Seasonal changes in water relations, photosynthesis and leaf anatomy of two species growing along a natural CO₂ gradient

Oranys Marín, Elizabeth Rengifo, Ana Herrera and Wilmer Tezara

Oranys Marín. Biologist, Universidad Central de Venezuela (UCV). Graduate student, Xerophyte Ecophysiology Laboratory, UCV. Address: Apartado 47114, Caracas 1041A. e-mail: omarin@tyto.ciens.ucv.ve

Elizabeth Rengifo. Biologist, UCV. D.Sc. in Botany, UCV. Researcher, Plant Ecophysiology Laboratory, Ecology Center, Instituto Venezolano de Investigaciones Científicas. e-mail: erengifo@ivic.ve

Ana Herrera: Biologist, UCV. Ph.D. in Plant Sciences, University of London. Professor, Laboratory of Xerophyte Ecophysiology (IBE-UCV). e-mail: aherrera50@yahoo.com.mx.

Wilmer Tezara. Biologist, UCV. D.Sc. in Botany, UCV. Professor, Laboratory of Xerophyte Ecophysiology (IBE-UCV). e-mail: wtezara@strix.ciens.ucv.ve.

 

Resumen

Se realizaron medidas del estado hídrico, intercambio gaseoso y características anatómicas foliares en dos especies (Brownea coccinea y Spatiphylum cannifolium) que crecen naturalmente a lo largo de un gradiente natural de CO₂, desde una concentración supra-atmosférica (CS) de 35000µmol·mol^-1 a una concentración ambiental (CA) de 435µmol·mol^-1, en el bosque ribereño del río Santa Ana (Estado Sucre, Venezuela). Las medidas se realizaron en dos épocas contrastantes, lluvia y sequía. El potencial hídrico (y) fue cerca de 60% mayor en lluvia que en sequía en ambas especies. El crecimiento en CS no ocasionó cambios en el y de B. coccinea, mientras que en S. cannifolium causó un descenso, estos cambios estuvieron acompañados en ambas especies con disminuciones en el potencial osmótico (y_p ) Las elevadas concentraciones de CO₂ causaron una mejora en la fotosíntesis (A) y la conductancia estomática (g_s) de B. coccinea, pero en S. cannifolium no se encontraron diferencias significativas. En las plantas de ambas especies que crecen en CS las hojas eran 10% más gruesas debido a un incremento del grosor de los tejidos del mesófilo en la misma proporción; además, las plantas de S. cannifolium que crecen en CS tiene lugar una mayor deposición de ceras epicuticulares durante la época de sequía.

Summary

In order to gain knowledge on the physiological and anatomical responses of plants to long-term growth under elevated CO₂ concentrations, measurements of water status, leaf gas exchange and leaf anatomical characteristics were made during the rainy and the dry season in plants of Brownea coccinea and Spatiphylum cannifolium growing along a natural CO₂ gradient, from 35000µmol·mol^-1 (supra-atmospheric CO₂ concentration, SC) to 435µmol·mol^-1 (ambient CO₂, AC) in the riparian forest of the Santa Ana river, Sucre State, Venezuela. Water potential (y) was higher during the rainy than the dry season in both species. Growth under SC did not cause changes in y of B. coccinea, whereas it decreased it in S. cannifolium; both drought and growth under SC decreased leaf sap osmotic potential (yp) in both species, helping maintain turgor pressure in B. coccinea but not in S. cannifolium. Elevated CO₂ increased photosynthetic rate (A) and leaf conductance (g_s) of B. coccinea under drought but no effect was found in S. cannifolium. Leaves of both species growing under SC were 10% thicker than under AC, owing to a proportional increase in the thickness of the mesophyll tissues; additionally, during the dry season a higher deposition of epicuticular waxes took place on leaves of S. cannifolium growing under SC. Changes in photosynthetic rate, leaf conductance and leaf anatomy suggest acclimation to growth under elevated CO₂.

