Abscisic acid and drought response of Canarian laurel forest tree species growing under controlled conditions

Abscisic acid and drought response of Canarian laurel forest tree species growing under controlled conditions

Environmental and Experimental Botany 64 (2008) 155–161 Contents lists available at ScienceDirect Environmental and Experimental Botany journal home...

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Environmental and Experimental Botany 64 (2008) 155–161

Contents lists available at ScienceDirect

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

Abscisic acid and drought response of Canarian laurel forest tree species growing under controlled conditions ´ Manuel Sanchez-D´ ıaz, Carolina Tapia, M. Carmen Antol´ın ∗ Departamento de Biolog´ıa Vegetal, Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea s/n, 31008 Pamplona, Spain

a r t i c l e

i n f o

Article history: Received 8 August 2007 Received in revised form 5 May 2008 Accepted 20 May 2008 Keywords: Leaf conductance Osmotic adjustment Photosynthesis Proline accumulation

a b s t r a c t We have studied interactions between leaf abscisic acid (ABA) and the response of gas exchange and solute synthesis in the three major species of the Canarian laurel forest (Laurus azorica (Seub.) Franco, Persea indica (L.) K. Spreng and Myrica faya Aiton). Trees were subjected to drought under controlled conditions by withholding water until leaf relative water content (RWC) reached 50–55%. Drought treatment reduced predawn leaf  together with  s in all the studied species. At moderate drought (RWC ca. 80%) L. azorica showed higher net assimilation rate than the other species. In addition, this species showed significant accumulation of ABA, total soluble sugars (TSS) and proline in leaves, contributing to lowered s100 that might result in maintenance of leaf turgor. By contrast, M. faya showed the lowest  that was not accompanied by changes in s100 , neither ABA concentration nor in leaf solutes. P. indica performed similarly to M. faya but it had the lowest rates of gas exchange indicating that this species was the most sensitive to drought treatment. All results clearly indicate that L. azorica exhibited higher capacity for drought acclimation than the other laurel species. This response seems to be mediated by ABA synthesis because significant relationships between leaf ABA, s100 and solute accumulation were found. These data provide new information about the role of ABA in the physiological responses of laurel tree species to drought. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The laurel forest is an evergreen forest relic of the Tertiary Mediterranean flora that occupied Southern Europe and Northern Africa about 20 million years ago and which today exists in the Canary Islands (Santos, 1990). This forest evolved in a humid mediterranean climate with moderate temperatures and very high air humidity. The Canarian laurel forest belt is altitudinally closely associated with the zone of orographic cloud formation, hence these ecosystems are also known as cloud forest. Dry season cloud cover is of vital importance to this forest because it creates a semihumid environment that allows the ecosystem to persist in what ¨ would otherwise be a semi-arid climate (Hollermann, 1981). The natural mixed hardwood forest is dominated by evergreen species, which are composed of about 20 tree species belonging to different families but the three major tree species present are: Laurus azorica (Seub.) Franco, Persea indica (L.) K. Spreng and Myrica faya Aiton. Early ecophysiological studies about this forest indicate that such species cannot endure strong environmental

∗ Corresponding author. Tel.: +34 948425600; fax: +34 948425649. E-mail address: [email protected] (M.C. Antol´ın). 0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2008.05.011

¨ stress (Losch, 1993). This may be one reason why the laurel forest is restricted to perhumid sites. Several years ago numerous studies concerning laurel trees have been performed in situ under ´ natural conditions (Jimenez et al., 1996; Morales et al., 1997). These authors have shown that laurel trees exhibited gas exchange characteristics that indicate a non-conservative use of water. This strategy could be appropriate for a species subjected to droughts ´ that are mild or of short duration (Gonzalez-Rodr´ ıguez et al., 2001, 2002a,b). The physiological mechanisms which allow a species to acclimate to prolonged periods of drought can involve numerous attributes. One means of increasing drought acclimation capacity is by the accumulation of osmotically active solutes (osmotic adjustment). These solutes include sugars, glycerol, amino acids such as proline, sugar alcohols like mannitol, and other low molecular weight metabolites (Morgan, 1984). Abscisic acid (ABA) is thought to play an important role in the acclimation of plants to environmental stress. In addition to its well established role in closing stomata (Assmann, 2003; Hartung et al., 2005), ABA has a role in regulating solute accumulation (LaRosa et al., 1987; Trewavas and Jones, 1991). There is evidence that ABA is required for proline accumulation at low leaf water potential (Ober and Sharp, 1994). At molecular level, ABA is involved in numerous changes in gene

