Stomatal and non-stomatal limitations to photosynthesis in seedlings and saplings of Mediterranean species pre-conditioned and aged in nurseries: Different response to water stress

Environmental and Experimental Botany 75 (2012) 235–247

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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Stomatal and non-stomatal limitations to photosynthesis in seedlings and saplings of Mediterranean species pre-conditioned and aged in nurseries: Different response to water stress Laura Varone a , Miquel Ribas-Carbo b , Carles Cardona c , Alexander Gallé b , Hipólito Medrano b , Loretta Gratani a , Jaume Flexas b,∗ a

Department of Environmental Biology, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy Research Group on Plant Biology Under Mediterranean Conditions, Departament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa Km 7.5, 07122 Palma de Mallorca, Illes Balears, Spain c Laboratori de Botànica, Departament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa Km 7.5, 07122 Palma de Mallorca, Illes Balears, Spain b

a r t i c l e

i n f o

Article history: Received 4 January 2011 Received in revised form 20 June 2011 Accepted 10 July 2011 Keywords: Mesophyll conductance Leaf mass area Photosynthetic limitation analysis Plant age Plant size Stomatal conductance

a b s t r a c t The aim of the present work was to compare the physiological responses to water stress and recovery of seedlings and saplings of three different Mediterranean species (Olea europaea var. sylvestris, Rhamnus alaternus and Cneorum tricoccon), pre-conditioning and aged in nursery and presenting different ages and pot sizes. Our hypothesis was that the ratio of plant size to soil volume (which constrains root development leading to low root-to-shoot ratios) rather than any of the two factors separately determines the seedling response to water stress. Seedlings (1-y) and saplings (3 to 4-y) were transplanted into pots bigger than those used during growth in the nursery and irrigation was stopped to each species × age/size combination. Leaf water potential ( ), net CO2 assimilation (AN ), stomatal (gs ) and mesophyll (gm ) conductances, and the rate of photosynthetic electron transport (ETR) were determined every few days. Plants were re-watered when AN dropped below 70% of control values. Saplings of each species presented larger total leaf area (TLA) and reached lower  than seedlings. Even under irrigation, saplings showed lower AN , which was not related to gs but to lower gm and ETR. During water stress, AN decreased slowly in seedlings due to stomatal limitations, while in saplings it decreased fast and mainly associated to non-stomatal limitations (gm and ETR). Upon re-watering, seedlings recovered maximum AN within a few days, while recovery was slow and incomplete in saplings. At the end of the experiment, significant leaf die-back occurred in saplings but not in seedlings except for Cneorum. The minimum  achieved during water stress was strongly linearly related to TLA when pooling all species and ages, and leaf die-back was strongly dependent on  and on the appearance of non-stomatal limitations to photosynthesis. Therefore, we conclude that the total amount of leaf area for a given volume of substrate (i.e., maximum water availability), rather than plants pre-conditioning in nurseries or plant age, determines seedling/sapling responses to water stress and re-watering in Mediterranean species. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Mitigation of desertification in Mediterranean areas is an important issue which requires reforestation and afforestation of former woodlands and abandoned agricultural lands. However, the achievement of successful plantations has technical problems imposed by the extreme aridity during summer, which leads to low establishment and high seedling mortality after summer (Vallejo et al., 2005; McDowell et al., 2008; Luis et al., 2009). Strategies based on pre-conditioning and/or hardening seedlings in nurseries

∗ Corresponding author. Tel.: +34 971 172365; fax: +34 971 173184. E-mail address: jaume.fl[email protected] (J. Flexas). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.07.007

have been proposed to improve seedling performance under field ˜ conditions (van den Driessche, 1991; Banon et al., 2006; Vilagrosa et al., 2006). Of the two, hardening (i.e., application of short term stress treatments) has been more often reported. In Mediterranean species, the most common hardening techniques include induced nutrient deficiency (Vilagrosa et al., 2006), acclimation of seedlings to low temperatures and, most often, water deficiency (Villar-Salvador et al., 1999, 2004a,b; Vilagrosa et al., 2003a). However, although hardening techniques are often reported to increase seedling resistance to environmental stress when tested in nurseries, their success for improving seedling survival and growth when planted in the wild is far from being demonstrated. For instance, recent studies suggest than nutrient fertilization rather than deficiency improves field performance (Villar-Salvador et al.,

