Plant, Cell and Environment (2003) 26, 857-865 Increases of chlorophyll alb ratios during acclimation of tropical woody seedlings to nitrogen limitation and high light K. KITAJIMA1 & K. P. HOGAN12 'Department of Botany, University of Florida, Gainesville, FL 32611, USA and 2The Evergreen State College, Olympia, WA ABSTRACT According to the theory of optimal nitrogen partitioning within a leaf, the chlorophyll (Chi) alb ratio is expected to increase when leaf N content decreases. Here, we report the first empirical support for this prediction. The Chi alb ratio increased while Chi content decreased in response to N limitation in photosynthetic cotyledons and leaves of seedlings of four tropical woody species in the Bignoni- aceae. The responses of all four species were in the same direction, but differed in magnitude. For Tabebuia rosea, the species that exhibited the greatest increase in Chi alb ratios (up to values of 5.9), detailed photosynthetic charac- teristics were also examined. Light and N availability were positively correlated with the light- and C02-saturated pho- tosynthetic 02 evolution rate, as well as with leaf carboxy- lation capacity (Fana*) and electron transport rate (Vj). Severe N limitation and high light did not cause chronic photo-inhibition (i.e. no change in quantum yield or in dark-acclimated FJFm). The observed change in the ratio of Fcmax to leaf N in response to N availability was consis- tent with likely functional reasons for change in the Chi al b ratio. Adjustment of the Chi alb ratio was apparently an integral feature of acclimation to high light conditions and low N availability. Key-words: Callichlamys latifolia; Pithecoctenium cru- cigerum; Tabebuia guayacan; Tabebuia rosea; Bignoni- aceae; carboxylation; fluorescence; nitrogen allocation; photo-inhibition; photosynthesis. INTRODUCTION The ability to acclimate to contrasting light environments is particularly important for tropical woody seedlings that initially establish in the deep shade and that later must take advantage of sudden openings in the canopy to grow to larger sizes (Whitmore 1989; Newell et al. 1993). Although maternal plants endow each seedling with sufficient N for initial development in shade (Milberg, Perez-Fernandez, & Correspondence: Kaoru Kitajima. E-mail: kitajima@botany. uft. edu Lamont 1998; Walters & Reich 2000; Kitajima 2002), low soil N may substantially constrain subsequent growth in gaps (Bungard, Press & Sholes 2000; Coomes & Grubb 2000). High irradiance in tropical latitudes can cause chronic photo-inhibition through impairment of photosys- tem II (PSII) reaction centres in leaves of plants that have experienced gap openings (Mulkey & Pearcy 1992; Araus & Hogan 1994; Lovelock, Jebb & Osmond 1994). Photo- damage is especially pronounced in plants with low N, although leaves may acclimate to higher light regimes by enhancing photoprotective mechanisms after several weeks (Bungard et al. 2000). Acclimation to a change in irradiance involves optimal N allocation at whole plant, leaf and cellular levels (Hirose & Werger 1987; Evans 1989; Hikosaka & Terashima 1995). Evans (1989, 1993) suggested that shade-leaf chloroplasts should increase the ratio of N invested in thylakoid proteins to that in soluble proteins in order to balance energy cap- ture and energy transfer. Hikosaka & Terashima (1995) further developed a theory of optimal N partitioning in chloroplasts by simulating effects of variable N partitioning to five chloroplast-protein components. The Chi alb ratio can be a useful indicator of N partition- ing within a leaf, because this ratio should be positively correlated with the ratio of PSII cores to light harvesting chlorophyll-protein complex (LHCII) (Terashima & Hiko- saka 1995). LHCII contains the majority of Chi b, and consequently it has a lower Chi alb ratio (1.3-1.4) than other Chi binding proteins associated with PSII (Evans 1989; Green & Durnford 1996). Thus, the Chi alb ratio is predicted to respond to light and N availability in the fol- lowing ways (Hikosaka & Terashima 1995); 1 Chi alb ratios should increase with increasing irradiance at a given N availability. 2 Chi alb ratios should increase with decreasing N avail- ability, especially under high light conditions. This second prediction has received much less attention than the first prediction. The logic underlying the second prediction can be explained as follows (Hikosaka & Terash- ima 1995). When N supply becomes limiting under high light, the proportional allocation to PSII should increase at the cost of decreased N allocation to Rubisco, whereas N ) 2003 Blackwell Publishing Ltd 857 858 K. Kitajima & K. P. Hogan allocation to LHCII is maintained at a similar level. Con- sequently, the ratio of PSII to LHCII (and the Chi alb ratio) should increase with decreasing N availability. The first prediction for the relationship between the Chi alb ratio and irradiance has been supported by many stud- ies at the leaf and vegetation-stand levels (e.g. Terashima & Evans 1988; Dale & Causton 1992). However, published data are equivocal regarding the second