JXB Advance Access originally published online on July 1, 2003
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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1957-1967,
August 1, 2003
© 2003 Oxford University Press
Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation
Received 23 December 2002; Accepted 16 April 2003
1 Plant Research International, PO Box 16, 6700 AA, Wageningen, The Netherlands
2 Agrotechnological Research Institute (ATO), PO Box 17, 6700 AA, Wageningen, The Netherlands
3 Wageningen University, Department of Plant Sciences, Horticultural Production Chains Group, Wageningen, The Netherlands
4 School of Plant Biology, The University of Western Australia, Crawley WA 6009, Australia
5 Plant Ecophysiology, Utrecht University, Utrecht, The Netherlands
* Present address and to whom correspondence should be sent: Bejo Zaden BV, PO Box 50, 1749 ZH Warmenhuizen, The Netherlands. Fax: +31 226 393504. E-mail: c.degroot{at}bejo.nl
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Abstract |
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The effects were studied of both nitrogen and phosphorus limitation and irradiance on the performance and operation of photosynthesis in tomato leaves (Lycopersicon esculentum Mill.). Plants were grown at low N, high N, low P or high P supply and at two irradiances. Using mature leaves, measurements were made of the irradiance dependencies of the relative quantum efficiencies of photosystems I and II, and of the rate of carbon dioxide fixation. Measurements were also made of foliar starch and chlorophyll concentrations. The results showed that photosynthetic light-harvesting and electron-transport activity acclimate to nutrient stress and growth irradiance such that the internal relationships between electron transport by photosystems I and II do not change; the linear relationship between
PSII, and PSI was not affected. It was also evident that under N stress photosynthesis was reduced by a decreased light absorption and by the decreased utilization of assimilates, while P stress mainly affected the carboxylation capacity. Under N stress foliar starch levels increased and the oxygen sensitivity of CO2 fixation decreased, whereas P stress resulted in decreased starch levels and increased oxygen sensitivity of CO2 fixation. The relationship between starch accumulation and oxygen sensitivity (increased starch correlated with decreased oxygen sensitivity) was always the same across the nutrient treatments. These results are consistent with N deprivation producing an increasing limitation of photosynthesis, possibly by feedback from the leaf carbohydrate pool, whereas, although P deprivation produces a decreased rate of CO2 fixation, this is accompanied by a increase in oxygen sensitivity, suggesting that feedback limitation is decreased under P stress. Key words: Carboxylation activity, feedback limitation, light harvesting, nitrogen, phosphorus.
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Introduction |
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The response of plant growth to nitrogen limitation differs from the response to phosphorus limitation (Burns et al., 1997; De Groot et al., 2002). This may be due to the different functions of nitrogen and phosphorus in the plant. A relatively large part of reduced N in a plant is associated with enzymes that are required for energy metabolism (photosynthesis, respiration), whereas a relatively large part of organically bound Pi is incorporated in structural compounds (phospholipids, nucleic acids) (Mengel and Kirkby, 1987; Marschner, 1995). These different roles of N and P would suggest that the effects of limitation of N or P supply on photosynthesis will differ. Nitrogen, more so than phosphorus, is a component of the photosynthetic machinery. Nitrogen limitation therefore affects CO2 fixation directly through effects on photosynthetic structures rich in nitrogen, for example, chlorophyll, light-harvesting complex and Rubisco (Evans, 1989c; Hikosaka, 1996; Evans and Poorter, 2001). Furthermore, nitrogen limitation may affect CO2 fixation indirectly due to the limitation of growth and the subsequent accumulation of carbohydrates and feedback limitation of photosynthesis (Rufty et al., 1988; Paul and Driscoll, 1997; Rogers et al., 1998). Finally, the rate of net CO2 fixation per unit leaf nitrogen is known to increase with decreasing leaf nitrogen concentration (Boot et al., 1992; Pons et al., 1994). In the photosynthetic apparatus, phosphate plays a regulatory role in starch/sucrose biosynthesis and Rubisco activation, and a role in metabolites as it is used to phosphorylate intermediates of the Calvin cycle, and in energy availability (ATP and NADPH) (Edwards and Walker, 1983; Sawada et al., 1992). Phosphorus limitation may affect photosynthesis through changes in the activity of Calvin-cycle enzymes, RuBP regeneration, and/or Rubisco activity (Brooks, 1986; Rao and Terry, 1989; Jacob and Lawlor, 1991; Sawada et al., 1992). As with nitrogen, a feedback limitation of photosynthesis has been suggested as a cause of decreased CO2 fixation at low P supply (Pieters et al., 2001). However, for tomato plants a decrease in starch accumulation with decreasing P supply suggested that the production, rather than the utilization of photosynthates, was limiting (De Groot et al., 2001).
