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Tree Physiology 29, 375–388 doi:10.1093/treephys/tpn032

Seasonal effects of deficit irrigation on leaf photosynthetic traits of fruiting and non-fruiting shoots in almond trees PEDRO A. NORTES,1 MARIA M. GONZALEZ-REAL,1,2 GREGORIO EGEA1 and ALAIN BAILLE1 1

Universidad Polite´cnica de Cartagena, Escuela Te´cnica Superior de Ingenieros Agro´nomos, A´rea de Ingenierı´a Agroforestal, Paseo Alfonso XIII, 48, 30203, Cartagena, Spain

2

Corresponding author ([email protected])

Received July 17, 2008; accepted October 27, 2008; published online January 13, 2009

Summary We investigated seasonal trends in, and relationships between, leaf structural properties, leaf nitrogen concentration, and maximum (Am) and potential (Ap) leaf net CO2 assimilation of 1-year-old fruiting (f) and currentyear non-fruiting (nf) shoots in 5-year-old almond trees (Prunus dulcis (Mill.) D.A. Webb cv Marta). These trees had been subjected in the previous 4 years to either full irrigation (FI regime) or sustained deficit irrigation (DI) at 50% of standard crop evapotranspiration during the entire growing season (DI regime) in the semiarid climate of southeast Spain. Measurements were made during an entire growing season on sun-exposed leaves. Leaf dry mass per unit area (Wa), area and dry-mass-based leaf N concentrations (Na and Nw, respectively), and area and dry-massbased Am (Ama and Amw, respectively) were lower in f-leaves than in nf-leaves. Changes in leaf structural attributes induced by DI were more pronounced in nf-leaves than in f-leaves, the latter being little affected. Over the entire growth season, Am and Ap were correlated negatively with Wa and positively with Nw for both the leaf classes and the irrigation regimes. When calculated with respect to total leaf N concentration, maximum photosynthetic nitrogenuse efficiency (PNUEm) was significantly higher in f-leaves than in nf-leaves, with no significant differences between the leaf classes among the irrigation regimes. However, when PNUEm was calculated with respect to photosynthetic N, no significant effect of leaf class or irrigation regime was observed. Overall, our results showed that DI and FI trees exhibited similar seasonal patterns of leaf structural properties and maximum and potential leaf net CO2 assimilation rates, but there were distinct N-allocation patterns between f- and nf-leaves. In the DI treatment, leaf structural adjustments appeared to operate to maintain a high N status in the leaves of fruit-bearing shoots, to the detriment of N resources allocated to vegetative shoots. Keywords: leaf N concentration, phenological stage, photosynthetic capacity, photosynthetic nitrogen-use efficiency, Prunus dulcis, specific leaf area.

Introduction Deficit irrigation (DI) strategies can regularize or increase, or both, almond productivity when a limited amount of irrigation water is available (Goldhamer et al. 2006). These strategies, which include regulated DI (Chalmers et al. 1981), sustained DI (Girona et al. 2005, Goldhamer et al. 2006), and partial root-zone drying (Dry and Loveys 1999, Kang et al. 2003), can substantially reduce tree water consumption without causing a significant negative effect on tree productivity (Girona et al. 1997, Goldhamer and Viveros 2000, Esparza et al. 2001a, 2001b, Romero et al. 2004, Girona et al. 2005). One of the prerequisites for valuation and further implementation of best DI management practices is a detailed knowledge of plant physiologic responses to abiotic stresses, while accounting for leaf age, plant phenology (Dungan et al. 2003, Xu and Baldocchi 2003, Muroaka and Koizumi 2005) and fruit load (DeJong 1986, Palmer et al. 1997, Syvertsen et al. 2003). Studies on the application of DI to the almond tree have provided valuable information, especially on leaf net CO2 assimilation (DeJong 1983, Wartinger et al. 1990, Higgins et al. 1992, Matos et al. 1998, Romero et al. 2004, Rouhi et al. 2007) and an influence of natural or imposed drought-induced stress on photosynthetic attributes (Klein et al. 2001, Heilmeier et al. 2002, Romero and Botı´ a 2006). Details related to nutrient deficiency (Basile et al. 2003), species, cultivar type and rootstocks (Matos et al. 1997, De Herralde et al. 2003, Rouhi et al. 2007) and leaf ontogeny (Wartinger et al. 1990, Matos et al. 1998, Klein et al. 2001, Romero and Botı´ a 2006) are also available. Knowledge about how water stress can modify the functioning of different types of shoots in the almond tree is needed, because it is likely to affect almond yield determinants. Studies on fruit trees have shown differences in leaf structural and physiologic traits between shoot types (Fujii and Kennedy 1985, Syvertsen et al. 2003, Zhang et al. 2005), associated with the presence or absence of fruits on shoots. Recently, Heerema et al. (2008) reported that the