prediction. The Chi alb ratio and the ratio of PSII to Chi are independent of N availability for spinach (Terashima & Evans 1988). Both leaf N and Chi alb ratios decreased slightly with leaf age in Ipomoea tricolor even when self-shading was prevented (Hikosaka 1996). For tropical rainforest tree species, Bun- gard et al. (2000) found little response in Chi alb ratios to light or N, whereas Thompson, Huang & Kriedemann (1992) found lower Chi alb ratios in low N plants, a response opposite to the theoretical prediction. Here we report the first empirical support for the predic- tion of increasing Chi alb ratios in response to N limitation, a result found during an experimental study that examined whole-plant growth responses to light and nitrogen avail- ability for seedlings of four Bignoniaceae species (Kitajima 2002). In order to assess whether the observed increases in Chi alb ratios were the result of optimal acclimation or were due to impaired physiology under high light in N- dehcient leaves, we quantified photosynthetic characteris- tics including quantum yields and chlorophyll fluorescence for the species (T. rosea) that showed the strongest response in Chi alb ratios. Changes in allocation to different components of photosynthetic system were examined indi- rectly via the ratios of various photosynthetic parameters to overall N and Chi content. METHODS Study site and species All experiments and measurements were conducted on Barro Colorado Island (BCI), Panama, using four woody species in Bignoniaceae that are common in this seasonal moist tropical forest. Tabebuia rosea DC. and Tabebuia guayacan (Seem.) Hemsl. are canopy trees, whereas Calli- chlamys latifolia K. Schum. and Pithecoctenium crucigerum A. Gentry are lianas whose adults reach the forest canopy. These species differ in the degree of shade tolerance as measured by recruitment probability of seedlings per dis- persed seed and by survival probability from the first to second year (Kitajima 2002; S. J. Wright unpublished results). Callichlamys latifolia is the most shade-tolerant, followed by P. crucigerum, T. rosea, and finally T. guayacan. Seedlings of T. rosea and T. guayacan germinate in the shaded understorey, but rarely survive until the end of the first year unless they are within treefall gaps. In contrast, >1-year-old seedlings of C. latifolia are common in the shaded understorey. The study species also differ in cotyle- don morphology. Cotyledons of T. rosea and T. guayacan expand to become leaf-like photosynthetic organs, whereas green cotyledons of C. latifolia remain thick and have just enough photosynthetic capacity to compensate for the cot- yledons' own dark respiration (Kitajima 1992, 2002). Cot- yledons of P. crucigerum remain inside the seed coat and serve only as storage organs. Additional ecological and tax- onomic information for these species is available in Croat (1978). Growth conditions Details of the growth conditions and results of functional growth analysis are given in Kitajima (2002). Summarized here is the relevant background information for the current study focusing on acclimation responses at the leaf level. At the time of radicle emergence, germinating seeds were transplanted singly into pots filled with sand and vermicu- lite (1 :1 ratio), and were randomly assigned to light and- N treatment combinations (40-120 plants per treatment combination for each species, reflecting differences in seed availability). Pot position was changed daily, and pots were rotated among three benches within each light treatment. Plants in the high light treatment received 27% of total daily photon flux density (FED) above the canopy (6.77 mol rrr2 d ' averaged over 12-24 d; daily maximum FED up to HOOjUmol nr2 s"1), whereas plants in the low light treatment were located beneath layers of shade cloth and received 1.2% of the total daily PFD (0.29 mol nr2 d'; daily maximum PFD below 100 ,umol rrr2 s"1), corre- sponding to deep shade in the forest understorey. Nitrogen treatments were created by saturating each pot daily with 20 mL of one of three nutrient solutions. One-fifth strength modified Johnson solution containing all macro- and micro- nutrients including 2.2 mM N03~ and 0.42 mM NH4+ (Epstein 1972) was used for the high N treatment. This N concentration is comparable to the highest N concentration recorded in stream water on BCI (M. Keller and R. Stal- lard, pers. comm.). The low N solution had the same com- position as the high N solution, except that 90% of the N03~ and NH4+ was replaced with Cl" and K+, respectively, whereas all N ions were replaced with Cl" and K+in the zero N solution. Cotyledons of all species expanded fully by 10-14 d after radicle emergence, whereas the first pair of opposite leaves expanded 10-28 d after radicle emergence. In all species, leaf area and seedling biomass growth responded to N supply in high light, but not in shade. In the two Tabebuia spp., seed N reserves supported the seedlings' N demands for 60 d in shade, but for only 14 d in high light. Plants grown in high light with zero N supply, and plants in shade at all nitrogen