Edited by J. I. Sprent
Annual changes in the concentration of minerals and organic compounds of Quercus suber leaves
Version of Record online: 18 APR 2006
Volume 127, Issue 1, pages 100–110, May 2006
How to Cite
Passarinho, J. A. P., Lamosa, P., Baeta, J. P., Santos, H. and Ricardo, C. P. P. (2006), Annual changes in the concentration of minerals and organic compounds of Quercus suber leaves. Physiologia Plantarum, 127: 100–110. doi: 10.1111/j.1399-3054.2006.00655.x
- Issue online: 18 APR 2006
- Version of Record online: 18 APR 2006
- Received 6 October 2005; revised 6 November 2005
Mature leaves are the primary source of sugars, which give rise to many secondary metabolites required for plant survival under adverse conditions. In order to study the interaction of field-grown cork oak (Quercus suber L.) with the environment, we investigated the seasonal variation of minerals and organic metabolites in the leaves, using inductively coupled plasma atomic emission spectrometry, elemental analysis and nuclear magnetic resonance spectrometry. Statistical analysis showed that the data strongly correlated with seasonal climate and were divided in three groups corresponding to: (1) spring-early summer, (2) summer and (3) autumn-winter. The concentration of N, P, K and leaf ash content were highest in spring (recently formed leaves), reached the minimum during the hot and dry summer and increased slightly during the rainy period of autumn-winter. Conversely, Na, Mg and Ca concentrations were lowest in spring-early summer and increased during summer and autumn-winter, the Ca concentration increasing five-fold. Two cyclitol derivatives, quinic acid and quercitol were the major organic metabolites of the leaves. Their concentration along the season followed opposite trends. While quinic acid predominated during spring-early summer, when it contributed 12% to the leaf osmotic potential, quercitol was predominant during autumn-winter, when its contribution to leaf osmotic potential was about 10%. This different preponderance of the two compounds is expressed by the quercitol/quinic acid ratio, which can be as low as 0.2 in early summer and as high as 9 in winter. Sucrose and glucose concentrations also increased during autumn-winter. Evidence for the quercitol protective role in plants during stress is discussed, and on the basis of structural similarity, it is suggested that quinic acid could have an identical importance, with a protective role against heat and high irradiance. It is concluded that the marked changes in Q. suber leaf composition throughout the year could have important implications in the plant capacity to endure climatic stress.
13C-nuclear magnetic resonance
bidimensional nuclear magnetic resonance
heteronuclear multiple bond connectivity
heteronuclear multiple quantum coherence
1H-nuclear magnetic resonance
nuclear Overhauser effect spectroscopy
photosynthetic active radiation
Cork oak (Quercus suber L.) is an evergreen tree of great importance in the Mediterranean area both for ecological reasons and for the economic value of cork. In Portugal, it occupies approximately 22% of the forests, which represents about 26% of the world area of Q. suber (Mendes 1977). This tree is adapted to a 4-month dry summer period with little or no rain, maximum temperatures reaching 35–40°C and midday irradiances exceeding 2000 µmol m−2 s−1 photosynthetic active radiation (PAR) (Faria et al. 1996).
In recent years, a marked decline in some cork oak stands has been observed, associated with increased number of trees showing ‘sudden death’ symptoms, which may be related to infection by the fungus Phytophthora cinnamomi (Brasier 1992, Brasier et al. 1993) and the repeated succession of extremely dry and hot years. Spring rain precipitation in Portugal has declined approximately 20% from the decades 1931–1960 to 1961–1990 (Instituto de Meteorologia 1997). Under Mediterranean climate, soil and atmospheric water deficits are indeed considered the most important environmental constraints to the physiology of forest trees (Pereira and Chaves 1995). This investigation is of relevance because little is known about Q. suber leaf metabolites and the influence of the fluctuating environmental agents on leaf composition.
Using 13C-nuclear magnetic resonance (13C-NMR), we have identified the major water-soluble organic metabolites of Q. suber leaves and have followed the variation in their content along the year. The concomitant changes in the concentrations of leaf minerals were analysed. We discuss the implications of the observed metabolic interconversions for the plant's adjustable responses to environmental stress conditions.