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expression (Bray, 2003; Shinozaki et al., 2003). For instance, ABA accumulation during osmotic stress has been determined to regulate expression of a gene involved in proline biosynthesis (Xiong et al., 2001). Although much is known about physiological responses to water limitation in woody plants (reviewed by Kozlowski and Pallardy, 2002; Larcher, 2003), drought studies on the physiology of Canarian laurel trees have been scarce. Therefore, our goal was to investigate the role of ABA on the response of gas exchange and solute synthesis on three Canarian laurel species when affected by increasing drought stress. Specifically, we sought to identify the extent of involvement of ABA on the response of gas exchange and osmotic adjustment capacity in drought-treated trees.

ing leaf samples at 85 ◦ C to constant mass. Specific leaf area was calculated as the ratio of leaf area to total leaf dry mass. 2.3. Gas exchange measurements Leaf conductance (gw ) and net assimilation rate (A) were measured 3 h after the onset of the photoperiod at ambient CO2 (330 ␮mol mol−1 ), PPF of 400 ␮mol m−2 s−1 , 80% RH and 25 ◦ C with a Walz model HCM-1000 minicuvette system (Effeltrich, Germany). For each sampling time and treatment, measurements were made on six young, current-year, fully expanded leaves collected from different plants. 2.4. Leaf solutes

2. Material and methods 2.1. Plant material and growth conditions We studied 2-year-old trees of three Canarian laurel forest species: L. azorica (Seub.) Franco, P. indica (L.) K. Spreng and M. faya Aiton, which were supplied by Viveros de la Viceconsejer´ıa de Medioambiente of Government of Canary Islands (Spain). Trees were transplanted to pots (30 L soil volume) containing a 1:1 (v/v) peat:soil potting mix. The soil was obtained from native Canarian forest and was classified as a colluvial andosol, derived from bedrock that is a mixture of olivine basalt and volcanic ash with high permeability and drainage (Morales et al., 1996). Plants were transferred to a greenhouse located in the University of Navarra (Pamplona, Spain), where they grew with a day/night regime of 20/15 ◦ C and 80/90% relative humidity (RH) in a 12 h photoperiod with natural daylight supplemented with light from high pressure sodium lamps (SON-T Agro Phillips, Eindhoven, The Netherlands), providing a minimum photosynthetic photon flux (PPF) of 300 ␮mol m−2 s−1 . The plants were watered twice a week with Hoagland’s nutrient solution and once a week with deionised water to avoid salt accumulation in the pots. At the start of the experiment, plants were transferred to a controlled environment chamber with a day/night regime of 25/15 ◦ C and 80/90% RH. A PPF of 400 ␮mol m−2 s−1 at the canopy level was provided by fluorescent lamps (Sylvania F 48T12 CW-WHO, ¨ Munchen, Germany) for a 12-h photoperiod. Plants were subjected to a single drought episode by withholding water until leaf relative water content (RWC) reached about 50–55%. Measurements were taken at the drought-intensified stage. 2.2. Leaf water status and specific leaf area (SLA) Leaves were collected, weighed, and rehydrated for 24 h at 4 ◦ C in darkness and subsequently oven-dried at 85 ◦ C until constant mass. RWC was calculated as 100 × (FM–DM)/(TM–DM), where FM is the fresh mass, TM is the turgid mass after leaf rehydration, and DM is dry mass. The leaf water potential ( ) was measured at predawn on fresh leaves using a pressure chamber (PMS Instruments, Oregon, USA). The osmotic potential ( s ) was measured with a thermocouple psychrometer Tru Psi SC10X (Decagon, WA, USA) after equilibrating frozen leaf tissue for 2 h at 20 ◦ C. Leaf osmotic potential at full turgor (s100 ) was calculated according to the expression s100 = s × RWC/100 (Irigoyen et al., 1996). Leaf sampling and manipulation were performed quickly to avoid tissue dehydration. All measurements were done at predawn on six recently fully expanded leaves from different plants for each sampling time and treatment. Leaf area was measured with a portable leaf area meter (Model LI-3000, LiCor, Lincoln, NE). Leaf dry mass was determined by dry-