Feedback limitation of photosynthesis can be assessed by measuring CO2 fixation at 2% (v/v) O2 and comparing it with CO2 fixation at 21% (v/v) O2 (Foyer and Galtier, 1996). The reason for this is that Rubisco catalyses the carboxylation of RuBP as well as its oxygenation; the further metabolism of the product of this oxygenation reaction, phosphoglycolate, leads to the release of CO2 via the process of photorespiration. Due to the competitive effect of O2 on CO2 fixation, the rate of CO2 fixation by a leaf in air is less than would be achieved under conditions of low oxygen, where photorespiration is decreased. It is possible to calculate the effect of O2 on CO2 fixation using models of CO2 fixation based on the biochemical properties of Rubisco (Von Caemmerer and Farquhar, 1981). Using this approach, Foyer and Galtier (1996) estimated that changing the O2 concentration from 21% to 2%, at a CO2 concentration of 360 µmol mol–1 and leaf temperature of 25 °C, should produce an increase in CO2 fixation of about 40%, provided that the extra carbohydrates produced can be used (Foyer and Galtier, 1996). A lower increase of CO2 fixation indicates that photosynthesis is more or less limited by the capacity of the plant to utilize photosynthates. This provides a tool for assessing the role of feed back limitation of carbohydrates on photosynthesis. This tool was used to test the hypothesis that photosynthesis under N-limited conditions is limited by the use of photosynthates while under P-limited conditions the production of assimilates is limited. Furthermore, the hypothesis that photosynthesis is more limited by the utilization of photosynthates at higher growth irradiance than it is at lower growth irradiance was tested.
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Materials and methods |
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Growth of plants
Two experiments were conducted in which N supply (N experiment) or P supply (P experiment) was varied by daily adding N to an N-free nutrient solution or P to a P-free nutrient solution. The composition of the macronutrients and trace elements of the nutrient solution was as described by Steiner (1984), with the exception that, in the N-free nutrient solution, nitrate was replaced by phosphate and sulphate without changing the ratio of these ions, and in the P-free nutrient solution, phosphate was replaced by nitrate and sulphate without changing the ratio of these ions (Table 1). In both the N and P experiments seeds of tomato (Lycopersicon esculentum Mill. cv. Capita) were germinated on moistened vermiculite at 21 °C. At 8 d after sowing (DAS), seedlings were transferred from the vermiculite to 2.7 dm3 containers with either N-free nutrient solution brought to 1 mM NO-3 by adding KNO3 (N experiment) or with P-free nutrient solution brought to 55 µM H2PO-4 by adding KH2PO4 (P experiment), one plant per container. The containers were placed in a growth chamber with a photosynthetically active radiation (PAR) of 300 µmol m–2 s–1 for 16 h d–1 produced by TL-D-HF lamps (Philips, Eindhoven, the Netherlands) followed by 30 min of incandescent light. The relative humidity of the growth chamber was 70%. The day/night temperature was set to 23/23 °C. For the P experiment the pH was readjusted to 6.0 when it dropped below 5.5, with a 1:1 mixture of sulphuric and nitric acid. In the N experiment the pH remained stable.
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Nutrient and light treatments
At the beginning of the treatments (15 DAS) the nutrient solution in the containers was replaced by either N-free or P-free solution. Two levels of irradiance (70 and 300 µmol m–2 s–1), in combination with two rates of N or P supply, 170 and 320 mg g–1 d–1 (mg N or P g–1 N or P in plant d–1, henceforth mg g–1 d–1), were applied. Throughout this paper 70 µmol m–2 s–1 will be referred to as low irradiance, and 300 µmol m–2 s–1 as high, since this was the highest growth irradiance used in the experiments presented in this paper. The nutrient treatments will be referred to as low and high N or P (nutrient) supply. The initial amount of N and P in the plants at the start of the treatments was estimated to be 1.4 mg and 0.24 mg, respectively. In order to shorten the time needed for the plants of the low-nutrient supply treatments to adjust to the lower supply, the low-N and P plants were starved for N or P for 2 d. To create the low-growth-irradiance level, the plants were shaded by four layers of cheesecloth.
Photosynthesis measurements
CO2 fixation, the relative quantum efficiency of photosystem II electron transport (PSII.660) and the relative quantum efficiency of photosystem I (PSI) were measured simultaneously at 31 DAS using equipment similar to that described by Kingston-Smith et al. (1997, 1999). The gas mixing system employed three mass-flow controllers (Brooks Instruments, Veenendaal, the Netherlands) that blended pure nitrogen, oxygen and 5% CO2 in N2 to produce the gas phase required. This was humidified to 70% relative humidity using a laboratory-built humidifier, and buffered in a 10 l buffer tank. Gas flow to the assimilation chamber was measured by a mass-flow controller, and gas was also bled off to serve as the reference gas for the CO2 and humidity measurements. The CO2 concentration of the analysis gas stream was continuously measured with an infrared gas analyser (Binos, Hanau, Germany) and the difference in CO2 concentration between the reference and analysis gas streams was measured by another infrared gas analyser (type Mk3, ADC, Hoddesdon, UK).