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2005; Trubat et al., 2006, 2010; Luis et al., 2009). Vilagrosa et al. (2006) reviewed fifteen drought-hardening assays in Mediterranean plants. Survival improved in three cases, decreased in another three and was not affected by drought-hardening in nine of the cases studied. On the other hand, growth decreased in four cases and only increased in one, being unaffected in the rest of the studies. Clearly, other effects than hardening may contribute to the observed differences among studies. For instance, plants grown in larger pots in nurseries and/or grown for longer, i.e., older and bigger saplings, are likely to reach better performance after transplanting into the field regardless of pre-hardening. The effects of pot volume/size on plant growth have been largely studied in annual crops (Kratky et al., 1982; Ismail et al., 1994; Ray and Sinclair, 1998), but fewer studies have been done in forest woody species (Appleton and Whitcomb, 1983; South, 2000). Particularly, only few studies have addressed the container effects in Mediterranean species and, in these, container material and depth rather than volume, have been addressed (Tsakaldimi et al., 2005; Pemán et al., 2006). However, while potted plants are expected to perform better in larger pots, this is not guarantee of better plant survival and growth after transplantation. For evergreen woody species showing year-to-year growth, the ratio between plant and pot size seems to matter more than pot size alone, because it determines the balance between water demand and water availability. When ageing seedlings occurs in commercial nurseries, pot size is often increased to a lesser extent than plant size, leading to increased ratios of aboveground to root biomass. Surprisingly, there are no studies on how this specific combination would affect seedling performance under drought conditions, although this has been recognized to be crucial in determining afforestation success besides pre-hardening (South, 2000). Besides morphological features, such as increased root surface and depth, the different plant survival ability in the field has often been attributed to differences in plant water relations, notably hydraulic conductance/hydraulic failure thresholds, either constitutive of a given species or conferred through hardening (Pemán et al., 2006; Trubat et al., 2006; Galmés et al., 2007; McDowell et al., 2008). However, in addition to plant water relations, seedling establishment and fitness of Mediterranean plants are tightly related to leaf carbon balance and to its response to water stress (Oechel et al., 1981; Larcher, 1994, 2000; Galmés et al., 2005; Gratani et al., 2008; Quero et al., 2008). Photosynthesis is one of the most negatively affected physiological processes during summer droughts in Mediterranean plants, and its decrease is mostly mediated by stomatal closure before hydraulic effects appear (Gratani, 1995; Galmés et al., 2007; Gulías et al., 2009). Photosynthesis responses to water stress differ among species (Gratani, 1995; Galmés et al., 2007; Gulías et al., 2009), and depend on plant age (Bond, 2000; Niinemets et al., 2005, 2006), size (Bond, 2000; Woodruff et al., 2008; Mullin et al., 2009) as well as on environmental conditions interacting with water stress, such as irradiance (Valladares et al., 2005, 1997; Niinemets et al., 2006), temperature and air humidity (Pérez-Martín et al., 2009). Moreover, the survival of Mediterranean species after summer water stress depends also on their recovery capacity after a rain pulse (Gratani and Varone, 2004a; Gallé et al., 2007). Thus, the net plant carbon gain during a period of water stress and recovery may depend as much on the velocity and degree of photosynthetic recovery as on the degree and velocity of photosynthesis decline during water depletion (Flexas et al., 2006; Xu et al., 2009). While stomatal closure is often invoked as the main cause for photosynthesis decline under water stress, CO2 uptake may be limited by non-stomatal limitations as well, especially when water stress intensifies and persists for long periods (Flexas et al., 2006; Peguero-Pina et al., 2008). Non-stomatal limitations include both diffusive (reduced mesophyll conductance) and metabolic

(photochemical and enzymatic limitations) processes (Grassi and Magnani, 2005; Galmés et al., 2007). In particular, mesophyll conductance plays an important role during water stress and rewatering (Galmés et al., 2007; Flexas et al., 2008; Xu et al., 2009), and it is specially limiting in species with high leaf mass per area (LMA), such as in Mediterranean sclerophylls (Di Marco et al., 1990; Flexas et al., 2008). High LMA is a recurrent leaf trait of Mediterranean species (Niinemets, 2001; Gratani and Varone, 2006; Paula and Pausas, 2006) with a special protection function for plants facing long periods of drought stress. Since high LMA is correlated with poorer resource environments (Turner, 1994; Wright et al., 2004), it is likely that low root-to-shoot ratios resulting from growing seedlings in containers limiting root growth may induce higher LMA. Moreover, LMA and mesophyll conductance (gm ) are affected by leaf development as well as by leaf and plant age (Niinemets et al., 2005, 2006; Marchi et al., 2008), for which both hardening and ageing seedlings at nurseries may have an effect on maximum photosynthetic capacity through modulating gm . Despite its importance, only few studies have addressed a quantitative analysis of the main photosynthesis limitations in response to drought and re-watering in seedlings of Mediterranean species (Grassi and Magnani, 2005; Galmés et al., 2007; JuárezLopez et al., 2008; Grassi et al., 2009) and, to the best of our knowledge, none of them has compared plants with different age and size. In summary, growth conditions at nurseries may have important implications for seedling performance in afforestation of Mediterranean areas. We hypothesize that, in seedlings aged and pre-conditioned in nurseries (i.e., saplings) the parameter that determines the most the seedling response to water stress and recovery after re-watering is the ratio of plant size to soil volume (which constrains root development leading to low root-to-shoot ratios) allowed during growth in nurseries, rather than any of the two factors separately. Moreover, we also hypothesize that these factors affect the relative stomatal versus non-stomatal limitations to photosynthesis, which results in differences in plant survival and velocity of recovery after a water stress. Therefore, the aim of the present work was to compare the physiological responses to water stress and recovery, with a special emphasis on photosynthesis limitations, in seedlings of different Mediterranean species typically used in afforestation, presenting different ages and pot size resulting in different ratios of plant size to soil volume. 2. Materials and methods 2.1. Study site and plant material The study was performed in the period May–June 2006 at the Universitat de les Illes Balears (UIB), sited in Palma de Mallorca (inland South of Mallorca, Spain) on three Mediterranean species: Olea europaea L. var. sylvestris, Rhamnus alaternus L. and Cneorum tricoccon L. These three species were selected because they are shrubs typically used in afforestation in Mediterranean areas, while they are well represented in the Balearic Islands along the whole gradient of environmental conditions found in the islands (Gulías et al., 2009). For each species, both seedlings (1 yr old) and saplings (3 yr old for Cneorum, 4 yr old for Rhamnus and Olea) were analyzed. Additionally, only for O. europaea, mid-aged saplings (3 yr old) were also analyzed. Until the onset of the experiment, plants were grown in a nursery in Sa Pobla (inland North of Mallorca, Spain). Plants were growing outdoors in containers filled with a mixture of 50% coconut fibre, 45% vegetal compost and 5% perlite, supplied with traces of slow-release fertilizer (Osmocote® 15-10-12, Sierra Chemicals Co.). Because germination and initial growth are critical stages which often require mild conditions, plants were

