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This study tests the hypothesis that diffusional limitation of photosynthesis, rather than light, determines the distribution of photosynthetic capacity in olive leaves under drought conditions. The crowns of four olive trees growing in an orchard were divided into two sectors: one sector absorbed most of the radiation early in the morning (MS) while the other absorbed most in the afternoon (AS). When the peak of radiation absorption was higher in MS, air vapour pressure deficit (VPD) was not high enough to provoke stomatal closure. In contrast, peak radiation absorption in AS coincided with the daily peak in VPD. In addition, two soil water treatments were evaluated: irrigated trees (I) and non-irrigated trees (nI). The seasonal evolution of leaf water potential, leaf gas exchange and photosynthetic capacity were measured throughout the tree crowns in spring and summer. Results showed that stomatal conductance was reduced in nI trees in summer as a consequence of soil water stress, which limited their net assimilation rate. Olive leaves displayed isohydric behaviour and no important differences in the diurnal course of leaf water potentials among treatments and sectors were found. Seasonal diffusional limitation of photosynthesis was mainly increased in nI trees, especially as a result of stomatal limitation, although mesophyll conductance (gm) was found to decrease in summer in both treatments and sectors. A positive relationship between leaf nitrogen content with both leaf photosynthetic capacity and the daily integrated quantum flux density was found in spring, but not in summer. The relationship between photosynthetic capacity and gm was curvilinear. Leaf temperature also affected to gm with an optimum temperature at 29 °C. AS showed larger biochemical limitation than MS in August in both treatments. All these suggest that both diffusional limitation and the effect of leaf temperature could be involved in the seasonal reduction of photosynthetic capacity of olive leaves. This work highlights the need for models of plant growth and ecosystem function to incorporate new parameters affecting the distribution of photosynthetic capacity in canopies.
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Distribution of photosynthetic capacity in leaves within a tree crown has been reported to be closely related to the daily-integrated quantum flux density (Qint) (Le Roux, Sinoquet & Vandame 1999; Meir et al. 2002; Niinemets, Kull & Tenhunen 2004a). This evidence supports the optimization theory (Anten 2000), whereby plants optimize the investment in the photosynthetic apparatus within the tree crown according to absorbed irradiance. Greater photosynthetic capacity is less likely in locations where light is the primary limiting factor, because of greater maintenance respiration costs. However, in locations where light is not a limiting factor, for example, the outer layer of a tree crown, other factors may influence the distribution of photosynthetic capacity.
The Mediterranean environment is characterized by hot, dry summers with high daily irradiance and evaporative demand and significant, long-term soil water deficit (Flexas & Medrano 2002a). To maintain a balance between water supply and demand, the most immediate response of plants to water stress is to limit leaf transpiration by stomatal closure. However, this also causes leaf intercellular CO2 concentration (Ci) to decline, thereby limiting photosynthesis. Stomatal limitation of photosynthesis mediated by water stress has been widely studied (Jones 1985; Flexas & Medrano 2002b; Grassi & Magnani 2005), and it is currently accepted as one of the main limitations to plant productivity in dry-land ecosystems. But under mild water stress, there are multiple evidences that mesophyll conductance also plays an important role in limiting photosynthesis (Grassi & Magnani 2005; Warren 2006a). Evans et al. (1986) and Loreto et al. (1992) demonstrated that mesophyll conductance is finite, and therefore, introduces another diffusive photosynthetic limitation in addition to stomatal conductance. The sum of both stomatal and mesophyll limitations represents the diffusional limitations of a leaf. However, under severe water stress, it has been reported also a down-regulation of biochemical capacity to assimilate CO2 (Flexas et al. 2004). This can be observed as a reduction in the maximum carboxylation capacity (Vcmax) measured. This biochemical limitation to photosynthesisplus mesophyll defines what it is called non-stomatal limitation of photosynthesis. In olive, all these three limitations have been already reported. Under drought conditions, it has been shown in olive stomatal limitation (Moriana, Villalobos & Fereres 2002; Diaz-Espejo et al. 2006), mesophyll limitation (Centritto, Loreto & Chartzoulakis 2003; Niinemets et al. 2005) and biochemical limitations (Giorio, Sorrentino & d'Andria 2003; Diaz-Espejo et al. 2006).