Resumo

Realizaram-se medidas do estado hídrico, intercambio gasoso e características anatômicas foliares em duas espécies (Brownea coccinea e Spatiphylum cannifolium) que crescem naturalmente ao longo de um gradiente natural de CO₂, desde uma concentração supra-atmosférica (CS) de 35.000mmol·mol^-1 a uma concentração ambiental (CA) de 435mmol·mol-1, no bosque marginal do rio Santa Ana (Estado Sucre, Venezuela). As medições se realizaram em duas épocas contrastantes, chuva e seca. O potencial hídrico (Y) foi aproximadamente 60% maior na chuva que na seca em ambas espécies. O crescimento em CS não ocasionou mudanças no Y de B. coccinea, enquanto que no S. cannifolium causou um descenso, estas mudanças estiveram acompanhadas em ambas espécies com diminuições no potencial osmótico (Yð?) As elevadas concentrações de CO₂ causaram uma melhora na fotossínteses (A) e a condutância estomática (g_s) de B. cocinea, mas em S. cannifolium não se encontraram diferenças significativas. Nas plantas de ambas espécies que crescem em CS as folhas eram 10% mais grossas devido a um incremento na espessura dos tecidos do mesófilo na mesma proporção; além disso, nas plantas de S. cannifolium que crescem em CS acontece uma maior deposição de ceras epicuticulares durante a época da seca.

KEYWORDS / Brownea coccinea / Leaf Anatomy / Photosynthesis / Spatiphylum cannifolium / Supra-atmospheric CO₂ Concentration /

Received: 09/07/2004. Modified: 12/04/2004. Accepted: 12/08/2004.

Introduction

The indiscriminate use of fossil fuels, the emission of chlorofluorocarbonate compounds and the change of land use from forests to cultivated lands have brought about an increase in atmospheric CO₂ concentration from 290µmol·mol^-1 during the second half of the 60s to 360µmol·mol^-1 at present (Houghton et al., 2001). This continued increase could eventually have an impact on the growth of wild plants and crops (Tipping and Murray, 1999).

The increase in atmospheric CO₂ concentration has resulted in an increase in global temperature, which will probably alter the global rain patterns (Houghton et al., 2001; Lawlor, 2001); this, together with an increased evapotranspiration, will produce a severe water deficit, thus affecting photosynthesis and growth (Chaves and Pereira, 1992).

Leaf anatomical characteristics are sensitive to changes in environmental variables. For example, structure, shape and some functions of the leaf may change (Long, 1998; Pritchard et al., 1998) in response to elevated CO₂. The leaf is a key organ for transpiration and photosynthesis; therefore, leaf morphology and anatomy must strongly influence physiological processes (Parkhurst, 1986; Sims et al., 1998). Changes in the number of cell layers in the mesophyll and/or cell dimensions affect photosynthesis (A), and modify internal CO₂ conductance and, consequently, water-use efficiency, i.e. the ratio A/transpiration rate (Romero-Aranda et al., 1997; Mediavilla et al., 2001).

In a number of experiments with CO₂-enriched atmospheres a large variation in the photosynthetic response has been found in C3 plants, from increases to decreases to no change (Drake et al., 1997). Nevertheless, only a few studies have evaluated the response of leaf gas exchange to long-term growth under naturally elevated CO₂; even fewer studies have addressed the issue of changes in leaf anatomy under these conditions.

Most studies have addressed the short-term plant response to elevated CO₂ concentrations. Even though some studies have been followed for longer periods (over one year), the response to chronic exposure to elevated CO₂ for generations is not well known, with a few exceptions (Miglietta and Raschi, 1993). Studying an ecosystem with an atmosphere naturally enriched in CO₂ provides the opportunity to study long-term responses to elevated CO₂ in a natural vegetation (Miglietta and Raschi, 1993).

On the site of the present study two natural cold sources of CO₂ exist (Hevia and Di Gianni, 1983). They consist of water-filled circular holes in the ground 0.3 and 0.8m in diameter each. The CO₂ molar fraction in the air above the sources is relatively constant during the day and decreases along a 24m long transect from 27000 (small source) and 35000 (large source) down to 435µmol·mol^-1 (Fernández et al., 1998). These authors reported that in plants growing in the proximity or at a distance from these two sources, stomatal characteristics varied with CO₂ concentration; stomatal density and index were 70% lower on the adaxial surface and 40% higher on the abaxial surface of leaves of Spatiphylum cannifolium growing near the sources, while in Bauhinia multinervia both stomatal density and index decreased more than 70% on the adaxial surface, whereas no changes due to elevated CO₂ were found on the abaxial surface.