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placed below a shelter restricting 30% of the incoming light during that period. After several months, although no specific hardening treatment was applied, the plants were subject to less optimal conditions than those typically endured in commercial nurseries, in order to induce some degree of acclimation to environmental stress. For instance, the light shelter was removed and plants were grown at full sunlight. Moreover, no irrigation was applied during growth to ensure some drought acclimation, although some water was sprayed with nebulizers for only 20 min at midnight, daily during summer (from mid-May to mid-September) and once per week during the rest of the year. This treatment allowed for deposition of some water in the plants’ and substrate’s surfaces, but it did not result in substrate water re-filling. We refer to these suboptimal growing conditions as ‘drought pre-conditioning’. Initially, plants were grown in 0.2-L forest alveoles during the first year, after which they were transferred to 2.8-L pot containers. Four-year old saplings were transferred to 10-L pots. Plants were transferred to the study site two months before the experiment and allowed to acclimate. Pots were irrigated at full capacity twice a week during this period. Two weeks before the onset of the experiments all plants (seedlings and saplings) were transferred to 12-L pots containing clay-calcareous soil taken from a proximal area occupied by a Mediterranean macchia ecosystem. This transfer into a pot somewhat larger than the pot of origin was done to avoid initial root growth restrictions during the experiment, although it also implied putting roots in contact with a contrasting substrate, while still constraining further root expansion, as it often occurs in the field due to the massive presence of stones in Mediterranean calcareous soils (Yaalon, 1997; Cerdà, 1998; Eugenio et al., 2006). Both the nursery site and the study site are characterised by a Mediterranean climate type with similar meteorology. The year mean air temperature is 17.4 ◦ C in Sa Pobla and 16.8 ◦ C in UIB, while total annual rainfall is 675 L m−2 and 580 L m−2 , respectively. During the experiments at UIB in June–July 2006, maximum mean air temperature peaked 31.5 ◦ C, and minimum 18.5 ◦ C, and precipitation events were infrequent and short (less than 10 L m−2 during the whole period). The selected measuring dates were all fully sunny to ensure the maximum homogeneity during measurements. This resulted in air temperatures ranging 26–32 ◦ C and vapour pressure deficits ranging 2.5–3.7 kPa for all measuring dates, with two exceptions: D.O.Y.s 135 and 150 were specially hot and dry, with air temperatures reaching 35 ◦ C and vapour pressure deficits 4.5–5 kPa during measurements. 2.2. Water stress treatments After transferring all 56 plants from the nursery to the study site they were allowed to acclimate for two months to the new site conditions, but in their original pots, being irrigated twice a week during this period. Then, after transferring all plants to 12-L pots, they were allowed to acclimate to their new pots for another 12 days, during which they were all watered regularly to field capacity to ensure plants under an irrigation treatment. Finally, on May 10th water was withheld to four plants of each species and age/size (n = 28) to induce water stress, while the remaining plants were kept under daily irrigation. During the experiment all pots were daily weighted to evaluate the water loss. Measurements corresponding to control plants were performed on the first day of the experiment when all the plants were still well watered. During water stress treatment, plants were covered by a clear plastic sheet during the night to prevent any influence of occasional rainfall. Measurements of gas exchange were performed every two days in each of the selected plant species and age classes, and water stress treatment was stopped by irrigation after the first day in which the recorded net assimilation rate was

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below 70% of that measured in control plants of each species and age. This criterion was chosen to ensure substantial but not lethal water stress conditions (Gulías et al., 2002; Gratani and Varone, 2004b), and resulted in similar photosynthetic reductions for all treatments (from 68% in Rhamnus seedlings to 100% in Olea and Rhamnus saplings, see Section 3). Only in one particular day (D.O.Y. 137) the reductions were greater than 100% (i.e., negative photosynthesis rates) in Olea saplings, but even these have been shown to not result in lethal conditions in these plants (Gallé et al., 2009). The day after water stress reached the selected level pots were re-watered to field capacity and were subsequently re-watered daily to follow the recovery of plants over a period of several days. 2.3. Leaf water potential, leaf relative water content and substrate water content Measurements of midday leaf water potential ( , MPa) and relative water content (RWC, %) were carried out in stressed plants, when AN dropped below 70% of the control and the first day after re-watering (i.e., early during the recovery period), as well as in control plants. Measurements were carried out on the four plants per species and age.  was determined using a Scholander pressure chamber. RWC was calculated as: (FW-DW)/(TW-DW)*100, where FW was leaf fresh weight, DW leaf dry weight and TW turgid weight. Leaf samples were weighed immediately after sampling to determine FW. Thereafter they were submerged in distilled water and kept in darkness at 4 ◦ C for 24 h to determine FW. DW was measured after oven drying these leaf samples for 48 h at 60 ◦ C. Pots were weighted the day of maximum stress level for each species and age. The corresponding days, pots containing irrigated (control) and water stressed plants were weighted (Pot weightsample ). At the end of the experiment, all pots were brought to field capacity by several consecutive cycles of watering and drainage (until constant weight) and weighted again (Pot weightfield capacity ). From the later values, the substrate water content (SWC) of both control and water stressed plants was calculated as percentage of field capacity as: SWC = (Pot weightsample /Pot weightfield capacity ) × 100. 2.4. Plant and leaf morphometry and leaf die-back Plant height (H, cm) and total leaf area (TLA, cm2 ), and leaf mass area (LMA, mg cm−2 ) were determined on the first day of the experiment. Leaf die-back (LDB) was determined during the entire experiment and computed the last day. H and TLA per plant were measured in four plants per species and age. TLA was calculated from the total number of leaves per plant and their mean leaf area, which was measured on 20 leaves per species and age using an AM-100 Area Meter (ADC, Herts, UK). LMA was determined in four individual and fully developed leaves per treatment as the ratio between their DM and leaf area. LDB was determined by the daily collection of the leaves fallen from each plant, which were immediately counted and oven dried for dry weight determination. At the end of the experimental period, total LDB was calculated as the sum of leaf dry weights determined daily from fallen leaves. Although we could not determine root characteristics at the end of the experiment (the clay nature of the substrate made it difficult to extract intact roots), the pot volume was the same for all species and ages (12 L). Therefore, relative variations of total leaf area were equivalent to relative variations in the ratio of leaf area to pot volume. Therefore, we used TLA as a proxy for the relative ratio of leaf area to water availability.

