Plant Soil (2007) 290:139–155 DOI 10.1007/s11104-006-9148-6

ORIGINAL PAPER

Water relations and stomatal characteristics of Mediterranean with different growth forms and leaf habits: responses to water stress and recovery

Jeroni Galme´s Æ Jaume Flexas Æ Robert Save´ Æ Hipo´ lito Medrano

Received: 10 July 2006 / Accepted: 18 October 2006 / Published online: 11 November 2006 Springer Science+Business Media B.V. 2006

Abstract The aim of this study was to extent the traits, such as StoD, LMA or e, provided no range of knowledge about water relations and general correlations when including all . It stomatal responses to water stress to ten Medi- is concluded that a high variability is present terranean plants with different growth forms and among Mediterranean plants reflecting a contin- leaf habits. Plants were subjected to different uum of leaf water relations and stomatal behav- levels of water stress and a treatment of recovery. iour in response to water stress. Stomatal attributes (stomatal density, StoD), stomatal conductance (gs), stomatal responsive- Keywords Drought Á Mediterranean Á ness to water stress (SR), leaf water relations Recovery Á Stomatal characteristics Á Stomatal (pre-dawn and midday leaf water potential and conductance Á Water relations Á Water stress relative water content), soil to leaf apparent hydraulic conductance (KL) and bulk modulus of elasticity (e) were determined. The observed Introduction wide range of water relations and stomatal characteristics was found to be partially depended Summer water deficit is considered the main on the growth form. Maximum gs was related to environmental constraint for growth and StoD and the stomatal area index (SAI), while gs survival in Mediterranean-type ecosystems. Un- evolution after water stress and recovery was der Mediterranean climatic conditions, the phys- highly correlated with KL. Relationships between iological regulation of water use in response to SR to water deficit and other morphological leaf soil water depletion is essential for species survival, productivity, distribution and competi- tive relationships (Joffre et al. 1999). A large J. Galme´s(&) Á J. Flexas Á H. Medrano number of studies have been performed describ- Grup de Recerca en Biologia de les Plantes en ing morphological and physiological adaptive Condicions Mediterra`nies, Universitat de les Illes responses displayed by the natural inhabiting Balears, Carretera de Valldemossa km 7.5, 07122 Palma de Mallorca, vegetation in response to water deficit (Levitt e-mail: [email protected] 1980; Chaves et al. 2003). However, many of these adaptive responses have been strictly focused on R. Save´ woody species, and particularly in sclerophyll and Departament de Tecnologia Hortı´cola, Institut de Recerca i Tecnologia Agroalimenta`ries (IRTA), semi-deciduous and winter deciduous trees Carretera de Cabrils s/n, 08348 Cabrils, Spain (Davis and Mooney 1986; Salleo and Lo Gullo

123 140 Plant Soil (2007) 290:139–155

1990; Acherar et al. 1991; Duhme and Hinckley different evergreen sclerophyll shrubs were 1992; Abril and Hanano 1998; Mediavilla and shown to present different strategies regarding Escudero 2003, 2004; Vilagrosa et al. 2003), which the regulation of leaf water relations. Similar withstand the most severe water stress conditions differences have been described between species of the Mediterranean climate. Nevertheless, in within a single genus, as in Quercus (Salleo and response to the diversity in the climatic conditions Lo Gullo 1990; Corcuera et al. 2002). Mediterranean vegetation has developed an array Stomatal closure in response to soil water of adaptations to water stress, resulting in a high shortage is a common response among plants, but diversity of life habits and growth forms. The the extent and velocity of this response may also vegetation then consists mostly of deep rooted differ among species (Schulze and Hall 1982). evergreen sclerophyll trees and shrubs, which Some general features have emerged regarding maintain green leaves during the summer period, the factors involved in the regulation of stomatal semi-deciduous shrubs, which lose a part of their conductance in species belonging to different leaves during summer, and geophytes and winter growth form groups (Schulze and Hall 1982; annual herbs, which escape seasonal water limi- Mediavilla and Escudero 2003). For instance, tation by finishing their annual cycle before stomatal size is smaller and stomatal density summer (Ehleringer and Mooney 1982). (StoD) is greater in species typical of xeric The stochastic distribution of rainfall in this environments (Dunlap and Stettler 2001; Pearce region leads to frequent episodic water deficit et al. 2006). Also, isohydric and anisohydric events in whatever season of the year. Thus, the behaviours have been discussed in terms of its capacity of withstanding a water limitation period underlying physiological mechanisms (Tardieu and the capacity for rapid recovery after rainfall and Simmonneau 1998; Schultz 2003) as well as may be adaptive to Mediterranean plants regard- regarding their adaptive value under water less of their growth forms and leaf habits, includ- stressed conditions. ing also winter annual herbs. Then, it can be Since the majority of studies about water hypothesised that the high diversity in growth relations and stomatal regulation in Mediterra- forms and leaf habits present in the Mediterra- nean plants have been focused strictly on woody nean type ecosystems may derive in a diversity species, a general analysis of the plant traits of leaf ecophysiological traits, resulting in a involved in such regulation, including Mediterra- continuum behaviour in response to water stress. nean species with broad range of life habits and Keeping cell water content within an opera- growth forms, is lacking. In addition, comparison tional range is essential for plant metabolism and among different studies within the literature is survival. The regulation of plant water relations in rather problematic, since the effects of water response to soil water depletion may be crucial to deficit on plant behaviour may rely on the specific overcome Mediterranean conditions. The varia- environmental conditions where the plants have tion of leaf water potential and/or leaf relative grown. Moreover, the interaction between water content (RWC) during the season has been water availability and stand structure, especially analysed, and important differences between when comparing plants growing in nature and species have been observed (Hinckley et al. grown in pots, adds more complexity to the 1980; Davis and Mooney 1986; Rhizopoulou and analysis. Mitrakos 1990; Abril and Hanano 1998; Salleo In present study, we test the variability present and Nardini 2000; Serrano et al. 2005). Generally, among Mediterranean plants on the water rela- drought semi-deciduous species attain lower leaf tions and stomatal characteristics, including a water potentials and contents during summer variety of species with different growth forms and than evergreen sclerophylls (Correia and Catari- leaf habits, and analysed in a controlled environ- no 1994; Werner et al. 1999). However, important ment. It is expected that, despite of well-known differences are also found among species within a common responses to water deficit, such variety single growth form. For instance, in a classical of growth forms and leaf habits will report a study by Lo Gullo and Salleo (1988), three continuum of leaf water relations and stomatal