The aim of the present study was to gain knowledge on the effects of elevated CO₂ concentration in a natural gradient on the water relations, leaf gas exchange and anatomy of a tree (Brownea coccinea) and an herb (S. cannifolium). Measurements were made in the field in two contrasting seasons, rainy and dry.

Materials and Methods

Field site and plant material

The study was conducted in the semi-evergreen forest (Huber and Alarcón, 1988) of the Santa Ana river (El Pilar, Municipio Benítez, Estado Sucre, Venezuela), located at 10º35'N and 63º08'W. Field trips were made, in May 2001 (dry season) and August 2001 (rainy season). The species studied were Brownea coccinea (Jacq.) Velasq. & Agostini (Caesalpinaceae), a 3-5m tall tree, and Spatiphylum cannifolium (Dryand.) Schatt. (Araceae), a 75cm tall herb which generally grows on the river bank. Individuals of both species were found growing near the supra-atmospheric CO₂ sources (SC) and 20m away from the sources, under atmospheric CO₂ concentrations (AC).

Microclimatic variables

Photosynthetic photon flux density (PPFD) was measured using a quantum sensor LI- 250 (LI-COR Biosciences, Lincoln, NE; USA); air temperature by means of a telethermometer (Yellow Springs Instruments, Yellow Springs, OH, USA); and relative humidity using a hygrometer AB167B (Abbeon Cal, Santa Barbara, CA, USA). Soil water content (SWC) was determined in samples taken at a 30cm depth near the sources and 20m away, weighed fresh (FM), oven-dried at 60ºC for 48h and weighed again (DM), and expressed as SWC=100(FM-DM)/DM.

Plant water status

Water potential (y) was measured at 06:00 (n=3) using a pressure chamber PMS200 (PMS Instruments, Corvallis, OR, USA). Leaf sap osmotic potential (y_p) was determined in the sap extracted from leaves previously used for y determination, frozen in liquid N2 and thawed and measured with a mod. 5500 osmometer (Wescor, Logan, UT, USA). Pressure potential was calculated as P=y-y_s.

Leaf gas exchange

For the measurements (n=3) of photosynthesis (A) and leaf conductance (g_s) an infrared gas analyzer CIRAS 1 connected to a PLC(B) assimilation chamber (PP Systems, Hitchin, UK) was used. Intrinsic water-use efficiency was calculated as IWUE=A/g_s. Measurements were made at 350 and 1000µmol·mol^-1 CO₂ under natural illumination; when PPFD<500µmol·m-²·s^-1 light from a halogen lamp was shone on the assimilation chamber.

Leaf anatomy

Samples (n=5) consisted of youngest fully expanded leaves exposed to similar conditions of irradiance and CO₂ concentration placed in a mixture of formol:acetic acid:ethanol (5:5:90). Leaves were hand-sectioned, clarified with 10% commercial bleach, stained with toluidine blue and semi-permanently mounted on slides with phenolated 50% glycerin. Observations were carried out on five fields per slide under a binocular microscope (Leica, DMLS, Sweden).

Statistics

The statistical package Statistica 4.0 was used to perform one- or two-way analysis of variance (ANOVA); significance was assessed at p<0.05. Plot adjustments and regressions were done using the SigmaPlot 5.0 package.

Results

Water relations

Two clearly identifiable seasons occur in the Santa Ana forest, a dry season from Dec to May and a rainy season spanning from Jun to Nov (Figure 1). Daily average air temperature was 24-34ºC and relative humidity (RH) was 45-95%. The highest photosynthetic photon flux density (PPFD) was 1500µmol·m-²·s^-1 during the dry season and 70µmol·m-²·s^-1during the rainy season, this lower value being due to a higher canopy cover (Figure 2). The soil water content (SWC; Figure 3) was higher during the rainy than the dry season on both sources, values on the large source being higher at 0 than at 20m from it on both seasons; no significant differences in SWC along the transect of the small source were found at any season.