Materials and methods
Two Q. suber trees T1 (trunk diameter at breast height of 80 cm) and T2 (trunk diameter of 20 cm) were the main subjects of this study. They grow 50 m apart at Tapada da Ajuda (Instituto Superior de Agronomia, Lisbon) on a clay loam Haplic Phaeozem soil (FAO 1998) at a location of 38°43′6′′N latitude, 9°11′27′′W longitude and 101 m altitude. The soil is derived from basalt, with an Ap C R profile; the Ap horizon is about 40 cm thick with high amounts of coarse fragments and an available water capacity around 10%; the C horizon extends to 150 cm, consisting of mixed weathered basalt and unaltered coarse rock fragments. Three other Q. suber populations in distinct locations were also studied: Montargil (95 km NE of Lisbon, Ing. Paula Firme farm), Coruche (65 km NE of Lisbon, Dr Isabel Carrasquinho clonal orchard at Escaroupim) and Évora (110 km SSE of Lisbon, Professor J. Santos Pereira field experiment).
Data concerning the weather variables over the period of observation (December 1998 to January 2000) at Tapada da Ajuda are shown in Fig. 1. Solar insolation data (hours), representing the sum of periods of direct solar radiation above 120 W m−2, were recorded with a Campbell-Stokes Heliograph and obtained from Instituto de Meteorologia (Lisbon). The remaining climatic data were recorded at the meteorological station of Tapada da Ajuda (Instituto Superior de Agronomia). The mean annual rainfall over the 1971–2000 period was 679.4 mm, precipitation occurring primarily from September to April. For the calculation of potential evapotranspiration, the Penman/Monteith method (Monteith and Unsworth 1990) was used.
Plant material, sampling and leaf parameters
Fully expanded leaves from the shoots of the year located at the southern surface of the canopy (sun leaves) were collected at about fortnight intervals, 2 h after sunrise. Leaf area was determined using a portable area meter (Li-Cor model LI-3000 A), and dry weight (DW) was calculated after drying for 48 h in an oven at 60°C. For the remaining determinations, samples were frozen in liquid N2 and stored at −80°C until subsequent use. Leaf osmotic potential was determined according to Mendes et al. (2001) in discs (0.7 cm in diameter) of frozen leaves, using C-52 chambers attached to a Wescor HR-33T dew-point microvoltmeter (Wescor Inc., Logon, UT). The contribution of solutes to the leaf osmotic potential was calculated from the solute concentration in the osmotic volume of leaves, using the Van't Hoff gas-low equation, so that 40 µmol solute g−1 H2O corresponds to −0.1 MPa. Fully ripe acorns were collected in autumn.
For all determinations, two distinct samples were analysed and the results presented are the mean values ± sd.
NMR analysis of extracts
Frozen leaf samples (10 g, 35–45 leaves) and acorn cotyledons (10 g) were ground to a fine powder on liquid N2 in a mortar. The powder was dropped into boiling water and boiled for 5 min. The slurry was centrifuged, the recovered supernatant lyophilized and the residue resuspended in 4 ml of an aqueous solution containing D2O (5.8 M), Na2 ethylenediaminetetraacetic acid (2.5 mM) and NaN3 (2.5 mM). Quantification of metabolites was carried out by 13 C-NMR using dioxan as an internal concentration standard. Spectra were obtained at 75.47 MHz on a Bruker AMX300 spectrometer using a 10 mm diameter broadband probe head with the following parameters: number of scans 1500; recycle time 2.9 s; sweep width 22.7 kHz; and pulse width 11 µs corresponding to a flip angle of 45°. Proton decoupling was applied during the acquisition only. The temperature of the probe head was kept at 300°K. Data were processed with 4 Hz exponential line broadening. Fully relaxed spectra were obtained using 31.4 s for the repetition delay and used to calculate correction factors for the areas of relevant peaks. Chemical shifts are expressed in p.p.m. relative to dioxan at 67.0 p.p.m. Resonances due to glucose, sucrose, fructose, citric and malic acids were identified from their chemical shifts. Resonances due to quercitol were assigned by comparison with chemical shifts of this compound in an extract of Q. suber acorns where quercitol is known to be abundant. Quinic acid resonances were identified by 2D-NMR techniques [1H-NMR, correlative spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple bond connectivity (HMBC) and heteronuclear multiple quantum coherence (HMQC)]. 1H-13C correlation spectra were acquired on a Bruker AMX500 spectrometer using standard pulse programs (Bruker, Rheinstetten, Germany). For 1H-13C HMQC spectra (Bax and Summers 1986), a delay of 3.5 ms was used for evolution of 1JCH. The HMBC spectrum was recorded collecting 4096 (t2) × 256 (t1) data points; a delay of 73.5 ms was used for evolution of long range couplings. Proton decoupling was applied during the acquisition time only, using the wideband alternating-phase low-power technique for zero-residue-splitting (WALTZ) sequence.