The light source was a quartz halogen lamp filtered with NIR and Calflex dichroic mirrors, and with metal-film neutral-density filters (Balzers, Liechtenstein) to adjust the irradiance level at the leaf surface (Kingston-Smith et al., 1997). Two excitation wavelengths (560 and 660 nm measuring beams) were used to excite the chlorophyll fluorescence in order to measure PSII; the PSII measured by using the 660 nm measuring beam will be referred to as PSII,660, and that by using the 560 nm measuring beam as PSII,560. The efficiency of PSI was measured using the irradiance-induced absorbance change around 820 nm.
Irradiance-response curves of the third leaf, counted from plant base to top, were made in air consisting of 21% (v/v) O2, 370 µmol mol–1 CO2 with the remainder N2, the temperature was 23 °C. Photosynthesis parameters were measured when photosynthesis was in steady-state after acclimation to the irradiance level, this typically occurred after 30 min. Once steady-state CO2 fixation was achieved, the chlorophyll fluorescence and light-absorbance change measurements required to calculate the photochemical efficiencies of photosystems I and II were made. A dark respiration measurement was then made to allow the estimation of the rate of gross CO2 fixation. When CO2 fixation was light saturated, the O2 concentration was changed to 2% (v/v) (370 µmol mol–1 CO2 with the remainder N2), and about 45 min later, when photosynthesis was again in steady-state, the photosynthetic parameters were measured again. The quantum efficiency of gross CO2 fixation (CO2) at each measuring irradiance is calculated as the ratio of CO2 fixation to incident irradiance.
The light-saturated rate of CO2 fixation and the curvature factor of the irradiance-response curves were calculated by fitting a non-rectangular hyperbola (Thornley and Johnson, 1990):
where A (µmol m–2 s–1) is the CO2 fixation rate, I (µmol m–2 s–1) is the irradiance, is an estimate of the maximum quantum efficiency (based on incident irradiance), Amax (µmol m–2 s–1) is the light-saturated rate of CO2 fixation at infinitely high irradiances, and 
is a term that describes the curvature of the CO2 fixation–irradiance relationship. The non-rectangular hyperbolic model assumes that the relationship between CO2 fixation and irradiance is non-linear over the whole range of measured irradiances, thus also under strictly light-limited conditions. However, under these conditions the irradiance–response curve of CO2 fixation has a clear linear phase, so the estimation of may be in error. The light-limited, maximum, quantum efficiency of CO2 fixation (max CO2) is estimated as the difference in CO2 fixation at zero irradiance and at 30 µmol m–2 s–1 divided by the difference in irradiance (30 µmol m–2 s–1), which is the initial slope of the light–response curve. The calculation of max CO2 takes no account of variations in leaf absorptance, which are likely to occur under nutritional stress, therefore the maximum absolute quantum efficiency for CO2 (max *CO2) was calculated.
The maximum absolute quantum efficiency for CO2 fixation (based on absorbed irradiance; (max *CO2) was calculated as the difference in CO2 fixation at zero irradiance and at 30 µmol m–2 s–1 divided by the increase in absorbed irradiance. Leaf absorptance was calculated from the measured chlorophyll a+b concentrations ([Chl], mmol m–2), as described below.
The 660 nm (red) measuring beam used in the fluorescence measurements, is more strongly absorbed by the leaf than the 560 nm (green) measuring beam (Cui et al., 1991) and will thus mainly reflect PSII of the upper layers of the leaf. The 560 nm measuring beam penetrates deeper into the leaf, and thus lower leaf layers will make a greater contribution to PSII. When measuring CO2 fixation the relative contribution of the leaf layers to the total CO2 fixation depends on the penetration of the actinic light (Nishio et al., 1994). For the measurement of light-induced absorbance changes, from which PSI was calculated, a measuring beam of 820 nm was used. This wavelength is hardly absorbed and penetrates deeply into the leaf. For these reasons the 660 nm measuring beam was used when comparing PSII to CO2 fixation and the 560 nm measuring beam for comparing PSII to PSI (Harbinson, 1994; Kingston-Smith et al., 1997).
Harvests and chemical analysis
On another set of identically grown plants (De Groot et al., 2001, 2002) starch and chlorophyll concentrations were measured. For the starch measurements two leaflets of the third leaf were sampled at the end of the light period, and two were sampled at the end of the dark period. Leaf area and fresh weight of these samples were determined. The samples were freeze-dried, weighed and stored for starch determination as described in De Groot et al. (2001). The top leaflet of the third leaf was used for chlorophyll analysis. Three samples with a diameter of 9.5 mm, two at the base and one at the top of this leaflet, were taken and extracted with dimethylformamide for 48 h in the dark at 4 °C. Subsequently, the absorbance of the chlorophyll extract was measured spectrophotometrically (Shimadzu UV 160-A; Shimadzu Scientific Instrument Corp., Columbia, Md., USA) at 647.0 and 664.5 nm and the chlorophyll concentration (a+b, mmol m–2) was calculated (Inskeep and Bloom, 1985).