123 Plant Soil (2007) 290:139–155 141 behaviour in response to water stress and ma subsp. marcosii) and an annual herb (Diplo- recovery. taxis ibicensis). Seeds of each species were collected in the field from natural populations and taken from several parent plants to obtain a Materials and Methods representative sample of populations in the nature. Seeds were germinated on filter paper Plant material moistened with deionised water in a controlled environment (germination chamber, at 18Cin Ten Mediterranean species naturally occurring in darkness). After germination and emergence of the Balearic Islands, some of them endemic to one true leaf, ten seedlings were transplanted into these islands, were selected for this study (Ta- pots (25 L, 40 cm) containing a 40:40:20 mixture ble 1). Special care was taken in the selection of of clay-calcareous soil, horticultural substrate the species, in order to include taxons represen- (peat) and pearlite (granulometry A13). Plants tative of different growth forms and leaf habits: were grown outdoors at the University of the two evergreen sclerophyll shrubs (Pistacia lentis- Balearic Islands (Mallorca, Spain). The experi- cus and Hypericum balearicum), two evergreen ment was performed in five rounds, each one with sclerophyll semi-shrubs (Limonium gibertii and one couple of species at the same time. The Limonium magallufianum), three summer semi- number of plants used was 10 per species, and the deciduous shrubs (Lavatera maritima, Phlomis age differed because of the different phenology of italica and albidus), two perennial herbs the species selected. Plants of P. lentiscus, H. (Beta maritima subsp. maritima and Beta mariti- balearicum, C. albidus, P. italica and L. maritima

Table 1 List of species considered for study with their growth form, family and a brief description Growth form Species Code Family Description

Herbs Diplotaxis ibicensis DI Brassicaceae Annual herb, endemic of the Balearic Islands and Pau inhabiting a few coastal locations Beta maritima L. MC Chenopodiaceae Perennial herb. Endemic of the Balearic Islands, inhabiting subsp. marcosii A. a few small islets subjected to strong saline spray Juan & M. B. Crespo Beta maritima L. MT Chenopodiaceae Perennial herb inhabiting coastal ecosystems. Widespread subsp. maritima in Mediterranean and temperate climates Semi- Lavatera maritima LA Malvaceae Semi-deciduous up to 2 m, densely covered by hairs. deciduous Gouan Inhabits in coastal locations shrubs Phlomis italica L. PI Labiatae Semi-deciduous shrub up to 1 m, densely covered by hairs. Endemic of the Balearic Islands. The biggest populations are found 500 m above the sealevel, where they co-exist with Cistus albidus Cistus albidus L. CA Semi-deciduous shrub up to 1 m. Commonly found in the Mediterranean garigue. Its leaves are densely covered by hairs Woody Hypericum HB Guttiferae Woody evergreen shrub up to 2 m, endemic of the Balearic evergreen balearicum L. Islands. The biggest populations are found in the garigue shrubs 500 m above the sea level, where competes with Pistacia lentiscus Pistacia lentiscus L. PL Anacardiaceae Woody evergreen shrub up to 5 m, commonly found in the Mediterranean garigue Woody Limonium LM Plumbaginaceae Woody evergreen semi-shrub, in cushion-like rosettes. evergreen magallufianum L. Endemic of the Balearic Islands, inhabiting just in one semi-shrubs Llorens coastal marsh located in Magalluf, Mallorca Limonium gibertii LG Plumbaginaceae Woody evergreen semi-shrub, in cushion-like rosettes. (Sennen) Sennen Occurring in West Mediterranean rocky and sandy coastal areas

