changed with leaf age
40
Our second hypothesis stated that a substantial change in P1500,
20
Pmax, F, gs and Rd would occur in Cullen species and M. sativa
during leaf development. P1500 and Pmax showed a tri-phasic
0 response to leaf age, with an initial rapid increase in values to
0 400 800 1200 1600 reach maxima across a short plateau, followed by a near-linear
Leaf age (°C days) decrease with leaf age (Fig. 3, S2), which supported our
Fig. 8. (a) Simulated leaf photosynthesis (Pleaf) with leaf age in a closed hypothesis. The leaf age at which these rates achieved a
canopy with LAI = 4, for an intercepted radiation of 677 mmol PPFD m–2 s–1 maximum coincided with the time at which a leaf achieved
at the upper layer (circles), 371 mmol PPFD m–2 s–1 at the second layer full expansion. Similar results have been obtained by Stirling
(squares), 204 mmol PPFD m–2 s–1 at the third layer (diamonds) and et al. (1994) and Boedhram (1998) for other crop species. This
112 mmol PPFD m–2 s–1 at the fourth layer (triangles) with either fixed F reduction in Pmax after full leaf expansion (down the canopy)
(closed symbols) or variable F (open symbols). (b) Estimated % reduction of is functional if resources such as photosynthetic leaf N are
Pleaf at variable F compared with that of fixed F with leaf age for Fig. 8a invested at the best light environment for photosynthesis
at the upper layer (circles), second layer (squares), third layer (diamonds) and (Charles-Edwards 1981; Evans and Seemann 1989; Sands
the fourth layer (triangles).
1995), thereby achieving the highest Pmax per plant. The
temporal change in Pmax observed in the present study
P toxicity. Thus, overall, our hypothesis that Cullen species and supports our second hypothesis and is in agreement with
M. sativa would show reduced P1500, Pmax, F, q, gs and Rd at findings for several other crop species (Marshall and Biscoe
low P supply at a given leaf age was not supported. This might 1980; Leech and Baker 1983; Dwyer and Stewart 1986;
be due to several reasons. Stirling et al. 1994).
One possible reason is that the lowest [P] of 3 mg P kg–1 dry Even though the temporal change in Pmax has been extensively
soil used in this study might not have been low enough to studied for many crops, there is a lack of literature on the change of
significantly reduce photosynthetic rate (Khamis et al. 1990; F and q with the complete leaf developmental period. In the
Jacob and Lawlor 1991). However, in a P response study Pang current study the response of F to increasing leaf age for Cullen
et al. (2010) found that both C. australasicum and M. sativa species was similar to that observed for Pmax (r = 0.46, P 0.001)
increased growth until soil [P] reached to 24 mg P kg–1 dry soil. and supported our second hypothesis. The peak estimates of F
Therefore, P3 and P30 in the present study represent low and obtained at full leaf expansion in the current study were similar to
optimum supply of P for the growth of C. australasicum and observations by Field and Mooney (1983), Dwyer and Stewart
M. sativa, respectively. It is also known that leaf growth under P (1986), Stirling et al. (1994) and Singsaas et al. (2001) for several
deficiency is reduced before any reduction in Pleaf (Rao and Terry other crop species. However, in contrast with these responses,
1989; Jacob and Lawlor 1991; Plénet et al. 2000b). During the a constant value of F with leaf age has also been observed
initial growth stages of the present experiment, leaf size did not (Field and Mooney 1983; Stirling et al. 1994). This constant F
decrease for any species at P3, suggesting that P was not limiting response was the same as that observed for M. sativa in the present
for the initial growth, even though the growth was reduced later at study, which did not support our second hypothesis. Reports
P3 (Fig. 1). This means that the photosynthetic rates of the 8th leaf suggest that F can vary due to environmental factors such as light
might not have been affected by P3. Even though the canopy [P] intensity and temperature (Groom et al. 1991), season (Field and
was measured, it was not possible to measure the [P] of the 8th leaf Mooney 1983; Niinemets et al. 2004), and leaf traits such as leaf
due to its small size. Therefore, [P] of the 8th leaf was not known transmittance (Ehleringer and Björkman 1977; Osborne and
and it may not have differed between P3 and P30. A second Garrett 1983). Stirling et al. (1994) suggested that limitation in
possible reason is that low light intensity might have limited Pleaf stomatal conductance, biophysical processes of energy transfer
more than the low P treatment. As a leaf develops, its position in and electron transport can also change F. However, the patterns
the canopy changes and it often becomes shaded by the upper of change of gs with leaf development for Cullen species and

10. 722 Functional Plant Biology L. D. B. Suriyagoda et al.