Leaf samples for solute analysis were collected at the end of water treatments and rapidly frozen at −80 ◦ C until analysis. Samples of 0.1 g of fresh leaves were ground in an ice-cold mortar and pestle containing potassium phosphate buffer (50 mM, pH 7.5). The homogenates were filtered through four layers of cheesecloth and centrifuged at 3500 × g at 4 ◦ C for 15 min. The supernatant was collected and stored 4 ◦ C. Total soluble sugars (TSS) were analysed by reaction 0.25 ml of the supernatant with 3 ml of freshly prepared anthrone and placing in boiling water for 10 min. After cooling, the absorbance at 620 nm was determined in a spectrophotometer (Yemm and Willis, 1954). Free proline determination of the supernatant was estimated by reacting 1 ml of ninhydrine and placed in boiling water for 45 min. The absorbance was read in a spectrophotometer at 515 nm (Paquin and Lechasseur, 1979). Free proline concentration was calculated from a calibration curve using proline as a standard. 2.5. Abscisic acid determinations Leaf samples for ABA measurements were frozen in liquid nitrogen and stored at −80 ◦ C until analysis. ABA was extracted following the protocol of Zhou et al. (2003) as described elsewhere (Antol´ın et al., 2006). The initial procedure involved preparing lyophilised plant tissues (0.03 g dry mass), which were crushed in liquid nitrogen using a mortar and pestle and extracted with 750 ␮l of acetone:water:acetic acid (80:19:1, v/v). The extracts were centrifuged at 10,000 × g for 2 min, the supernatant was collected and the residues were re-extracted with 750 ␮l of the extraction solvent. The second extract was centrifuged and supernatants were then combined and dried under a stream of nitrogen. The dried sample was reconstituted in 200 ␮l of acetonitrile–water (15:85, v/v) containing 12 mM acetic acid (pH 3.3). A portion (10–15 ␮l) of the sample was loaded onto a PerkinElmer Series 200 HPLC system (Wellesley, MA, USA) equipped with a 50 mm × 2.1 mm, 3.5 ␮m LC/MS column (a Sunfire, Waters, Milford, MA, USA), using a flowrate of 0.6 ml/min and a binary solvent system comprising 12 mM acetic acid in water (A) and 12 mM acetic acid in acetonitrile–water (90:10, v/v) (B). Typically, the solvent gradient was programmed to change linearly from 15% B over the first 10 min and then to 100% B over the next 6.2 min, before returning to the initial composition at 12 min. The retention time was 13.5 min. Analysis was performed with a triple quadrupole mass spectrometer API 3000 LC/MS/MS (Applied Biosystems, Foster City, CA, USA). The solvent was removed with a 180-L/h flow of nitrogen gas heated to 400 ◦ C. The ion pair 263/153 was monitored using the multiple reaction-monitoring mode (MRM). Quantification was done by the standard addition method by spiking control plant samples with ABA solutions (ranging from 10 to 200 ng ml−1 ). Endogenous ABA was quantified by the comparison of peak areas

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with those obtained from the respective calibration curves. Six samples were collected from different plants for each treatment. 2.6. Statistical analysis Data were submitted to a two-factor analysis of variance (ANOVA). Variance was related to the main treatments (species and irrigation treatment) and to the interaction between them. Means ± standard errors (S.E.) were calculated, and when F ratio was significant, least significant differences were evaluated by Tukey’s test, as available in the SPSS statistical package version 13.0 programs for Windows XP. Using the same data, a correlation analysis was also calculated, to evaluate the extent of association and its significance. All values shown in figures are means ± S.E. 3. Results 3.1. Leaf water status and gas exchange Drought treatment reduced predawn RWC and leaf  at the same extent in all studied laurel species (Table 1). Drought also induced a marked decrease of leaf osmotic potential ( s ), which was significantly lower in L. azorica followed by M. faya and P. indica (Fig. 1, Table 1). Two-way ANOVA analysis revealed significant interactions between species and water treatment for all parameters analysed. Leaf conductance (gw ) and net assimilation rate (A) decreased in drought-treated trees (Fig. 2). However, this reduction followed different patterns for the investigated species. At moderate leaf desiccation (RWC ca. 80%), L. azorica showed higher gw than P. indica reaching 28% and 8% of gw measured at high RWC, respectively (Fig. 3A). In general, net assimilation rate (A) performed similarly to gw as evidenced by the strong relationship detected between both parameters (r2 = 0.87, p < 0.001) (Fig. 2). However, L. azorica and M. faya exhibited always higher A than P. indica. The comparison of L. azorica and M. faya at moderate RWC (ca. 80%) showed that the former reached 45% of values measured at high RWC, whereas in M. faya, it decreased to ca. 22% (Fig. 3B). Results showed that a moderate level of water stress reduced more A in M. faya than in L. azorica. The intracellular CO2 concentration (Ci ) significantly decreased at moderate leaf desiccation in L. azorica and M. faya. However, Ci strongly increased in the case of P. indica (Fig. 3C). The drought treatment decreased SLA in all studied trees (Table 2). This change was less pronounced in L. azorica (Table 2) because this species showed higher SLA than others.