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2.5. Gas exchange, chlorophyll fluorescence and photosynthesis limitation analysis Gas exchange and chlorophyll fluorescence measurements were performed using a portable infrared gas analyser (Li-6400; Li-Cor Inc., Lincoln, NE, USA) equipped with the Li-6400-40 leaf chamber fluorometer. Measurements were carried out in the morning hours (from 10 to 13 a.m.) on attached, fully expanded leaves (4 replicates per species and age). CO2 concentration in the Li-6400 leaf chamber (Ca ) was set at 400 ␮mol CO2 mol−1 air and photosynthetic photon flux density (PPFD) was set at 1500 ␮mol m−2 s−1 with a 10% of blue light, while air temperature ranged 27–35 ◦ C and the relative humidity of the incoming air ranged between 20 and 50%, depending on the day of measurement. Light-saturated net assimilation rate (AN , ␮mol CO2 m−2 s−1 ), stomatal conductance (gs , mol H2 O m−2 s−1 ) and sub-stomatal CO2 concentration (Ci , ␮mol CO2 mol air−1 ) were measured following Li-cor standard protocols. From the fluorescence measurements, the actual quantum efficiency of the photosystem II (PSII)-driven electron transport (˚PSII ) was determined according to Genty et al. (1989) as: ˚PSII =

 −F Fm s ,  Fm

where Fs is the steady state fluorescence in the light  the maximum fluorescence obtained (1500 ␮mol m−2 s−1 ) and Fm with a light-saturating pulse (∼8000 ␮mol m−2 s−1 ). As ˚PSII represents the number of electrons transferred per photon absorbed by PSII, the rate of electron transport (ETR) can be calculated as: ETR (␮mol e− m−2 s−1 ) = ˚PSII · PPFD · ˛, where the term ˛ includes the product of leaf absorptance and the partitioning of absorbed quanta between photosystems I and II. Leaf absorptance was measured using a spectroradiometer (HR2000CGUV-NIR, Ocean Optics Inc., Dunedin, FL, USA) as described by Schultz (1996), using the light source from the Li-6400 and making the measurements inside a dark chamber. The results ranged from 0.93 to 0.95 for all species and ages, in agreement with reports by Niinemets et al. (2005, 2006) and Flexas et al. (2008) for other Mediterranean evergreen species. The partitioning of absorbed quanta between photosystems I and II was assumed to be 0.5 (Laisk and Loreto, 1996). From combined gas-exchange and chlorophyll a fluorescence measurements, the mesophyll conductance for CO2 (gm ) was estimated according to Harley et al. (1992) as: gm =

AN , Ci − ( ∗ (ETR + 8 · (AN + Rd )))/(ETR − 4 · (AN + Rd ))

where AN and Ci were obtained from gas-exchange measurements and ETR from chlorophyll fluorescence measured, as described above. A value of 45 ␮mol mol−1 for the CO2 compensation point under non-respiratory conditions ( *) was used, which was calculated from Rubisco specificity factor ‘␶’determined in Mediterranean evergreen sclerophyll species (Galmés et al., 2005) and according to Brooks and Farquhar (1985):  = 0.5O/ * (O denotes for the oxygen molar fraction at the oxygenation site, assumed to be 21%). The temperature dependency for  * was taken from Bernacchi et al. (2002). Concerning values for mitochondrial respiration (Rd ) these were estimated as a function of leaf temperature from previously determined respiration rates using the Li-6400, and applying a Q10 of 2.2 (Valentini et al., 1995). Calculated values of gm were used to calculate the chloroplast CO2 concentration (Cc ) according to the following equation: Cc = Ci −

A  N

gm

To assess the limitations imposed by water stress and recovery on photosynthesis, a quantitative limitation analysis of photosynthesis according to Grassi and Magnani (2005) was applied as modified by Gallé et al. (2009). The modification consisted in using ETR, together with estimates of AN , gs , and gm , as a proxy for biochemical limitations instead of the maximum velocity of carboxylation (Vc,max ) proposed in the original reference. Since actual electron transport rate (i.e., fluorescence derived ETR) is tightly coupled with Vc,max (e.g. Galmés et al., 2007; Gallé et al., 2009). This is particularly recommendable in the present study because AN –Cc curves were not performed to properly asses Vc,max . Hence, AN , gs , gm and ETR were used to calculate the proportion of the three major components of total limitation (TL) for CO2 assimilation: stomatal limitation (SL) and mesophyll conductance limitation (ML), as well as biochemical limitation (BL). In the current study, the maximum assimilation rate, concomitantly with gs , gm and ETR, was achieved in young plants under well-watered conditions for all species. Therefore the values corresponding to irrigated young plants were used as the reference for “zero” TL and for the estimation of photosynthetic limitations in all other days-treatments of both plant ages. 2.6. Statistical analysis Pearson’s correlation analyses were performed to evaluate the correlation among the considered physiological variables. Moreover, regression analysis was carried out to evaluate the relationship between: maximum gm and LMA, minimum Y and TLA, LMA and TLA, leaf die-back and minimum Y, leaf die-back and SL, leaf die-back and NSL. When non-linear fits were used the goodness of fit compared to those of linear model was evaluate by a variation of the Akaike Information Criteria (AIC, Akaike, 1973), namely AICc . AICc was a correction of AIC used for small sample size and it was calculated according to Miao et al. (2009) as: AICc = nLn

 RSS  n

+

2nk n−k−1

where RSS is the residual sum of squares, k the number of estimated parameters and n the sample size. In this case the RSS was used instead of likelihood function because of the data followed a normal distribution (Miao et al., 2009). The model which gave the minimum AICc was considered as the best model. One- and two-way ANOVAs were performed to analyze either the effect of species and age either combined or separated and their interaction on the considered variables. A post hoc Tukey’s test was performed to compare differences among means (p < 0.05). Kolmogorov–Smirnov and Levene tests were used to verify the assumptions of normality and homogeneity of variances, respectively. All statistic tests were performed by a statistical software package (Statistica, Stasoft, USA). 3. Results 3.1. Plant and leaf morphometric characteristics There were significant differences in plant height (H), total plant leaf area (TLA) and leaf mass area (LMA) among species and ages (Table 1). O. europaea and R. alaternus saplings presented the largest H values (up to ca. 1.5 m), while O. europaea and C. tricoccon seedlings presented the smallest (ca. 30 cm). R. alaternus seedlings and old C. tricoccon saplings showed similar, intermediate values. Mid-aged O. europaea plants showed intermediate values between young and old Olea for H and for all the other parameters analyzed, as well as for water stress and recovery responses (data not show, except for correlations between