M. sativa were similar (Fig. 2). The relationship between gs and F with full leaf expansion, it is reasonable to use leaves at full
was significant for Cullen species (r = 0.41, P 0.05), but not expansion for comparison purposes. However, extrapolation
for M. sativa (r = 0.18, P 0.05). Therefore, our observation for of leaf photosynthetic rates at full expansion to different leaf
M. sativa was not in agreement with the suggestion by Stirling layers of a plant requires consideration of light microclimate
et al. (1994) that stomatal limitation affects the change in F with (proportional Pmax to local irradiance), leaf characteristics
leaf age. We believe that the different response of F of Cullen (leaf age and N status) and plant age (Charles-Edwards 1981;
species and M. sativa to leaf age might be due to limitations in Sands 1995, 1996; Hollinger 1996). This is why it was important
biophysical processes of energy transfer and electron transport to test our third hypothesis: that taking into consideration the
caused by N nutrition. Leaf [N] reflects the amount of the primary way that estimated changes of Pmax, F and q with leaf age will
carboxylating enzyme in C3 plants (Friedrich and Huffaker 1980; reduce estimates of Pleaf. This has direct relevance to modelling
Kitajima et al. 2002). In the present study, leaf [N] of Cullen the growth of these species as pastures. It may be particularly
species at the final harvest was lower than that of M. sativa and important when modelling the effects of grazing, where younger
this was partly associated with a lower SLA of Cullen species leaves higher in the canopy will be removed first and the leaves
than that of M. sativa. This lower leaf [N] might have been most likely to remain will be the older and less efficient ones.
associated with a reduced carboxylating capacity in Cullen In the present study, lower estimates of Pleaf at variable F
species with leaf age, resulting in a lower value of F. compared with a fixed F, for a horizontally placed leaf
However, further investigation is required to establish such a (k = 1), under different light intensities (1500, 1000 and
relationship. 500 mmol m–2 s–1, Fig. 7a, b) showed that using a fixed F
Even though Pmax and F changed with leaf age supporting our results in overestimates of Pleaf at any given time and that
second hypothesis, q was unchanged. The range of q observed for greater over-estimation of Pleaf occurs at lower light intensities
Cullen species and M. sativa in this study (0.75–1.0) was similar (500 mmol m–2 s–1 compared with 1500 mmol m–2 s–1). Similarly,
to that observed by Stirling et al. (1994) and Lizaso et al. (2005) when considering a canopy with an angular leaf orientation
for other crop species. The fact that we found q unchanging with (k = 0.6) the % reduction of Pleaf at variable F compared with
leaf development during the current study might be due to the a fixed F was greater for deeper leaf layers and further increased
very large variability of q observed among leaves for a given during very early expansion and with leaf age after full expansion
species  P treatment combination and the ability of the other two (Fig. 8a, b). Therefore, when Pmax and F were considered as
parameters to account for much of the change in the shape of the functions of leaf age and treated as variables in the PLRC,
PLRC. different estimates of Pleaf resulted. Our results show that
A decline in Pmax after full leaf expansion was highly when using a fixed-parameters approach in actual stand-level
correlated with the decline in Rd (r = 0.73, P 0.001). Lower modelling, overestimation of Pleaf could occur when the LAI is
Rd at lower Pmax is expected to be due mainly to lower respiratory very high, if the stand is mature (during later growth stages of the
costs involved in sugar export (Bouma et al. 1995). After full pasture stand or at dormancy), soon after grazing (most of the
expansion of a leaf, the ratio of Rd to Pmax was ~0.10, in agreement mature leaves are still retained but younger more efficient leaves
other published results of 0.05–0.12 (Angus and Wilson 1976, removed), during cloudy days compared with sunny days, during
Hordeum vulgare L. (barley); Marshall and Biscoe (1980); winter compared with summer, for shade-grown stands
Stirling et al. (1994), T. aestivum; Noguchi et al. 1996; (understory crops or pastures), at higher plant densities and on