Fig. 1. Leaf water potential ( ) (A) and solute water potential ( s ) (B) measured at predawn as a function of the leaf relative water content (RWC) in leaves of three Canarian laurel species (Laurus azorica, Myrica faya and Persea indica) grown under well-watered (control) or soil water deficit (drought) conditions. Values are means (n = 6) and bars indicate standard error (S.E.) of the mean. Values of S.E. lower than 10% are not shown. Different letters indicate significant differences (p ≤ 0.05) between treatments and species according to a Tukey’s test.

3.2. Leaf solutes and osmotic adjustment capacity Drought induced a significant decrease in s100 only in leaves of L. azorica that were not evident in the other tested species (Table 2). This differential pattern was emphasized by two-way ANOVA because there was significant interaction between species and water treatment. Leaf solute content, which was determined at the end of drought period, showed that L. azorica had the highest concentration of TSS. Whatever the plant water status, the pool of TSS remained constant in all analysed species, and no differences between treatments were detected. Finally, proline concentration increased sharply in drought-treated plants only in L. azorica. Two-way ANOVA analysis revealed significant interactions between species and water treatment for proline concentration.

Table 1 Relative water content (RWC), water potential ( ) and solute water potential ( s ) measured at predawn in leaves of three Canarian laurel species (Laurus azorica, Myrica faya and Persea indica) grown under well-watered (control) or soil water deficit (drought) conditions Species

Treatments

RWC (%)

Laurus azorica

Control Drought Control Drought Control Drought

81.8 58.4 79.49 57.3 93.6 59.33

Myrica faya Persea indica

Species Water treatment Interaction

± ± ± ± ± ±

 (MPa) 4.4 a 3.9 b 6.0 a 4.5 b 0.7 a 0.9 b

−1.27 −3.12 −0.93 −3.30 −0.99 −2.83

± ± ± ± ± ±

 s (MPa) 0.04 a 0.30 b 0.04 a 0.18 b 0.02 a 0.16 b

−2.36 −3.99 −2.28 −3.57 −2.17 −3.23

nsa

ns

***

***

***

***

**

**

**

± ± ± ± ± ±

0.06 a 0.15 c 0.04 a 0.09 b 0.03 a 0.15 b

Determinations were made at the end of experimental period. Within each column, means followed by different letters are significantly different (p < 0.05) according to a Tukey’s test. Values are means (n = 6). a Not significant. ** Significance at 0.01 probability level. *** Significance at 0.001 probability level.

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Fig. 2. Leaf conductance, gw (A) and net assimilation rate, A (B) as a function of the leaf relative water content in leaves of different Canarian laurel trees subjected to different soil water moisture. Measurements were made at ambient CO2 (330 ␮mol mol−1 ), PPF of 400 ␮mol m−2 s−1 , 80% RH and 25 ◦ C. Otherwise as in Fig. 1.

3.3. Abscisic acid concentration Bulk leaf ABA determinations were made at the end of experimental period (Fig. 4). In well-watered plants L. azorica had the highest leaf ABA concentration. In drought-treated plants, leaf ABA concentration increased in L. azorica but M. faya and P. indica exhibited no change in response to drought. Combining all measurements, leaf ABA was significantly related to proline (r2 = 0.54, p < 0.001), TSS (r2 = 0.37, p < 0.001) and s100 (r2 = 0.25, p < 0.01) (Fig. 5).

Fig. 3. Leaf conductance, gw (A), net assimilation rate, A (B) and intracellular CO2 concentration, Ci (C) measured at completely leaf hydration (RWC ca. 92%) and at moderate leaf desiccation (RWC ca. 80%) in leaves of different Canarian laurel trees. Otherwise as in Fig. 1.