Seasonal changes in the parameters of water relations are shown in Table I. The y values for both species were higher during the rainy than the dry season, as expected, but y was lower in SC than in AC during the dry season. The values of y_p for plants of both species were higher during the rainy than the dry season, and higher under AC than SC. Values of P in B. coccinea did not show significant differences due to either season or CO₂ concentration but turgor was maintained during the dry season. In contrast, plants of S. cannifolium growing under AC had higher values of P during the dry season, when P£0, than plants growing under SC; these negative values are possibly due to an artifact created by dilution with apoplastic water of the cellular contents.

Leaf gas exchange

In Figure 4 the changes in leaf gas exchange parameters of plants of B. coccinea due to growth and measurement CO₂ concentrations, and season, are compared. Measurements during the rainy season were made at 350 and 1000µmol·mol^-1 CO₂, and only at 350 µmol·mol^-1 CO₂ during the dry season; measurements at 1000µmol·mol^-1 CO₂ could not be made during the dry season because of the extremely low g_s. Values of A, g_sand IWUE measured during the rainy season at 350µmol·mol^-1 CO₂ were not significantly different between SC and AC. During the rainy season, A measured at 1000µmol·mol^-1 CO₂ in SC plants was 11 times higher than A measured at 350µmol·mol^-1 CO₂ and twice as high as A measured in AC plants at 1000µmol·mol^-1 CO₂. During the dry season A measured at 350µmol·mol^-1 CO₂ was five times higher in SC than in AC. No statistically significant differences due to growth CO₂ concentration were found during the rainy season in g_s measured at either 350 or 1000µmol·mol^-1 CO₂, whereas during the dry season g_s measured at 350µmol·mol-1 CO₂ was four times higher in SC than in AC. The IWUE was consequently higher in SC than in AC during the rainy season, no differences occurring between SC and AC during the dry season.

Similarly to B. coccinea, in plants of S. cannifolium values of A, g_s and IWUE measured during the rainy season at 350µmol·mol^-1 CO₂ were not significantly different between SC and AC (Fig. 5). Also, the A measured during the rainy season at 1000 relative to 350µmol·mol^-1 was four times higher in SC as well as AC; during the dry season A was slightly higher in AC than in SC. No differences due to either measurement or growth CO₂ level, nor to season, were found in g_s. Due to differences in A, IWUE during the rainy season was ten times higher in SC plants measured at 1000 than at 350µmol·mol^-1 CO₂ and ten times higher during the rainy than the dry season. The same holds for plants growing at AC, except that values were four times higher. No differences due to growth CO₂ level were found in values of IWUE measured at 350µmol·mol^-1 during the dry season.

Leaf anatomy

Both season and growth CO₂ concentration had effects on total leaf and tissue thickness. Leaves of both species growing under SC were nearly 10% thicker than those of plants growing under AC, regardless of season (Figure 6, Table II). The number of mesophyll cell layers did not change with either season or CO₂ level; in both species the epidermes as well as the palisade parenchyma had one cell layer. The spongy parenchyma had three and seven cell layers in B. coccinea and S. cannifolium, respectively.

 

In B. coccinea the palisade parenchyma in leaves was 10% thicker during the rainy than the dry season but no differences due to CO₂ concentration were found. The spongy parenchyma was thicker under SC than AC, with no effect of CO₂ level. Both epidermes in the two growth CO₂ concentrations were 10% thicker during the rainy than the dry season, the lower epidermis being 5% thicker under AC than SC. The thickness of both cuticles was unresponsive to either season or CO₂.

In leaves of S. cannifolium no significant differences in palisade parenchyma due to either season or CO₂ were found. The spongy parenchyma was 12% thicker in plants growing under SC than AC, regardless of season. No differences due to either CO₂ or season were found in the thickness of both epidermes. Both cuticles were thicker in SC than AC plants during the dry season.