Z. mays, Spinacia oleracea L. (spinach) and Colocasia fertile soils (higher growth rate).
esculenta L. (Asian taro)). However, as leaf age increased, the The q parameter did not change with leaf age in the present
ratio tended to increase, due to a greater reduction of the study. Therefore, the impact of a variable q on Pleaf could not be
photosynthetic capacity of leaves as a result of leaf senescence. studied to test our hypothesis. In cases where q is found to change
In the present study, gs also showed a tri-phasic response with with leaf development, the impact of a variable q on Pleaf should
leaf age, supporting our second hypothesis and was highly be investigated and accounted for in simulation models in order
correlated with P1500 (r = 0.92, P 0.001) and Pmax (r = 0.89, to estimate Pleaf more accurately.
P 0.001). Similar responses of gs with leaf age have been
observed for maize (Dwyer and Stewart 1986). However,
PWUE was unchanged with leaf age (data not shown), due to P affected leaf area and leaf appearance in M. sativa
proportional reduction of P1500 and gs as also found by Field and compared with Cullen species
Mooney (1983) and Shirke (2001). Lower gs and PWUE are Leaf size and appearance rate of Cullen species and M. sativa at P3
often, but not always, observed with leaf aging (Field and Mooney were reduced compared with P30 with the response for M. sativa
1983; Witkowski et al. 1992; Kitajima et al. 2002). being much quicker than for the Cullen species, which supports
our hypothesis. Reduced leaf size at low P supply has been
obtained in previous studies for other crop and tree species
Estimates of Pleaf were affected by changes (Kirschbaum et al. 1992; Plénet et al. 2000a). The lower leaf
of F with leaf age [P] at harvest for P3 compared with P30 for all the species
The results of the present study indicate that the parameters of indicates that decreased leaf area and leaf appearance at P3
the light response curve are subject to change with leaf age. were due to lower availability of P. Furthermore, the decline in
Therefore, photosynthetic capacity of a leaf varies with leaf the leaf area was earlier for M. sativa (at 7th nodal position) than
age, or for leaves at different depths of a canopy, at a given for Cullen species (after 11th nodal position). Possible reasons for
time. Since peak values of P1500, Pmax, gs and F were associated the earlier response of M. sativa could include a higher sensitivity

11. Leaf age, phosphorus supply and photosynthesis Functional Plant Biology 723
to low P supply or a faster decline of P reserves due to a higher Chiera J, Thomas J, Rufty T (2002) Leaf initiation and development in
growth rate. However, in this study all species took up less than soybean under phosphorus stress. Journal of Experimental Botany 53,
15% of the total P available in the pot. Also, since M. sativa did not 473–481. doi:10.1093/jexbot/53.368.473
Cocks PS (2001) Ecology of herbaceous perennial legumes: a review of
have a higher growth rate than the Cullen species at P3, the decline
characteristics that may provide management options for the control of
in leaf area and leaf appearance of M. sativa at P3 compared with
salinity and waterlogging in dryland cropping systems. Australian
the Cullen species was not due to the lower availability of P, but Journal of Agricultural Research 52, 137–151. doi:10.1071/AR99170
was due to a higher sensitivity of M. sativa to P supply. Leaf size Constable GA, Rawson HM (1980) Effect of leaf position, expansion and
and appearance rate are important drivers in models predicting age on photosynthesis, transpiration and water use efficiency of cotton.