levels, did not develop the second pair of leaves. Subsequent growth in zero and low N treat- ment in high light was accompanied by dilution of tissue nitrogen concentration. In the other two species (which have storage cotyledons) the response of seedling biomass to light occurred later (11-14 d) than in Tabebuia spp. (7 d), whereas the response to N occurred either at a similar time (12 dm. P. crucigerum) or much later (> 35 d in C. latifolia, which exhibits a high nitrogen concentration in its seed). > 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 Photosynthetic acclimation and Chi a/b ratios 859 Chi and N measurements For all species, Chi was extracted in 90% acetone to deter- mine Chi contents and Chi alb ratios of fully expanded cotyledons and the first pair of leaves. For T. rosea, we sampled fully expanded cotyledons (of 30-day-old seed- lings), the first pair of leaves (of 30-day and 50-day-old seedlings), and leaves used in physiological measurements. For the other three species, the seedling age at sampling varied (24 d and 50 d for leaves of C. latifolia; 50 d for leaves of P. crucigerum; 65 d for cotyledons and leaves of T. guayacan). Seedling age at sampling was older for T. guayacan because of differences in the timing of growth experiments and in the sample size, but its leaves and cot- yledons appeared similar to those of 50-day-old T. rosea. None of these leaves or cotyledons exhibited colours that would indicate anthocyanin or delayed greening. Each sam- ple (a whole cotyledon or a 10-cm2 leaf disc) was ground with a chilled mortar and a pestle in 1 ml of pure acetone with a pinch of MgC02 to prevent pheophytin formation (Linder 1974), and the pigment extract in 90% acetone was analysed for Chi a and Chi b following Jeffrey & Humphrey (1975). The other sample from each pair of cotyledons or leaves was dried at 60 ?C for subsequent determination of dry mass per area (for all species). Total Kjeldahl N was determined only for T. rosea, by using Nessler colorimetry (Allen 1974). Gas exchange measurements Photosynthetic gas exchange characteristics were examined only for T. rosea, the species that showed the strongest response in Chi alb. Photosynthetic light curves were determined with a leaf-disc 02 electrode (Model LD2; Hansatech, Norfolk, England) for the first true leaves of 50- day-old-seedlings. Light was supplied to the adaxial side with a computer-controlled array of light-emitting diodes (LS3; Hansatech). After induction at 100 /mrol irr2 s"1 with a continuous supply of air containing 5% C02 for 5-10 min to reach a steady state, the chamber was closed and 02 evolution rates were measured in steps from 100 up to 400 jiimol rrr2 s_1, and then down from 100 to 0 /imol m 2 s_1. At each step, the rate was recorded as soon as it reached quasi-steady state (60-200 s). Light saturation occurred around 200 ,umol m 2 s_1, and the linear region of the light curves at 0-70 jimoi m 2 s"1 was used to calculate the appar- ent quantum yield (= initial slope; mol 02 moL1 incident photons) and dark respiration (the intercept). Gross photosynthetic rates at 400 umol rrr2 s_1 are reported as light- and C02-saturated photosynthetic rates (-Pmax). Absorptance of PFD by cotyledons and leaves was deter- mined with a LI-1800 spectroradiometer (Li-Cor, Lincoln, NE, USA) equipped with an integrating sphere to calculate true quantum yields. The F/Fm ratio of dark-acclimated leaves of 55-day-old- seedlings was measured with a pulse amplitude modulated Huorometer (PAM-101; Waltz, Effeltrich, Germany) as described by Schreiber (1986). Plants were brought into the laboratory before dawn and kept in dark at a high humidity until measurements were made in the morning. Using an open-system gas analyser (LCA3; ADC, Hod- desdon, UK), carboxylation capacity was estimated from measurements of net C02 assimilation rates (A) at different C02 concentrations under saturating light provided by a metal halide lamp and adjusted by neutral filters (650 and 430 /imol m 2 s_1 for high- and low-light grown plants, respectively). On the day of measurements, plants were brought into a growth chamber in which air temperature and relative humidity were kept constant (28 ?C and 75%). The ambient C02 was adjusted so that the estimated inter- cellular C02 pressure (pj was increased from 34 to 70- 80 Pa, and then was decreased from 34 to 5 Pa in 10-15 Pa intervals. During measurements, conductance to water vapour was between 50 and 200 mmol m 2 s"\ whereas leaf temperature averaged at 29 ?C Carboxylation capacity (Kmax) and electron transport rates (Vj) were estimated from non-linear curve fitting to the A-p{ relationship as described in Kirschbaum & Farquhar (1984) and Sims & Pearcy (1989), with the following parameters for Rubisco at 29 ?C: Km, the Michaelis-Menten constant for C02 by Rubisco after taking the competitive inhibition of 02 into account = 86.7 Pa; iQ, Michaelis-Menten constant for acti- vation of Rubisco = 4.0 Pa; and GSTAR, the C02 compen- sation point in the absence of non-photorespiratory respiration = 4.1 Pa. Statistical analysis of the effects of