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survival of almond rosette-like short shoots (spurs) over winter was strongly related to their leaf area per unit of leaf mass. Although a clear seasonal trend in leaf traits, which can vary considerably over time with leaf age, has been observed (Wilson et al. 2000), it has not been clearly established if leaf age dominates other factors such as soil water availability in the almond tree. To our knowledge, the long-term effects of DI strategies on the response of leaf structural and physiologic traits of fruit-bearing shoots and vegetative shoots, and on the pattern of N allocation to these classes of foliage, have not been documented. Such responses might induce changes in the pool of carbohydrate reserves, which are known to affect the renewal of fruiting position, as well as the amount of reserves available at the onset of a new growth cycle, leading to subsequent yield reduction in the following year (Esparza et al. 2001a, 2001b). In early foliating species, growing shoots and developing fruits are strong sinks for assimilates, which compete for the reserves stored in woody plant parts during the previous growing season (Weinbaum et al. 1987, Millard 1996, Nii 1997, Rosecrance et al. 1998, Youssefi et al. 2000, Bi et al. 2003). The influence of fruits (sinks) in attracting assimilates from the proximal leaves (Nii 1997, Syvertsen et al. 2003) and competition for N resources between leaves and fruits (Youssefi et al. 2000) can lead to lower N concentration in leaves near the fruit, as observed in citrus trees (Syvertsen et al. 2003). In walnut trees, a net decrease in leaf N concentration as a result of protein degradation (resorption) can occur in response to the demands of developing fruits (Weinbaum et al. 1994), but this pattern has not clearly been demonstrated in almond trees (Weinbaum and Muraoka 1986). Stress conditions have been reported to affect leaf N concentration (Reich et al. 1989). Because leaves are an important source of N for tree reserves, restricting the water supply by DI might have a negative effect on N accumulation in woody structures (Bi et al. 2003). It has been observed that, after harvest, N storage in perennial tree parts depends much more on N recycling from leaves than on soil N uptake (Rosecrance et al. 1998, Esparza et al. 2001a), and this storage can provide up to 50% of the N used over the growing season (Weinbaum et al. 1987). Romero et al. (2004) reported a significant reduction in the photosynthetic capacity of young branches of almond trees under pre-harvest conditions of mild to moderate soil water deprivation, whereas the accumulation of dry matter in the kernel was little affected compared with the fully irrigated trees. Because shoots appear to function as autonomous structures with respect to assimilation and use of carbon in many tree species (Sprugel et al. 1991), the sustained sink activity of fruits under conditions of water stress might indicate that the almond tree tends to preserve the photosynthetic capacity of fruit-bearing shoots to the detriment of the vegetative shoots. This suggestion is supported by the observed differences in nonstructural carbohydrate

concentration between shoot types of dormant almond trees that had previously been subjected to water stress during the harvest period (Esparza et al. 2001a). The results of the cited studies suggest that a DI strategy applied for several consecutive years could lead to significant changes in the morphologic and physiologic traits of almond leaves. We hypothesized that vegetative and fruitbearing shoots exhibit a differential response to water stress through changes in leaf structural and physiologic traits. To test this hypothesis, we characterized the seasonal trend in leaf traits, and investigated whether they vary (1) with leaf age and class (fruiting versus non-fruiting shoots) and (2) between irrigation regimes (full irrigation (FI) versus sustained DI), and investigated to what extent the relationships between leaf traits are conserved or co-vary across leaf classes and irrigation regimes. We also examined the effect of irrigation treatment on the photosynthetic nitrogen-use efficiency (PNUE) of the two leaf classes.

Materials and methods Site description The study was conducted during the 2004 growing season in a 1-ha plot of 5-year-old almond (Prunus dulcis (Mill.) D.A. Webb cv Marta) trees that had been grafted on ‘Mayor’ rootstock and planted in December 1999 at a spacing of 6 m · 7 m in an almond orchard at the Agricultural Experimental Station of the University of Cartagena (3735 0 N and 059 0 W). The climate at the site is mediterranean with low cloudiness, high temperature, low humidity and virtually no rainfall during the summer. The winter is mild, with a mean air temperature of about 10 C during the coldest month (January). Mean annual temperature was 18.2 C in 2004 and precipitation was 325 mm. These values are close to climatic means determined over 20 years at a nearby weather station (mean air temperature was 18.3 ± 0.9 C and mean precipitation was 355 ± 90 mm). The corresponding value of reference crop evapotranspiration (ETo), calculated with the FAO Penman–Monteith equation (Allen et al. 1998), is 1180 ± 60 mm. The soil texture was a deep silt-clay-loam with low available potassium and organic matter concentrations and high phosphorus concentration. Electrical conductivity (EC) of the saturation extract was 1.4 dS m1 in 2004. The bulk density of the surface layer was 1.4 ± 0.1 g cm3 and available water-holding capacity of soil was about 0.18 mm1. Irrigation regimes In 2000, two irrigation treatments, FI and sustained DI were applied to the experimental plots until the end of 2004, following a completely randomized statistical design with three replicates per treatment and 12 trees per

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replicate. Trees were irrigated by a single drip line equipped with six pressure compensated drippers (4 l h1 per dripper) per tree. In the FI plots, an amount of water corresponding to 100% of the standard crop evapotranspiration, ETc, was supplied weekly. We calculated ETc as the product of ETo and the crop coefficient for almond trees (Doorenbos and Pruitt 1977), and corrected with a localization factor (Fereres et al. 1982). During the first 3 years (2000–2002), the trees in the DI plots were irrigated following a sustained DI strategy, receiving 60% of ETc in the entire irrigation season. In 2003 and 2004, the water supply was further restricted such that the trees received about 40% of ETc. The EC of irrigation water was 1.2 dS m1 with chloride and sodium concentrations of 4.6 and 5.41 meq l1, respectively. Trees were fertilized with 35, 35, 57 kg ha1 year1 of N, P, K and were managed according to current commercial practices (i.e., a routine pesticide program was maintained, pruning was applied manually in December, and all weeds were removed from the orchard). Measurement period In 2004, leaf characteristics were measured from the beginning of April (day of year (DOY) 97, when fruits had reached 92% of their final size) until post-harvest, corresponding to the period of reserve accumulation in woody structures. Fruit harvest occurred in mid-August (DOY 220) for trees in the DI treatment and about 10 days later for trees in the FI treatment. Figure 1 gives a schematic representation of the stages of tree development as a function of DOY for trees in the FI regime. The curves of relative cumulative values of fruit growth, young branch growth and fruit dry mass accumulation (kernel-filling) were normalized against their maximum values. Before the experimental period (February 2004), mean trunk diameter of the trees was 14.08 ± 0.28 cm in FI and 12.02 ± 0.15 cm in DI, and canopy height was 3.56 ± 0.09 m in FI and 3.19 ± 0.07 m in DI. At the end of the 2003 growing season, crown leaf area was 68.11 ± 2.48 m2 tree1 in FI and 41.94 ± 3.58 m2 tree1 in DI.

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Figure 1. Schematic representation of P. dulcis tree developmental Stages I–V. The curves show the relative cumulative values of fruit growth, young branch growth and fruit dry mass accumulation (kernel-filling), with respect to their maximum, for the cultivar studied (cv Marta) in the FI regime in southern Spain, as a function of DOY. Vertical lines delimit the beginning and the end of each developmental stage.