Estimation of leaf absorptance
The absorptance of 29 leaves in the spectral range 400–800 nm was measured at 2 nm intervals using a Taylor Sphere (for a non-diffuse incident irradiance) (Li-Cor, Lincoln, Nebraska, USA) and an Instaspec CCD spectrometer (Oriel Scientific, Stratford, CT, USA). The chlorophyll concentration ([Chl], mmol m–2) of these leaves was changed by withholding nitrogen from the plants for 0, 1, 2, 4 or 7 d. The chlorophyll concentration of the leaves was measured as described above. The relative spectral distribution of the light source used to provide irradiance for the photosynthesis measurements was measured at 2 nm intervals. The leaf absorptances and lamp spectrum were then normalized and multiplied together to provide a measure of the absorptance of the leaves for the total incident irradiance of the light source, which was all in the PAR range of the spectrum. The formula of Evans (1993) was fitted to the leaf absorptance for the incident irradiance and chlorophyll a+b concentration data of those 29 leaves (Fig. 1). This equation
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absorptance=[Chl]/([Chl]+0.074)
was used to estimate the leaf absorptance for the incident irradiance of the leaves used in the photosynthesis measurements described in this paper.
Statistics
Both the N and the P experiment were conducted twice, each time with two replicate plants per treatment, which gives a total of four replicate plants for the photosynthesis measurements. Chlorophyll and starch were measured on a different set of plants (n=6), grown at the same time, in the same growth chamber, using the same procedure. Data were analysed at a significance level of 
=0.05 with an ANOVA using GENSTAT 5 release 4.2 (Lawes Agricultural Trust, IACR-Rothamsted, UK). Differences were tested separately for the N and P experiment.
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Results |
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Light response curves
The maximum rate of CO2 fixation per unit leaf area (Amax) was highest for plants grown at high nutrient (N or P) supply and high growth irradiance (Fig. 2; Table 2). Low growth irradiance decreased Amax more than low N supply, and the combination of low light and low N supply decreased Amax even further (P <0.05; Fig. 2A; Table 2). Although at low growth irradiance a low N supply decreased Amax compared with high N supply, this effect was not statistically significant (Table 2). Compared with high growth irradiance and high P supply, low growth irradiance decreased Amax of plants grown at high P supply to the same extent as the combination of low growth irradiance and low P supply (P <0.05; Fig. 2B; Table 2). At low growth irradiance, Amax of plants grown with either high or low P supply was the same and this value was slightly higher than Amax of plants grown at low P supply and high growth irradiance, although this difference was not statistically significant (Table 2).
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Net CO2 fixation measured at growth irradiance (actual photosynthesis, Aact) was independent of the nutrient treatments for the plants grown at low growth irradiance (P >0.05; Table 2). Plants grown at a high growth irradiance showed a decrease in Aact when grown with a low N and a low P supply, compared with high N and high P supply (P <0.05). This effect was more pronounced for low P than for low N (Table 2).
No statistically significant differences were found for the curvature factor () of the light-response curves for the N experiment (Table 2). The curvature factor was significantly lower for plants grown at high P supply and high growth irradiance compared with the other treatments in the P experiment (P <0.05, Table 2).
Quantum efficiency for electron transport by PSII, PSI and CO2 fixation
With increasing measuring irradiance, both PSII,560 and PSI decreased (data not shown). This relationship was largely independent of the treatments (Fig. 3A, B). At low measuring irradiances (high efficiencies) PSII,560 decreased relatively more than PSI. This decrease was followed by a parallel decline in both PSII,560 and PSI with increasing measuring irradiance (Fig. 3A, B).
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With increasing measuring irradiance both
CO2 and PSII,660 decreased (data not shown). At low growth irradiance, the CO2 decreased in parallel with PSII,660, and this relationship was independent of the nutrient treatments (Fig. 4A, B). However, at high growth irradiance, the high N and high P treatments had, with the same PSII,660, a larger CO2 than the low nutrient treatments. From the relationship between PSII,660 and CO2 (Fig. 4) it is clear that the maximum value of CO2 (measured at 30 µmol m–2 s–1) is very close to an estimate of CO2 by extrapolating to the maximum PSII,660 at zero irradiance (Table 2).