N and light availabil- ity on N contents and physiological characteristics were examined using analysis of covariance (ANCOVA) imple- mented in JMP software (SAS Institute, Cary, NC, USA). RESULTS Chi response to light and nitrogen In T. rosea, leaf N content was positively correlated with N supply within any given light treatment (Table 1). Leaves under low and zero N supply in high light were yellow- green and had lower PFD absorption relative to other treatments (Table 1). N limitation decreased the total Chi per unit leaf area, [Chi], and Chi per unit mass, but increased the Chi alb ratio in cotyledons and leaves (Fig. 1, Table 1). Under low and zero N supply, [Chi] was lower whereas the Chi alb ratio was higher for plants grown in high light than for those grown in low light (Fig. 1). In contrast, [Chi] and the Chi alb ratio were similar between the two light treatments under high N supply (Fig. 1). Shade leaves of T. rosea had a higher [Chi] than did sun leaves for a given leaf N per unit area (Fig. 2a), and [Chi] was posi- tively correlated with leaf N within each light environment. The Chi alb ratio was negatively correlated with leaf N in sun, but not in shade (Fig. 2b). Figure 3 shows the relationship between the Chi alb ratio and [Chi] for the four study species. On average, shade leaves had higher [Chi] and correspondingly lower Chi alb ratios than did high-light leaves. The slope of the correla- tion between [Chi] and the Chi alb ratio did not differ ) 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 860 K. Kitajima & K. P. Hogan Table 1. Physiological characteristics of the first pair of true leaves from 55-day-old seedlings of Tabebuia rosea grown under six treatment combinations, including photosynthetic light-curve characteristics determined with a leaf-disc oxygen electrode with saturating C02, and dark acclimated F/Fm determined by chlorophyll fluorescence Light treatment High light Low light Two-way ANOVA P Light N Nitrogen treatment High Low Zero High Low Zero (interaction) N per leaf area 53.2 42.6 30.3 37.4 33.0 27.5 0.0001 0.0001 (mmol nf2) (1.6) (6.9) (2.5) (3.2) (0.9) (27.5) (0.02) Leaf mass per area 29.9 30.4 29.5 12.8 10.8 11.8 0.0001 NS (gm-=) (2.1) (0.4) (3.1) (0.2) (0.2) (0.3) (NS) Chi per unit leaf area 0.267 0.161 0.085 0.243 0.224 0.169 0.0001 0.0001 (mmol nf2) (0.010) (0.011) (0.008) (0.006) (0.006) (0.013) (0.0001) PAR absorption 0.877 0.808 0.706 0.852 0.852 0.815 0.0001 0.0001 (0.004) (0.012) (0.030) (0.005) (0.005) (0.014) (0.001) Quantum yield 0.091 0.087 0.087 0.098 0.094 0.089 NS NS (mol 02 moL1 photon) (0.004) (0.001) (0.001) (0.009) (0.009) (0.006) (NS) Dark respiration -1.26 -1.15 -1.52 -1.27 -1.26 -1.42 NS NS (,umol 02 nf2 s_1) (0.13) (0.24) (0.09) (0.07) (0.20) (0.36) (NS) Fmax per unit leaf area 8.94 5.76 3.91 3.96 3.67 2.69 0.0001 0.0009 (,umol 02 m 2 s_1) (1.77) (1.70) (0.37) (0.50) (0.35) (0.25) (0.02) Fmax per unit Chi 38.2 43.3 64.1 21.5 22.0 24.4 0.001 0.002 (mmol Q2 mol ChL1 s"1) (6.5) (10.3) (2.9) (1.7) (2.6) (1.1) (0.05) Fmax per unit N 0.194 0.160 0.180 0.140 0.150 0.145 0.007 NS (mmol Q2 mol N_1 s_1) (0.035) (0.033) (0.010) (0.003) (0.012) (0.012) (NS) FJFm 0.78 0.78 0.76 0.76 0.77 0.76 NS NS (0.01) (0.01) (0.01) (0.02) (0.01) (0.01) (NS) Mean (standard deviation) is indicated for three plants. Statistical significance levels of treatment effects are indicated as F-values from a two-way ANOVA (NS: P > 0.05). Cotyledons 0.3- | 0.2 E, Z 0.1- O J3 (a) 7- 6- 5- 4 3 2 6 1- A X I (c) # # # %%%. 0.3- 0.2- 0.1- 7- 6- 5- 4- 3 2 H 0 Leaves (b) A (d) Light ? High 11 Low # # # Zero Low High Zero Nitrogen supply rate Low High Figure 1. Effects of N supply on Chi con- tent per unit area (a, b) and Chi alb ratios (c, d) for cotyledons of 30-d seedlings (left side, a, c) and leaves of 50-d seedlings (right side, b, d) of Tabebuia rosea grown under high- and low-light treatments (open and closed symbols, respectively). Mean with standard deviation. i 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 Photosynthetic acclimation and Chi a/b ratios 861 0.30 o0-20 E E O 0.10 0.00 6 4 2 ia)J^ <. oo / o I o (b) o^^^^ o ^^ ? ? o?~? - -? ^^? I I I I i o E o E, g "5 n IS sz O 25 30 35 40 45 50 55 Total leaf nitrogen (mmol m"2) Figure 2. Relationship of N per unit area with Chi per unit area (a) and Chi alb ratio (b) for leaves of 50-d seedlings of Tabebuia rosea grown under high- and low-light treatments (open and closed symbols, respectively). Significant regressions are shown as solid lines. significantly between the two light treatments (no signifi- cant light-by-[Chi] interaction in ANCOVA with light as the main factor and [Chi] as the covariate; P > 0.05). In all species, the Chi alb ratio was negatively correlated with [Chi] across light treatments, although species differed in the strength of correlation, ranging from a strong negative correlation in T. rosea to a weak correlation in T. guayacan (Fig. 3, Table 2). In T. guayacan, [Chi] was not a significant covariate in the result of ANCOVA (P > 0.05), but N treat- ment had a significant effect on Chi alb ratio in a two-way analysis of variance (ANOVA) (P = 0.04 for N treatment effect, P = 0.005 for light effect). The negative correlation for P. crucigerum was due to the values of three plants under high light conditions and zero nitrogen supply (the three points with the lowest [Chi]). The relationship between the Chi alb ratio and [Chi] was not significantly different between cotyledons and leaves for the two species with photosynthetic cotyledons, T. rosea and T. guayacan (ANCOVA, organ type as the main factor and [Chi] as the covariate, P > 0.05). Gas exchange and fluorescence in T. rosea Light and N treatments significantly influenced light- and C02- saturated photosynthetic rates (Pmax), but not quan- tum yields and dark respiration rates of T. rosea (Table 1). Pmux was positively correlated with leaf N in both light treatments (Fig. 4a P < 0.005). Photosynthetic N use effi- ciency (= the ratio of _Pmax to N) was not affected by N treatment, but was higher in the higher light treatment (Table 1). Pmax per mol Chi was higher at the higher light treatment (Fig. 4b, Table 1). Pmax per mol Chi increased o "to .a IS xi O 8 7 6 h 5 4 3 2 (a) T. rosea (P = 0.0001) 0 8 0 0.1 0.2 0.3 7 (c) C. latifolia (P = 0.0001) 6 5 n 4 3 - . o o o cboo S o m ???> ill 0.1 0 D __ool 0 0 7 6 5 4 3 2 8 7 6 5 4 3 2 0.2 0.3 0 0.1 Total chlorophyll (mmol m"2) (b) T. guayacan (P = 0.018) ) 0.1 0.2 0.3 - (d) P. crucigerum (P = 0.0001) Q 0 0 - I 0.2 0.3 Figure 3. Relationships between the Chi al b ratio and Chi per unit area [Chi] for seed- lings of four species [(a) Tabebuia rosea, (b) T. guayacan, (c) Callichlamys latifolia, (d) Pithecoctenium crucigerum] grown under high (open symbol) and low (closed symbol) light treatments. Each point represents either a cotyledon (circle) or leaf (square for 24?30 d seedlings, and diamond for 50-65 d seedlings). As there is no significant light-by- [Chl] interaction in ANCOVA (Table 2), the regression line for the pooled data is shown for each species. ) 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 862 K. Kitajima & K. P. Hogan d.f. ^-values Variable T. rosea T. guayacan C. latifolia P. crucigerum Light [Chi] 1 1 1.38 (NS) 63.56*** 2.87 (NS) 0.37 (NS) 19 09*** 18.40*** 4.64* 13.0** Light x [Chi] interactions were non-significant for all four species. F-values are shown with the level of significance indicated as follows: ***, P< 0.001; **, P< 0.005; *, P < 0.05; NS, P>0.05. Table 2. Results of ANCOVA with Chi alb ratio as the dependent variable, light treat- ment as the main factor, and total chloro- phyll contents per unit leaf area, [Chi], as the covariate with decreasing leaf N in high light (P < 0.005) but not in low light (P > 0.1, Fig. 4b). Dark-acclimated Fv/Fm was high and did not differ among light and N treatments (mean across treatments = 0.77; Table 1). Quantum yields were also high and did not differ among treatments (mean = 0.091, Table 1). Thus, chronic photo-inhibition was not observed even in severely N- deficient leaves at high light. N treatment affected electron transport rate (Vj) and carboxylation capacity (Vcm!ix) estimated from the A-p{ rela- tionships more strongly in high than in low light (Fig. 5, Table 3). Overall, shaded plants had lower Vj and Vcmm. A measure of N use efficiency, Vcmax per mol N, significantly increased with a decrease in N supply in high light, but an 0 w o 3 E CO bU X sz 5(1 CO U E en o 40 CO t o 1 CM O 30 o 20 E F 10 0 25 30 35 40 45 50 Leaf nitrogen (mmol m"2) 55 Figure 4. Relationship of N per unit area with light- and C02- saturated gross photosynthetic oxygen evolution rate (Fmax) per unit area (a) and per unit mol Chi (b) for leaves of 50 d seedlings of Tabebuia rosea. Significant and non-significant regressions are shown as solid and broken lines, respectively. opposite trend was observed in the low light treatment (Table 3). C02 compensation point (p; at which point A is zero) did not differ significantly among light and N treat- ments (Table 3). DISCUSSION The results of this study supported predictions made by theoretical models of optimal nitrogen partitioning among photosynthetic components (Evans 1989; Hikosaka & Terashima 1995). The unique finding in this study was that the Chi alb ratio increases in response to N limitation, a theoretical prediction not previously demonstrated empiri- cally. In other aspects, these results corroborate existing empirical studies (Terashima & Evans 1988; Evans 1989; Sims & Pearcy 1989; Hikosaka & Terashima 1996; Bungard etal. 2000) as well as certain theoretical predictions. For example, acclimation to high light was accompanied by increases in electron transport rate (Vj), in Rubisco carbox- ylation capacity (Vcmax) per unit leaf area and unit chloro- phyll, and in the Chi alb ratio. Within each light treatment, Vj and Kmax both increased in response to N supply (Table 3). The Chi alb ratio should respond to N availability more strongly under high light than in shade (Hikosaka & Terashima 1995,1996). In shade, where optimal Pmax is con- strained by photon availability, optimal nitrogen allocation strategy suggests a consistently low ratio of PSII to LHCII across leaf