123 142 Plant Soil (2007) 290:139–155

were 3 years old, plants of L. magallufianum and The RWC at pre-dawn (RWCPD) and midday L. gibertii were a year and half old and plants of (RWCMD) were determined as follows: D. ibicensis, B. maritima subsp. marcosii and RWC = (Fresh weight - Dry weight)/(Turgid B. maritima subsp. maritima were 6 months old at weight - Dry weight) · 100. To determine the the onset of the experiments. turgid weight of the samples, these were kept in Four weeks before starting the experiment, distilled water in darkness at 4C to minimise plants were placed in a controlled growth cham- respiration losses, until they reached a constant ber with a 12-h photoperiod (26C day/20C weight (full turgor, typically after 24 h). Their dry night), ambient vapour pressure deficit of 1.0– weight was obtained after 48 h at 60C in an oven. 1.5 kPa, and a photon flux density at the top of Four replicates per species and treatment were the leaves of about 600 lmol m–2 s–1. obtained from different individuals. Plants were daily fertirrigated with 50% Hoa- Pressure–volume (P–V) curves were not deter- gland’s solution (Epstein 1972). Measurements mined in this experiment. However, plotting for corresponding to control treatments were made each species the inverse of the w against RWC, during the first day of the experiment, when all including all the data collected during the exper- the plants were well watered. Thereafter, irriga- iment, was taken as a surrogate for P–V curves. tion was stopped in five plants for each species. Values from the re-watering treatment were not Pots were weighted every day to determine the considered. These plots allowed getting some amount of water available for plants with respect insights into the tissue-water relations parameters to the control. To measure at different degrees of typically evaluated using P-V curves: osmotic water stress, measurements were made on days 4, potential at full (Yp100) and at zero turgor (Yp0) 8 and 13–17 after the last irrigation, when plants and the RWC at zero turgor (RWC0) (Sobrado were subjected to mild, moderate and severe 1986; Turner 1988). Leaf volumetric elastic mod- water stress intensities, respectively. The treat- ulus (e) was estimated as the slope of the ment was stopped when gs was close to zero (for relationship between 1/w and RWC above the more details see Gas Exchange Measurements), turgor-loss point (Turner 1988). These values 13–17 days after water withholding, depending on should to be considered as ‘average’ for the species. Once achieved such gs values, pots were species during the entire experiment. Since these again irrigated at field capacity, and considered were short-term experiments (i.e. about 2 weeks), for the re-watering treatment on the next day. we considered that changes in leaf hydraulic Control plants were watered daily during all the properties, as occur along the season under field experiment and measured to ensure that they conditions (Lo Gullo and Salleo 1988; Serrano maintained constant values of each parameter et al. 2005), did not happen. For the two Limo- during the experiment. nium species, the values obtained at full satura- tion were not considered for the calculation of e, Plant water status since the leaf RWC increased from to control to mild drought treatment, suggesting the existence

Leaf pre-dawn (wPD) and midday (wMD) water of flooding stress at full irrigation (Folzer et al. potentials were determined in fully expanded 2006). leaves with a Scholander chamber (Soilmoisture Equipment Corp., Goleta, CA, USA). For three Leaf mass area of the species, C. albidus, L. maritima and H. balearicum, because their very short petiole Leaf mass area (LMA) was calculated in four difficult measuring single leaves, water potentials fully expanded leaves from different individuals were measured in small apical branches including per species under the well-watered treatment, as two or three leaves. Four replicates per species the ratio of leaf dry mass to leaf area. First, the and treatment were obtained from different leaf area was determined with an AM-100 Area individuals. Meter (Analytical Development Company, Herts,

123 Plant Soil (2007) 290:139–155 143

UK). Then, the dry mass of these leaves was (1994), and expressed in mm stomata · number determined after oven drying for 48 h at 60C. of stomata mm–2 leaf.

Gas exchange measurements Statistical analysis

Instantaneous determinations of stomatal con- Regressions coefficients were calculated with the ductance (gs) and transpiration rate (E) at satu- 8.0 Sigma Plot software package (SPSS). Differ- rating light (1,500 lmol photon m–2 s–1), 25C ences between means were revealed by Duncan –1 and 400 lmol mol CO2 were performed at analyses (P < 0.05) performed with the SPSS 12.0 mid-morning, using a Li-6400 (Li-Cor Inc., Lin- software package (SPSS, Chicago, IL, USA). The coln, NE, USA) in one fully expanded leaf of four step-wise method was used to calculate the SR, as different plants per treatment and species. Rela- the slope between gs and wPD. tive humidity was kept at 50 ± 5% during mea- surements.

For each one of the species, gs was plotted Results and Discussion against wPD, resulting in a non-linear biphasic relationship with steeper decreases in gs at high Water relations in response to water stress wPD (Mediavilla and Escudero 2004). The initial slope of such relationship (i.e. at high wPD) was Among Mediterranean plants, the response of calculated as an indicator of stomatal responsive- leaf water relations and stomatal conductance to ness to water stress (SR). These slopes were water stress has been studied in woody species, obtained using the step-wise method, adding mostly in sclerophyll and semi-deciduous shrubs points down the slope until the regression coef- (Hinckley et al. 1980; Davis and Mooney 1986; ficient was maximized. Rhizopoulou and Mitrakos 1990; Abril and Ha- Soil to leaf apparent hydraulic conductance nano 1998; Serrano et al. 2005). In the present (KL) was estimated from the slope of the study, we include an annual and two perennial relationship between leaf transpiration rate (E, herbs, three summer semi-deciduous shrubs and –2 –1 mmol H2Om s ) and w, and was calculated as four evergreen sclerophylls, in order to compare -E/(wMD - wPD) (Sperry and Pockman 1993). water relations and their response to water deficit in Mediterranean species with a broader range of Stomatal density and size growth forms and life habits. The different species did not reach the same Fully exposed mature leaves were detached from water stress level in terms of soil water content each plant. StoD was determined using the silicon (SWC) at the end of the drought period (Fig. 1), leaf impression method (Weyers and Johansen which was probably due to differences in transpi- 1985) on the abaxial lamina immediately to the ration rate and/or leaf area among species. right of the mid-vein. All the species analysed However, all species presented similar stomatal were hypostomatic. The numbers of stomata were conductance values, i.e. close to zero, the last day counted with a microscope at 400 · magnification of measurements. Therefore, all species suffered on four different vision fields of separate impres- from gradual to severe water stress under similar sions of the lamina obtained from four different environmental conditions. leaves of four well-watered plants per species (i.e. Maximum pre-dawn leaf water potential ran- 16 different vision fields). Guard cell length was ged between –0.30 and –0.55 MPa for all the measured on 16 randomly selected stomata from species analysed, but there was a much larger the same impressions used for StoD determina- variation under water stress, the lowest values tions. Stomatal area index (SAI) was calculated ranging from –1 MPa in D. ibicensis to –5 MPa in by taking the product of the mean stomatal length P. lentiscus (Fig. 1). A significant correlation and the StoD according to Ashton and Berlyn between SWC and wPD was found only for wPD