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The maximum quantum efficiency of photosystem II electron transport (max
PSII,660, also known as the dark-adapted Fv/Fm) was highest for plants grown at high N or high P supply and high growth irradiance (Table 2). Low growth irradiance decreased max PSII,660, and for plants grown at this low growth irradiance there was no statistical difference between high N or P and low N or P supply. Max PSII,660 was lowest for plants grown at high growth irradiance with a low N or P supply. The maximum apparent quantum efficiency for CO2 fixation (max CO2) was highest at high N and P supply for plants grown at high growth irradiance (Table 2). Max CO2 was most reduced by low N and P supply for plants grown at high growth irradiance. Also low growth irradiance and the combination of low growth irradiance and low N or P supply decreased max CO2, though to a lesser extent than low N or P supply at high growth irradiance (Table 2).
When grown at high growth irradiance max PSII,660 was decreased by 9% (P <0.05) with a low P supply compared with a high P supply (Table 2). However, this decrease was relatively small compared with the 29% decrease in max CO2 produced by the same change in P nutrition (P <0.05; Table 2). At high growth irradiance, low N supply decreased maxPSII,660 by just 4% (P <0.05), while max CO2 decreased by 27% (P <0.05; Table 2). When plants were grown at low growth irradiances, low N and P supply did not significantly affect max PSII,660 and max CO2 (Table 2). Furthermore, at high P supply effects of growth irradiance on max PSII,660 and max CO2 were also not statistically significant (Table 2). At high N supply, low growth irradiance caused a small, but significant (P <0.05) decrease in max PSII,660 and max CO2. At low N supply max CO2 increased significantly with decreasing growth irradiance (P <0.05), max PSII,660 was not affected (Table 2).
Chlorophyll a+b concentration (mmol m–2) decreased with decreasing nutrient availability and decreasing growth irradiance (P <0.05), and the interaction between growth irradiance and nutrient supply was significant (P <0.05). The decrease in chlorophyll concentration was more pronounced for N than for P (Table 2). At high growth irradiance, low N supply decreased the chlorophyll concentration by 51%, while at low growth irradiance the chlorophyll concentration was decreased 35% (P <0.05) by low N supply. At high N supply, chlorophyll concentration decreased by 30% with decreasing growth irradiance. However, at low N, no significant effect of a decreased growth irradiance could be detected (Table 2).
At high growth irradiance decreasing P supply decreased the chlorophyll concentration by 27% (P <0.05). At low growth irradiance there was a non-significant increase of 13% at low P. At high P supply a decrease in growth irradiance decreased the chlorophyll concentration (P <0.05), whereas at low P supply the decrease in chlorophyll concentration with decreased growth irradiance was not significant (Table 2).
The absolute maximum quantum efficiency of CO2 fixation (max *CO2) showed the same pattern as the apparent maximum quantum efficiency (max CO2), though the treatment effects were smaller (Table 2). At high growth irradiance changes in leaf absorptance could explain 10 percentage points of the 27% decrease of max CO2 with decreasing N supply. From the 29% decrease in max CO2 with decreasing P supply for plants grown at high irradiance only three percentage points could be explained by changes in leaf absorptance (Table 2). When plants were grown at low growth irradiance nutrient supply did not have a significant effect on max CO2. Despite that, the effect of leaf absorptance on max CO2 was larger at low N than at high N supply (P <0.001); max *CO2 was not significantly different at low N from that at high N (Table 2).
For plants grown at high N supply five percentage points of the decrease in max CO2 with decreased growth irradiance could be explained by differences in light absorptance by the leaves and max *CO2 was not significantly different for low and high growth irradiances at high N supply (Table 2). At high P supply nine percentage points of the decrease in max CO2 with decreasing growth irradiance could almost completely be explained by differences in light absorptance; max *CO2 was the same for low and high growth irradiance (Table 2). At low N or P supply max CO2 was higher at low irradiance than at high irradiance. This difference could not be explained by differences in light absorptance; the relative difference between low and high irradiance was the same for max *CO2 as max CO2 (Table 2).
Because of the sequential character of the processes of photosynthesis, a decrease in PSII quantum efficiency will cause the quantum efficiency of CO2 fixation to decrease to the same extent. If the quantum efficiency of CO2 fixation decreases relatively more than the PSII quantum efficiency this implies that the quantum efficiency of CO2 fixation is being affected by factors other than, or in addition to, PSII quantum efficiency. For plants grown at high growth irradiances, eight percentage points of the 29% decrease in max CO2 with decreasing P supply can be explained by the decrease in max PSII,660, whereas, when N is considered, only three percentage points of the 27% decrease in max CO2 can be explained by the decrease in max PSII,660 (Table 2).