During leaf ontogeny, nitrogen is allocated to newly developing leaves as a function of their light microenvironment, such that leaves in sunny positions have greater photosynthetic capacity than leaves in shaded locations (Niinemets, Tenhunen & Beyschlag 2004b). However, high diffusional limitations of photosynthesis during leaf ontogeny could cause a similar down investment in leaf nitrogen to the one described earlier related to the light available. Grassi et al. (2005) showed that a high proportion of the seasonal variability in photosynthetic capacity found in oak and narrow-leaved ash was a function of nitrogen and soil water potential, demonstrating the importance of including a water stress function for the accurate modelling of forest net ecosystem exchange. On the other hand, Medlyn, Lousteau & Delzon (2002) suggested that the seasonal change in the photosynthetic temperature response of maritime pine could be mediated by the seasonal change in the stomatal response to air vapour pressure deficit (VPD). Stress caused by soil water deficit and/or high VPD could be one of the main factors responsible for the usually found deviations from the theoretical optimum values of distribution of photosynthetic capacity and leaf nitrogen in canopies as a function of maximizing light capture (Hollinger 1996; Meir et al. 2002). Stomata of olive leaves respond strongly to soil water deficit and especially to increasing VPD (Loreto & Sharkey 1990; Fernández et al. 1997; Moriana et al. 2002). Therefore, in summer when VPD is high, stomatal limitation of photosynthesis commonly occurs. Soil water stress enhances the stomatal response to atmospheric demand. But also, mesophyll conductance has been reported to decrease under salinity stress (Centritto et al. 2003), suggesting that its role in gas exchange studies of water stress must be considered.
In this study, we have tested the hypothesis that distribution of nitrogen and photosynthetic capacity of new leaves in a crown of an olive tree is primarily determined by Qint, only when there is not any stronger limitation imposed by another environmental factor. The main factor considered in this work influencing the photosynthetic capacity distribution was the effect of diffusional limitation to photosynthesis. We chose different locations in crowns of olive trees where the peak of Qint was reached at different time of the day, when atmospheric demand was very contrasting. Furthermore, we imposed two irrigation treatments to study whether the combination of water deficit and high VPD increased their influence in the photosynthetic capacity.
MATERIALS AND METHODS
Field site and plant material
Measurements were carried out during 2002 at La Hampa experimental farm (37° 17′ N, 6° 3′ W; altitude 30 m), near Seville (SW Spain). The farm contained a 0.5 ha olive orchard with 34-year-old trees planted at 7 × 5 m spacing. The tree crowns had a spherical shape of about 4.5 m diameter, 4 m height and a single trunk with two main branches. Trees were subjected to normal crop loads, and there were not significant differences in crop load among experimental trees. The soil is a sandy loam (Xerochrept) of homogeneous texture with depth and average values of 14.8% clay, 7.0% silt, 4.7% fine sand and 73.5% coarse sand. Soil water content values at field capacity (−0.03 MPA) and wilting point (−1.5 MPA) were 0.21 and 0.10 m3 m−3, respectively.
Two irrigation treatments were imposed: (1) irrigation treatment (I) where an earthern dyke was built around the trees and enough water was supplied biweekly to reach soil water capacity. This irrigation method ensured a complete watering of the whole root zone; (2) Non-irrigation treatment (nI) in which rainfall was the only source of water. Each treatment was applied to two representative trees within the orchard.
Meteorological measurements (net radiation, global radiation, photosynthetically active radiation, wind speed, rainfall, air temperature and relative humidity) were monitored continuously, and 30 min average values were logged to an automatic weather station (Campbell Scientific Ltd, Shepshed, UK) located 50 m away from the experimental trees. Volumetric soil water content (θ) was measured in the nI treatment by neutron probe (Troxler 3300, Research Triangle Park, NC, USA), except in the top 0.2 m where θ was estimated by gravimetric measurements and averaged values of soil bulk density. Four access tubes were installed in each nI tree at distances of 0.5, 1.5, 2.5 and 3.5 m from the trunk, along the tree row and down to the maximum depth of the root zone (2 m). In the I trees, θ was estimated from gravimetric measurements up to 2 m, to avoid installing access tubes that may lead to preferential channels for water along the wall of the access tubes. Measurements were conducted every 2 weeks, and in the case of the irrigated trees just before and a day after irrigation took place.