123 od vrre shrubs, evergreen woody shrubs, deciduous herbs, abbreviations: form Growth replicates. four of error standard ± means represent Values species. selected the ten for values) control to ( content water soil and potential symbols semi-shrubs WESS idy( midday symbols filled ewe r-an( pre-dawn between 1 Fig. 144 123 SDS od evergreen woody Relationship efwater leaf ) w SWC MD semi- , and ) respect % , empty WES w HE PD ,

Ψ Ψ Ψ Ψ Ψ PD (MPa) PD (MPa) PD (MPa) PD (MPa) PD (MPa) -5 -4 -3 -2 -1 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 WES H. balearicum WESS L. magallufianum HE maritima B. SDS P. italica HE D. ibicensis 100 SWC (% of control) 80 subsp. subsp. 60 marcosii 40 20 HE maritima B. WES P. lentiscus SDS maritima L. WESS L. gibertii SDS C. albidus 100 SWC (% of control) 80 subsp. 60 ln ol(07 290:139–155 (2007) Soil Plant maritima 40 20 -5 -4 -3 -2 -1 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5

Ψ Ψ Ψ Ψ Ψ ΜD (MPa) ΜD (MPa) ΜD (MPa) ΜD (MPa) ΜD (MPa) Plant Soil (2007) 290:139–155 145 values above –1.5 MPa, i.e. from mild to moder- 2003). However, comparing the physiological ate water stress (R2 = 0.55, P < 0.01). Further responses of plants belonging to different func- decreases of wPD during severe stress were tional groups under field conditions is confusing, present for all species, but associated to small since plants can be exploring different soil depths. decreases in the SWC, and may therefore reflect The present results, obtained in plants under differences between species in their ability to identical conditions and exploring a soil volume explore all soil volume in the pots or to adjust at least more similar than wild vegetation, suggest their osmotic potential to continue extracting that the reported differences between functional available water from the soil. groups in minimum leaf water potential under Clearly, the minimum water potential achieved field conditions are not species constitutive. during water stress did not depend on specific Although we did not perform true P–V curves growth forms or life habits. For instance, the in the present study, we plotted all the data of 1/w highest wPD under severe stress was found in the and RWC collected during the experiment as a annual herb D. ibicensis as well as in the two surrogate of P–V curves (Fig. 2). From these evergreen semi-shrubs L. gibertii and L. magal- plots, a series of water relation parameters, such lufianum. Lower values (i.e. below –3.5 MPa) as osmotic potential at full and zero turgor, the were found in the perennial herb B. maritima RWC at turgor loss, and the bulk elastic modulus subsp. maritima, in the three summer semi- (e), were calculated (Table 2). These should be deciduous shrubs and in the evergreen sclerophyll considered as ‘average’ for the species during the P. lentiscus. Previous studies have shown that, entire experiment. Since these were short-term under field conditions during the dry season, experiments (i.e. about 2 weeks), we assume that summer deciduous and semi-deciduous shrubs changes in e, as occur along the season under field usually attain lower wPD than evergreens (Duhme conditions (Lo Gullo and Salleo 1988; Serrano and Hinckley 1992; Correia and Catarino 1994; et al. 2005), should be irrelevant. From these Werner et al. 1999; Mediavilla and Escudero relationships, it is clear that all the species

Fig. 2 Relationship 3.5 3.5 between the reciprocals of D. ibicensis 3.0 L. maritima 3.0 leaf water potential (1/w) B. maritima ssp. marcosii P. italica B. maritima ssp. maritima C. albidus and relative water content 2.5 2.5 RWC ) ( ). These are not ) 1 1 - - 2.0 2.0 a true P–V curves, but a P surrogates obtained P M M ( ( 1.5 1.5 including data both from Ψ Ψ / / 1 predawn and midday for 1.0 1.0 1 - - all treatments except recovery. Values are 0.5 0.5 means ± standard errors 0.0 0.0 of four replicates. Growth HE SDS form abbreviations: HE 3.5 3.5 70 60 50 40 herbs, SDS semi- H. balearicum L. magallufianum 3.0 P. lentiscus 3.0 deciduous shrubs, WES L. gibertii woody evergreen shrubs, 2.5 2.5 ) ) 1 WESS woody evergreen 1 - - 2.0 2.0 a semi-shrubs a P P M M ( ( 1.5 1.5

Ψ Ψ / / 1 1.0 1.0 1 - -

0.5 0.5

0.0 0.0 WES WESS

90 80 70 60 50 90 80 70 60 50 40 RWC (%) RWC (%)

123 146 Plant Soil (2007) 290:139–155

Table 2 Parameters derived from water potential versus relative water content plots for the ten species subjected to study, reflecting the relationship between the reciprocals of the leaf water potential and the relative water content (Fig. 2)

Growth form Species wp100 (MPa) wp0 (MPa) RWC0 (%) e (MPa)