Feedback limitation of photosynthesis
The ratio between the light-saturated rates of CO2 fixation at 21% O2 and at 2% O2, after eliminating photorespiration, gives information about feedback limitation of photosynthesis (Foyer and Galtier, 1996). When compared for the same nutrient supply treatments plants grown at low growth irradiance showed a smaller stimulation of CO2 fixation, than plants grown at high growth irradiance (Fig. 5A, B). Eliminating photorespiration resulted in a 30% stimulation of photosynthesis for plants grown at high growth irradiance and high N and P supply (Fig. 5A, B). CO2 fixation of plants grown at low N and high growth irradiance was stimulated to the same extent as CO2 fixation of plants grown at high N and low growth irradiance (22% and 21%, respectively; Fig. 5A). Photosynthesis was least stimulated at the low growth irradiance and low N treatment (14%, Fig. 5A). For the N experiment the effect of growth irradiance on the ratio between the light-saturated rates of CO2 fixation at 2% and 21% O2 was significant (P <0.05), the effect of N supply was not significant (P=0.07) and also the interaction between light and N supply was not significant.
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CO2 fixation of leaves grown at high growth irradiance and low P supply increased by 38% after eliminating photorespiration, which is higher than the increase measured on plants grown at high growth irradiance and high P supply (29%). Also in leaves of plants grown at low growth irradiance CO2 fixation was more stimulated in P-limited plants (23%) than in P-sufficient plants (14%, Fig. 5B). The interactive effects of growth irradiance and P supply on the ratio between light-saturated CO2 fixation at 2% and 21% O2 were not significant; however, the main effects of light treatment (P <0.01) and P supply treatment (P <0.05) were.
The ratio between the light-saturated rates of CO2 fixation at 2% and 21% O2 correlated well with starch concentration, measured at the end of the light period (r2=0.79). The fitted lines differed for high and low growth irradiance only for the overall level of the line (P <0.01); the difference in slope was not significant (Fig. 6).
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Discussion |
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This discussion consists of two parts. The first part discusses the acclimation of photosynthetic light-harvesting and electron-transport activity to nutrient stress and growth irradiance. The second part of this discussion deals with the regulation of photosynthesis in relation to feedback limitation under low N, P and irradiance conditions. Throughout this paper 70 µmol m–2 s–1 was referred to as low irradiance and 300 µmol m–2 s–1 was referred to as high, since 300 µmol m–2 s–1 was the highest irradiance used in the experiments presented in this paper. However the reader should note that 300 µmol m–2 s–1 is less than the irradiance required to light-saturate CO2 fixation in the leaves used in these experiments. So, the ‘high irradiance’ is high relative to the 70 µmol m–2 s–1 treatment, but not in terms of the light-response of photosynthesis.
Quantum efficiency for electron transport by PSII, PSI and CO2 fixation
The linear relationship between PSII,560 and PSI (Fig. 3) suggests that the control of the development of the photosystems is very robust; growth irradiance and N and P supply had large effects on the rates of light-saturated CO2 fixation and the foliar chlorophyll concentrations (Table 2), but the linear relationship between PSII,560 and PSI was not affected. Furthermore, this linear relationship between PSII and PSI (Fig. 3) shows that electron transport is largely non-cyclic and that if cyclic electron transport occurs, it does so as a constant percentage of the rate of linear electron transport (Harbinson, 1994). The relationship between PSII and PSI has been shown before to be independent of temperature stress in maize (Kingston-Smith et al., 1999) and photorespiration (Genty et al., 1990). It has been shown that growth irradiance and N and P supply do not affect the relationship between PSII and PSI. There was also no evidence of photoinhibition of PSII, except for plants grown at high growth irradiance and low P. These data show the capacity of the photosystems to acclimate to stress and retain stable operation.
The maximum apparent quantum efficiency of CO2 fixation (max CO2) was significantly decreased at low nutrient supply compared with that at high nutrient supply, at high growth irradiance (Table 2). Decreases in max CO2 as a result of low N and low P supply have been reported before (Brooks, 1986; Jacob and Lawlor, 1991; Plesni
ar et al., 1994; Hikosaka and Terashima, 1995). To gain an insight into the possible causes of the decrease in max CO2 the effect of leaf absorptance was first estimated. The calculation of max CO2 takes no account of variations in leaf absorptance, and changes in the latter are likely to occur under nutritional stress. Leaf absorptance, calculated from the measured chlorophyll concentration, was used to estimate the maximum absolute quantum efficiency of CO2 fixation (max *CO2; Table 2). At high growth irradiances, the decrease in max CO2 at low N compared with high N supply was mainly due to a decrease in light absorptance (chlorophyll concentration), rather than due to a decrease in max PSII. The opposite was true for low P compared with high P supply at high growth irradiance. This applies to the whole range of measured irradiances (Fig. 4) and shows that N limitation mainly affects chlorophyll concentration and thus absorptance and light harvesting, while P limitation mainly affects the functioning of PSII. Nonetheless, the functioning of PSII and PSI remain co-ordinated under both N and P limitation. Stomatal conductance and internal leaf CO2 concentration are two factors determining CO2, and may explain part of the decrease in max CO2 with N and P limitation at high growth irradiance. At high nutrient supply max *CO2 was not significantly different for low and high irradiance. This indicates that the difference in max CO2 was caused by the decrease in chlorophyll concentration and, thus, light absorptance.