4. Discussion As expected, drought imposition resulted in reduced predawn leaf  ,  s and RWC in all the studied laurel species (Fig. 1, Table 1), which further induce stomatal closure (Fig. 2A). Progressive decrease in RWC decreased net assimilation rate (A) of leaves in all tested species, eventually inhibiting it (Fig. 2B). In general there was a concomitant decrease on both leaf conductance to water

vapour (gw ) and A, suggesting that stomatal closure was the main factor determining A. This relationship was especially evident in L. azorica and M. faya, where the lower stomatal conductance induced by water stress seems to limit CO2 supply to mesophyll cells, as evidenced by the low Ci (Fig. 3C). Previous field studies reported that the annual course of transpiration in laurel forest was very variable, owing to weather conditions, and was mainly controlled

Table 2 Specific leaf area (SLA), osmotic potential at full turgor (s100 ) and some organic solutes in leaves of three Canarian laurel species (L. azorica, M. faya and P. indica) grown under well-watered (control) or soil water deficit (drought) conditions Species

Treatments

SLA (m2 kg−1 )

Laurus azorica

Control Drought Control Drought Control Drought

13.23 10.50 12.97 7.95 12.61 8.16

Myrica faya Persea indica

Species Water treatment Interaction

± ± ± ± ± ±

0.62 a 0.85 b 0.31a 0.04 c 0.21 a 0.09 c

s100 (MPa)

TSS (mg g−1 DM)

−1.88 −2.45 −1.82 −1.95 −2.02 −1.92

74.7 77.1 55.2 55.5 59.2 60.3

± ± ± ± ± ±

0.12 a 0.20 b 0.15 a 0.09 a 0.04 ab 0.07 a

± ± ± ± ± ±

4.8 a 8.1 a 2.1 b 2.1 b 2.4 ab 4.0 ab

Proline (␮mol g−1 DM) 1.1 38.0 3.0 3.7 1.3 5.9

*

**

***

***

***

nsa

***

ns

***

ns ns

± ± ± ± ± ±

0.1 b 4.0 a 0.2 b 0.2 b 0.1 b 0.5 b

***

Determinations were made at the end of experimental period. TSS: total soluble sugars. Within each column, means followed by different letters are significantly different (p < 0.05) according to a Tukey’s test. Values are means (n = 6). a Not significant. * Significance at 0.05 probability level. ** Significance at 0.01 probability level. *** Significance at 0.001 probability level.

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Fig. 4. Bulk leaf ABA in different Canarian laurel trees subjected to different water treatments. Otherwise as in Fig. 1.

´ by the evaporative conditions, not by the stomata (Jimenez et al., 1996). Under natural conditions, these trees have high water availability because of deep roots and accessible water table, therefore they show low stomatal control; but when they were grown in pots with decreasing water availability the roots do not have sufficient available water and the stomata showed more sensitivity (Fig. 2A). In the case of P. indica, moderate drought induced a severe inhibition of A and increased Ci , which might result in a strong

Fig. 5. Relationship between leaf ABA concentrations and osmotic potential at full turgor (s100 ) (A), and some leaf solutes as proline (B) and total soluble sugars (TSS) (C) in three Canarian laurel trees subjected to different water treatments. Straight lines fitted for the joint data of all determinations. The corresponding equations were: (A) y = −0.024x–1.62; (B) y = 1.24x–7.40; (C) y = 1.01x + 52.16 (n = 36).