Photosynthetic photon flux density (PPFD) measurements
The crowns of the experimental trees were divided into two sectors, one in which the peak of daily radiation was received early in the morning (MS), and the other in which the peak of radiation was in the afternoon (AS). PPFD was measured using a hand-held line-integrating ceptometer (AccuPAR, Decagon Devices, Inc., Pullman, WA, USA) in one location within each sector (two replicates). Measurements were made in April and August in the outer part of the crown on clear days at 2 m height, where leaf gas exchange measurements were also carried out, as explained as follows. Measurements were conducted every hour from sunrise to sunset to characterize the Qint, which was averaged over a certain number of days providing the final value of Qint.
Leaf water status, gas exchange measurements and leaf temperature
Measurements were made in April and in August in leaves nearby the sites where Qint was measured. In both cases, sampled leaves were 50–70 days old. We chose fully mature leaves (Marchi et al. 2005) to avoid the affect of leaf ontogeny on gas exchange (Niinemets et al. 2005). Leaf water status was quantified by measuring leaf water potential (ψ) with a pressure chamber (Soilmoisture Equipment Corp., Santa Barbara, CA, USA). Diurnal courses of ψ were obtained after measuring 2 leaves per sector in each tree (four replicates). Survey measurements of gas exchange were conducted with a portable photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA), to determine net CO2 assimilation rate (A) and stomatal conductance (gs). Three leaves per sector in each tree (six replicates) were sampled. Leaf temperature (Tleaf) was measured with the thermocouple inside the cuvette of the LI-6400. Photosynthetic capacity of leaves was estimated from the analysis of A–Ci response curves. Measurements were made with a 2 × 3 cm broadleaf chamber and an integrated light source (LI-6400-02B; Li-Cor). The curves were performed under saturating irradiance (1600 µmol m−2 s−1), and under constant leaf temperature (20 °C in April and 25 °C in August) by controlling the CO2 concentration of inlet air in 11 steps from 50 to 1400 µmol mol−1 (see Diaz-Espejo et al. 2006 for details). Diffusion leaks when performing the curves were taken into account by applying the manufacturer's equation to determine the diffusion coefficient (Anonymous 2005). Six leaves per treatment and sector were measured in April, and 4 leaves per treatment and sector in August. Photosynthetic parameters maximum rate of carboxylation by ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) (Vcmax), the maximum rate of electron transport (Jmax) and Rd (the rate of non-photorespiratory CO2 evolution in the light) were determined according to the Farquhar model of leaf photosynthesis (Farquhar, von Caemmerer & Berry 1980; von Caemmerer & Farquhar 1981; Harley & Tenhunen 1991). The temperature dependencies of photosynthetic parameters were calculated according to Bernacchi et al. (2002), using parameters specifically determined for olive leaves (Diaz-Espejo et al. 2006) and modified taking into account the effect of mesophyll conductance (gm). To do this, the whole data set of A–Ci curves measured at different temperatures under controlled conditions in Diaz-Espejo et al. (2006) was reanalysed. The response of gm to temperature was also obtained from this analysis. Once calculated, the photosynthetic parameters derived from A–Ci curves under field conditions, both in April and August, were normalized to 25 °C. A–Ci curves were fitted with a non-rectangular hyperbola version of the biochemical model of leaf photosynthesis following Ethier & Livingston (2004) (Fig. 1). The main premise of this method is that gm reduces the curvature of the Rubisco-limited portion of an A–Ci response curve. Values of the Michaelis–Menten constant for CO2 (Kc), and oxygen (Ko) and the chloroplastic CO2 photocompensation point (Γ*) and their temperature responses were the Cc-based in vivo values of Bernacchi et al. (2002). The Ci cut-off point was determined based on the method proposed by Ethier et al. (2006). From this analysis Vcmax, Jmax and gm were determined. Following gas-exchange measurements, leaves were sampled and immediately returned to the laboratory for leaf area measurement using a Delta-T Image Analysis System (Delta-T Devices, Cambridge, UK). Leaf samples were dried for 2 d at 70 °C before weighing to determine dry mass, and then leaf total nitrogen concentration was measured by a micro-Kjeldahl assay.
The relative photosynthetic limitations were partitioned into their functional components following the method by Jones (1985) implemented by Grassi & Magnani (2005) to take into account gm. This approach, which requires the measurement of A, gs, gm and Vcmax, allows to partition photosynthesis limitations into components related to stomatal conductance (SL), mesophyll conductance (MCL) and leaf biochemical characteristics (BL), assuming that a reference treatment in which the maximum assimilation rate, gs, gm and Vcmax, can be defined. These maximum values were observed in April in nI–AS trees, and therefore used as the reference to calculate partial photosynthetic limitations in old plants. SL plus MCL give the total diffusional limitations (DL). MCL plus BL give the non-stomatal limitations (NSL).