Measurements of leaf N concentration and leaf area Foliar N concentration was measured from April to September 2004 in the fully expanded leaves of non-fruiting branches (current-year branches) and fruiting branches (1-year-old branches) – hereafter nf- and f-leaves, respectively – in the two irrigation treatments. Samples consisted of at least 12 leaves in each class taken from the upper third of sun-exposed branches. Leaf area was measured with an area meter (Delta-T Image Analysis System (DIAS), Delta-T Devices, Cambridge, UK). Mean leaf N concentration was determined after Kjeldahl digestion of leaves that had been oven-dried at 80 C for 48 h, weighed and ground. For each class of leaves, leaf N concentration is expressed per unit leaf area (Na, in g m2) and per unit leaf dry mass (DW) (Nw, in g g1). Leaf dry mass per unit leaf area (Wa) is expressed in g m2. Gas exchange measurements Leaf gas exchange was measured from April to September on trees in the FI and DI irrigation regimes. The criteria adopted for selecting leaves on the two classes of branches were similar to those described for the leaf N concentration measurements. Only healthy branches were selected from the periphery of the tree crown. Area-based leaf net CO2 assimilation (A, lmol m2 s1), transpiration rate (E, mmol m2 s1), stomatal conductance to water vapor (gs, mmol m2 s1) and intercellular CO2 concentration (Ci, lmol mol1) were measured with a portable gas exchange system CIRAS2 (PP Systems, Hitchin, Hertfordshire, UK). The desired photosynthetic photon flux (I, lmol m2 s1) was provided by an internal red/blue LED light source (PC 069-1). A CIRAS2 injection system controlled the ambient CO2 concentration (Ca, lmol mol1) in the chamber by adjusting the flow of CO2 from a CO2 cylinder, and also controlled the leaf temperature (Tl) in the chamber. Air temperature (Ta) and actual vapor pressure of the chamber air were continuously monitored by the CIRAS2. Measurements were made at periodic intervals ( 7 to 10 days) throughout the growth season on at least six leaves (one leaf per branch, two branches per tree

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and three trees per irrigation treatment). All leaf gas exchange measurements were made between 1000 and 1300 h (local time). Light-saturated leaf net CO2 assimilation (Am) and maximum stomatal conductance (gsm) were measured at I  1400 lmol m2 s1, near constant Ca ( 360 lmol mol1) and Tl ( 30 C). Area- and dry-mass-based values of Am are denoted as Ama (lmol m2 s1) and Amw (lmol g1 s1), respectively. Light-saturated, PNUEm, was computed as the ratio Amw/Nw (lmol g1 s1). Leaf potential net CO2 assimilation or maximum metabolic capacity (Ap) was measured from April to September in trees in both FI and DI regimes, at saturating I ( 1400 lmol m2 s1) and saturating Ca ( 1400 lmol mol1), and at near optimum Tl (35 C). This Tl value was derived in spring from the measurements of Ap in FI leaves over a wide range of Tl (18–42 C). Tree water status Every 2 weeks, we measured predawn leaf water potential (Wpd) with a pressure chamber (Model 3000, Soil Moisture Equipment, Santa Barbara, CA) on mature leaves (12 leaves per irrigation treatment, i.e., six trees and two leaves per tree) taken before dawn from the upper third of the branches. Data for the f- and nf-leaves were pooled. Statistical analysis We analyzed (1) leaf N concentration (area- and dry-massbased) as a dependent variable against Wa and (2) the photosynthetic parameters (area- and dry-mass-based) as dependent variables against Wa and against leaf N concentration (area- and dry-mass-based) by using linear regression. Significant differences between slopes and non-zero intercepts were evaluated by analyses of covariance, by comparing (1) FI and DI per each leaf class, (2) FI leaves and DI leaves with pooled data for the two leaf classes, (3) the two leaf classes with pooled data from FI and DI for each leaf class and (4) all datasets. To discriminate the main treatment effects on leaf structural properties and on photosynthetic parameters analysis of variance (ANOVA) was used. All analyses were performed using Statgraphics software (Statgraphics Plus for Windows Version 4.1). Results Climate conditions Maximum absolute values of air temperature (Ta) and vapor pressure deficit (VPD) increased rapidly from spring (23 C and 2.2 kPa, respectively) to summer (36.6 C and 4.4 kPa, respectively) (Figure 2A), corresponding to the increase in the daily integral of global solar radiation over the same period (Figure 2C). During sunny days, mean values of Ta and VPD remained below 18 C and 1.1 kPa, respectively, in spring and below 29 C and 2.1 kPa,

respectively, in summer (Figure 2B). The calculated reference crop evapotranspiration (ETo) was typical for the location, with maximum daily values in summer of about 8 mm day1 (Figure 2D). Because of the high amount of precipitation in spring 2004 (Figure 2D), irrigation started on May 3 in all plots. Practically no rain occurred from the end of June until post-harvest (September). For the entire irrigation season (May until the end of November), the total amount of irrigation water was 410 and 170 mm for FI and DI, respectively, and rainfall was 355 mm. Seasonal variation in predawn leaf water potential In both irrigation treatments, predawn leaf water potential Wpd was 0.30 ± 0.03 MPa during the active vegetative growth period (i.e., Stages II and III; Figures 1 and 3), indicating high soil water availability during this period. The FI trees maintained Wpd within a small range from the onset of the kernel-filling stage (0.35 ± 0.02 MPa) until postharvest (0.45 ± 0.01 MPa) compared with the range of variation observed in DI trees over the same period (from 0.47 ± 0.05 to 0.83 ± 0.06 MPa, respectively). Seasonal trends in leaf structural traits The seasonal trends in leaf dry mass per unit area (Wa) (Figure 4A) and area-based nitrogen concentration (Na) (Figure 4B) were characterized by two seasonal peaks that were more marked in nf-leaves than in f-leaves. For both leaf traits, the first peak occurred near DOY 140–145 (onset of the kernel-filling stage), whereas the absolute maximum was reached just before harvest (DOY 218–220). Dry matter accumulated in the leaf blade except for two periods coinciding with the first half of kernel-filling and the onset of reserve accumulation (Figure 4A). Over time, Wa showed a greater increase in FI trees (about 36% in both leaf classes) than in DI trees (21% and 29% in f- and nfleaves, respectively) (Figure 4A). Independently of irrigation treatment, Wa was generally greater in nf-leaves than in f-leaves. The FI trees maintained differences between the two leaf classes when Wa was averaged over the growth season (P = 0.02), whereas the corresponding differences in DI trees were significant only across Stages IV and V (P  0.04). Restriction of water supply moderately – but significantly – reduced Wa in nf-leaves (about 10%) during post-harvest compared with nf-leaves of FI trees (P < 0.01), but the DI treatment did not influence Wa in f-leaves during the growing season. Values of Wa were highly correlated between f- and nf-leaves within each irrigation regime (R2 > 0.90, data not shown). Area-based leaf N concentration (Na) was significantly higher in nf-leaves than in f-leaves (Figure 4B) from the onset of kernel-filling stage onward in both irrigation treatments. In FI trees, Na averaged over the measurement period was 27% higher in nf-leaves (3.1 ± 0.3 g m2) than in f-leaves (P < 0.01), a greater difference than that observed for Wa (17%). In DI, the difference in Na among leaf classes