Chromatographic separation of extract constituents
Leaf extracts were fractionated by ion exchange chromatography using Dowex 50 W X8 (H+) and AG1-X2 (Cl–) resins to obtain neutral, anionic and cationic fractions by elution with water, 2 M HCl and 5 M NH4OH. The anionic fraction was further purified using a Hiload system (Pharmacia, Uppsala, Sweden) separation on QAE-Sephadex A25 eluted with pH 8 NH4HCO3 (gradient 0.05–1 M).
Ash content and mineral and elemental analysis
Ash content was determined by dry combustion of 1 g of dry tissue in an oven at 470°C for 24 h. Ashes were dissolved in 0.1 M HCl and the mineral elements analysed in an inductively coupled plasma atomic emission spectrometer (Jovin-Yvon 24, Longjumeau, France). The total N, H and C contents were measured in thinly powdered dried leaves in a CHNOS Elemental Analyzer (Elementar Vario EL, Mt. Laurel, NJ).
Canonical discriminant analysis
Using statistica 6.1 program (StatSoft Inc., Tulsa, OK), we applied a canonical discriminant analysis (Huberty 1975) to the concentration data of leaf organic metabolites and mineral elements, referring to the several sampling dates which were considered as independent variables.
Leaf development and survival
Observation of leaf longevity in the Q. suber trees revealed that new leaves were produced in spring (April), when remaining leaves from the previous year were shed.
Dry matter content of new leaves was low, but increased with age, to reach the maximum value around August/September. Ash content was slightly above 5% in young leaves; it decreased slightly with time, reaching a minimum by August/September, and recovered in autumn (Fig. 2). Specific leaf area followed a similar trend. Its value was 8.05 ± 0.39 m2 kg−1 during April to July, 7.44 ± 0.41 m2 kg−1 during July to September and 8.98 ± 0.37 m2 kg−1 during September to February.
Identification of leaf organic metabolites
In order to identify the major soluble metabolites of Q. suber leaves, 13C-NMR spectra were run from water extracts. Using leaves collected in July 1998, we easily identified sucrose (S), glucose (G) and fructose (F) (Fig. 3A). Resonances detected in the region 110–170 p.p.m. (aromatic-C and phenolic-C) were possibly due to low concentrations of water-soluble phenolic compounds. To assign the unknown peaks numbered 1–13 in Fig. 3(A,B), we fractionated the leaf extracts by ion exchange chromatography and recovered the neutral, anionic and cationic fractions. The resonances of sugars plus those numbered 8–13 were observed in the 13C-NMR spectrum of the neutral fraction (data not shown). The fact that quercitol (2-deoxy-d-chiro-inositol), usually named ‘acorn-sugar’, is an important constituent of Quercus acorns prompted us to find out whether those resonances, at 33.9, 69.2, 69.5, 71.6, 72.9 and 75.2 p.p.m. of the leaf extracts, were due to the presence of this compound. Indeed, in a 13C-NMR spectrum of the water extract of Q. suber acorns, we detected the resonances of sucrose and solubilized starch and, additionally, only six other resonances, at 33.9, 69.2, 69.5, 71.6, 72.9 and 75.2 p.p.m., coincident with peaks numbered 8–13 in the leaf extracts (Fig. 3B), which we assigned to quercitol.
Concerning the anionic fraction, the 13C-NMR spectrum revealed the peaks corresponding to malate and citrate and those numbered 1–7 in Fig. 3(A). To elucidate the nature of these unassigned peaks, we purified the anionic fraction further by passing through a QAE-Shephadex A25 column. An abundant compound was recovered and subjected to 1H-NMR, COSY, NOESY, HMBC and HMQC (data not shown). Using these techniques, it was concluded that the resonances 1–7 of Fig. 3(A), at 37.6, 41.1, 67.4, 70.9, 75.6, 77.6 and 181.2 p.p.m., corresponded to the 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid (quinic acid).