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239

Table 1 Plant and leaf traits of the considered species (H, plant height, TLA, total leaf area, LMA, leaf mass area and leaf die-back). Values correspond to well-watered plants. Mean values (±S.E.) are shown. Mean values with different letters are significantly different (Tukey’s test, p < 0.05). H (cm)

O. europaea R. alaternus C. tricoccon O. europaea R. alaternus C. tricoccon

Saplings Saplings Saplings Seedlings Seedlings Seedlings

142 152 48 29 44 29

± ± ± ± ± ±

LMA (g cm−2 )

TLA (cm2 )

6c 2c 7b 6a 6ab 5a

9634 6928 1129 244 342 216

76d 31c 75b 22a 48a 9a

194 153 224 145 136 140

± ± ± ± ± ±

Leaf die-back (g DW) 53.7 ± 3c 63.3 ± 1.5d 8.0 ± 1.0b 0 0 1.1 ± 0.2a

4b 6a 3c 9a 2a 8a

A. Olea europaea

20

15

-2

Table 2 Substrate water content as % of the field capacity of control (SWCc ) and stressed (SWCs ) plants. Mean values (±S.E.) are shown. Mean values with different small letters indicate significant differences among species and age/size; mean values with different capital letters indicate significant difference among treatment (Tukey’s test, p < 0.05).

± ± ± ± ± ±

-1

Age/size

AN (μmol μ CO2 m s )

Specie

Specie

Age/size

SWCc (%)

O. europaea R. alaternus C. tricoccon O. europaea R. alaternus C. tricoccon

Saplings Saplings Saplings Seedlings Seedlings Seedlings

86 87 86 86 89 86

± ± ± ± ± ±

4aA 1aA 3aA 6aA 1bA 6aA

SWCs (%) 68 58 49 45 46 43

± ± ± ± ± ±

4bB 3bB 4aB 5aB 1aB 7aB

10

5

0

20

130

Seedlings Saplings

B. Rhamnus alaternus 150 160

140 X Data

15

-2

-1

AN (μmol μ CO2 m s )

parameters depicted in Fig. 7). O. europaea saplings had the highest TLA, followed by R. alaternus saplings and, to a lesser extent, by C. tricoccon saplings. Seedlings had 80–95% less TLA than saplings in all species (Table 1). LMA significantly increased with age/size in all species except Rhamnus (Table 1). On average saplings had a 36% higher LMA than seedlings (Table 1). Among species, C. tricoccon had the highest range of LMA (140–224 g m−2 ) and R. alaternus the lowest (136–153 g m−2 ). At the end of the water stress and recovery cycle, most plants presented significant leaf die-back (LDB), except Rhamnus and Olea seedlings (Table 1). Cneorum seedlings presented only little LDB (1. g DW), and Cneorum saplings somewhat higher values (8 g DW), while Rhamnus and Olea saplings presented large LDB (63 and 54 g DW, respectively). In percentage of total leaf area, LDB represented 28% in Olea saplings, 32 and 37% in Cneorum saplings and seedlings, respectively, and as much as 60% in Rhamnus saplings.

10

5

0

20

130

150Cneorum tricoccon 160 C.

140

-1

15

-2

3.2. Substrate and leaf water status

AN (μmol CO2 m s ) μ

X Data

By the time the treatment AN was reached, substrate water content (SWC) had significantly dropped to 58–68% in Olea and Rhamnus saplings, and to 45–49% in the other species and ages (Table 2), while SWC of control plants ranged between 86 and 89% considering all species and treatments. In addition, midday leaf water potential ( ) and relative water content (RWC) also decreased in both seedlings and saplings (Table 3), with larger decreases in saplings than in seedlings. Nevertheless, decreases of  were proportionally larger than decreases of RWC in all species. The first day after re-watering, the three species showed only partial recovery of both  and RWC, for saplings typically to about half the control values (Table 3). In seedlings  decreased under water stress to a lesser extent than in saplings, being larger in Cneorum than in the other two species (Table 3). RWC also decreased more in Cneorum than in Rhmanus, but in Olea RWC was unchanged after water stress. The three species also showed differences during the first day of recovery. Olea fully recovered  while RWC was kept constant. Cneorum also fully recovered  but with only partial recovery of RWC. Finally, Rhamnus did not show any recovery in either  or RWC (Table 3).

10

5

0

130

135

140

145

150

155

160

D.O.Y. Fig. 1. Net assimilation rate trend (AN ) of the considered species during the study period. Each point is the mean (±S.E.) of four replicates. In each plot the arrows indicate the day of re-watering.

3.3. Net CO2 assimilation and photosynthesis limitations In all species, AN of control plants was significantly lower in saplings than in seedlings (Fig. 1). AN was highest in

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Table 3 Leaf water potential ( ) and relative water content of the considered species corresponding to the control (c), stress (s) and recovery (r) treatment. Mean values (±S.E.) are shown. Mean values with different small letters indicate significant differences among species and age/size; mean values with different capital letters indicate significant difference among treatment (Tukey’s test, p < 0.05). Specie

Age/size

 c (MPa)

 s (MPa)

 r (MPa)

RWCc (%)

O. europaea R. alaternus C. tricoccon O. europaea R. alaternus C. tricoccon

Saplings Saplings Saplings Seedlings Seedlings Seedlings

−2.2 −2.6 −1.6 −1.3 −0.9 −0.9

−8.0 −8.1 −3.9 −2.1 −1.7 −3.0

−6.6 −4.9 −2.7 −1.2 −1.5 −1.1

73.9 69.1 72.5 75.6 83.1 89.7

± ± ± ± ± ±

0.1cC 0.1cC 0.1bC 0.1bB 0.1aB 0.1aB

± ± ± ± ± ±

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2.6aC 0.3aB 1.3aC 1.8aA 0.6bB 2.5bB

36.8 39.2 54.2 70.8 70.1 68.8

± ± ± ± ± ±

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1.5aA 2.9aA 1.2bA 1.2cA 1.3cA 3.7cA

0,3

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53.9 46.1 63.8 73.4 73.9 78.9

± ± ± ± ± ±

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D.O.Y. Fig. 2. Stomatal conductance trend (gs ) of the considered species during the study period. Each point is the mean (±S.E.) of four replicates. In each plot the arrows indicate the day of re-watering.