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Figure 3. Time course of (Wpd) in P. dulcis trees through the 2004 growing season. Vertical bars indicate standard deviations. Each value is the mean of 12 measurements per irrigation treatment (pooled data of f- and nf-leaves). Vertical dotted lines delimit the beginning and the end of each developmental stage (cf. Figure 1). Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation; DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season and DOY, day of year.

was much lower (about 13% higher in nf-leaves) (P = 0.01) and was of the same order of magnitude as that observed for Wa. Thus, on average over the growth season,

the amount of N in nf-leaves was 15% higher in FI trees than in DI trees (P < 0.01). The N concentration in f-leaves was not significantly affected by irrigation regime. The seasonal variations in Wa and Na resulted in a sustained decrease in Nw (=Na/Wa) over time (Figure 4C). Differences in Nw between leaf classes were observed in FI trees only during Stages IV and V (14% higher in nfleaves than in f-leaves, P = 0.03), i.e., about twofold lower than those observed for Na. The nf-leaves maintained a higher Nw over the growth cycle in the FI treatment (0.0274 ± 0.003 g g1) than in the DI treatment (P = 0.02), a pattern similar to that observed for Na. Regression analyses: N versus Wa relationships The results of the regression analyses between leaf N concentration (Na or Nw) and Wa are shown in Table 1, for all combinations of leaf class and irrigation treatment over the entire growth cycle. There were significant relationships between Na and Wa in nf-leaves in both FI and DI treatments (R2 = 0.61 and R2 = 0.43, respectively), but not in f-leaves (R2  < 0.01) (Table 1). For the pooled dataset, there was a highly significant relationship (P < 0.001) between Na and Wa (R2 = 0.37), as well as between Nw and Wa (R2 = 0.29). For each developmental stage, significant relationships (P < 0.001) were obtained when

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irrespective of leaf class and irrigation regime. No significant differences were observed between the slopes for the irrigation treatments, but the non-zero intercepts differed significantly (Table 1). For the pooled data for FI and DI in each leaf class, the relationships between Nw and Wa were significant for both nf- (R2 = 0.41, P < 0.005) and f-leaves (R2 = 0.83, P < 0.001). The data for f-leaves in the FI and DI treatments displayed a unique relationship, suggesting that the DI treatment did not modify the partitioning of N in f-leaves, whereas the nf-leaves had higher Nw in the FI treatment than in the DI treatment for a given Wa (Figure 6).

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Figure 4. Seasonal trends for (A) area-based leaf dry mass (Wa), (B) area-based leaf N concentration (Na) and (C) mass-based leaf N concentration (Nw) in the FI (open symbols) and DI (closed symbols) regimes, in P. dulcis f-leaves (circles) and nf-leaves (triangles) as a function of DOY. Pooled standard deviations of the mean (not shown for clarity) were typically ± 5 g m2, ± 0.015 g m2 and ± 0.0012 g g1 for Wa, Na and Nw, respectively. Vertical dotted lines delimit the beginning and the end of each developmental stage (cf. Figure 1). Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

pooling data from Stages IIIII (Figure 5A), Stage IV (Figure 5B) and Stage V (Figure 5C). Forcing the relationships through the origin provided a slope (i.e., the mean Nw over a given stage) of 0.0285 and 0.0211 g g1 for Stages II, III and V, respectively, indicating a substantial decrease in Nw for both leaf classes and in the two irrigation regimes during the post-harvest stage. For comparison, the relationship reported by DeJong and Doyle (1985) for Prunus persica (L.) Batsch (Na = 0.027 Wa + 0.002) is shown in Figure 5A–C. In contrast to Na, Nw decreased with increasing Wa in both leaf classes (Figure 6), with significant relationships (0.60 < R2 < 0.93) over the entire growth season,

Light-saturated photosynthetic attributes showed a nonuniform trend over the growth cycle (Figure 7A–C), with most of the attributes tending to reach a more or less clear maximum. Area-based Am was highest during Stages II and III, decreasing rapidly after the onset of Stage IV (Figure 7A). In both irrigation regimes, the differences in Ama between leaf classes were not significant, except in the DI treatment during Stage IV when nf-leaves had significantly higher values than f-leaves (20.0 ± 1.4 versus 17.6 ± 0.5 lmol m2 s1) (P < 0.01). The f-leaves had similar Ama in both irrigation treatments, whereas the nfleaves had significantly lower Ama in the DI treatment than in the FI treatment during Stages II, III and V (17% and 38%, respectively). Mean Ama was not significantly affected by irrigation regime during the kernel-filling stage, regardless of leaf class. Mass-based Am tended to decrease during the growing season (Figure 7B), declining in FI leaves during Stages II and III (from about 0.30 ± 0.05 to 0.22 ± 0.02 lmol g1 s1) and then reaching a plateau until the first half of kernel-filling stage, coinciding with the decline in Wa (Figure 4A). In contrast, Amw was rather constant in DI leaves over the same period ( 0.20 lmol g1 s1); however, after harvest, it declined to low values ( 0.10 lmol g1 s1, pooled data for both irrigation regimes). The relative decline over the growing season was greater for Amw ( 61%) than for Nw (37%), and the differences in Amw between leaf classes and between irrigation regimes were significantly reduced during the kernel-filling stage. The seasonal trend in gsm (Figure 7C) showed maximum absolute values during the kernel-filling stage (323 ± 35 and 273 ± 20 mmol m2 s1 for nf-leaves in FI and DI trees, respectively). Peak values were more marked in FI trees than in DI trees, because of lower variability in gsm in the former. After reaching a maximum, gsm more or less paralleled the downward trend observed for Ama. The magnitude of this decrease was lower in FI trees than in DI trees, with a mean reduction in gsm for the two leaf classes of 85 mmol m2 s1 in the FI treatment and 153 mmol m2 s1 in the DI treatment for the period DOY 154– 245. The greatest differences in gsm between irrigation regimes were observed during the post-harvest stage.