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limitation at the photosynthetic apparatus level in this species (Fig. 3). A high sensitivity of P. indica to changes in the environmental conditions was reported previously under natural (Tausz ´ et al., 2004), and controlled conditions (Sanchez-D´ ıaz et al., 2007). This last study showed that under severe drought, P. indica had low maximum efficiency of PSII and low capacity for energy dissipation, indicating photoinhibitory damages at photosynthetic apparatus level. When comparing the three species under moderate stressed conditions (RWC ca. 80%) it was evident that L. azorica maintained higher photosynthetic rate than other laurel species (Fig. 3B). The higher SLA detected in drought-stressed L. azorica in comparison to M. faya and P. indica (Table 2), could contribute to better photosynthetic rate under moderate water deficits apparently due to greater light interception per unit leaf area in this species. Our results are partially consistent with measurements made in the Canarian laurel forest stand, where M. faya resulted in higher sensitivity to ´ moisture stress than L. azorica (Gonzalez-Rodr´ ıguez et al., 2001, 2002a). These authors also indicated that L. azorica have leaves with high plasticity, enabling them to adjust their morphology and phys´ iology to the microclimatic conditions (Gonzalez-Rodr´ ıguez et al., 2001). Our data showed that M. faya and P. indica were also able to adjust their morphology to imposed conditions by increasing leaf thickness (low SLA) (Table 2). Osmotic adjustment through active accumulation of solutes and/or changes of tissue elasticity are some interrelated mechanisms serving turgor maintenance, which is very important for plant growth and survival under conditions of restricted water availability (Tyree and Jarvis, 1982). Osmotic adjustment occurs when consists in there is an accumulation of solutes in response to low  that decreases cellular  s and functions to sustain partially turgor despite cellular water loss (Zhang et al., 1999). L. azorica was the sole species that displayed a significant drop of s100 during drought (Table 2). In a previous study with the same Canarian laurel species subjected to drought under controlled conditions, pressure–volume curves constructed in L. azorica showed a significant capacity for osmotic adjustment and increased maximal ´ bulk modulus of elasticity (Gonzalez-Rodr´ ıguez et al., 1999). Such response indicates a decrease in tissue elasticity with increasing drought. Although no measurements of bulk modulus of elasticity were made, previous results basically agree with those reported in our study (Table 2). One of the most common stress responses in plants is enhanced production of different types of compatible organic solutes (Serraj and Sinclair, 2002). Drought response of laurel trees did not seem to be related to accumulation of TSS (Table 2). On the other hand, proline accumulation is an important response to low  although its function and regulation are not well understood (see revision in Ashraf and Foolad, 2007). Proline is a compatible solute that can have a major role in osmotic adjustment (Delauney and Verma, 1993) but may also have a number of other protective roles. These include protecting protein and membrane structure (Chen and Murata, 2002), eliminating excess reductant or regulating cellular redox status (Hare et al., 1998). Drought induced proline accumulation only in L. azorica (Table 2), suggesting that this increase plus high TSS concentrations detected in this species could have a significant role for decreased s100 (Table 2). Besides, proline could also perform other protective role as scavenger of reactive oxygen species (Smirnoff and Cumber, 1989; Koca et al., 2007). This view agrees with the observed resistance of L. azorica to photoinhibition under severe ´ drought due to high antioxidant capacity (Sanchez-D´ ıaz et al., 2007). The role of ABA as a root-to-shoot stress signal is now well established (Sauter et al., 2001; Hartung et al., 2005). Results

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showed that leaf ABA concentration was constitutively higher in L. azorica than in the other species and it increased under drought (Fig. 4). In addition to the well-established role of ABA in stomatal closure, it has been reported that increased concentration of ABA in response to drought was parallel with increased concentration of proline (Ober and Sharp, 1994). The response of L. azorica to drought could agree with recent evidences showing that ABA is required for induced proline accumulation at low  (Verslues and Bray, 2006). It seems that proline accumulation in plants is mediated, at least in part, by an ABA-dependent signalling pathway (Ashraf and Foolad, 2007). In this same way, the lack of leaf ABA increase in drought stressed M. faya and P. indica coincided with no significant accumulation of proline (Table 2). These data were reinforced with the highly significant relationship between leaf ABA and proline content (Fig. 5B). It has also been reported that osmotic adjustment may occur independently of ABA accumulation (Alves and Setter, 2004; Verslues and Bray, 2006). However, this lack of relationship may not be universal, because in our case there were significant relationships between leaf ABA and TSS (Fig. 5C) and s100 (Fig. 5A), suggesting a possible involvement of ABA in the response of laurel trees to severe drought. In conclusion, this study demonstrated that even, within the same environment, species of Canarian laurel forest display different responses to limited water supply. At moderate water deficit, L. azorica showed higher net assimilation rate than M. faya and P. indica, suggesting a better acclimation to drought in this species. In addition, L. azorica was the sole species able to accumulate osmotic solutes (especially, proline) and ABA in leaves. Osmotic adjustment and proline synthesis seems to be related to ABA because significant relationships between leaf ABA, s100 and proline accumulation were found. Thus, it could be expect L. azorica to perform better than the other species under more stressful environments. Species distribution in the Canarian forest showed that L. azorica may play a role in the succession from degraded sites to closed ´ canopy stands (Gonzalez-Henr´ ıquez et al., 1986). To our knowledge, this is the first study reporting on changes in leaf gas exchange, solute and ABA concentrations in drought stressed Canarian laurel trees. Acknowledgements ´ The authors thank Drs. M.S. Jimenez and D. Morales (Departamento de Biolog´ıa Vegetal, Universidad de La Laguna, Tenerife, Spain) for providing plants and for helpful discussions about results. ´ has been Technical support during the experiments by A. Urdiain most valuable. This project was supported by DGICYT (PB 94-0580). ´ de Amigos de C. Tapia was the recipient of a grant from Asociacion la Universidad de Navarra. References Alves, A.A.C., Setter, T.L., 2004. Abscisic acid accumulation and osmotic adjustment in cassava under water deficit. Environ. Exp. Bot. 51, 259–271. ´ Antol´ın, M.C., Ayari, M., Sanchez-D´ ıaz, M., 2006. Effects of partial rootzone drying on yield, ripening and berry ABA in potted Tempranillo grapevines with split roots. Aust. J. Grape Wine Res. 12, 13–20. Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216. Assmann, S.M., 2003. OPEN STOMATA1 opens the door to ABA signalling in Arabidopsis guard cells. Trends Plant Sci. 5, 151–153. Bray, E.A., 2003. Abscisic acid regulation of gene expression during water-deficit stress in the era of the Arabidopsis genome. Plant Cell Environ. 25, 153–161. Chen, T.H.H., Murata, N., 2002. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant Biol. 5, 250–257. Delauney, A.J., Verma, D.P.S., 1993. Proline biosynthesis and osmoregulation in plants. Plant J. 4, 215–223.