N content, because the N cost per mole of chlo- rophyll is almost three times as high for PSII than for LHCII (Hikosaka & Terashima 1996). The result is a uni- formly low Chi alb in shade leaves independent of leaf N content (Fig. 2b). In contrast, in high light environments, N availability may affect partitioning among Rubisco, PSII and LHCII. In high light environments, as long as N supply is not limited, leaves should achieve very high Pmax through disproportionate investment of N into Rubisco relative to PSII, while maintaining sufficient [Chi] through a relatively low ratio of PSII to LHCII. However, when Pmax is con- strained by low N in the same high light environment, pro- portional N allocation to PSII should increase at the cost of decreased N allocation to Rubisco and LHCII (see Fig. 6 of Hikosaka & Terashima 1996). In support of this view, we observed a decrease in V^max per N at higher N availability in sun, but not in shade (Table 3). This difference between high- and low-N leaves exists because Pmax is related lin- early to PSII, but curvilinearly with Rubisco (i.e. less > 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 Photosynthetic acclimation and Chi a/b ratios 863 20 15 CO 10 CM E 5 CM o o 0 o E -9 ZL C o 10 CO 1 8 CO CO 6 CO "55 4 (a) High light0o0 o , ^o I I I 20 40 60 80 100 120 (b) Low light 20 40 60 P,. (Pa) 80 Figure 5. Relationship between net C02 assimilation rate (A) versus intercellular C02 partial pressure (/>;) for leaves of 50-day- old seedlings of Tabebuia rosea in high (a) and low light (b). Circles, high N; triangles, low N; squares, zero N. See Table 3 for the age of plants used in measurements. The scales differ in both axes between (a) and (b). increase in Pmax per unit increase in Rubisco at higher Rubisco content). Ultimately, the curvilinear relationship of Pmax to Rubisco exists because the rate of internal diffu- sion of C02 can become limiting to photosynthesis when Rubisco content per unit leaf area is high (Evans 1999). For all study species, the Chi alb ratio was negatively correlated with [Chi]. Variation in [Chi] within a light treat- ment for a given species was presumably due to variation in leaf N content, as in T. rosea (Fig. 2). Despite the expected difference between sun and shade in responsive- ness of the Chi alb ratio to N limitation, as discussed in the preceding paragraph, the slope of the linear correlation between Chi alb and [Chi] did not differ between high and low light treatments for any species. The responsiveness of the Chi alb ratio to N limitation, however, differed among the four species, ranging from a strong response in T. rosea to a weak response in T. guayaca (Fig. 3). Studies of other tropical trees and temperate herbs reported either no response or a slight decrease in the Chi alb ratio in response to N limitation under high light (Terashima & Evans 1988; Thompson et al. 1992; Hikosaka 1996). However, leaves of some Malaysian tree seedlings grown under lower N supply exhibited a slight but non-significant increase in Chi alb ratio as they recovered from photodamage over the course of 20 d (Bungard et al. 2000). Comparison of a larger num- ber of plant species from various habitats is necessary to understand which species characteristics, such as ecological habitat or life history traits, may be related to the respon- siveness of the Chi alb ratio to N limitation. The Chi alb ratios observed under the high light treat- ment in this study were higher than those reported in many other studies (Terashima & Evans 1988; Dale & Causton 1992; Turner, Ong & Tan 1995). This was partly because the Table 3. Summary of leaf traits for leaves of Tabebuia rosea grown under six treatment combinations of light and nitrogen availability High Low Light treatment N treatment One-way ANOVA P High Low Zero High Low Zero Plant age (days) 42 56 42 54 55 55 High Low Sample size 3 3 3 3 3 3 (HZ) (H,L,Z) N per leaf mass 2.71 1.41 1.14 2.92 3.06 2.33 0.0004 0.0012 (mmol g_1) (0.15) (0.31) (0.23) (0.23) (0.03) (0.12) Leaf mass per area 26.0 28.0 26.3 11.4 12.0 11.2 0.04 NS (gm-=) (2.1) (1.6) (2.0) (0.6) (1.2) (0.4) Initial slope 0.36 0.20 0.19 0.18 0.18 0.12 0.002 0.04 (umol C02 nT2 s"1 Pa"1) (0.02) (0.01) (0.02) (0.03) (0.03) (0.01) Vj (umol C02 nr2 s"1) 24.4 12.1 118 8.1 9.1 5.7 0.03 0.05 (4.5) (0.4) (1.1) (1.5) (1.6) (0.7) Kmax (umol C02 m2 s"1) 57.1 34.6 31.7 38.2 24.1 16.9 0.006 0.0004 (7.5) (0.4) (3.3) (2.9) (42) (1.2) T^cmux per Nitrogen 0.78 0.81 1.05 1.02 0.73 0.61 0.04 0.004 (mmol C02 mol N"1 s"1) (0.10) (0.01) (0.11) (0.08) (0.13) (0.04) Pi compensation point 7.05 4.66 5.94 7.04 6.86 6.99 NS NS (Pa) 1.13 (1.11) (0.50) (0.36) (1.56) (1.62) Characteristics of assimilation (A) - internal C02 partial pressure (p,) relationships: initial slope (0 Pa 0.05 for NS). ) 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 864 K. Kitajima & K. P. Hogan widely used Arnon's equations (Arnon 1949) tend to underestimate the Chi alb ratio (Porra, Thompson & Kried- mann 1989). For example, the Chi alb ratios of 2.7 and 4.8 reported in this study correspond to Chi alb ratios of 2.1 and 