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Table 1. Linear regression coefficients for Na versus Wa and Nw versus Wa for the two leaf classes (f- and nf-leaves) and the two irrigation regimes (FI and DI). Significance values: ns, not significant; *P < 0.05; **P < 0.01; and ***P < 0.001. For each leaf class · irrigation treatment, different letters within the same column indicate significant differences between the non-zero intercepts and between the slopes (P < 0.05). Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season. Units: Wa, g m–2; Na, g m–2 and Nw, g g–1. Na = bo + b1 Wa

f-leaves nf-leaves f + nf-leaves FI + DI All datasets

FI DI FI DI FI DI f-leaves nf-leaves All datasets

Nw = bo + b1 Wa

bo

b1

R

bo

b1

R2

2.131***a 2.808**a 1.744**a 1.467*b 0.952*a 1.508**a 2.338***a 1.423**b 1.105**

0.0015ns a 0.0049ns a 0.0117**a 0.0107*a 0.0165***a 0.0094*a 0.0004ns a 0.0128**b 0.0142***

0.03 ns 0.06 ns 0.61** 0.43* 0.48** 0.23* 0.00ns 0.40** 0.37***

0.0495***a 0.0521***a 0.0423***a 0.0377***b 0.0362***a 0.0388***a 0.0503***a 0.0385***b 0.0367***

0.00026***a 0.00029**a 0.00013***a 0.00012**a 0.00010*a 0.00014**a 0.00027***a 0.00011**b 0.00011***

0.93*** 0.71** 0.78*** 0.60** 0.26* 0.43** 0.83*** 0.41** 0.29***

The ratio of intercellular CO2 concentration to ambient CO2 concentration (Ci/Ca) was lowest during the active vegetative growth period (data not shown). The ratio tended to increase from Stages II and III (mean value in FI leaves: 0.52 ± 0.07) until the second half of kernel-filling stage and then remained more or less constant (mean value in FI leaves beyond Stage V: 0.61 ± 0.04). Area-based leaf potential net CO2 assimilation (Apa) exhibited a seasonal trend similar to that of Ama, with highest rates during Stages II and III (Figure 7D) and a decrease over the growing season that was independent of leaf class and irrigation regime. The differences in Apa among all combinations of leaf class and irrigation treatment were similar to or lower than in Ama. Analyses of the relationships between Am and Wa and between Am and leaf N concentration Regression analyses revealed highly significant relationships between Am and Wa, and between Am and Nw (P < 0.001) for all combinations of leaf class and irrigation treatment, as well as for the pooled datasets. Two examples of Amw versus Wa relationships are given in Figure 8A and B. For the pooled data for f- and nf-leaves per irrigation treatment (relationships for FI leaves and DI leaves, Figure 8A), no significant differences between irrigation regimes were observed in slopes or in non-zero intercepts. For the pooled data for the FI and DI treatments per leaf class (relationships for f- and nf-leaves, Figure 8B), there were significant differences in slopes and non-zero intercepts between leaf classes. The relationships between Amw and Wa (Figure 8B), and between Nw and Wa (Figure 6) were similar. For all pooled datasets, Wa explained about two-thirds of the seasonal variability in Amw (R2 = 0.65, P < 0.001). The regression analysis for Ama versus Wa yielded lower R2 values than for Amw versus Wa. Seasonal changes in Amw and Ama were poorly correlated to changes in Na (R2 < 0.10), but were correlated with Nw

2

(0.69 < R2 < 0.90,) with P < 0.001 for the linear regressions between either Amw or Ama versus Nw, for all combinations of leaf class and irrigation regime. Two examples of Am versus Nw relationships are shown in Figure 9A (Ama versus Nw for FI leaves and DI leaves, pooled data of f- and nf-leaves) and Figure 9B (Amw versus Nw for f- and nf-leaves, pooled data of FI and DI). PNUE The seasonal trend in light-saturated PNUEm reached a plateau from Stages II and III until the second half of kernel-filling stage (Figure 10A) and then decreased dramatically, reaching a minimum after harvest. Regression analyses of the PNUEm versus Wa, PNUE versus Na, and PNUE versus Nw relationships indicated that PNUEm was strongly correlated with Wa and Nw but loosely correlated with Na (data not shown). The relationship between PNUEm and Nw (Figure 10B) shows that, for a fixed value of Nw, PNUEm was generally higher in f-leaves than in nfleaves, irrespective of irrigation regime. Pooling all the data revealed that PNUEm was more closely correlated with Wa (R2 = 0.78; Figure 10C) than with Nw (R2 = 0.49). The three outliers in Figure 10C correspond to the post-harvest period in the DI regime. Nitrogen-use efficiency versus photosynthetic N An estimate of the amount of leaf N concentration diverted to the photosynthetic apparatus (i.e., dry-mass-based photosynthetic N, Nwp) was derived from Eq. (1) (Field and Mooney 1983), based on the pooled data for FI and DI for each leaf class Amw ¼ mðN w  N wo Þ ¼ mN wp ;

ð1Þ

where Nwo is the residual value of leaf N concentration when Amw = 0 and the slope m represents the mean PNUE related to photosynthetic leaf N concentration,

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A

0.035 f+nf-leaves, FI+DI

0.030

3.0

1

Nw (g g )

Na (g m 2 )