´ ´ ´ Gonzalez-Henr´ ıquez, M.N., Rodrigo Perez, J.D., Suarez Rodr´ıguez, C., 1986. Flora y ´ del Archipielago ´ Vegetacion Canario. Editora Regional Canaria, Las Palmas de Gran Canaria, Spain. ´ ´ Gonzalez-Rodr´ ıguez, A.M., Morales, D., Jimenez, M.S., 2001. Gas Exchange characteristics of a Canarian laurel forest tree species (Laurus azorica) in relation to environmental conditions and leaf canopy position. Tree Physiol. 21, 1039–1045. ´ ´ Gonzalez-Rodr´ ıguez, A.M., Morales, D., Jimenez, M.S., 2002a. Leaf gas exchange characteristics in relation to leaf canopy position of Myrica faya in its native environment (Tenerife Canary Islands). Plant Biol. 4, 576–583. ´ ´ Gonzalez-Rodr´ ıguez, A.M., Morales, D., Jimenez, M.S., 2002b. Leaf gas exchange characteristics of a Canarian laurel forest tree species [Persea indica (L.) Spreng.] under natural conditions. J. Plant Physiol. 159, 695–704. ´ ´ ¨ Gonzalez-Rodr´ ıguez, A.M., Jimenez, M.S., Morales, D., Aschan, G., Losch, R., 1999. Physiological responses of Laurus azorica and Viburnum rigidum to drought stress: osmotic adjustment and tissue elasticity. Phyton (Horn, Austria) 39, 251–263. Hare, P.D., Cress, W.A., Staden, J. van, 1998. Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ. 21, 535–553. Hartung, W., Schraut, D., Jiang, F., 2005. Physiology of abscisic acid (ABA) in roots under stress—a review of the relationship between root ABA and radial water and ABA flows. Aust. J. Agric. Res. 56, 1253–1259. ¨ Hollermann, P., 1981. Microenvironmental studies in the laurel forests of the Canary Islands. Mount. Res. Dev. 1, 193–207. ´ ´ Irigoyen, J.J., Perez de Juan, J., Sanchez-D´ ıaz, M., 1996. Drought enhances chilling tolerance in a chilling-sensitive maize (Zea mays) variety. New Phytol. 134, 53– 59. ˇ ´ ´ J., Kucera, J., Morales, D., 1996. Laurel forests in Tenerife, Jimenez, M.S., Cerm ak, Canary Islands: the annual course of sap flow in Laurus trees and stand. J. Hydrol. 183, 307–321. ¨ ¨ Koca, H., Bor, M., Ozdemir, F., Turkan, I., 2007. The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ. Exp. Bot. 60, 344–351. Kozlowski, T.T., Pallardy, S.G., 2002. Acclimation and adaptive response of woody plants to environmental stresses. Bot. Rev. 68, 270–334. Larcher, W., 2003. Physiological Plant Ecology, 4th ed. Springer, New York. LaRosa, P.C., Hasegawa, P.M., Rhodes, D., Clithero, J.M., Watad, A.E.A., Bressan, R.A., 1987. Abscisic acid stimulated osmotic adjustment and its involvement in adaptation of tobacco cells to NaCl. Plant Physiol. 85, 174–181. ¨ Losch, R., 1993. Water relations of Canarian laurel forest trees. In: Borghetti, M., Grace, J., Raschi, A. (Eds.), Water Transport in Plants Under Climatic Stress. Cambridge University Press, Cambridge, pp. 243–245. ´ ´ Morales, D., Gonzalez-Rodr´ ıguez, A.M., Tausz, M., Grill, D., Jimenez, M.S., 1997. Oxygen stress and pigment composition in Canarian laurel forest trees. Phyton (Horn, Austria) 37, 181–186. ˇ ´ ´ ´ J., 1996. Laurel forests Morales, D., Jimenez, M.S., Gonzalez-Rodr´ ıguez, A.M., Cerm ak, in Tenerife, Canary Islands. I. The site, stand structure and stand leaf area distribution. Trees 11, 34–40. Morgan, J.M., 1984. Osmoregulation and water stress in higher plants. Ann. Rev. Plant Physiol. 