Changes in leaf organic metabolites along the year
Extracts of Q. suber leaves collected along the year (at about 2-week intervals) were analysed by 13C-NMR in order to monitor the changes that occurred in the contents of the major soluble organic compounds. Figs 4 and 5 show the results obtained from mid-April 1999 until February 2000. Quinic acid, quercitol, sucrose and glucose were the most relevant organic metabolites, but malate, fructose and citrate were also present, although at much lower concentrations. Quinic acid was the most abundant metabolite in the recently formed leaves (mid-April) and remained so for a long period. It reached the maximum of 0.23 mmol g−1 DW during the hot weather period (June to July), after which it started to decline steadily.
Sugars (sucrose, glucose and fructose) were low in recently formed leaves and their concentration rose with time, although less intensely during the summer period (June to September). Sucrose was the predominant sugar and reached the highest concentration (about 0.09 mmol g−1 DW) in autumn-winter, while fructose was always very low. Quercitol was the second most abundant organic metabolite, and its content in recently formed leaves was very high (0.09 mmol g−1 DW), but it steadily declined with time until the hottest month (July). From then onwards, the concentration sharply increased, behaving similarly to sucrose and glucose and reaching the maximum value (0.10 mmol g−1 DW) in autumn-winter.
Malic and citric acids both followed in a similar annual pattern which was distinct and opposite to that observed for quinic acid. Their concentrations were much lower for most of the year, remaining very low during the hot summer period and increasing slightly with the autumn rains. Citrate concentration was then highest (0.04 mmol g−1 DW), but the highest malate concentration (0.05 mmol g−1 DW) had been reached in newly formed leaves.
Changes in leaf mineral contents along the year
Figs 6 and 7 show the leaf contents of the mineral elements N, P, K, Ca, Mg and Na from mid-April 1999 to February 2000. Three distinct patterns of variation were observed: (1) Ca and Mg were low in newly formed leaves and increased steadily with time to reach the maximum value in old leaves; Ca was always at much higher concentration than Mg and it even surpassed the highest value of K; (2) N, P and K were high in recently formed leaves but decreased with age to stabilize during the summer period (July to September), although a slight increase with the autumn rains was apparent for N and P; (3) Na was always at a low concentration but appeared to also slightly increase with the autumn rains.
Relationship between the concentration of leaf constituents, leaf age and climate
Fig. 8 shows the result of a canonical discriminant analysis applied to the data of the leaf constituents (organic metabolites and minerals) for the period from mid-April 1999 to February 2000. It is evident that the data are clustered in three groups that correspond to three distinct seasons of the year: (1) from mid-April to July (spring-early summer period); (2) from July to September (summer period); and (3) from September to February (autumn-winter period). This confirms the existence of a close relationship between the composition of the Q. suber leaves and climate.
When the changes of the two major organic constituents (quinic acid and quercitol) were analysed in more detail, it was found that the pattern of the quinic acid concentrations closely relates to the annual changes of maximum air temperature and insolation (see Fig. 1). Indeed, a positive correlation (coefficient of 0.56 at P < 0.05) was observed between quinic acid and maximum temperature, so that maximal concentration was observed during the hottest period of the year (June/July) (Fig. 5). Quercitol was then at its minimum (Fig. 4), and an inverse relationship between the seasonal concentrations of quercitol and quinic acid was observed, as expressed by a high negative correlation (coefficient of −0.84 at P < 0.001 for the data shown in Figs 4 and 5). Identical results were consistently obtained with other trees. In order to substantiate these observations, we determined the quercitol/quinic acid ratio, which is presented in Table 1 for the T1 and T2 Lisbon trees and in Table 2 for trees at three other locations in the country (Montargil, Coruche, Évora). The ratio for newly formed leaves (April) was already less than one and continued to decrease with time, to reach the lowest value (approximately 0.2) in the hottest period of the year, when quinic acid was highest. During the subsequent autumn and winter period the ratio increased, until leaf fall (March/April) when its value was highest (approximately 10.0). The high contribution made by these two cyclitol derivatives to the leaf osmotic potential (Table 3) further reinforces the importance of these compounds in the Q. suber leaves. During the May to June period, quinic acid contributed 12% and quercitol 2%, while during November to December, the values were approximately 7% and 10%, respectively.