3.5

2.5 2.0

Na (g m 2 )

R2 = 0.60

R2 = 0.34

f(FI+DI)

nf-FI

nf-DI

100

125

0.010 50

3.5 3.0

stage IV y = 0.0260x R2 = 0.66

1.0 3.5

f+nf-leaves, FI+DI

3.0 stage V 2.5

y = 0.0211x R2 = 0.75

2.0 1.5 1.0 25

50

75

150

Figure 6. Relationship between leaf N concentration (Nw) and leaf dry mass per unit leaf area (Wa) in P. dulcis nf-leaves in the FI and DI irrigation treatments and for f-leaves based on pooled data from the FI and DI treatments. Regression parameters and significance level of slopes and non-zero intercepts are given in Table 1. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

2.5 2.0

75

Wa (g m 2 )

f+nf-leaves, FI+DI

1.5

Na (g m 2 )

R2 = 0.83

0.020 0.015

y = 0.0285x

1.0

C

0.025

stages II-III

1.5

B

R2 = 0.78

100

125

150

2

Wa (g m )

Figure 5. Relationship, forced through the origin, between leaf N concentration per unit leaf area (Na) and leaf dry mass per unit leaf area (Wa) for different stages of development of P. dulcis trees (A, B and C) (pooled data for f- and nf-leaves in the FI and DI treatments). Standard deviations of the mean (data not shown) were typically ± 5 g m2 and ± 0.015 g m2 for Wa and Na, respectively. The dashed line is the linear relationship reported by DeJong and Doyle (1985) for P. persica (y = 0.027x + 0.002). In (A), (B) and (C), the continuous lines correspond to the respective linear regressions. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

i.e., PNUEmp = Amw/Nwp. Assuming that Eq. (1) is valid over the entire range of Nw, and that, for a given leaf class, Nwo is constant throughout the growing season and is independent of the irrigation regime (Figure 9B), the regression analysis yielded for f-leaves: Nwo = 0.0127 ± 0.004 g g1 and PNUEmp = 15.62 ± 1.78 lmol g1 s1; and for nf-leaves: Nwo = 0.0153 ± 0.005 g g1, and PNUEmp = 16.14 ± 2.38 lmol g1 s1. Thus, the values of Nwo differed significantly between leaf classes (P < 0.05), whereas PNUEmp did not differ significantly

between leaf classes, in contrast to what was observed for PNUEm (Figure 10B). The trend in photosynthetic N as a fraction of total leaf N concentration (fpN = Nwp/Nw) indicated that, in both irrigation regimes, the f-leaves had a high fpN during Stages II and III ( 0.60 on DOY 90) compared with nf-leaves ( 0.50 and 0.40 in FI and DI, respectively) (Figure 11). Thereafter, fpN tended to decline in both leaf classes, the greatest decrease occurring during the second half of the kernel-filling stage. In the post-harvest stage, fpN ranged from 0.25 (nf-leaves in DI) to 0.40 (f-leaves in FI).

Discussion Effects of irrigation treatment on leaf structural traits in fruiting versus non-fruiting shoots Our study highlighted two main characteristics of the seasonal pattern of leaf structural traits. First, there was a close similarity in the seasonal patterns of Wa, Na and Nw between FI and DI trees (Figure 4A–C). The greatest similarity was in Nw, which exhibited a decrease of about 40% throughout the season, independently of leaf class and irrigation treatment. This reduction was greater than that reported for other temperate deciduous trees (Niinemets et al. 2004) but similar to that found in almond leaves by Weinbaum and Muraoka (1986). There are several possible explanations for this seasonal pattern: (1) dilution of N in the leaf blade, because of the accumulation of non-N compounds; (2) N allocation to fruits and woody structures and (3) changes in N partitioning between metabolic and structural components within the leaf (Yasumura et al. 2006). Second, independently of irrigation treatment, the f-leaves

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2

20

nf-FI f-FI nf-DI f-DI

B

15

0.4

0.3

0.2

10 0.1

1

1

Ama (μmol m s )

25

nf-FI f-FI nf-DI f-DI

A

1 Amw (μmol g s )

30

383

5 Stages II-III

C

Stage IV

Stage V

0.0 80

D

70 60

2

300

50 40

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30 20

100 0

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Stages II-III

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Stage IV

1

Stage V

2

Stage IV

1

gsm (mmol m s )

400

Stages II-III

Apa (μmol m s )

0

10

Stage V

0

250

100

150

DOY

200

250

DOY

Figure 7. Seasonal trends in (A) area-based (Ama) and (B) dry-mass-based (Amw) light-saturated net CO2 assimilation, (C) maximum stomatal conductance (gsm) and (D) potential net CO2 assimilation (Apa) in FI (open symbols) and DI (closed symbols) regimes, in P. dulcis f-leaves (circles) and nf-leaves (triangles) as a function of DOY. Pooled standard deviations of the mean (not shown for clarity) were typically close to (A) ± 2.0 lmol m2 s1, (B) ± 0.052 lmol g1 s1, (C) ± 47 mmol m2 s1 and (D) ± 4.2 lmol m2 s1. Vertical dotted lines delimit the beginning and the end of each developmental stage (cf. Figure 1). Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

A

0.4

B

FI DI

f-leaves nf-leaves

0.3

0.3

0.2

0.2

0.1

0.1

0.0

Amw (μmol g 1 s 1 )

Amw (μmol g 1 s 1 )

0.4

0.0 50

75

100

125

150 50

Wa (g m 2 )

75

100

125

150

Wa (g m 2 )