C. Cneorum tricoccon 130

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D.O.Y. Fig. 3. Mesophyll conductance trend (gm ) of the considered species during the study period. Each point is the mean (±S.E.) of four replicates. In each plot the arrows indicate the day of re-watering.

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Fig. 4. Electron transport rate trend (ETR) of the considered species during the study period. Each point is the mean (±S.E.) of four replicates. In each plot the arrows indicate the day of re-watering.

Fig. 5. The relationship between net assimilation rate (AN ) and the estimated chloroplast CO2 concentration (Cc ). Data for each sampling date along the experiment are included. Each point is the mean (±S.E.) of four replicates.

C. tricoccon and lowest in R. alaternus. Saplings reached the threshold of 70% AN of controls faster during water stress than seedlings. In R. alaternus, O. europaea, and C. tricoccon, these levels were achieved 2, 9 and 14 days after withholding water, respectively. In seedlings similar decreases were only achieved after 20–25 days after withholding water (Fig. 1). However, seedlings almost fully recovered AN within five days, regardless of the species. In contrast, saplings reached only 50–70% recovery in five to ten days, this being very different between species (Fig. 1). Hence, the velocity was fastest in C. tricoccon, which recovered about 50% of the control AN only two days after re-watering, while R. alaternus was slowest, which initially showed a decline of AN for five days upon re-watering, and a slow and incomplete recovery thereafter, reaching 50% of controls only after 12 days (Fig. 1).

For both sapling and seedlings of all species, stomatal conductance (gs ) followed a water stress-recovery pattern very similar to that of AN , declining during water stress and recovering to different extents after re-watering (Fig. 2). Control saplings had significantly lower gs than seedlings, and these differences were partly maintained when water stress was achieved. Mesophyll conductance to CO2 (gm ) also followed a similar pattern, particularly in saplings (Fig. 3). In seedlings, however, this pattern was not so apparent, and gm showed a tendency to decline less (on average by 62%) under water stress than in saplings (on average by 89%), being kept within a narrower range of variation than gs . While this was observed in all species, it was particularly clear in Rhamnus. The electron transport rate (ETR) was largely depressed during water stress in saplings of Olea and Rhamnus (less marked for Cneorum), and partially recovered after re-watering, while it was

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Fig. 6. Quantitative limitation of photosynthesis during water stress and recovery in all three species and for both age/size combinations. SL, MCL and BL denote for stomatal, mesophyll and biochemical limitations, respectively.

kept almost constant through the entire experiment in seedlings (Fig. 4). As a consequence of these differences between saplings and seedlings, photosynthetic parameters correlated differently among them (Table 4). While in saplings AN correlated significantly and similarly with gs , gm , ETR and the chloroplast CO2 concentration (Cc ) and all these parameters were significantly correlated to each other, in seedlings AN was much strongly correlated with gs than with any other parameter, although other correlations were also significant. Moreover, not all parameters were inter-correlated in seedlings, as gm showed no significant correlation with gs , and ETR with neither gs or Cc (Table 4). These differences are reflected in Fig. 5. While AN was strongly correlated with Cc in all species and ages except Rhamnus saplings at any given Cc , AN was generally larger in seedlings than in saplings. From the above data, photosynthetic limitations were calculated (Fig. 6). In seedlings, total limitation of AN (TL) slowly increased from 0 at the beginning of the experiment to about 70% after 20–25 days of water stress. R. alaternus presents an exception to this pattern in the early days of water stress, in which TL suddenly increased to ca. 50%, to decrease back in about 5 days and then continue its slow increase similarly to the other two species. This anomaly corresponds with decreased values of AN (Fig. 1) strongly associated with large decreases of gs (Fig. 2) which took place the week after the beginning of water stress treatments. Among TL components in seedlings, stomatal limitation (SL) accounted for at least two thirds of TL in all cases during water stress with SL increasing in parallel with TL (Fig. 6). Of the other two limitations, mesophyll conductance limitation (MCL) was the most Table 4 Pearson’s coefficients between the considered physiological variables (AN , net assimilation rate, gs , stomatal conductance, ETR, electron transport rate, gm , mesophyll conductance and CC , CO2 concentration in the chloroplast). AN Saplings AN gs ETR gm Seedlings AN gs ETR gm *

gs

ETR

gm

CC

0.95*

0.86* 0.78*

0.82* 0.70* 0.71*

0.77* 0.70 0.47* 0.66*

0.87*

0.56* 0.30n.s.

0.57* 0.23n.s. 0.48*

0.64* 0.72* 0.12n.s. 0.35*

p < 0.05, n.s.: not significant.