3.4, respectively, according to Arnon's equations. But, many Chi alb ratios observed in this study are high even in comparison to those reported for N-limited tropical tree seedlings in other studies that used equivalent spectropho- tometric equations (Thompson et al. 1992) and pigment determination with high-performance liquid chromatogra- phy (HPLC: Bungard et al. 2000). These differences possi- bly arise because N limitation was more severe in our study ([Chi] < 0.1 mmol irr2) than in the other two studies ([Chi] > 0.2 mmol irr2). An increase in the Chi alb ratio for N-deficient leaves may be viewed as a response to higher intracellular light intensity, because thylakoids of N-deficient leaves experi- ence greater PFD per PSII core for given incident light (Terashima & Hikosaka 1995). The PFD experienced by thylakoids depends on many factors, including the intraleaf light gradient, mesophyll cell arrangement, chloroplast ori- entation, and thylakoid stacking patterns (Terashima, Sak- aguchi & Hara 1986; Evans 1999). Excess light per PSII core must be dissipated in order to avoid photodamage. Chronic and severe N stress imposed upon seedlings in this experiment did not impair pre-dawn quantum yields or FvIFm ratios, suggesting that PSII reaction centres were largely intact. Xanthophyll cycle carotenoids, carboxylation capacity, and electron transport rates are all important in avoiding excess energy build-up in the PSII (Demming- Adams & Adams 1992; Horton, Ruban & Walters 1996; Hogan et al. 1997). Photodamage to the PSII reaction cen- tre may be exacerbated under nutrient deficiency because of constraints on the carboxylation capacity (Bungard et al. 2000). Nitrogen-deficient plants increase non-photochemi- cal dissipation of energy through an increase in xanthophyll cycle pigments per unit chlorophyll (Verhoeven, Demmig- Adams & Adams 1997; Bungard et al. 2000). It will be inter- esting to examine how the responses of the Chi alb ratio of species to nitrogen stress under high light may be corre- lated with production of xanthophyll pigments with HPLC measurements of pigments. More attention should be paid to N limitation in studies of growth and survival responses of tropical woody species relative to light availability. Interpretations of trenching experiments suggest that low soil N availability due to root competition significantly constrains seedling growth, espe- cially in light gaps (Coomes & Grubb 2000; Lewis & Tanner 2000; Ed Tanner, personal communication). Chi alb ratio can be easily measured in studies of plant response to N limitation as an indicator of N partitioning among different functional groups of photosynthetic components for both tropical and temperate plants. ACKNOWLEDGMENTS We thank the late Alan Smith for his mentorship, the Smithsonian Tropical Research Institute for logistical and fellowship supports, T Kursar, R. Pearcy, D. Sims, I. Terash- ima for useful suggestions during data collection and anal- ysis, D. Alvarez and A. Salazar for laboratory assistance, and K. Hikosaka, G. Bowes, R. Dudley and an anonymous reviewer for constructive comments. REFERENCES Allen S.E. (ed.) (1974) Chemical Analysis of Ecological Materials. Blackwell Scientific, Oxford, UK.. Araus J.L. & Hogan K.P. (1994) Leaf structure and patterns of photoinhibition in two neotropical palms in clearings and forest understory during the dry season. American Journal of Botany 81,726-738. Arnon D.J. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24,1-15. Bungard R.A., Press M.B. & Sholes J.D. (2000) The influence of nitrogen on rain forest dipterocarp seedlings exposed to a large increase in irradiance. Plant, Cell and Environment 23, 1183- 1194. Coomes D. A. & Grubb P.J. (2000) Impacts of root competition in forests and woodlands: a theoretical framework and review of experiments. Ecological Monographs 70,171-207. Croat T.B. (1978) Flora of Barro Colorado Island. Stanford Uni- versity Press, Stanford, CA, USA. Dale M.P. & Causton DR. (1992) Use of the chlorophyll a/b ratio as a bioassay for the light environment of a plant. Functional Ecology 6,190-196. Demming-Adams B. & Adams W.W. (1992) Photoprotection and other responses of plants to high light stress. Annual Review of Plant Physiology and Plant Molecular Biology 43, 599-626. Epstein E. (1972) Mineral Nutrition of Plants: Principles and Per- spectives. Wiley, New York, USA. Evans JR. (1989) Partitioning of nitrogen between and within leaves grown under different irradiances. Australian Journal of Plant Physiology 16, 533-548. Evans JR. (1993) Photosynthetic acclimation and nitrogen parti- tioning in a lucerne canopy. II. Stability through time and com- parison with a theoretical optimum. Australian Journal of Plant Physiology 20, 69-82. Evans JR. (1999) Leaf anatomy enables more equal access to light and C02 between chloroplasts. New Phytologist 14, 93-104. Green B.R. & Durnford D.G. (1996) The chlorophyll-carotenoid proteins of oxygenic photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 47, 685-714. Hikosaka K. (1996) Effects of leaf age, nitrogen nutrition and photon flux density on the