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+, Cl−, 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. However—except for P. crassifolia—proline 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.

Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus–both halophytes–and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na⁺ and Cl^- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K⁺ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K⁺ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600–800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K⁺ transport to the leaves of the plants.

Osmolytes, used for maintaining osmotic balance and as ‘osmoprotectants’, are synthesized in plants as a general, conserved response to abiotic stress, although their contribution to stress-tolerance mechanisms remains unclear. Proline, the most common osmolyte, accumulates in many plant species in parallel with increased external salinity and is considered a reliable biochemical marker of salt stress. We have measured proline levels in two halophytic, closely related Juncus species under laboratory and field conditions to assess the possible relevance of proline biosynthesis for salt tolerance and therefore for the ecology of these two taxa. Proline was quantified in plants treated with increasing NaCl concentrations and in plants sampled in two salt marshes located in the provinces of Valencia and Alicante, respectively, in southeast Spain. Electrical conductivity, pH, Na⁺ and Cl⁻ concentrations were measured in soil samples collected in parallel with the plant material. Treatment with NaCl inhibited growth of J. acutus plants in a concentration-dependent manner, but only under high salt conditions for J. maritimus. Salt treatments led to proline accumulation in both species, especially in the more salt-tolerant J. maritimus. The results, obtained under laboratory conditions, were confirmed in plants sampled in the field. In all the samplings, proline contents were significantly lower in J. acutus than in the more tolerant J. maritimus growing in the same area. No direct correlation between soil salinity and proline levels could be established, but seasonal variations were detected, with increased proline contents under accentuated water deficit conditions. Our results suggest that proline biosynthesis is not only an induced, general response to salt stress but also an important contributing factor in the physiological mechanisms of salt tolerance in Juncus, and that it therefore correlates with the ecology of both species.

Limonium is a genus represented in the Iberian Peninsula by numerous halophytic species that are affected in nature by salinity, and often by prolonged drought episodes. Responses to water deficit have been studied in four Mediterranean Limonium species, previously investigated regarding salt tolerance mechanisms. The levels of biochemical markers, associated with specific responses—photosynthetic pigments, mono- and divalent ions, osmolytes, antioxidant compounds and enzymes—were determined in the control and water-stressed plants, and correlated with their relative degree of stress-induced growth inhibition. All the tested Limonium taxa are relatively resistant to drought on the basis of both the constitutive presence of high leaf ion levels that contribute to osmotic adjustment, and the stress-induced accumulation of osmolytes and increased activity of antioxidant enzymes, albeit with different qualitative and quantitative induction patterns. Limonium santapolense activated the strongest responses and clearly differed from Limonium virgatum, Limonium girardianum, and Limonium narbonense, as indicated by cluster and principal component analysis (PCA) analyses in agreement with its drier natural habitat, and compared to that of the other plants. Somewhat surprisingly, however, L. santapolense was the species most affected by water deficit in growth inhibition terms, which suggests the existence of additional mechanisms of defense operating in the field that cannot be mimicked in greenhouses.