|1998/99||T1||1.77 ± 0.95||3.90 ± 1.10||9.19 ± 0.57||9.91 ± 2.61|
|1999/00||T1||0.53 ± 0.14||0.43 ± 0.07||0.19 ± 0.02||0.33 ± 0.07||0.39 ± 0.06||0.88 ± 0.02||0.77 ± 0.11||1.34 ± 0.03||1.58 ± 0.22||2.73 ± 0.18|
|2004/05||T1||0.66 ± 0.10||0.80 ± 0.02||1.10 ± 0.03||1.03 ± 0.12||3.57 ± 0.49||2.94 ± 0.26||3.30 ± 0.32||4.29 ± 0.70|
|2004/05||T2||0.63 ± 0.09||1.98 ± 0.90||1.39 ± 0.05||1.30 ± 0.06||2.48 ± 0.18||4.36 ± 0.40||6.38 ± 1.75||7.28 ± 1.17|
|Year||Location||Number of trees||May||August||February||April|
|1999||Coruche||2 (C1–C2)||4.56 ± 1.45a|
|2000||Coruche||5 (C3–C7)||4.74 ± 2.61a|
|2002||Montargil||3 (M1–M3)||0.48 ± 0.09|
|2003||Évora||6 (E1–E6)||0.79 ± 0.18|
|Calculated solute osmotic potential (MPa)a and the contribution (%) to the measured leaf osmotic potential|
|Time of the year||Measured leaf osmotic potential (MPa)||Quinic acid||Quercitol||Sucrose||Glucose||Fructose||Malic acid||Citric acid|
|May to June||−2.20 ± 0.03||−0.261||−0.044||−0.033||−0.027||−0.005||−0.027||−0.011|
|November to||−2.02 ± 0.10||−0.150||−0.200||−0.200||−0.125||−0.025||−0.045||−0.075|
Q. suber is usually considered an evergreen tree (Natividade 1950), but most of its leaves only persist for 1 year, the average leaf longevity being 252 days (Pereira et al. 1987). High leaf mortality has been observed as early as the juvenile state (Pereira et al. 1987), and spells of intense leaf shedding in summer due to the stressful arid conditions have also been reported (Natividade 1950). This period is characterized by the conjunction of high insolation (10–12.7 h) and radiation (reaching 2000 µmol m−2 s−1 PAR), persistent elevated temperature (25–30°C), absence of rain and soil water deficit. The decline in leaf ash content and in the specific leaf area observed during this period appears as a result of these stressful conditions. The subsequent increment in leaf ash content following the autumn rains might result from increased mineral absorption associated with the production of new fine roots, as described for Quercus ilex (López et al. 2001).
The quite low mineral concentrations of Q. suber leaves in summer as compared with crop plants are near the lower sufficiency ranges reported (Marschner 1995). However, the values we observed are identical to those reported for Q. suber and other Quercus species (Orgeas and Bonin 1996, Damesin et al. 1998, Rambal 2001) and also for the avocado tree (Castillo-Gonzalez et al. 2000). It is noteworthy that K content of Q. suber leaves remains low in summer. K+ is considered the major osmolyte in agricultural crops, and some trees, but its accumulation in trees in response to water stress is not common (Gebre and Tschaplinski 2002). The importance of Ca2+ for the acid and charge balance should not be neglected because in vacuolated leaf cells a large proportion of Ca2+ is localized in the vacuole, where it contributes to the cation–anion balance (Kinzel 1989). The high Ca content of Q. suber leaves, particularly after June/July, is indicative of its great importance in this tree.
Considering the leaf organic metabolites, it should be noted that organic acids are normally accumulated in plant tissues as metabolically cheap sources of solutes needed for osmotic adjustment and cation balance. In glycophytic plants, malate is the most common organic anion, reaching high levels in many tissues (MacLennan et al. 1963). This acid is considered the natural glycolytic end product, because a high proportion of phosphoenolpyruvate (PEP) formed in glycolysis is diverted for its synthesis (Bryce and ap Rees 1985).