HE D. ibicensis –0.91 ± 0.21 –1.05 ± 0.31 71.5 ± 4.1 2.28 ± 0.32 B. maritima subsp. marcosii –1.07 ± 0.13 –2.00 ± 0.08 61.9 ± 1.4 1.74 ± 0.24 B. maritima subsp. maritima –1.06 ± 0.12 –1.61 ± 0.06 68.3 ± 3.1 2.28 ± 0.35 SDS L. maritima –1.58 ± 0.25 –2.39 ± 0.18 66.1 ± 2.1 3.08 ± 0.34 P. italica –1.52 ± 0.24 –1.76 ± 0.25 72.9 ± 2.6 4.08 ± 1.07 C. albidus –1.71 ± 0.12 –2.20 ± 0.14 71.7 ± 1.6 4.32 ± 1.08 WES H. balearicum –1.08 ± 0.10 –1.21 ± 0.11 85.6 ± 0.7 6.40 ± 1.28 P. lentiscus –2.39 ± 0.25 –2.75 ± 0.20 83.6 ± 2.1 12.16 ± 2.17 WESS L. gibertii n.d. n.d. <61.4 ± 0.1 1.38 ± 0.11 L. magallufianum n.d. n.d. <66.5 ± 3.8 0.98 ± 0.13

Osmotic potential at full turgor (wp100), at zero turgor (wp0), relative water content at zero turgor (RWC0) and leaf bulk elastic modulus (e). Data obtained both from predawn and midday measurements were considered, excepting values from re-watering treatment. Values are means ± standard errors of four different plants per species Growth form abbreviations: HE herbs, SDS semi-deciduous shrubs, WES woody evergreen shrubs, WESS woody evergreen semi-shrubs, n.d. not determined

except the two Limonium reached turgor loss R2 = 0.750, P < 0.01 14 point during the experiment, most of them under ) a

P PL severe water deficit. These species may be M 12 ( s u considered as drought-tolerant, since they all l u 10 d survived and recovered leaf water potential to o m

c 8 i some extent after re-watering. In contrast, the two t s HB a l

Limonium may be considered as drought-avoi- e 6

dant species, since they did not show the typical bulk CA

f 4

a PI inflexion in the 1/w versus RWC curve, and e MT DI LA L 2 LM therefore they overcome the experimental condi- MC LG tions above the turgor loss point. Clearly, for a 40 60 80 100 120 140 160 180 200 given soil water depletion level Limonium species LMA (g m-2) were able to keep leaf water potential and RWC at higher values than the other species. Fig. 3 Relationship between leaf bulk elastic modulus (e) and leaf mass area (LMA) for the species analysed. Values Values for the leaf elastic modulus (e) were are means ± standard errors of four replicates. Species obtained, being of around 1 MPa in evergreen codes as in Table 1. Growth form symbols: d herbs, n semi- semi-shrubs, 2 MPa in herbs, 3–4 MPa in semi- deciduous shrubs, h woody evergreen shrubs, s woody deciduous shrubs, and higher than 6 MPa in evergreen semi-shrubs evergreen shrubs (Table 2). The RWC at the turgor loss point (RWC0) ranged from a low value Groom and Lamont 1997; Salleo et al. 1997). of 62% for B. maritima subsp. marcosii to a high Therefore, it is clear that e-values followed a value of 86% for H. balearicum (Table 2). These pattern that was to some extent dependent on data are consistent with values already published, growth forms and life habits, particularly on the particularly for evergreen Mediterranean species degree of sclerophylly. Previous works (Robich- (Lo Gullo and Salleo 1988; Save´ et al. 1999; aux et al. 1986) showed that differences in tissue Serrano et al. 2005). Differences between species elasticity can be attributable to cell wall compo- in osmotic potential at full turgor and turgor loss sition, mainly in the ratio pectin (including point were less important than differences in e hemicellulose)/cellulose. This phenomenon has (Table 2). Bulk modulus of elasticity was closely been related to sclerophylly (also aging) and must correlated (P < 0.01) with LMA (Fig. 3), as be attributable to resistance mechanisms against already described (Salleo and Lo Gullo 1990; environmental stresses. It is remarkable that the

123 Plant Soil (2007) 290:139–155 147

Table 3 Maximum (irrigated plants), minimum (severe drought conditions) and range of variation of stomatal conductance (gs) for the ten species analysed –2 –1 Growth form Species gs (mol H2Om s )

Maximum Minimum Range

HE D. ibicensis 0.510 ± 0.035 0.059 ± 0.012 0.451 B. maritima subsp. marcosii 0.450 ± 0.017 0.009 ± 0.040 0.442 B. maritima subsp. maritima 0.704 ± 0.087 0.008 ± 0.002 0.696 SDS L. maritima 1.022 ± 0.076 0.052 ± 0.010 0.970 P. italica 0.357 ± 0.041 0.016 ± 0.001 0.341 C. albidus 0.318 ± 0.037 0.022 ± 0.004 0.296 WES H. balearicum 0.330 ± 0.025 0.023 ± 0.004 0.307 P. lentiscus 0.122 ± 0.020 0.014 ± 0.002 0.109 WESS L. magallufianum 0.246 ± 0.016 0.017 ± 0.005 0.229 L. gibertii 0.187 ± 0.021 0.029 ± 0.007 0.158 Values are means ± standard error of four replicates Growth form abbreviations: HE herbs, SDS semi-deciduous shrubs, WES woody evergreen shrubs, WESS woody evergreen semi-shrubs two Limonium species did not follow the general P. lentiscus. The latter was the only of the ten LMA-e relationship found for the others. That species analysed for which previous determina- not all Mediterranean species fit this general tions of StoD were available, differing between relationship has been already pointed out by 287 (Meister and Bolha`r-Nordenkampf 2001) and Salleo and Nardini (2000). A similar conclusion 325 stomata mm–2 (Gratani and Varone 2004). was reached by Zobel (1996) for temperate trees The present results show that there is a high intra- of the Appalachian Mountains. specific variability in StoD. A highly significant negative relationship was found between stomatal Stomatal traits and stomatal conductance length and StoD (Fig. 4b), as previously described responsiveness to water stress (Larcher 1995; Hetherington and Woodward 2003; Gratani and Varone 2004; Pearce et al. Under well-watered conditions, stomatal conduc- 2006). The maximum stomatal conductance tance (gs) strongly differed among species and (gsmax) was positively correlated to StoD in a growth forms, approximately in a ten-fold range significant relationship (P < 0.05), but only at low –2 (Table 3). L. maritima showed the highest gs StoD values, i.e. up to 350 stomata mm –2 –1 values (1.022 mol H2Om s ) and P. lentiscus (Fig. 4c), as already shown for Mediterranean –2 –1 the lowest (0.122 mol H2Om s ). The maxi- (Gratani and Varone 2004) and non-Mediterra- mum gs were significantly higher for herbaceous nean species (Pearce et al. 2006). However, this and semi-deciduous shrubs than for evergreens. commonly accepted relationship was not followed However, H. balearicum presented similar values by P. lentiscus and L. maritima, both species with to the semi-deciduous P. italica and C. albidus. the highest StoD values, which behaved as out-

