Plant and Soil 230: 175–183, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 175 Comparison of proline accumulation in two mediterranean shrubs subjected to natural and experimental water deficit F. Ain-Lhout, M. Zunzunegui 1 , M.C. Diaz Barradas, R. Tirado, A. Clavijo & F. Garcia Novo Departamento de Biolog´ ıa Vegetal y Ecolog´ ıa. Universidad de Sevilla. Apartado 1095. 41080 Sevilla. Spain 1 Corresponding author ∗ Received 9 November 1999. Accepted in revised form 6 November 2000 Key words: drought, Halimium halimifolium, leaf water potential, Pistacia lentiscus, plant responses, stomatal resistance Abstract The effect of water stress on proline accumulation was tested in two contrasted species of Mediterranean scrub: Halimium halimifolium (L.) Willk and Pistacia lentiscus L. Leaf water potential, stomatal resistance and proline content have been measured both in experimental and in natural water stress conditions. Both species accumulated proline in their leaves when leaf water potential dropped below a threshold value of −3.0 MPa, under natural as well as under experimental conditions. In the field, however, a time-lag between decrease of leaf water potential and proline accumulation could be observed. In Halimium halimifolium, proline accumulation appeared to be associated with severe stress conditions as most plants with high proline contents suffered irreversible wilting, especially in the greenhouse. P. lentiscus showed a different pattern, accumulating proline at two different times of the year, as a response to cold or to drought. The results of our study indicated that the role of proline in this species, rather than an osmotic agent, seems to be more related to a protective action in cases of severe stress conditions. Introduction Under Mediterranean type climate, the performance and distribution of shrub species depend on their abil- ity to withstand drought, high temperatures and strong irradiance (Poole and Miller, 1975). Under these cli- matic conditions, two functional types of shrub species persist (Oliveira and Peñuelas, 2000; Verdú, 2000; Werner et al., 1998), having received different names: evergreen sclerophylls, chaparral, maquis, Herrera’s type II (1984, 1992); versus drought semi-deciduous, malacophyllous, coastal sage scrub, phrygana, garriga and Herrera’s type I. Semi-deciduous species partially avoid water stress through major reductions of their foliage area, thus restricting their growth to the more favourable seasons. In contrast, evergreen sclerophylls go through the stress conditions with intact green leaves, by ex- hibiting a variety of morphological adaptations such ∗ FAX No: +34-954626308. E-mail: zunzu@cica.es as small, highly sclerophyllous leaves, thick cuticles, deep root systems and effective stomatal control of water loss (Margaris, 1981; Mooney, 1981; Werner et al., 1998). The increase of proline levels during drought stress is unique compared to other free amino acids in the same tissue (Aspinall and Paleg, 1981; Handa et al., 1983; Sivaramakrishnan et al., 1988), although similar to other low molecular weight solutes such as sugars and organic acids (Ford, 1984; Newton et al., 1986; Sivaramakrishnan et al., 1988). These compounds may act as osmotic solutes (Hare et al., 1998). Proline may protect protein structure and mem- branes from damage, and to reduce enzyme denatur- ation (Iyer and Caplan, 1998; Rajendrakumar et al., 1994; Saradhi et al., 1995; Smirnoff and Cumbes, 1989). Alternatively, proline accumulation has also been proposed to be nitrogen storage (Gil, 1995; Hare et al., 1998; Larcher, 1995). Under the stressing summer conditions of Medi- terranean climate (high temperature and irradiance,