Statistical analysis was performed using analysis of variance (anova). Significant differences between values were determined at P < 0.05, according to Tukey's test. Leaf temperature response of Vcmax was fitted using a three-parameter log normal function similar to the one used by Warren & Dreyer (2006). The data were analysed using SPSS 11.5, and Sigma-Plot 8.0 software package.
Figure 2 shows the average diurnal distribution of PPFD for both sampling dates and canopy sectors. Peak irradiance in MS was at 1000 h GMT, while in AS it was at 1300 h GMT. Daily-integrated values in MS were 24.2 and 26.7 mol m−2 s−1 in April and August, respectively. In AS, daily-integrated irradiance was 39.1 in April and 37.4 mol m−2 s−1 in August. Because of the higher position of the sun in the afternoon and the shading effect of neighbor trees, it was not possible to find identical Qint values in MS and AS, and AS showed greater values than MS. Air temperature (Fig. 3) fluctuated between 7 and 32 °C in April, and between 15 and 38 °C in August. Mean VPD when PPFD was maximum at each sector (Fig. 3) was 0.42 kPa for MS and 1.06 kPa for AS, in April. Values in August were 1.25 kPa for MS and 2.65 kPa for AS. In April, the daily maximum VPD was usually lower than 1.5 kPa, while in August it was generally around 4 kPa, although on some days reached over 5 kPa. The average θ decreased gradually in the nI trees from April (0.175 m3 m−3) to August (0.124 m3 m−3). For the I trees, θ was 0.162 m3 m−3 in April, just before irrigation commenced. In August, θ fluctuated between 0.171 and 0.19 m3 m−3, just prior to and just after irrigation events, respectively.
Plant water status and diurnal courses of leaf gas exchange
Figure 4 shows the diurnal evolution of gs for both treatments and canopy sectors, recorded in April and August. In all cases, maximum values of gs were achieved around noon. Both in April and August, gs before midday was greater in MS than in AS. The opposite happened in the afternoon, although differences between MS and AS were not as large. Trees of the nI treatment had similar evolution of gs than I trees in April, when θ was similar between treatments and atmospheric demand was relatively low. In August, the greater difference in θ between irrigation treatments was clearly reflected in the differences in gs between treatments, at the two explored sectors. In MS, the maximum value of gs measured in the nI trees was 60% of that measured in the I trees, while in AS, the value was only 34%. It is noteworthy that, despite large differences in θ between treatments in August stomata closed around midday in both the I and nI trees. In April, the timing of maximum A coincided with the timing of maximum irradiance (Fig. 5). In August, however, maximum A was recorded earlier in the day than maximum irradiance. Differences in A between sectors were evident, both for nI and I trees. This was especially clear in April, when the highest A values were recorded before midday in MS and after midday in AS. In all cases, maximum values of A were recorded in MS.
Leaf water potential ψ (Fig. 6) did not show large differences between irrigation treatments either in April or August. The only significant differences (P ≤ 0.01) between irrigation treatments in August were found at predawn (0600 h GMT) and late in the afternoon (1800 h GMT), indicating a greater degree of stress in nI trees. In April, ψ was always greater than –2 MPA in both treatments and sectors, while in August minimum ψ values of ca. −3 MPA were recorded in nI trees at midday. For both treatments and sectors, diurnal evolution of ψ in August paralleled the evolution of gs. In April, this tendency was less related to gs, and more related to the radiation evolution. The similar evolution of ψ in both treatments highly contrast with the large difference observed in gs between treatments, mainly in August.
Photosynthetic capacity, nitrogen and irradiance
Seasonal changes in leaf nitrogen in mass basis (Nm) and area basis (Na) were driven mostly by differences in leaf mass area (LMA) (Fig. 7), although a highest correlation was found for Na (r2 = 0.86; P < 0.001) than for Nm (r2 = 0.33; P < 0.001). The three mentioned variables decreased their values in August compared to April. Seasonal change in Nm was not so significant as the decline in Na and LMA about 50 and 30%, respectively. No differences were found in these variables between sectors, except for slightly highest values in April at nI–AS.