The relationship between the maximum gs and liers (i.e. values much higher or lower than the degree of sclerophylly, approached as the expected) for this relationship. This introduce LMA, was not significant (Fig. 4a). Although controversy in the current literature, showing that non-significant, the negative trend of such rela- higher StoD permits a luxury water consumption tionship is consistent with the typically described and, in other species an extraordinary water water-saving behaviour of Mediterranean ever- saving behaviour. As a result of the significant green sclerophylls (Ehleringer and Mooney relationship between stomatal length and StoD,

1982). the relationship between the SAI and gsmax was Stomatal density, ranged from 60 stomata mm–2 also significant (Fig. 4d). Hence, species with in L. magallufianum to 420 stomata mm–2 in higher proportion of the leaf surface being

123 148 Plant Soil (2007) 290:139–155

2 a R = 0.256, n.s. R2 = 0.773, P < 0.01 b LG 1.0 LA 40 ) ) 1 - s

LM m 35 2 µ - 0.8 (

m

MT th g O 30 2

PI n e H

0.6 LA l

l l o DI 25 a CA t m a ( DI

x 0.4 MC m a PI

HB o m t

20 s PL S g LM CA HB MC 0.2 PL 15 LG MT

50 100 150 200 100 200 300 400 LMA (g m-2) StoD (stomata mm-2)

c R2 = 0.558, P < 0.05 d R2 = 0.593, P < 0.05 LA LA ) 1.0 1.0 1 - ) s 1

- 2 - s

2 m -

0.8 0.8 m O

MT 2 MT O H 2

l H

o 0.6 0.6

DI l m o

( DI

MC m x ( a MC

x m

0.4 0.4

CA a s m g

LM PI PI s

HB HB g CA LM 0.2 LG PL LG PL 0.2

100 200 300 400 246810 StoD (stomata mm-2) SAI

Fig. 4 a Relationship between the maximum stomatal replicates for the stomatal characters. Regression coeffi- conductance (gsmax) and leaf mass area (LMA). b cients and significance of each relationship are shown Relationship between the stomatal length and the stomatal in the correspondent figure. The regression coefficient of density (StoD). c Relationship between the maximal Fig. 4c was obtained from values of StoD up to –2 stomatal conductance (gsmax) and the StoD. d Relationship 350 stomata mm . Species codes as in Table 1. Growth between the maximum stomatal conductance (gsmax) and form symbols: d herbs, n semi-deciduous shrubs, h woody the stomatal area index (SAI). Values represent evergreen shrubs, s woody evergreen semi-shrubs, n.s. means ± standard errors of four replicates for gs and 16 non-significant stomata presented a higher capacity to transpire which occurred in semi-deciduous and woody water through leaves. evergreen shrubs, were not related to gs, since Soil to leaf apparent hydraulic conductance there was no correlation between maximum gs –2 –1 (KL) ranged between 6 and 27 mol m s MPa and maximum KL in these species (not shown). in irrigated plants, the two extremes correspond- Although transpiration rates could not be esti- ing to the evergreen sclerophylls L. magallufia- mated by an independent technique, and there- num and H. balearicum, respectively. KL declined fore gs and KL are not completely independent during water deficit in all the species, and up to a variables, Fig. 5 suggests that these two parame- –2 –1 –1 KL of 8–10 mmol m s MPa the relationship ters are also physiologically co-regulated. with gs was linear for all the species (Fig. 5), as During water stress, gs decreased proportion- described by Schultz (2003) in grapevines. The ally to SWC and wPD (not shown) in all the species present results support that gs variations during analysed. The relationship between gs and wPD water stress are highly determined by hydraulic was biphasic, and the slope of the initial phase was conductance in Mediterranean species, as already taken as an indicator of stomatal responsiveness suggested (Salleo et al. 2000; Serrano and Pen˜ u- (SR) to water stress (Acherar et al. 1991; Media- elas 2005). However, further increases in KL, villa and Escudero 2003). A large variability was

123 Plant Soil (2007) 290:139–155 149

Fig. 5 Relationship HE SDS between the stomatal 120 120 ) ) l

conductance (g ,in l s 100 100 o o r r t percentage respect to t n n o control values) and soil to o c c 80 80

o o t leaf apparent hydraulic t

t t c conductance (KL) along c 60 60 e e p p s the drought experiment s e e r r 40 40 for the ten selected % % ( ( species. Values of g

s s s D. ibicensis L. maritima 20 20 g g represent B. maritima ssp. marcosii P. italica means ± standard errors B. maritima ssp. maritima C. albidus 0 0 of four replicates. Growth form abbreviations: HE 120 WES WESS 120 herbs, SDS semi- ) ) l deciduous shrubs, WES l 100 100 o o r r t t n woody evergreen shrubs, n o o c c 80 80