Discussion

Drought caused a reduction in y of both species, whereas growth CO₂ concentration did not affect it in B. coccinea and diminished it in S. cannifolium. These responses of y to seasonal period were accompanied by decreases in y_p  of both species. The differences in SWC did not help explain the changes in y of S. cannifolium, suggesting that the decrease of y in elevated CO₂ are due to a negative effect of SC on the water status. Values of y were higher in wild plants growing near natural sources of CO₂ (Tognetti et al., 1996, 1999, 2000); in contrast, elevated CO₂ had a negative impact on y of several species (Bunce, 1996; Centritto et al., 1999; De Luis et al. 1999). Growth CO₂ concentration did not affect y or y_p  of the xerophytes Alternanthera crucis and Jatropha gossypifolia (Rengifo et al., 2002) or of Quercus ilex (Tognetti et al., 1996). Turgor pressure, though, was as high during the dry season in plants of B. coccinea growing under SC than under AC, suggesting that osmotic adjustment under drought occurred and that elevated CO₂ ameliorated plant water status. Since no amelioration was observed in S. cannifolium, our results corroborate that there is not a clear trend in the effect of CO₂ concentration on the parameters of water relations.

Leaf gas exchange

Elevated CO₂ caused an increase in A of B. coccinea and S. cannifolium, indicating a positive acclimation of photosynthesis. Similar results have been found in B. multinervia and S. cannifolium (Fernández et al., 1998), Cercis canadensis, Acer rubrum, Carya glabra and Liquidambar styracicflua (DeLucia and Thomas, 2000) and Prunus avium (Centritto et al., 1999). Contrasting evidence was found in Quercus petraea, in which A did not change in response to elevated CO₂ during growth (Epron et al., 1994). The occurrence in both species growing at SC of higher A measured at ambient CO₂ than in AC indicates a positive acclimation.

The g_s of plants measured during the rainy season was unresponsive to growth CO₂ concentration, but during the dry season g_s measured at ambient CO₂ was higher at SC than at AC in plants of B. coccinea, again evidencing acclimation. Although stomatal closure has been recognized as a direct effect of elevated CO₂ (Hogan et al., 1991), it has been shown that the stomatal response to increases in growth CO₂ concentration is very variable, sensitivity varying among species (Eamus and Jarvis, 1989). The g_s was lower under elevated CO₂ in plants of cassava (Fernández et al., 2002), whereas it was higher in plants of Ipomoea carnea, Talinum triangulare (Fernández et al., 1999) and Fagus sylvatica (Heath and Kersteins, 1997). The increase in g_s measured at ambient CO₂ of plants of B. coccinea growing at elevated CO₂ did not result in an increase in water saving, since IWUE was lower in both growth CO₂ levels as compared to the rainy season. A similar situation was found in S. cannifolium, in which a lack of change in g_s with growth and measurement CO₂ levels and season did not result in a higher IWUE of droughted plants in SC. The acclimation to growth CO₂ concentration of the leaf gas exchange response in both species during the dry season may be evidence of changes in the developmental stage, rather than in water status.

Leaves of both species growing at SC were 10% thicker than at AC, similarly to needles of Pinus sylvestris growing experimentally under elevated CO₂ (Lin et al., 2001); in B. coccinea this increase owed to a proportional increase in the thickness of the mesophyll, while in S. cannifolium it was due solely to an increased spongy parenchyma. In leaves of soybean an increase in leaf thickness under elevated CO₂ was the result of an increased mesophyll, mostly the spongy parenchyma (Sims et al., 1998). The higher values of A in plants of B. coccinea at SC during the dry season than the rainy season are probably the result of developmental changes that took place after the end of the rainy season and increased mesophyll conductance. In the case of a higher A in plants of S. cannifolium at AC during the dry season relative to the rainy season developmental changes may have taken place independently of CO₂ concentration.