important, while biochemical limitation (BL) was almost negligible except in Cneorum seedlings. MCL and BL recovered very fast upon re-watering, but SL recovered somewhat slower. In saplings, even under control conditions TL was 40–60%, reflecting the fact that AN was always lower than in seedlings. Also in this case SL accounted for about two thirds of TL. However, contrary to seedlings, SL did not increase during water stress in saplings, although TL increased to values as high as 80% in Olea and Cneorum and 100% in Rhamnus (Fig. 6). This was associated with significant increases of BL and, most importantly, MCL. These two limitations recovered very slowly after re-watering, as did TL (Fig. 6). 3.4. Correlations between morphometry, water relations and photosynthesis limitations A two-way ANOVA revealed a significant effect of age/size, species, and of age/size × species interaction on most variables considered. In particular, the interaction age/size × species significantly affected LMA (F = 13.1; p = 0.002),  (F = 163.8; p = 0.0001), RWC (F = 14.5; p = 0.0003) and gm (F = 4.1; p = 0.0351). Although all species increased LMA in response to increased total leaf area (TLA) – i.e., form seedlings to saplings – the slope of this response largely differed among species (Fig. 7A). While in Rhamnus a large increase in TLA resulted only in a subtle, non-significant increase in LMA, in Cneorum a slight increase in TLA resulted in a large increase in LMA with Olea presenting an intermediate response (Fig. 7A). Increases in LMA were correlated with decreases in gm (Fig. 7B), although even here the relationship was species-dependent. While in Rhamnus a subtle increase in LMA resulted in largely reduced gm , in Olea and Cneorum the dependency was more proportional and highly significant when pooling both species (Fig. 7B). The minimum leaf water potential ( min ) achieved during water stress was also strongly, negatively and linearly correlated with TLA when pooling all species together (Fig. 7C), and leaf die-back was strongly and negatively correlated to  min , although the best fit was curvilinear in this case (Fig. 7D) as showed by the lowest AICc (AICc was −8.7 and −1.8 for non linear and linear model, respectively). As for photosynthesis limitations, leaf die-back was not related with SL (Fig. 7E) but strongly and positively (following a sigmoid function as the best fit, AICc was −33.6 and 8.8 for non linear and linear model, respectively) with NSL (Fig. 7F). Of the two components of NSL, MCL was the most important to explain the latter correlation, since it presents a sigmoid-like correlation with leaf die-back,

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Fig. 7. Relationships between some parameters analyzed. In all plots, circles represent Olea, triangles represent Rhamnus and squares represent Cneorum. (A) Leaf mass area (LMA) and total leaf area (TLA) – lines represent different relationships for the three species; (B) maximum mesophyll conductance (gm ) under control conditions and LMA – the regression shown is for Olea and Cneorum pooled together; (C) minimum leaf water potential ( min ) achieved during the experiment and TLA; (D) leaf die-back at the end of the experiment and  min ; (E) leaf die-back and stomatal limitations to photosynthesis (SL); and (F) leaf die-back and non-stomatal limitations to photosynthesis (NSL). The regressions shown in C, D and F are for all species and ages pooled.

while BL and leaf die-back were not correlated (Supplemental Fig. 1). 4. Discussion Reforestation projects in Mediterranean semi-arid or perturbed areas are often performed using seedlings grown in nurseries and later transplanted to the field (Vallejo et al., 2005). As seedlings

are sensitive to stress conditions, such as limited water availability and elevated temperature, their physiological response to stress has been proposed as a tool to assess seedling quality/potential for afforestation studies (Vilagrosa et al., 2003a, 2005; Pardos, 2005). In Mediterranean seedlings most ecophysiological studies have focused on water relations and plant hydraulics (Fernández et al., 1999; Villar-Salvador et al., 1999; Vilagrosa et al., 2010) rather than on CO2 assimilation. However, the latter is an important driver

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of plant colonization and growth. Moreover, to the best of our knowledge there are no studies comparing small and young plants (seedlings) grown in small pots – as usually coming from nurseries – with bigger and older plants (saplings) grown in somewhat bigger pots – as would be the normal condition for plants aged in nurseries. Therefore, the aim of the present study was to compare the performance of 1-year old plants (seedlings) with that of several-years-old plants (saplings) grown and pre-conditioned in a nursery. In particular, we focus on CO2 assimilation and its main determining factors. In the present study, nursery-grown plants reached their maximum photosynthetic capacity already after one year when compared to adult plants grown in the field (Gulías et al., 2003, 2009). Moreover, with regard to larger decreases of  than of RWC during water stress, it is likely that these plants have developed the capacity for osmotic adjustment, which has been shown to be characteristic of drought-acclimated plants (Villar-Salvador et al., 2004a,b). Therefore, growing conditions in the nursery were optimal for the development of fully functional, drought-acclimated seedlings/saplings. However, saplings having aged for years in the nursery presented much lower photosynthetic capacity than younger seedlings (on average 30% less AN ). They also showed a significantly higher LMA than seedlings, probably related to a thick cuticle, high mesophyll packaging or density and more cell wall compounds (Witkowski and Lamont, 1991; Niinemets et al., 2005, 2006; Fleck et al., 2010). While large LMA may be protective for plants facing water stress (Gratani and Varone, 2006), it may also be related to a more packed mesophyll cells determining a larger leaf internal CO2 resistance, i.e., a lower gm (Kogami et al., 2001; Niinemets et al., 2005, 2006; Fleck et al., 2010), thus being detrimental for photosynthesis. In fact, a negative correlation was observed between maximum gm and LMA, in agreement with previous reports in several species, including Mediterranean evergreens (Flexas et al., 2008; Fleck et al., 2010). According to photosynthesis limitation analysis, lower gm explains most of the observed lower maximum AN rates of saplings as compared to seedlings under irrigation. Moreover, plant size increased when ageing plants in the nursery, resulting in increased height and TLA. Because all plants were changed to pots of identical size (simulating what indeed happens when transplanting plants of any size to the field), a higher TLA may have led to increased water and nutrient restrictions in saplings, which may have also contributed to the observed higher LMA (Turner, 1994). Most importantly, higher TLA implies a much larger transpiring leaf area to water available ratio. Therefore, saplings may suffer larger water deficits than seedlings, particularly at midday when maximum evaporative demand occurs. Perhaps because of this, even under control conditions, saplings showed significantly lower midday  than seedlings. This difference becomes even larger when plants are subject to water stress, so that saplings reach  as low as −4 to −8 MPa, depending on the species, while seedlings remain at values of −1.7 to −3 MPa. As a consequence of lower photosynthetic capacity, and possibly of imbalanced water demand to supply resulting in much lower  , saplings presented a faster negative response to the imposition of water stress than seedlings. In fact, AN , gs and gm rapidly dropped during the early 5–10 days of water stress imposition, whereas ETR declined to a lesser extent. In contrast, AN and gs declined very slowly in seedlings (reaching values similar to that of stressed saplings only after 25 days of water stress imposition), while gm was reduced to a lesser extent, with almost no decline of ETR during the entire experiment. Consequently, under water stress photosynthesis in seedlings was mostly limited by stomatal regulation, while in saplings it was mostly dependent on non stomatal limitations. As an additional difference, recovery after rewatering was very slow in saplings, especially Olea and Rhamnus,