The seasonal decrease of Vcmax was correlated with a seasonal decrease in Na (r2 = 0.71; P < 0.05) (Fig. 8). Na ranged from 2.75 to 6.28 g m−2, and Vcmax responded positively to Na ranging from 33.16 to 131.14 µmol m−2 s−1. Furthermore, Jmax responded positively to Na (Fig. 8) ranging from 95 to 220 µmol m−2 s−1 (r2 = 0.78; P < 0.05) under the same range of Na mentioned earlier. It was not possible to estimate Jmax in August, except for three of them in the I treatment (two in I–MS and one in I–AS, which suggest an effect of water stress). This means that there was a direct transition from Vcmax limitation to triose phosphate utilization (TPU) limitation. However, photosynthesis was never limited by TPU at ambient CO2 concentration, even in the case of the highest values of gs measured in the I treatment, when maximum values of Ci were achieved. No differences in Vcmax were found in April between either treatments or sectors (Fig. 8). In August, Vcmax values were 40–60% lower than in April, for both treatments and sectors. In addition, Vcmax values in nI–AS were significantly lower (P < 0.05) than those measured in I–MS and nI–MS. Difference with I–AS was not so significant (P = 0.19) probably because of the high variability showed by gm. The estimates of gm are very variable despite the method used (Warren 2006b), and this was also our case. Average values of gm dropped from 0.247 mol m−2 s−1 in April to 0.156 mol m−2 s−1 in August (Fig. 9). There were not significant differences in gm between either treatments or sectors in April or August. Vcmax was related to gm in a non-linear fashion (r2 = 0.55). Vcmax tended to increase almost linearly with gm up to values of 130–140 µmol m−2 s−1 and gm about 0.25 mol m−2 s−1, from which further increases in gm did not produced increases in Vcmax. Leaves in April showed the highest values of both variables.
The Jmax : Vcmax ratio could be studied only in April. This ratio showed the lowest average value in nI–MS, 1.32, and the highest in I–AS, 1.61. We found a very good linear positive relationship between Qint and Jmax : Vcmax (Jmax : Vcmax = 0.014 Qint + 1.05; r2 = 0.98; P < 0.05), which means that the more the radiation intercepted the more investment the leaf does in electron transport capacity related to carboxylation capacity. In April, variations in Na were positively and linearly related to Qint (r2 = 0.70; P < 0.0001) (Fig. 10a), which suggests that Qint highly influenced leaf N content. Accordingly, and as it can be inferred from Figs 8 and 10a, Vcmax was also positively and linearly related to Qint (r2 = 0.46; P < 0.002) (Fig. 10b). In the relationship of both variables with Qint, it was observed a decline in Vcmax at the highest Qint values, which were not included in the former lineal correlations reported, because they reduced considerably the coefficient of determination in the regression. In August, however, neither Na nor Vcmax showed any relationship with Qint. This suggests that other factors apart from PPFD were limiting the photosynthetic capacity of olive leaves in summer.
Limitations to photosynthesis, leaf temperature and mesophyll response to temperature
Table 1 shows SL, MCL and BL, relative to nI–AS in April when maximum A was measured. In April, there were not large differences between treatments or sectors in any of the limitations calculated. However, in August there was an increase in most of them with respect to April. SL experienced a large increase in nI trees, ca. 30%, as expected by the reduction in gs as a consequence of soil water content depletion. MCL increased in a similar fashion for both treatments, although slightly more in nI, but no significant differences were found between sectors of the same treatment. A different pattern was observed for BL, and MS showed lower limitation than AS, about 50%, independently of water treatment. Therefore, olive leaves of both water treatments experienced a seasonal increase in their NSL, which was more accentuated in the AS, as a consequence of their larger BL. DL, although increased in both treatments, was, however, clearly higher in nI trees, especially because of their highest SL.
Table 1. Limitations of Amax, expressed as a percentage as compared to the maximum values found in nI–AS in April
Month, treatment and sector
Data show values for irrigated (I) and non-irrigated (nI) trees, and morning (MS) and afternoon (AS) sectors. The stomatal (SL) plus the mesophyll conductance limitation (MCL) give the total diffusional limitations (DL). The MCL plus the biochemical limitation (BL) give the non-stomatal limitations (NSL).