photosynthesis of whole plants and canopies and it would, Australian Journal of Plant Physiology 7, 89–100. doi:10.1071/
therefore, be important to include the observed differences due PP9800089
to P supply in such models when predicting and comparing Cordell D, Drangert JO, White S (2009) The story of phosphorus: global
the productivity of Cullen species and M. sativa in low P food security and food for thought. Global Environmental Change 19,
environments. 292–305. doi:10.1016/j.gloenvcha.2008.10.009
Dear BS, Li GD, Hayes RC, Hughes SJ, Charman N, Ballard RA (2007)
Conclusions Cullen australasicum (syn. Psoralea australasica): a review and some
preliminary studies related to its potential as a low rainfall perennial
P3 did not reduce the photosynthetic rate for any of the species at pasture legume. The Rangeland Journal 29, 121–132. doi:10.1071/
a given leaf age compared with P30. Pmax and gs of all the species RJ06039
and F of Cullen species increased until full leaf expansion, were de Groot CC, Van den Boogaard R, Marcelis LFM, Harbinson J, Lambers H
steady for ~150C days, and then gradually decreased with (2003) Contrasting effects of N and P deprivation on the regulation of
increasing leaf age. Rd decreased with leaf age for all species photosynthesis in tomato plants in relation to feedback limitation. Journal
while F of M. sativa and q of all species did not change with leaf of Experimental Botany 54, 1957–1967. doi:10.1093/jxb/erg193
age. When Pmax and F were considered as functions of leaf age Denton MD, Sasse C, Tibbett M, Ryan MH (2006) Root distributions of
and treated as variables when estimating the temporal changes of Australian herbaceous perennial legumes in response to phosphorus
placement. Functional Plant Biology 33, 1091–1102. doi:10.1071/
Pleaf, estimates of Pleaf obtained using variable F were lower
FP06176
than those obtained using a fixed F at any given leaf age. The
Dwyer LM, Stewart DW (1986) Effect of leaf age and position on net
% reduction of Pleaf was higher at lower light intensities as well as photosynthesis rates in maize (Zea mays L.). Agricultural and Forest
during very early and very late stages of leaf growth. Both leaf size Meteorology 37, 29–46. doi:10.1016/0168-1923(86)90026-2
and rate of leaf appearance were reduced at P3 for all the species, Ehleringer J, Björkman O (1977) Quantum yields for CO2 uptake in C3 and C4
while the response for M. sativa was considerably faster. plants. Dependence on temperature, CO2, and O2 concentration. Plant
Therefore, it is important to incorporate the effects of leaf age Physiology 59, 86–90. doi:10.1104/pp.59.1.86
and light intensity on photosynthetic parameters into carbon- Evans JR, Seemann JR (1989) The allocation of protein nitrogen in the
assimilation models for these particular species. More generally, photosynthetic apparatus: cost, consequences, and control. In
these results indicate that it is important to consider such effects ‘Photosynthesis’. (Ed. WR Briggs) pp. 183–205. (Alan R Liss: New York)
Fick GW, Holt DA, Lugg DG (1988) Environmental physiology and crop
when modelling the performance of any plant species.
growth. In ‘Alfalfa and alfalfa improvement. Agronomy Monograph
Acknowledgements No. 29’. (Eds AA Hanson, DK Barnes, RR Hill) pp. 164–194.
(American Society of Agronomy: Madison, WI)
We thank two anonymous referees for their valuable suggestions, which Field F, Mooney HA (1983) Leaf age and seasonal effects on light, water, and
greatly improved the original manuscript. This study was supported by nitrogen use efficiency in a California shrub. Oecologia 56, 348–355.
the School of Plant Biology and the Future Farm Industries Cooperative doi:10.1007/BF00379711
Research Centre, The University of Western Australia. LDB Suriyagoda Fletcher AL, Moot DJ, Stone PJ (2008) Radiation use efficiency and leaf
also appreciates the SIRF/UIS Scholarship awarded by the University of photosynthesis of sweet corn in response to phosphorus in a cool
Western Australia and further scholarship support from the late Frank Ford. temperate environment. European Journal of Agronomy 29, 88–93.
doi:10.1016/j.eja.2008.04.002
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