Responses of five Mediterranean halophytes to seasonal changes in environmental conditions

Ricardo Gil 2 Inmaculada Bautista 1 Monica Boscaiu 0 Antonio Lido n 1 Shantanu Wankhade 2 He ctor Sa nchez 2 Josep Llinares 3 Oscar Vicente 2 Guest Editor: Adele Muscolo 0 Instituto Agroforestal Mediterra neo (IAM), Universitat Polite`cnica de Vale`ncia , Camino de Vera s/n, 46022 Valencia, Spain 1 ReForest, Departamento de Ingeniera Hidra ulica y Medio Ambiente, Universitat Polite`cnica de Vale`ncia , Camino de Vera s/n, 46022 Valencia, Spain 2 Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Universitat Polite`cnica de Vale`ncia , Camino de Vera s/n, 46022 Valencia, Spain 3 Instituto de Investigacio n para la Gesti on Integral de Zonas Costeras (IGIC), Universitat Polite`cnica de Vale`ncia , C/Paranimf 1, 46730 Grao de Gandia, Valencia, Spain In their natural habitats, different mechanisms may contribute to the tolerance of halophytes to high soil salinity and other abiotic stresses, but their relative contribution and ecological relevance, for a given species, remain largely unknown. We studied the responses to changing environmental conditions of five halophytes (Sarcocornia fruticosa, Inula crithmoides, Plantago crassifolia, Juncus maritimus and J. acutus) in a Mediterranean salt marsh, from summer 2009 to autumn 2010. A principal component analysis was used to correlate soil and climatic data with changes in the plants' contents of chemical markers associated with stress responses: ions, osmolytes, malondialdehyde (MDA, a marker of oxidative stress) and antioxidant systems. Stress tolerance in S. fruticosa, I. crithmoides and P. crassifolia (all succulent dicots) seemed to depend mostly on the transport of ions to aerial parts and the biosynthesis of specific osmolytes, whereas both Juncus species (monocots) were able to avoid accumulation of toxic ions, maintaining relatively high K+/Na+ ratios. For the most salt-tolerant taxa (S. fruticosa and I. crithmoides), seasonal variations of Na+, Cl2, K+ and glycine betaine, their major osmolyte, did not correlate with environmental parameters associated with salt or water stress, suggesting that their tolerance mechanisms are constitutive and relatively independent of external conditions, although they could be mediated by changes in the subcellular compartmentalization of ions and compatible osmolytes. Proline levels were too low in all the species to possibly have any effect on osmotic adjustment. Howeverexcept for P. crassifoliaproline may play a role in stress tolerance based on its 'osmoprotectant' functions. No correlation was observed between the degree of environmental stress and the levels of MDA or enzymatic and non-enzymatic antioxidants, indicating that the investigated halophytes are not subjected to oxidative stress under natural conditions and do not, therefore, need to activate antioxidant defence mechanisms. Introduction Soil salinity is, together with drought, one of the most important environmental conditions that reduce crop yields worldwide and determine the distribution of wild plants in nature (Boyer 1982; Bartels and Sunkar 2005; Watson and Byrne 2009). Studies of plant responses to saltas well as to other abiotic stress factorsand the elucidation of stress tolerance mechanisms have become one of the most active areas of research in plant biology due to their academic and practical interest. There is now substantial evidence that all plants react against adverse environmental conditions by activating a series of conserved responses which are common to different abiotic stresses. One of these basic stress responses involves ion homoeostasis and the maintenance of osmotic balance to counteract cellular dehydration caused by, for example, high soil salinity, drought, cold or high temperatures: limitation of water losses, sequestration of toxic ions in the vacuole (according to the so-called ion compartmentalization hypothesis; Flowers et al. 1977; Wyn Jones et al. 1977; Glenn et al. 1999) and synthesis and accumulation of compatible solutes or osmolytes in the cytoplasm (Munns and Termaat 1986; Zhu 2001; Munns and Tester 2008; Kronzucker and Britto 2011). These latter compounds, apart from contributing to osmotic adjustment, play osmoprotectant roles by acting as low-molecular-weight chaperones to stabilize proteins, membranes and other macromolecular structures under stressful conditions, and also as scavengers of reactive oxygen species (ROS) (Ashraf and Foolad 2007; Chen and Murata 2008; Flowers and Colmer 2008; Hussain et al. 2008; Szabados and Savoure 2010). Most stressful environmental factors cause oxidative stress in plants through generation of ROS; consequently, another fundamental and conserved response to abiotic stress consists of the activation of enzymatic and non-enzymatic antioxidant systems to avoid or reduce oxidative damage of proteins, membranes and DNA (Apel and Hirt 2004; Halliwell 2006; Miller et al. 2008; Turkan and Demiral 2009). Paradoxically, the vast majority of studies on salt stress responses and salt tolerance mechanisms have been performed using glycophytes (salt-sensitive plants), many with the model species Arabidopsis thaliana (e.g. Zhu 2000, 2002; Koiwa et al. 2006; Horie et al. 2009; and references therein). Nevertheless, there is a growing interest in the study of salt-tolerant plantshalophyteswhich a priori seem to be more appropriate models to elucidate these mechanisms. Research on halophytes responses to salt stress has indeed increased in recent years and has provided information on the molecular, biochemical and physiological bases of their tolerance to high soil salinity, which appear to rely on the activation of the aforementioned general stress responses also used by salt-sensitive species, albeit with much lower efficiency. Yet important aspects of these mechanisms remain largely unknown, especially regarding the ecological relevance of distinct response mechanisms and their relative contribution to salt tolerance in particular halophytic taxa. Most studies on halophytes behaviour under high salinity conditions have been conducted in artificial laboratory or greenhouse environments, with the obvious advantage of allowing strict experimental control, but which cannot reflect the real conditions of plants in nature. In their natural habitats, halophytes must react dynamically to changing, uncontrollable environmental conditions. Halophytes must cope simultaneously with different abiotic stresses, not only soil salinity, which may activate the same, overlapping and/or specific responses, interacting in complex ways rather than showing simple additive effects (Ungar 1991; Krasensky and Jonak 2012; Ben Hamed et al. 2013). For these reasons, the interpretation of data obtained in the field is much more difficult than the analysis of laboratory results. It is, therefore, not surprising that very few studies have been published dealing with the stress responses of halophytes under varying environmental conditions in their natural ecosyste (...truncated)