In S. cannifolium thickness of both cuticles increased with growth CO₂ concentration and drought. In P. palustris elevated CO₂ increased cuticle thickness in plants growing in spring with a low N supply (Prior et al., 1997) and in soybean it increased cuticle thickness (Thomas and Harvey, 1983), whereas in Agave deserti elevated CO₂ decreased it (Graham and Nobel, 1996). These results highlight both the variability in the deposition of epicuticular waxes in response to elevated CO₂ and its dependence on different environmental factors. In the case of plants of S. cannifolium at SC during the dry season an increase in cuticle thickness may help prevent further desiccation.

ACKNOWLEDGEMENTS

This work was financed by grants FONACIT S1-99000054 and CDCH-UCV 03-33-4342-2000.

REFERENCES

1. Bunce J (1996) Growth at elevated carbon dioxide concentration reduces hydraulic conductance in alfalfa and soybean. Global Change Biol. 2: 155-158.        [ Links ]

2. Centritto M, Lee H, Jarvis P (1999) Interactive effects of elevated [CO₂] and drought on cherry (Prunus avium) seedling_s. I Growth, whole-plant water use efficiency and water loss. New Phytol. 141: 129-140.        [ Links ]

3. Chaves MM, Pereira JS (1992) Water stress, CO₂and climate change. J. Exp. Bot. 43: 1131-1139.        [ Links ]

4. De Luis I, Irigoyen J, Sánchez-Díaz M (1999) Elevated CO₂ enhances plant growth in droughted N2-fixing alfalfa without improving water status. Physiol. Plant. 107: 94-89.        [ Links ]

5. DeLucia E, Thomas R (2000) Photosynthetic responses to CO₂ enrichment of four hardwood species in a forest understory. Oecologia 122: 11-19.        [ Links ]

6. Drake BG, González-Meler MA, Long SP (1997) More efficient plants: a consequence of rising atmospheric CO₂? Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 609-639.        [ Links ]

7. Eamus E, Jarvis G (1989) The direct effects of increases in the global atmospheric CO₂ concentration on natural and commercial temperate trees and forests. Adv. Ecol. Res. 19:1-47.        [ Links ]

8. Epron D, Dreyer E, Picón C, Guehl J (1994) Relationship between CO₂-dependent O₂ evolution and photosystem II activity in oak (Quercus petraea) trees grown in ambient or elevated CO₂. Tree Physiol. 14: 725-733.        [ Links ]

9. Fernández MD, Pieters A, Donoso C, Tezara W, Azkue M, Herrera C, Rengifo E, Herrera A (1998) Effects of a natural source of very high CO₂ concentration on the leaf gas exchange, xylem water potential and stomatal characteristics of plant of Spatiphylum cannifolium and Bauhinia multinervia. New Phytol. 138: 689-697.        [ Links ]

10. Fernández MD, Pieters A, Azkue M, Rengifo E, Tezara W, Woodward FI, Herrera A (1999) Photosynthesis in plants of four tropical species growing under elevated CO₂. Photosynthetica 37: 587-599.        [ Links ]

11. Fernández MD, Tezara W, Rengifo E, Herrera A (2002) Lack of downregulation of photosynthesis in a tropical root crop, cassava, grown under elevated CO₂ concentration. Funct. Plant Biol. 29: 805-814.        [ Links ]

12. Graham EA, Nobel PS (1996) Long-term effects of a doubled atmospheric CO₂ concentration on the CAM species Agave deserti. J. Exp. Bot. 47: 61-69.        [ Links ]

13. Heath J, Kersteins G (1997) Effects of elevated CO2 on leaf gas exchange in beech and oak at two levels of nutrient supply: consequences for sensitivity to drought in beech. Plant Cell Envir. 20: 57-67        [ Links ]

14. Hevia A, Di Gianni N (1983) Inventario geotérmico del Edo. Sucre. Tesis. Universidad Central de Venezuela. Caracas. Venezuela. 220 pp.        [ Links ]

15. Hogan K, Smith A, Ziska L (1991) Potential effects of elevated CO₂ and changes in temperature on tropical plants. Plant Cell Envir. 14: 763-778.        [ Links ]

16. Houghton JT, Ding Y, Grigg_s DJ, Noguer M, van der Linden PJ, Xiaosu (2001) Climate Change 2001. Intergovernmental Panel on Climate Change. Cambridge University Press. London, UK. 944 pp.        [ Links ]