WESS woody evergreen o o t t

semi-shrubs t t c c 60 60 e e p p s s e e r r 40 40

% % ( (

s s

20 g

g 20 H. balearicum L. magallufianum P. lentiscus L. gibertii 0 0

0102030 0102030 -1 -2 -1 -1 -2 -1 KL (mmol H2O MPa m s ) KL (mmol H2O MPa m s ) found in SR, which was not related to growth species, while C. albidus showed one of the lowest forms or leaf habits (Table 4). For instance, both (0.128) and P. italica displayed an intermediate the maximum (1.351 and 1.229 in L. magallufia- value (0.176). Herbaceous species presented con- num and L. gibertii, respectively) and the mini- sistently high values of SR and on average their mum (0.035, P. lentiscus) values were found in SR did not differ from that of woody species due evergreen sclerophyll species (Table 4). Among to the large variability of the latter. semi-deciduous species, L. maritima presented The above results do not support the idea that one of the highest values (0.684) found for all the Mediterranean evergreen sclerophylls have a

Table 4 Stomatal responsiveness to water stress (SR), calculated as the slope of the initial phase of gs declining versus wPD for the ten selected species Growth form Species SR R2 P

HE D. ibicensis 0.582 ± 0.073 0.851 <0.001 B. maritima subsp. marcosii 0.538 ± 0.127 0.562 <0.001 B. maritima subsp. maritima 0.781 ± 0.212 0.531 <0.005 SDS L. maritima 0.684 ± 0.084 0.858 <0.001 P. italica 0.176 ± 0.022 0.878 <0.001 C. albidus 0.128 ± 0.026 0.626 <0.001 WES H. balearicum 0.637 ± 0.082 0.883 <0.001 P. lentiscus 0.035 ± 0.005 0.748 <0.001 WESS L. gibertii 1.229 ± 0.349 0.488 <0.05 L. magallufianum 1.351 ± 0.223 0.501 <0.05 To calculate the SR step-wise method was used, adding points down the slope until the regression coefficient was maximized. Values represent means ± standard errors Growth form abbreviations: HE herbs, SDS semi-deciduous shrubs, WES woody evergreen shrubs, WESS woody evergreen semi-shrubs

123 150 Plant Soil (2007) 290:139–155

b a Fig. 6 Relationship between the stomatal responsiveness to water stress (SR) and a leaf mass area (LMA), b leaf e StoD WESS bulk elastic modulus ( ), and c stomatal density ( ). 1.5 Values represent means ± standard errors. Species codes LM as in Table 1. Growth form symbols and abbreviations: d and HE herbs, n and SDS semi-deciduous shrubs, h and LG s 1.0 WES woody evergreen shrubs, and WESS woody evergreen semi-shrubs

R MT S

0.5 LA HB MC DI higher SR than malacophyll species or annuals SDS (Gratani and Varone 2004), but rather that a high HE WES PI variability is present among Mediterranean plants 0.0 CA PL reflecting a continuum of stomatal behaviour in response to water stress that is independent of the growth form and leaf habit (Joffre et al. 1999). In 40 60 80 100 120 140 160 180 fact, a lower SR in evergreen oaks as compared to -2 LMA (g m ) winter deciduous and malacophyll species has already been reported (Acherar et al. 1991; b Mediavilla and Escudero 2003, 2004). WESS Figure 6 shows the relationship between SR 1.5 and three morphological characters of the species: LM LG LMA, e and StoD. Contrarily to what expected no

1.0 general relationships were observed among these parameters. While this result suggests a high

R MT LA S HE HB diversity in the morphological traits governing gs 0.5 MC among Mediterranean species, further studies DI SDS WES including more species are required to better CA discern the existence of leaf morphological char- 0.0 PI PL acters influencing the capacity to regulate water lose through stomata. Nevertheless, interesting differences were observed when comparing 2 4 6 8 10 12 14 growth forms. For instance, on average, herba- ε (MPa) ceous species, with among the lowest values for LMA and e, and among the highest for StoD, c showed an improved SR. Limonium species presented a higher SR than the other species for 1.5 a given value of LMA, e and StoD. The fact that LM LG the two Limonium species had the highest SR is WESS HE 1.0 consistent with and explains that they were the only among the ten species analysed showing an

R MT S LA isohydric behaviour (Fig. 1). In previous surveys DI 0.5 HB MC including the same pool of species, the two SDS Limonium already showed some other ecophys- CA WES PI iological characteristics that make them different 0.0 PL from the other species. For instance, they dis- played the highest Rubisco specificity factor

among higher C3 plants, which may allow them 0 100 200 300 400 500 to sustain a somewhat higher photosynthesis with StoD (stomata mm-2) their stomata almost totally closed (Galme´s et al.

123 Plant Soil (2007) 290:139–155 151

Fig. 7 Relationship between the percentage of recoveryc 2 a R = 0.342, n.s. of the stomatal conductance (gs) and a the percentage of recovery of midday leaf water potential (w ), b the leaf MD 100 bulk elastic modulus (e), and c the percentage of recovery MC of the soil to leaf apparent hydraulic conductance (KL). Values represent means ± standard errors. The regression 80 s coefficients and significance of each relationship are g LA y shown. The regression coefficient of Fig. 7b was obtained r e MT v 60 DI excluding both Limonium species (empty circles). Species o c codes as in Table 1. Growth form symbols: d herbs, n semi- e r h s

deciduous shrubs, woody evergreen shrubs, woody % evergreen semi-shrubs, n.s. non-significant 40 LM PI CA LG 20 PL

HB

2005a). Similarly, Limonium spp. were the only in 20 40 60 80 Ψ which water stress-induced decreased relative % recovery MD growth rate was strongly associated to decreased net assimilation rate, and not to morphological 2 b R = 0.824, P < 0.01 adjustments as in the other species (Galme´s et al.