Figure 8. Relationships between mass-based maximum leaf net CO2 assimilation (Amw) and leaf dry mass per unit area (Wa) in P. dulcis trees (A) in the FI and DI regimes, using pooled data from f- and nf-leaves and (B) for f- and nf-leaves, using pooled data from FI and DI regimes. Regression lines in (A) y = 0.475  0.0028x (R2 = 0.73, P < 0.001) and y = 0.470  0.0030x (R2 = 0.64, P < 0.001) for FI and DI, respectively, and in (B) y = 0.623  0.0046x (R2 = 0.87, P < 0.001) and y = 0.472  0.0027x (R2 = 0.72, P < 0.001) for f- and nf-leaves, respectively. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

had lower Wa, Na and Nw than the nf-leaves (Figure 4A–C). The DI regime did not significantly affect Wa in f-leaves but decreased Wa in nf-leaves by 10–15%, mainly from pre-harvest onward (Figure 4A). Stress conditions have been reported to affect leaf N concentration (Reich et al. 1989). This was confirmed in our study, but only for the

nf-leaves, which had significantly lower Na in DI trees than in FI trees, whereas Na in f-leaves was little affected by the DI treatment (Figure 4B). A similar conclusion can be drawn for Nw (Figure 4C). Variations in Na (=WaNw) may result from variations in Wa, in Nw, or in both. A single relationship characterized the whole dataset when the

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Ama (μmol m 2 s 1 )

A

FI DI

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0.4

0.3 20 15

0.2

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Amw (μmol g 1 s 1 )

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5 0 0.010

0.015

0.020

0.025

0.030

0.035 0.010

0.015

Nw (g g 1)

0.020

0.025

0.030

0.0 0.035

Nw (g g 1)

Figure 9. Relationships (A) between area-based maximum leaf net CO2 assimilation (Ama) and mass-based leaf N concentration (Nw) in P. dulcis trees in the FI and DI regimes, using pooled data from f- and nf-leaves and (B) between dry-mass-based Am (Amw) and Nw for the f- and nf-leaves, using pooled data from FI and DI regimes. Regression lines in (A) y = 4.01 + 868.91x (R2 = 0.76, P < 0.001) and y = 13.05 + 1217.9x (R2 = 0.80, P < 0.001) for FI and DI, respectively, and in (B) y = 0.199 + 15.62x (R2 = 0.89, P < 0.001) and y = 0.247 + 16.04x (R2 = 0.80, P < 0.001) for f- and nf-leaves, respectively. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation and DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season.

8 6 4 2 Stages II-III

Stage IV

150

B

8 6 4 f-leaves nf-leaves

2

Stage V

0 100

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1

nf-FI f-FI nf-DI f-DI

1 PNUEm (μmol g s )

10

12

A

1

PNUEm (μmol g s 1 )

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200

0 0.010

250

0.015

0.020

C

10

PNUEm (μmol g

0.030

0.035

nf-FI f-FI nf-DI f-DI

1

s 1)

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Nw (g g 1 )

DOY

8 6 Postharvest DI regime

4 2 0 50

75

100

125

150

175

Wa (g m 2 )

Na versus Wa relationship was analyzed separately for each stage of development (Figure 5A–C), but the slope value (giving the mean Nw over a stage) shifted as the growing season progressed, indicating that the Na versus Wa relationship is stage-dependent in almond trees, in contrast to nectarine trees (Rosati et al. 1999). Independently of irrigation treatment, the presence of fruits near the leaves increased the slope and the intercept of the Nw versus Wa relationship compared with the

Figure 10. (A) Seasonal trend in PNUEm in P. dulcis trees. Symbols as in Figure 4. Pooled standard deviation of the mean was typically close to ±1.0 lmol g1 s1. The (B and C) relationships between PNUEm and Nw and between PNUEm and Wa, respectively. Regression lines showed in (B) for f-leaves (1.21 + 346.9Nw, R2 = 0.70, P < 0.001) and nf-leaves (3.8 + 396.0Nw, R2 = 0.60, P < 0.001), using pooled data from FI and DI regimes, and in (C) for all datasets (15.14  0.080Wa, R2 = 0.78; P < 0.001) excluding the three outliers corresponding to the post-harvest period in the DI regime. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation; DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season and DOY, day of year.

corresponding values in nf-leaves, implying that f-leaves experienced a greater decrease in Nw for a given Wa and had a lower nitrogen concentration at high Wa (Figure 6). This may be ascribed to a higher depletion of N in the leaf blade to cope with the demand of the fruits during the kernel-filling stage and that of the perennial organs after harvest. A unique linear Nw versus Wa relationship was found for f-leaves in the FI and DI treatments, whereas this relationship differed significantly between irrigation

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fpN

0.6

0.4

0.2 Stages II-III

Stage IV

Stage V

0.0 100

150

200

250

DOY

Figure 11. Seasonal trend in photosynthetic-N fraction, fpN (=Nwp/Nw) in P. dulcis trees. Same symbols as Figure 4. Pooled standard deviation of the mean (data not shown) was typically ± 0.040. Abbreviations: f- and nf-leaves were obtained from 1-year-old fruiting (f) and current-year non-fruiting (nf) shoots, respectively; FI, full irrigation; DI, sustained deficit irrigation at 50% of standard crop evapotranspiration throughout the growing season and DOY, day of year.

treatments for nf-leaves (Figure 6), suggesting that, in the DI regime, leaf structural adjustments operated to maintain a conservative N status in the leaves of fruit-bearing shoots, but to the detriment of N resources allocated to vegetative shoots. Effects of irrigation treatment on leaf physiologic traits in fruiting versus non-fruiting shoots Almond species are known to exhibit high rates of photosynthesis among woody Rosaceae (DeJong 1983, Wullschleger 1993, Higgins et al. 1992, Lakso 1994, Gonza´lez-Real and Baille 2000). We observed maximum absolute Ama values close to 24 lmol m2 s1 (Figure 7A), consistent with the values reported for almond leaves (DeJong 1983, Higgins et al. 1992, Matos et al. 1997, De Herralde et al. 2003, Rouhi et al. 2007). Independently of the irrigation regime, leaves of fruiting and non-fruiting shoots exhibited high Ama during the active vegetative growth period when the values of gsm were below their maximum (Figure 7A–C), therefore maintaining a high intrinsic water-use efficiency, as observed by Heilmeier et al. (2002). Low Ci/Ca ratios over this period (about 0.52) confirmed that highest photosynthetic activity occurred in spring. The finding that the decreases in both Ama and gsm from the second half of kernel-filling stage onward occurred concomitantly with the increasing values of Ci/Ca suggests that internal mesophyll limitations dominated over stomatal limitations (Jifon and Syvertsen 2003). The decreasing trend in both Am and Ap (Figure 7A–D) during the stage of fruit dry mass accumulation was consistent with the results reported by Walcroft et al. (2002) for peach trees. However, this pattern is not a general characteristic of fruit-bearing trees. Several studies have reported a positive effect of fruit demand for assimilates on the