As expected, Tleaf (Fig. 11) was higher in August than in April, as a consequence of the higher air temperature (Fig. 3). We found a maximum Tleaf in August (nI–AS) of 40.19 °C. In April, maximum Tleaf was 32 °C, and in August it was over 35 °C, with peak values close to 40 °C in the case of leaves at the AS. There were no significant differences between treatments at the same sector (MS or AS) at any time of the day either in April or August, except early in the afternoon (1400 h GMT) in August when Tleaf in nI–AS was significantly higher than Tleaf in I–AS (P ≤ 0.05). At that time of the day in August, leaves of the I treatment kept Tleaf close to air temperature; meanwhile, nI leaves reached up to +2.78 °C over air temperature (Fig. 11).
Temperature response of gm (Fig. 12) was found to increase from 20 to 30 °C, and then it declined from 30 to 40 °C. The temperature response of gm was described by a three-parameter log normal function, with a maximum at 29.61 °C and a mean value at this temperature of 0.224 mol m−2 s−1.
MS received peak radiation before noon, a time of the day at which olive trees can exploit their maximum photosynthetic capacity, because it coincides with maximum daily gs values (Moriana et al. 2002; Diaz-Espejo et al. 2006). At the time of the day that PPFD in AS was maximum, VPD was often high enough to cause a decrease in gs. This would limit CO2 diffusion to the carboxylation sites, and thereby limit A. Olive trees have an effective control of transpiration (Fernández et al. 1997), displaying both avoidance and tolerance mechanisms to cope with drought depending on the level of water stress experienced (Connor 2005). This control is mainly exerted by stomata. This is clearly shown in Fig. 4, and it explains the maintenance of ψ in narrow limits in summer (Fig. 6), even though soil water availability was low in nI treatment, which suggests an isohydric behaviour of olive. Reduction of gs increased SL slightly in summer at AS (Table 1). Photosynthetic capacity was also reduced both in I and nI trees (Fig. 8), indicating that it was not dependent upon soil water availability. Meteorological conditions during leaf ontogeny could have imposed restrictive conditions for gas exchange to the leaves sampled in August, when soil water content was low and VPD was high. Despite differences in θ between treatments in August, significant differences were not found either in Vcmax or Na between I and n–I trees in MS, although nI–AS leaves showed a tendency to decrease Vcmax more than other sectors. This suggests that soil water availability was not the main limiting factor in reduced photosynthetic capacity. The initial hypothesis of stomatal limitation to photosynthesis as a result of a stomatal response to high VPD could explain a reduction in nI, but not in I trees (Table 1). The other diffusional limitation, MCL, affected I and nI trees similarly, with slightly higher values in nI. However, gm was about 40% lower in August than in April, which was similar to the reduction in Vcmax. The photosynthetic capacity of leaves and gm have often been found to be correlated, which has been interpreted as indicative of coordination between both (Evans & Loreto 2000; Flexas et al. 2004; Grassi & Magnani 2005). This relationship has been reported to be affected by leaf age in Mediterranean species, including olive (Niinemets et al. 2005). They reported also a curvilinear relation between photosynthetic potential and gm, suggesting non-coordinated changes between them. Our data also suggest a seasonal curvilinear relationship between gm and Vcmax (Fig. 9), although leaf age was not involved because all sampled leaves were of the same age. This relationship agrees with the usually found relation between A and gs (Flexas et al. 2002; Medrano et al. 2002). There was not a strong correlation between gm and gs, (r2 = 0.15; P > 0.6; considering the maximum gs measured at each sector and treatment in Fig. 4), although some works have highlighted the correlation between gm and gs in olive trees (Centritto et al. 2003). In this study, the relationship was especially uncorrelated in I trees in August when gm was decreased; meanwhile, gs was maintained at high values like in April.