2005b). It would be interesting to perform further 100 MC studies in these species to understand how differ- ent ecophysiological traits combine to provide 80 s g specific adaptations to adverse environments.

y LA r e

It was also remarkable the existence of a v 60 MT o c

negative trend between SR and e (Fig. 6b), i.e. the e DI r

higher the e the lower the SR. These data % 40 contradicts the general assumption that, because LM PI stomatal closure is activated by the loss of cellular LG CA turgor, the greater the e the quicker the stomatal 20 PL closure (Corcuera et al. 2002). Similarly, contrary HB to what is usually assumed (Larcher 1995), a 2 4 6 8 10 12 14 higher StoD did not result in a higher stomatal ε (MPa) control for all the species. While a positive relationship is observed for seven of the ten species, Limonium and P. lentiscus where clearly c outliers (Fig. 6c). To the best of our knowledge 100 this is the first time that a direct relationship MC among different species is described between 80

StoD and a parameter reflecting SR to water s g

LA y stress, and interestingly there are important r 60 MT e

v DI exceptions showing that the assumed relationship o c e r

is not general. The apparent contradictions 40 % LM between some of the results of the present work PI and previous studies could rise from differences in LG CA 20 the environment where the plants have grown and PL HB in the velocity of water deficit imposition. While 0 2 most of the studies drawn from literature have R = 0.737, P < 0.01 been performed with plants growing in the field, 020406080 in the present survey plants were maintained in a % recovery KL

123 152 Plant Soil (2007) 290:139–155 growing chamber, which could induce some both parameters are not independent, but clearly changes in the stomatal behaviour. co-regulated. The regulation of KL depends on cavitation and recovery of xylem vessels, from Recovery of leaf water relations and stomatal which leaf veins seem the most sensitive (Cochard conductance after re-watering et al. 2002; Brodribb and Holbrook 2003). According to the ratio leaf water potential/turgor After severe water deficit stress, plants were re- loss point water potential, showed in Lo Gullo watered at field capacity, and water relations and and Salleo (1993), xylem embolism could happen gs were determined after 24 h to assess recovery. in evergreen oaks when such ratio was close to wPD recovered to values ranging from 11 (P. 0.9. This occurred at low water stress level and the lentiscus) to 78% (B. maritima subsp. maritima) losses in hydraulic conductivity were about 30%, of the initial (data not shown). The extent of wMD which were easily recovered with moderate water recovery ranged from 20 to 82%, the extremes supply. When the stress level increased the losses corresponding to the same species as for wMD in hydraulic conductivity could raise 85% of (Fig. 7a). No clear pattern of water relations initial values, and the recuperation percentage recovery was observed among growth forms and felt dramatically (Lo Gullo and Salleo 1993). If leaf habits. For instance, maximum recovery (70– we take in consideration this approach, in present 80%) was achieved by the two perennial herbs study, the ratio midday leaf water potential/turgor (Beta), a semi-deciduous shrub (L. maritima) and loss point water potential was only lower than 0.9 two evergreens (Limonium). under severe water stress, suggesting lower pos- The range of stomatal conductance recovery sibilities of embolism problems. However, for was similar to that of leaf water potential some species this ratio raised values between 1.50 (Fig. 7a), from 13 (H. balearicum) to 93% (B. and 2.50, which correlated with a lower capacity maritima subsp. marcosii). In this case, a certain of recovery of hydraulic conductivity after re- effect of growth form and leaf habit was observed, watering. The mechanisms leading to KL recovery with herbs showing the highest recovery, the after cavitation are not fully understood and semi-deciduous showing an intermediate recov- constitute an active area of research. Recent ery, and evergreens showing the lowest recovery. reports suggest the involvement of aquaporins in

A similar trend for recovery was proposed by KL regulation (Morillon and Chrispeels 2001; Gratani and Varone (2004) for sclerophyll versus Nardini et al. 2005). A better knowledge about malacophyll shrubs. However, this trend cannot these mechanisms may deserve better attention, be generalised since, as shown in Fig. 7a, the and the present results suggest that it would be semi-deciduous L. maritima aligns with the herbs crucial for the understanding of stomatal regula- and the two evergreens Limonium align with the tion in response to water stress in Mediterranean semi-deciduous shrubs. plants. A significant, negative relationship between Figure 8 shows a cluster analysis of the the extent of gs recovery and e was found when species considered in the present survey. Such considering the anisohydric species only, but analysis, which included nine physiological and again the two Limonium did not follow the same morphological parameters, reflects the existence trend (Fig. 7b). The negative relationship found of differences among growth form groups, but between gs recovery and e contradicts the idea also shows the presence of similar behaviours that low cell-wall elasticity would allow a rapid between groups, especially between semi-decid- recovery after stress (Corcuera et al. 2002). uous and herbaceous species. The two woody

As occurred with gs to increasing water stress, evergreen groups represented to two extremes of the extent of gs recovery showed a general, highly the range. While woody evergreen semi-shrubs significant relationship with the extent of KL were more related to semi-deciduous shrubs, the recovery (Fig. 7c). However, because KL is cal- observed traits for woody evergreen shrubs were culated on the basis of transpiration, as is in fact found to be more similar to those of herbaceous gs, the high correspondence could only reflect that species.

123 Plant Soil (2007) 290:139–155 153

UIB. This work was partly funded by Projects REN2001- 3506-CO2-O2 and BFU2005-03102/BFI (Plan Nacional, Spain).

References

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