385

photosynthetic rates of peach (DeJong 1986) and apple (Palmer et al. 1997) trees. There was a steep reduction in Amw (Figure 7B) from the second half of kernel-filling stage onward that was independent of irrigation regime and leaf class. However, after harvest, this declining trend slowed in the nf-leaves in the FI treatment but was still marked in the DI treatment. Because translocation of assimilates to perennial tree organs occurs post-harvest, the decrease in Am in DI trees during this stage indicates that DI trees could be subjected to carbohydrate shortage in the following growth season (Esparza et al. 2001a). This decrease probably explains the lower Ama in DI trees than in FI trees, as well as their smaller trunk diameter at the beginning of the growing season (see section Materials and methods). The lower Am observed in DI trees compared with FI trees, along with the sustained decrease in Am observed in trees in both treatments after Stages II and III, should be analyzed to determine to what extent gs (Figure 7C) determines this pattern by restricting CO2 availability in the mesophyll. Comparison of the seasonal trends in areabased Ap (Figure 7D) and Am (Figure 7A) indicated a rather similar behavior. Maximum Ap occurred in the spring but declined during the remainder of the growing season, independently of irrigation treatment. These results provided indirect evidence that, in both irrigation treatments, gs was not the main determinant of the decrease in photosynthetic capacity (Jifon and Syvertsen 2003) observed in our almond trees. A negative correlation was observed between Am (areaand dry-mass-based values) and Wa in both classes of leaves, which seemed to be independent of irrigation regime (Figure 8A and B). Higher values of Am in FI trees than in DI trees could be related to differences in Wa (Figure 4A), i.e., to differences in leaf thickness (Wa = leaf thickness · leaf density). Thicker leaves are likely to have more chloroplasts per unit of leaf area (Rieger and Duemmel 1992, Evans and Porter 2001) and therefore higher photosynthetic capacity. Our results demonstrated that maximum assimilation varied considerably over time with leaf age (Wilson et al. 2000), and that this seasonal effect dominated over other factors such as leaf class and soil water availability. This finding is supported by the data showing that Nw, which is often used to describe the effects of leaf age (Field and Mooney 1983, Reich et al. 1991), explained between 70% and 92% of the seasonal variability in both Ama and Amw (Figure 9A and B). The marked decline of PNUEm throughout the growth season (Figure 10A), irrespective of the irrigation treatment, and the concomitant decrease in Ap (Figure 7D) together suggest the seasonal dependence of N allocation within the leaf. It is known that the allocation patterns of chlorophyll and Rubisco can vary with leaf ontogeny (Porter and Evans 1998, Rey and Jarvis 1998, Wilson et al. 2000). The finding that seasonal changes in Ama and

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Na were poorly correlated, whereas both area- and massbased values of Am and leaf N concentration were strongly related (Figure 9A and B), could be ascribed to changes in the attribution of N within the leaf (Kull et al. 1998, Dungan et al. 2003, Yasumura et al. 2006). The finding that PNUEm was highly negatively correlated to Wa (Figure 10C) tends to support the hypothesis of a seasonal decrease in mesophyll conductance. The tight positive correlations between PNUEm and Nw (Figure 10B), and between PNUEm and Wa (Figure 10C) suggest that both leaf classes had an adequate N concentration and were functionally developed (Reich et al. 1991) in both irrigation treatments. Because non-photosynthetic N (Nwo) was lower in f-leaves, PNUEm was significantly higher in f-leaves than in nf-leaves (Figure 10A) even though there were no significant differences between leaf classes in the irrigation regimes. However, when PNUEm was calculated with respect to photosynthetic N (Nwp), no significant effect of leaf class or irrigation regime was observed. We found that the fraction of photosynthetic N (fpN) decreased over the growing season (Figure 11), which might support the hypothesis advanced by Wilson et al. (2000) of a seasonally dependent fractional allocation of leaf N to Rubisco. Effect of soil water status imposed by the irrigation strategy Lower Ama and Apa in nf-leaves of DI trees (17% and 15%, respectively) than in FI trees prevailed throughout the spring (Stages II and III) despite several heavy rainfall (Figure 2) that maintained soil water near field capacity in both irrigation treatments. This pattern, along with the lower crown leaf area of DI trees compared with FI trees (see section Materials and methods), suggested a carryover effect of DI on whole-tree potential for CO2 uptake and resource acquisition. Thus, after 4 years of sustained DI, the DI trees appear to have acclimated to water stress but at the cost of a lower photosynthetic capacity at the start of the active vegetative growth stage. We found that the photosynthetic capacity of almond leaves was unaffected by a  100% decline in predawn leaf water potential, likely indicating acclimation to the sustained conditions of mild water stress (minimum value of Wpd about 1 MPa) imposed during the previous 4 years. The mild conditions of soil water deficit imposed on the DI trees were not responsible for the seasonal reduction in photosynthetic capacity. Although the interaction between leaf class and irrigation regime was limited, with both leaf classes exhibiting rather similar patterns of Am and Nw in both irrigation regimes, the nf-leaves were more responsive to soil water deficit than the f-leaves, with the responses being mediated through leaf structural changes (see Figure 6). We conclude that, under the DI regime we applied, leaf structural adjustments appeared to operate to maintain a similar allocation of N resources in the leaves of fruit-bearing

shoots, but to the detriment of N resources allocated to vegetative shoots. Foliar structural changes, along with changes in nitrogen partitioning within the leaf – which might be related to concomitant decreases in photosynthetic leaf N concentration and the fractional allocation of leaf N to Rubisco – were probably responsible for the decrease in photosynthetic capacity throughout the growth cycle. Acknowledgments This study was supported by the Spanish Ministry of Science and Technology (MCYT, grant AGL2002-04048-C03-02) and European Commission (IRRIQUAL project, FP6-2004-FOOD-3BC023120). Gregorio Egea was granted a research fellowship from the FPU programme of the Spanish Ministry of Education.

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