Malate is an important osmoticum during the stress responses of most plants. For instance, during the growing season of Fraxinus excelsior, a close relationship between diurnal variations in leaf water potential and changes in malate, mannitol and K+ levels was observed (Peltier et al. 1998). In Quercus robur, a similar importance was attributed to malate, although in this species, quinic and shikimic acids were reported to be also involved in osmotic balancing (Peltier et al. 1998). What seems peculiar in the Q. suber leaves is the predominance of quinic acid and the low levels of malic acid, which reached the highest concentration in young leaves, but decreased during the most stressful period (June to September) and increased (together with citric acid) in autumn/winter when the concentration of quinic acid was low. Therefore, malate does not seem to be important for Q. suber under drought and the predominant acid is quinic acid, which is one of the early intermediates of the shikimate pathway. It is formed in the chloroplast from dehydroquinate by the action of quinate dehydrogenase (Gamborg 1967, Leuschner et al. 1995) and also from shikimate by quinate hydrolyase (Leuschner et al. 1995, Herrmann and Weaver 1999). This acid can attain relatively high concentrations in angiosperms (Boudet 1973, Yoshida et al. 1975), but its precise metabolic functions in higher plants have not been elucidated. In the gymnosperm Picea abies, quinic acid is intensively produced in the needles in spring and metabolized in summer, apparently participating in lignification processes (Dittrich and Kandler 1971 in Leuschner et al. 1995). The presence of quinic acid in Q. suber has already been reported (Boudet 1973), but our results show a pattern of distribution during the year quite distinct from that described for Q. robur (Peltier et al. 1998). Most striking is the close parallelism which exists between quinic acid concentration in Q. suber leaves and the insolation and temperature data.
In the light of the well-known importance of cyclitols in osmotic adjustment and osmoprotection (Bohnert et al. 1995), the intense Q. suber leaf accumulation of quinic acid (a cyclitol carboxylic acid) during the highly stressfull summer period could be a protective advantage for the tree. Because quinic acid is formed in the chloroplast, its synthesis is dependent on the amount of PEP entering this organelle, and thus on the functioning of the PEP/inorganic phosphate translocator (Fischer et al. 1997, Streatfield et al. 1999), which responds to changes in light intensity (Streatfield et al. 1999).
The very high concentrations of the other cyclitol derivative, quercitol, in the Q. suber leaves during the colder part of the year recall the cryoprotective action of this compound on the cell membranes (Orthen and Popp 2000). It was also shown that the transcription of the inositol O-methyltransferase gene involved in quercitol production is induced by low temperature (Bohnert et al. 1995). Furthermore, quercitol has recently been implicated in osmotic balancing in Eucalyptus species during responses to salt and water deficit stress (Arndt et al. 2005, Merchant and Adams 2005). Hence, quercitol could provide a wide protective action in Q. suber leaves. Considering the structural analogy of quinic acid and quercitol, we suggest that this acid could, possibly, also play some protective role in the Q. suber leaves, particularly in spring-early summer when its concentration is highest and that of quercitol is lowest.
In conclusion, we suggest that the accumulation of specific organic metabolites in the Q. suber leaves in certain periods of the year (most particularly quinic acid and quercitol) could be important for protection against stress. There have been reports on the protective action of several compounds in Quercus species and other trees. For instance, leaf flavonoids and hydroxycinnamates (Tattini et al. 2004), terpenoids (Peñuelas and Llusià 2002, Llusià et al. 2004) and carotenoids (Faria et al. 1996, García-Plazaola et al. 1997) have been associated with responses to drought, light and temperature stress. Our results suggest that quinic acid and quercitol could be added to the list of protective compounds of Quercus leaves. It should be noted that some of these compounds are located in the cytoplasm and others are in the chloroplast and that membrane transport systems are also affected by the environment. It therefore seems important to study the coordinated functioning of the cytoplasm and the chloroplast in response to environment. Particular attention should be given to the chloroplast membrane transporters. The specific participation of the shikimate and inositol pathways in the leaf metabolic interconversions that take place under adverse conditions could be most relevant for survival under stress. Considering the successive years of prolonged dry summers and increasing signs of decline in Q. suber forests and that this species behaves as a drought avoider (Nardini et al. 1999), further studies of leaf metabolism under stress should now be undertaken, so that the precise involvement of the several metabolites in plant survival can be evaluated.
Acknowledgements – We thank Mrs Ma Helena Flores and MaÂngela Pires (EAN) and Mrs Ma Conceição Almeida (ITQB) for performing mineral and elemental analyses; Mrs Ma José Moreira for determination of the osmotic potentials; Professor Francisco Abreu (ISA) for advice; the student Ana Passarinho for the calculations of evapotranspiration; Professor Rui P Ricardo (ISA) for help in collecting environmental data; Professor João Santos Pereira (ISA) and Ing. Ma Lucília Rodrigues for scientific comments and advice; and Dr Phil Jackson for the critical revision of the manuscript. Financial support from FCT (PRAXIS XXI/3/3.2/FLOR/2100/95) and INIA (PAMAF 4027) is acknowledged.
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