The strong relationships of Na and Vcmax with Qint found in April (Fig. 10) agree with results from the literature. Relationships between leaf photosynthetic capacity and the light regime at the location where the leaf develops have often been found in evergreen trees (Hollinger 1996; Warren & Adams 2001). The increase of Vcmax, LMA and Na with light has been reported before (Niinemets & Tenhunen 1997; Rosati et al. 1999). The commonly observed correlation of Na with A is based on the fact that up to three-quarters of foliar nitrogen may be invested in the photosynthetic apparatus (Field, Merino & Mooney 1983). Niinemets et al. (2004) demonstrated that canopy differences in the rate of development of leaf photosynthetic capacity were mainly controlled by the rate of change of LMA. We obtained similar results in olive leaves, and the seasonal decrease in Na was driven by LMA, because Nm was maintained nearly at constant levels (Fig. 7). Although the general impression is that leaves in summer have higher LMA (because there is usually more light available), Wirtz (2000) showed that LMA can increase or decrease over a season depending on environmental conditions, and the rate of leaf growth based on photosynthesis rate plays an important role. In April, AS is probably the most favourable location of the tree, because leaves can intercept more radiation and Tleaf is higher than in MS and closer to the optimum temperature for photosynthesis (Diaz-Espejo et al. 2006). However, leaves of nI–AS can intercept a similar amount of radiation in August than in April, but under soil water stress conditions, stomata close in summer, causing an increase in Tleaf beyond their optimum values (Fig. 11). This sector showed the lower values of Nm, pointing to Tleaf as another possible cause of the observed seasonal reduction on photosynthetic capacity.
In April, the relationships of Na and Vcmax with Qint were linear for the range shown, except at the highest values of Qint, where Na and Vcmax decreased slightly. We have not found in the literature examples of non-linearity in the relationship between Qint and Na or Vcmax at high Qint. Lemaire et al. (1991) found a gradual decrease of Na with decreasing PPFD, and when the PPFD value was close to the light compensation point of net photosynthesis, an abrupt decline of Na was observed. These results suggest that some other phenomenon is involved. In other studies carried out in Mediterranean environments, no seasonal decline on the relationship between Vcmax or Na and Qint was observed (Niinemets et al. 2004b). In this study, however, there was a general decrease of Vcmax in August, and there was no relationship between Vcmax and Na with Qint (Fig. 10). The different responses of Vcmax and Na to Qint in April and August cannot be attributed to leaf age (Niinemets et al. 2005), because leaves were of similar age at both sampling dates. In canopies, when leaves develop under different light conditions, other variables such as wind speed, air temperature and VPD may also covary (Niinemets & Valladares 2004). Therefore, one or more of these variables could limit photosynthesis besides light. Under conditions where several environmental variables covary, Anten (2000) suggested that it could be more advantageous for the plant to have more uniform nitrogen distribution in the canopy, which may explain the results in Fig. 10. Down-regulation of photosynthetic capacity in olive leaves in August is difficult to attribute to water stress conditions because I trees maintained gs of greater than 0.150 mol m−2 s−1. Seasonal evolution of leaf nitrogen in olive trees, and its relation with phenological stage, is typically reported on a mass basis (Fernández-Escobar, Moreno & García-Creus 1999), which is the variable that shows less seasonal reduction showed in this work. Similar to our findings, these authors also reported a decrease in Nm in summer compared to spring or autumn samplings. We cannot attribute the reduction in leaf nitrogen to soil nitrogen availability, because all trees were well fertilized.
Determination of Jmax during leaf development has been proposed to be proportional to Qint (Niinemets et al. 2004a). Diaz-Espejo et al. (2006) showed a seasonal decline both in Jmax and in the Jmax : Vcmax ratio for olive trees in this same orchard. These ratios, Ci based, have been reduced when expressed Cc based, and at 20 °C Jmax : Vcmax ratios were 1.82 and 1.49, respectively. Our data also suggest a seasonal down-regulation of Jmax. Although we have only three values of Jmax in August in leaves of I treatment, they show clearly lower values than in April. Wilson, Baldocchi & Hanson (2000) reported that autumn leaves of oak and maple had higher Jmax : Vcmax ratio than summer leaves. A similar result was found in ash (Grassi et al. 2005). A reduction in the Jmax : Vcmax ratio decreases the Ci at which photosynthesis is colimited by RuBP carboxylation and RuBP regeneration. This makes sense if we consider the reduction in gs and gm during summer, which reduces the values of Ci and Ca at which leaves operate, despite reducing the leaf's total photosynthetic capacity. Diaz-Espejo et al. (2006) showed that, in addition to the seasonal decrease in this ratio, temperature highly affected the ratio itself, with values varying from about 2.5 at 15 °C to less than 1.0 at 40 °C. Reduction in the electron