18. Huber O, Alarcón C (1988) Mapa de vegetación de Venezuela. Ministerio de los Recursos Naturales Renovables y Conservación de la Naturaleza. Oscar Todtmann. Caracas, Venezuela.        [ Links ]

19. Lawlor DW (2001) Photosynthesis. Bios. Oxford, RU. 398 pp.        [ Links ]

20. Lin J, Jach M, Ceulemans R (2001) Stomatal density and needle anatomy of Scots pine (Pinus sylvestris) are affected by elevated CO₂. New Phytol. 150: 665-674.        [ Links ]

21. Long SP (1998) Understanding the impact of rising CO₂: contribution of environmental physiology. In Press M, Scholes M, Baker M (Eds.) Physiological Plant Ecology. Blackwell. Oxford, UK. pp. 263-282.        [ Links ]

22. Mediavilla S, Escudero A, Heilmeier H (2001) Internal leaf and photosynthetic resource-use efficiency: interspecific and intraspecific comparisons. Tree Physiol. 21: 251-259.        [ Links ]

23. Miglietta F, Raschi A (1993) Studying the effect of elevated CO₂ in the open in a naturally enriched environment in central Italy. Vegetatio 104/105: 391-400.        [ Links ]

24. Parkhurst D (1986) Internal leaf structure: a three dimensional perspective. In Givnish T (Ed.) On the Economy of Plant Form and Function. Cambridge University Press. London, UK. pp. 215-249.        [ Links ]

25. Prior SA, Pritchard SG, Runion GB, Rogers HH, Mitchel RJ (1997) Influence of atmospheric CO₂ enrichment, soil N, and water stress on needle surface wax formation in Pinus palustris (Pinaceae). Am. J. Bot. 84: 1070-1077.        [ Links ]

26. Pritchard SG, Mosjidis C, Peterson CM, Brett G, Rogers HH (1998) Anatomical and morphological alterations in longleaf pine needles resulting from growth in elevated CO₂: Interactions with soil resource availability. Internat. J. Plant Sci. 159: 1002-1009.        [ Links ]

27. Romero-Aranda R, Bondada BR, Syvertsen JP, Grosser JW (1997) Leaf characteristics and net gas exchange of diploid and autotetraploid citrus. Ann. Bot. 79: 153-160        [ Links ]

28. Rengifo E, Urich R, Herrera A (2002) Water relations and leaf anatomy of the tropical species, Jatropha gossypifolia and Alternanthera crucis, grown under an elevated CO₂ concentration. Photosynthetica 40: 397-403.        [ Links ]

29. Sims D, Seemann J, Luo Y (1998) Elevated CO₂ concentration has independent effects on expansion rates and thickness of soybean leaves across light and nitrogen gradients. J. Exp. Bot. 49: 583-591.        [ Links ]

30. Thomas J, Harvey C (1983) Leaf anatomy of four species grown under continuous CO₂ enrichment. Bot. Gaz. 144: 303-309.        [ Links ]

31. Tipping C, Murray DR (1999) Effects of elevated atmospheric CO₂ concentration on leaf anatomy and morphology in Panicum species representing different photosynthetic modes. Internat. J. Plant Sci. 160: 1063-1073.        [ Links ]

32. Tognetti R, Giovannelli A, Longobucco A, Miglietta F, Raschi A (1996) Water relations of oak species growing in the natural CO₂ spring_s of Rapolano (Central Italy). Ann. Forestry Sci. 53: 475-485.

33. Tognetti R, Giovannelli A, Longobucco A, Miglietta F, Raschi A (1999) Water relations, stomatal response and transpiration of Quercus pubescens trees during summer in a mediterranean carbon dioxide spring. Tree Physiol. 19: 261-270.        [ Links ]

34. Tognetti R, Minnocci A, Peñuelas J, Raschi A, Jones M (2000) Comparative field water relations of three Mediterranean shrub species co-occurring at natural CO₂ vent. J. Exp. Bot. 51: 1135-1146.        [ Links ]