organization of the photosynthetic apparatus in leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves. Planta 198,144- 150. Hikosaka K. & Terashima I. (1995) A model of the acclimation of photosynthesis in the leaves of C3 plants to sun and shade with respect to nitrogen use. Plant, Cell and Environment 18, 605- 618. Hikosaka K. & Terashima I. (1996) Nitrogen partitioning among photosynthetic components and its consequence in sun and shade plants. Functional Ecology 10, 335-343. Hirose T. & Werger M.J.A. (1987) Nitrogen use efficiency in instantaneous and daily photosynthesis of leaves in the canopy of Solidago altissima stand. Physiologia Plantarum 70, 215- 222. Hogan K.P., Fleck I., Bungard R., Cheeseman J.M. & Whitehead D. (1997) Effect of elevated C02 on the utilization of light energy in Nothofagus fusca and Pinus radiata. Journal of Exper- imental Botany 48,1289-1297. > 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865 Photosynthetic acclimation and Chi a/b ratios 865 Horton P., Ruban A.V. & Walters R.G. (1996) Regulation of light harvesting in green plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 655-684. Jeffrey S.W. & Humphrey G.F. (1975) New spectrophotometric equations for determining chlorophyll a, b, c: and c2 in higher plants, algae and natural phytoplankton. Biochemie und Physi- ologie der Pftanzen 167,191-194. Kirschbaum M.U.F. & Farquhar G.D. (1984) Temperature depen- dence of whole-leaf photosynthesis in Eucalyptuspauciflora Sieb. ex Spreng. Australian Journal of Plant Physiology 11, 519-538. Kitajima K. (1992) Relationship between photosynthesis and thickness of cotyledons for tropical tree species. Functional Ecology 6, 582-589. Kitajima K. (2002) Do shade-tolerant tropical tree seedlings depend longer on seed-reserve? Functional growth analysis of three Bignoniaceae species. Functional Ecology 16, 433^144. Lewis S.L. & Tanner E.V.J. (2000) Effects of above- and below- ground competition on growth and survival of rain forest tree seedlings. Ecology 81, 2525-2538. Linder S. (1974) A proposal for the use of standardized methods for chlorophyll determination in ecological and ecophysiological investigations. Physiologia Plantarum 32,154-156. Lovelock C.E., Jebb M. & Osmond C.B. (1994) Photoinhibition and recovery in tropical plant species: response to disturbance. Oecologia 97, 297-307. Milberg P., Perez-Fernandez M.A. & Lamont B.B. (1998) Seedling growth response to added nutrients depends on seed size in three woody genera. Journal of Ecology 86, 624-632. Mulkey S.S. & Pearcy R.W. (1992) Interactions between acclima- tion and photoinhibition of photosynthesis of a tropical forest understory herb, Alocasia macrorrhiza, during simulated canopy gap formation. Functional Ecology 6,719-729. Newell E.A., McDonald E.P., Strain B.R. & Denslow J.S. (1993) Photosynthetic responses of Miconia species to canopy openings in a lowland tropical rain forest. Oecologia 94, 49-56. Porra R.J., Thompson WA. & Kriedemann P.E. (1989) Determi- nation of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chloro- phyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Ada 975, 384-394. Schreiber U. (1986) Detection of rapid induction kinetics with a new type of high- frequency modulated chlorophyll fluorometer. Photosynthesis Research 9, 261-272. Sims D.A. & Pearcy R.W. (1989) Photosynthetic characteristics of a tropical forest understory herb, Alocasia macrorrhiza, and a related crop species, Colocasia esculenta grown in contrasting light environment. Oecologia 79, 53-59. Terashima I. & Evans JR. (1988) Effects of nitrogen nutrition on electron transport components and photosynthesis in spinach. Australian Journal of Plant Physiology 14, 59-68. Terashima I. & Hikosaka K. (1995) Comparative ecophysiology of leaf and canopy photosynthesis. Plant, Cell and Environment 18, 1111-1128. Terashima I., Sakaguchi S. & Hara N. (1986) Intra-leaf and intra- cellular gradients in chloroplast ultrastructure of dorsiventral leaves illuminated from the adaxial of abaxial side during their development. Plant Cell Physiology 27,1023-1031. Thompson W.A., Huang L.K. & Kriedemann P.E. (1992) Photo- synthetic response to light and nutrients in sun-tolerant and shade-tolerant rain-forest trees. 2. Leaf gas-exchange and com- ponent processes of photosynthesis. Australian Journal of Plant Physiology 19,19-42. Turner I.M., Ong B.L. & Tan H.T.W. (1995) Vegetation analysis, leaf structure and nutrient status of a Malaysian heath commu- nity. Biotropica 27, 2-12. Verhoeven A.S., Demmig-Adams B. & Adams W.W. Ill (1997) Enhanced employment of the xanthophyll cycle and thermal energy dissipation in spinach exposed to high light and N stress. Plant Physiology 113, 817-824. Walters M.B. & Reich P.B. (2000) Seed size, nitrogen supply and growth rate affect tree seedling survival in deep shade. Ecology 81,1887-1901. Whitmore T.C. (1989) Canopy gaps and two major groups of trees. Ecokgy 66,682-687. Received 18 July 2002; received in revised form 21 November 2002; accepted for publication 25 November 2002 i 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26,857-865