transport rate at high temperature has been proposed to be responsible for the functional limitation of photosynthesis (Wise et al. 2004). Onoda, Hikosaka & Hirose (2005) also found a lower Jmax : Vcmax ratio in summer, and they ascribed it to a seasonal temperature range. In our case, air temperatures around 40 °C were recorded on many summer days. This, together with the reduction of the cooling effect of transpiration caused by stomatal closure, caused Tleaf to be greater than air temperature, especially in nI treatment. This would have affected the electron transport rate significantly, even in a species like olive, well adapted to high temperature. However, Haldimann & Feller (2004) showed that in Quercus pubescens leaves grown under natural conditions, the photosynthetic electron transport was well protected against heat stress and that inhibition of photosynthesis at high temperature was closely related to a reversible reduction of the Rubisco activation state. Figure 11 shows that AS reached the highest values of Tleaf regardless of the water treatment or time of year. It is known that temperature affects both Vcmax and Jmax (Medlyn et al. 2002), which can explain why in August AS experienced the largest BL (Table 1), and also the reduced correlation in the relationship between Vcmax and Qint when radiation is high, as shown in Fig. 10. Leaf temperature and Qint correlate within the canopy because leaves intercepting more irradiance have larger temperature (Niinemets et al. 2004a). These observations might be interpreted as another piece of evidence in favour of a leaf photosynthesis response to temperature rather than photosynthesis limitation by gs and gm solely, although they are interrelated. The decrease of gm observed in August (Fig. 9) could be related to Tleaf. Although gm was measured under optimal conditions when performing A–Ci curves, Tleaf reached supraoptimal conditions in August (Fig. 11). The limitation of the photosynthetic rate imposed by gm has been demonstrated to increase with leaf temperature and be greater than the limitation by stomata at any temperature (Yamori et al. 2006). There are only three reports of gm response to Tleaf in the literature. Bernacchi et al. (2002) reported in Nicotiana tabacum an increase in gm from 10 °C to a maximum of 36 °C, before declining at higher temperatures. But, Warren & Dreyer (2006) found in Quercus canariensis that gm doubled from 10 to 20 °C and then was nearly temperature independent from 20 to 35 °C. Finally, Yamori et al. (2006), working with Spinacia oleracea leaves, found a similar response to that showed in Fig. 11 for Olea europaea with optimum temperatures that varied depending on the growth temperature conditions. Tleaf effects on gm, electron transport rate and Rubisco activation state deserve more attention in the future to explain their influence on the seasonal adjustment of photosynthetic capacity of olive leaves.
Olive leaves experienced a seasonal decrease in their photosynthetic capacity that was unrelated to light availability. During spring, there were good relationships between both Vcmax and Na and Qint, which were not maintained in summer. Leaves of non-irrigated trees maintained ψ at similar values as leaves of well-irrigated trees by reducing stomatal conductance, at the expense of reducing A. Stomatal limitation of photosynthesis may explain why, for non-irrigated plants, photosynthetic capacity distribution in the canopy was not related to Qint during summer. However, a similar decline in Na and Vcmax in well-irrigated plants was observed in summer. Although there was some stomatal limitation of A in I plants, it was considerably lower than in nI plants, which makes insufficient, from our point of view, the hypothesis of stomatal limitation to explain the data observed. However, mesophyll conductance was found to decrease in all treatments in August, suggesting a potential role in Vcmax down-regulation. We do not know the mechanisms that could make this possible, but in a similar way that Vcmax is regulated as a function of available light to optimize resources, gm could act like a limiting factor affecting the down-regulation of Vcmax. Seasonal increases of leaf temperature could also help to explain our results, and these effects would be in addition to the commented earlier.
This work highlights the need for models of plant growth and ecosystem function to incorporate new parameters describing the distribution of photosynthetic capacity in canopies, which currently are mainly implemented as a function of light distribution. These parameters could explain why current models dealing with the distribution of photosynthetic capacity in canopies do not always match actual observations. Other factors than light, such as diffusional limitation caused by water stress, drought, high temperatures or photoinhibition may negatively affect the photosynthetic carbon gain of leaves. There is increasing evidence in the literature supporting their inclusion in models (Grassi & Magnani 2005; Niinemets et al. 2005, 2006; Warren & Adams 2006).
This research was supported by grants of the Spanish CICYT, Project AGL2002-04048-CO3-01, and research fellowship (MEC, Program FPI) to E. Nicolás. We really appreciate the constructive comments of A.S. Walcroft, X. Le Roux, U. Niinemets and J. Flexas on an early version of this manuscript.