Though the increase from max CO2 to max *CO2 of leaves grown at low growth irradiance is significantly higher for low N leaves than for high N leaves (P <0.001, data not shown), the difference between max CO2 at high N compared to low N leaves, and max *CO2 at high N compared to low N leaves were not significant (Table 2). This is explained by the fact that max CO2 for low N leaves is less than that for high N leaves, whereas the max *CO2 for low N leaves is greater than for high N leaves. This also implies that at low growth irradiance of light, absorption by leaves is significantly decreased by N limitation.
Feedback limitation of photosynthesis
At low growth irradiance, the response of light-saturated CO2 fixation to a decrease in O2 concentration was less than at high growth irradiance (Fig. 5); the cytosolic carbohydrate metabolism was less capable of processing the extra carbohydrates theoretically made available by eliminating photorespiration. CO2 fixation was determined less by the competitive effects of O2 for carboxylation of RuBP by Rubisco at low than at high growth irradiance, i.e. factors other than the properties of Rubisco were influencing the regulation of CO2 fixation in plants grown at low growth irradiances. At low growth irradiance plants invest more in chlorophyll–protein complexes rather than into Calvin-cycle enzymes (including Rubisco) (Evans, 1989a, b; Hikosaka and Terashima, 1995). The reduced investment in Rubisco and other Calvin-cycle enzymes in the chloroplast and in enzymes and structures associated with carbohydrate processing in the cytosol, can gain important savings to the plant without affecting CO2 fixation (Björkman, 1981). A relative reduction in cyto solic enzymes and structures can produce feedback limitation of photosynthesis when measured at saturating light and low O2 concentration because it decreases the capacity to process carbohydrates. Therefore, the photosynthesis of leaves grown at low irradiance may be more feedback limited than the photosynthesis of high-irradiance-grown leaves when measured at saturating light and low O2 concentration. These conditions produce large amounts of extra carbohydrates for which the low-irradiance-grown leaves are less equipped to process than the high-irradiance-grown leaves. A reduced investment in Rubisco and other Calvin-cycle enzymes and an increased investment in chlorophyll–protein complexes at low growth irradiances has been shown for several species (Hikosaka, 1996; Evans and Poorter, 2001).
Besides low growth irradiance, a low N supply also decreased the ability of the photosynthetic apparatus to process extra carbohydrates. A large portion of N in a plant is associated with the machinery of the plant’s energy metabolism (e.g. photosynthetic apparatus, respiratory system) and low N leads to a decrease in this machinery (Mengel and Kirkby, 1987). This means that the increase in the availability of carbon at 2% O2, by eliminating photorespiration, cannot stimulate photosynthesis, simply because the extra machinery needed to process this extra carbon is not available at low N, leading to the photosynthesis being more oxygen insensitive at low N than at high N supply. This is supported by the increase in starch accumulation at low N as reported elsewhere (De Groot et al., 2002), which showed that with decreasing N supply the transport and/or utilization of assimilates is reduced, and photosynthesis thus becomes limited by the lack of a direct sink for photoassimilates produced (Rufty et al., 1988; Paul and Driscoll, 1997; Rogers et al., 1998).
For both growth irradiances plants grown at low P supply showed a larger response to eliminating photorespiration than plants grown at high P supply (Fig. 5B). The low P plants appeared to be better capable of processing the extra carbohydrates gained from eliminating photorespiration than high P plants. This implies that photosynthesis was not limited by the utilization of photosynthates, and thus not sink limited, but that the production of assimilates was reduced at low P (Brooks, 1986; Sharkey, 1985; Sharkey et al., 1986).
The present results are in contradiction with experiments performed on tobacco (Nicotiana tabacum L.) and Arabidopsis from which it was concluded that, during the development of Pi deficiency, photosynthesis was limited by low sink demand and thus by the accumulation of photosynthetic products (Pieters et al., 2001; Ciereszko et al., 2001). An explanation for these contrasting results may be found in the different methods used to apply P limitation in these studies. Due to the method of nutrient addition used in this study, plants are considered to be acclimated to the low P supply, while Ciereszko et al. (2001) and Pieters et al. (2001) measured sink limitation during the development of Pi deficiency in P-deprived plants. Totally depriving a plant of phosphorus does not allow the plant to acclimate. Furthermore, sink limitation was studied at the leaf level, while in the studies mentioned above the whole plant was considered (Ciereszko et al., 2001; Pieters et al., 2001). Finally, the response of the photosynthetic apparatus of tomato leaves may be inherently different from the response of tobacco leaves. These differences might offer an explanation for the contradiction between the results presented here and by Ciereszko et al. (2001) and Pieters et al. (2001).