that needed 10–12 days to reach 50% AN of controls. In contrast, seedlings reached full recovery of AN in only 5 days. Slow recovery of AN in saplings was caused by slow recovery of gs , gm and ETR, while recovery of AN in seedlings was primarily delayed by gs , since gm was fully restored in two days after re-watering. This is confirmed by the fact that at any given Cc (i.e., scaled at the same diffusional limitations) AN is generally lower in saplings in seedlings plants. The larger MCL of saplings may be related to their higher LMA and the fact that they reach very low RWC (<55%), during stress. The capacity to avoid the damaging effects of water stress depends on the length and intensity of the stress period (Gratani and Varone, 2004b; Flexas et al., 2003, 2006; Gallé et al., 2007). According to this, seedlings having a smaller TLA may be in better position to avoid the damaging effects of drought than saplings, which present an excess TLA in respect to the development of their roots, because the length of water stress imposition is larger in the former, allowing more time for acclimation, while the intensity of stress (in terms of minimum  and RWC) is much higher in the latter. Despite these general patterns when comparing saplings and seedlings, some differences appear among species. Most notably, while C. tricoccon showed the largest differences in LMA between saplings and seedlings, it showed the smallest differences in water relations and photosynthetic responses to water stress. This is possibly due to the different morphology of C. tricoccon compared to the other two. In addition to grow shorter, it has also much lower lateral branching, which resulted in a very compact canopy, i.e., a large number of leaves per canopy volume. This may have resulted in large auto-shading, which in turn reduces the plant average evaporative demand. As mentioned earlier, improved water stress physiological tolerance of seedlings in nurseries may not necessarily imply better performance in terms of growth and survival, particularly under field conditions. In the present experiment, the water stress levels applied where moderate so that we prevented any plant mortality. However, Vilagrosa et al. (2003b) have suggested that leaf dieback (LDB) can be taken as a proxy for the capacity of survival of plants. In this sense, saplings showed much larger LDB than seedlings, and the species showing the smallest physiological differences between saplings and seedlings (C. tricoccon) also showed the smallest differences in LDB. Correlation analysis show that LDB is triggered by threshold values of  (−4 MPa) and non stomatal limitations to photosynthesis (25%). These two threshold values are probably very similar among other Mediterranean evergreen species. For instance, for Quercus ilex it has been shown that −4 MPa is the threshold value for decreasing the maximum photochemical efficiency of photosystem II (Méthy et al., 1999), and similar water potential thresholds have been shown for other Mediterranean Quercus (Epron et al., 1993; Damesin and Rambal, 1995; Méthy et al., 1999) or Cedrus species (Epron, 1997). 5. Concluding remarks Drought pre-conditioning in the nursery may result on plant acclimation to water stress, through mechanisms including osmotic adjustment, root development, increased photosynthetic capacity, etc. (Fig. 8). Those mechanisms are expected to result in better performance when seedlings are transplanted into the field in drought-prone areas. However, long-term pre-conditioning also implies that seedlings will age in the nursery. Ageing often results in increased LMA, and pot volume restrictions coupled with TLA increases may eventually result in low nutrient availability, also contributing to high LMA. This results in low gm . On the other hand, a high TLA for a constant pot volume may reflect a low root-to-shoot ratio. Under high evaporative demand and particularly under water stress, this results in large water expenses that cannot be com-

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Fig. 8. Diagram showing how plant ageing can cancel out the expected benefits of drought pre-conditioning in the nursery. On one hand (top of the diagram), pre-conditioning may result on plant acclimation to water stress, through mechanisms including osmotic adjustment, increased photosynthetic capacity, etc. Those mechanisms are expected to result in better performance in afforestation. On the other hand (bottom of the diagram), pre-conditioning also implies that plants age in the nursery, resulting in increased leaf mass area (LMA) and total leaf area (TLA), which may eventually result in low nutrient availability also contributing to high LMA. High LMA results in low mesophyll conductance to CO2 (gm ) and high TLA (reflecting a low root-to-shoot ratio) results in largely drops of leaf water potential ( ) during water stress. In consequence, in aged plants with high TLA non-stomatal limitations of photosynthesis occur, cancelling out the benefits of pre-conditioning and resulting in substantial leaf die-back and, eventually, seedling mortality, i.e., a decreased performance in afforestation.

pensated by water supply from the soil, and so  largely drops during water stress. In consequence, in aged plants with high TLA non-stomatal limitations of photosynthesis occur, cancelling out the benefits of pre-conditioning and resulting in substantial leaf die-back and, eventually, plants’ mortality, i.e., a decreased performance in afforestation. In summary, the present results suggest that to increase the success of drought pre-conditioning in nurseries, this should be restricted to short periods (e.g., a few months) to avoid plants’ ageing and excessive growth, or alternatively consider TLA as a critical factor, which implies adjusting the pot size to match plant aerial part growth during ageing. Nevertheless, because the study was performed in potted plants, these findings should be confirmed in future experiments in which seedlings and saplings are transplanted into the field. Acknowledgements This study was financed by the Spanish Ministry of Education and Research - projects BFU2005-03102/BFI “Effects of drought on photosynthesis and respiration: acclimation and recovery”, BFU2008-01072/BFI “Regulation of mesophyll conductance to CO2 in relation to plant photosynthesis and respiration”. We acknowledge two anonymous reviewers for detailed comments having contributed largely to improve this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2011.07.007. References Appleton, B.L., Whitcomb, C.E., 1983. Effects of container size and transplanting date on the growth of tree seedlings. J. Environ. Hortic. 1, 89–93. ˜ Banon, S., Ochoa, J., Franco, F.A., Alarcón, J.J., Sánchez-Blanco, M.J., 2006. Hardening of oleander seedlings by deficit irrigation and low air humidity. Environ. Exp. Bot. 56, 36–43.

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