35, 299–319. Ober, E.S., Sharp, R.E., 1994. Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. I. Requirement for increased levels of abscisic acid. Plant Physiol. 105, 981–987. ´ Paquin, R., Lechasseur, P., 1979. Observations sur une methode de dosage de la proline libre dans les extraits des plantes. Can. J. Bot. 57, 1851–1854. ´ Sanchez-D´ ıaz, M., Tapia, C., Antol´ın, M.C., 2007. Drought-induced oxidative stress in different Canarian laurel forest tree species growing under controlled conditions. Tree Physiol. 27, 1415–1422. Santos, A., 1990. Evergreen Forest in Macaronesian Region. Council of Europe, Strasbourg. Sauter, A., Davies, W.J., Hartung, W., 2001. The long-distance abscisic acid signal in the droughted plant: the fate of the hormone on its way from root to shoot. J. Exp. Bot. 52, 1991–1997. Serraj, R., Sinclair, T.R., 2002. Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant Cell Environ. 25, 333–341. Shinozaki, K., Yamaguchi-Shinozaki, K., Seki, M., 2003. Regulatory network of gene expression in the drought and cold stress responses. Curr. Opin. Plant Biol. 6, 410–417. Smirnoff, N., Cumber, Q.J., 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060. ´ Tausz, M., Gonzalez-Rodr´ ıguez, A.M., Wonish, A., Peter, J., Grill, D., Morales, D., ´ Jimenez, M.S., 2004. Photostress, photoprotection, and water soluble antioxidants in the canopies of five Canarian laurel forest tree species during a diurnal course in the field. Flora 199, 110–119. Trewavas, A.J., Jones, H.G., 1991. An assessment of the role of ABA in plant development. In: Davies, W.J., Jones, H.G. (Eds.), Abscisic Acid: Physiology and Biochemistry. BIOS Scientific Publishers Limited, Oxford, UK, pp. 169–188. Tyree, M.T., Jarvis, P.G., 1982. Water in tissues and cells. In: Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H. (Eds.), Water Relations and Carbon Assimilation. Encyclopedia of Plant Physiology, 12B. Springer-Verlag, Berlin, pp. 35–77. Verslues, P.E., Bray, E.A., 2006. Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation. J. Exp. Bot. 57, 201–212.

M. S´ anchez-D´ıaz et al. / Environmental and Experimental Botany 64 (2008) 155–161 Xiong, L., Ishitani, M., Lee, H., Zhu, J.-K., 2001. The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress and osmotic stress responsive gene expression. Plant Cell 13, 2063–2083. Yemm, E.W., Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J. 57, 508–514.

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Zhang, J., Nguyen, H.T., Blum, A., 1999. Genetic analysis of osmotic adjustment in crop plants. J. Exp. Bot. 50, 292–302. Zhou, R., Squires, T.M., Ambrose, S.J., Abrams, S.R., Ross, A.R.S., Cutler, A.J., 2003. Rapid extraction of abscisic acid and its metabolites for liquid chromatography–tandem mass spectrometry. J. Chromatogr. 1010, 75–85.