The near-maximal increase in CO2 fixation of 38%, in the low P treatment, at high growth irradiance (Fig. 5), suggests that, under these circumstances, the responses of CO2 fixation were determined by the biochemical properties of Rubisco, especially the competitive effects of O2 for carboxylation of RuBP by Rubisco. Decreases in the activity and amount of Rubisco due to P limitation have been shown for other C3 plants, for example, spinach, sunflower and soybean (Brooks, 1986; Jacob and Lawlor, 1991; Sawada et al., 1992). From experiments with sugar beet it has been concluded that low P affects photosynthesis through an effect on RuBP regeneration rather than through Rubisco activity (Rao and Terry, 1989; Rao et al., 1989). However, the response of the plants used by these authors was different from that of this study as they found an increase in starch accumulation at low P while in this study’s tomato plants starch accumulation in the leaves decreased at low P (Fig. 6; De Groot et al., 2001). The reason for this difference in response to low P remains unclear, but it is not impossible for low chloroplastidic Pi to produce an inhibition of Rubisco or a regeneration of RuBP and at the same time produce no accumulation of starch: it is just a matter of the regulatory balance between the regulation of carbohydrate formation on the one hand and starch synthesis on the other.
Despite differences in the responses to nutrient limitation of the tomato plants reported here and responses to nutrient limitation reported by others, the response of photosynthesis to a change in O2 concentration and starch accumulation in the leaves was consistent for this study’s plants (Fig. 6). The ratio between maximal CO2 fixation at 2% and 21% oxygen correlates well with starch accumulation at high light, regardless of the nutrient treatments (Fig. 6). A low ratio of maximal CO2 fixation at 2% and 21% O2 results from the limitation of triose-phosphate utilization in the cytosol (Sharkey et al., 1986; Micallef et al., 1995; Stitt, 1991), which consequently may limit triose-phosphate export from the chloroplast. Triose-phosphate that cannot be exported to the cytosol remains in the chloroplast and is converted into starch. This may explain the clear relationship between maximal CO2 fixation at 2% and 21% oxygen (Fig. 6). At low growth irradiance the relationship is less strong; however, the slope of this relationship was not significantly different from the relationship at high growth irradiance. At low growth irradiance, light was the most important determinant of acclimation, while the effect of low nutrient supply was of only marginal importance. This small effect of low nutrient supply on CO2 fixation at low growth irradiance as shown by the light-response curves (Fig. 2; Table 2) might explain the weak relationship at low growth irradiance (Fig. 6).
There are several hypotheses or models proposed to explain how feedback limitation of photosynthesis may be regulated. Sawada et al. (1992) proposed a model for Pi regulation of Rubisco activity in sink-limited plants. When photosynthesis is sink limited the production of triose-P exceeds the capacity to transport triose-P out of the chloro plast and/or the capacity to process the triose-P in the cytosol. Consequently, triose-P, and other phosphorylated intermediates of photosynthetic carbon metabolism, will accumulate and Pi will decrease (Sawada et al., 1992). This decrease in Pi may restrict ATP synthesis, which may cause deactivation of Rubisco-activase, and the deactivation of Rubisco results in an accumulation of RuBP (Streusand and Portis, 1987; Butz and Sharkey, 1989). Alternatively, decreased Pi levels in the chloroplast may inhibit the activity of Rubisco directly and, subsequently, reduce CO2 fixation (Mächler and Nösberger, 1984; Parry et al., 1985; Sawada et al., 1992). However, in the low P treatment described in this paper, Pi was low all the time, suggesting that this would limit photosynthesis and cause oxygen-insensitive photosynthesis (Pieters et al., 2001). However, photosynthesis was more responsive to low oxygen at low P than at high P (Fig. 5B), suggesting that photosynthesis was limited by the properties of Rubisco as discussed above. Starch concentration (Fig. 6; De Groot et al., 2001) and total soluble sugar concentration (De Groot et al., 2003) were decreased with P limitation. It has been hypothesized that increased leaf carbohydrate levels and decreased turnover may provide a signal to down-regulate photosynthesis at sink limitation through the modulation of photosynthetic genes, for example, the gene coding for the small subunit of Rubisco (Cheng et al., 1998; Paul and Foyer, 2001). It is suggested that the carbohydrate level, rather than Pi, provides the initial signal for feedback regulation of photosynthesis.
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In this paper it was shown that the photosynthetic light-harvesting and electron-transport adjusts to nutrient stress and light, and retains stable operation. It was shown that under N stress photosynthesis was reduced, presumably by a decreased absorption efficiency and by decreased utilization of assimilates, whereas P stress mainly affected the carboxylation capacity and thus the production of assimilates. Furthermore, at low growth irradiance leaves invested in light-absorption efficiency rather than in enzymes and structures associated with carbohydrate processing in the cytosol, as shown by the increased feedback limitation of photosynthesis at low irradiance compared with high irradiance, when measured at low O2 concentration and saturating irradiance.
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We are grateful to Manon Mensink for her help with the measurements and to her and Els Otma for fitting the light response curves.
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