Monica Boscaiu

Breeding and Domesticating Crops Adapted to Drought and Salinity: A New Paradigm for Increasing Food Production

World population is expected to reach 9.2 × 109 people by 2050. Feeding them will require a boost in crop productivity using innovative approaches. Current agricultural production is very dependent on large amounts of inputs and water availability is a major limiting factor. In addition, the loss of genetic diversity and the threat of climate change make a change of paradigm in plant breeding and agricultural practices necessary. Average yields in all major crops are only a small fraction of record yields, and drought and soil salinity are the main factors responsible for yield reduction. Therefore there is the need to enhance crop productivity by improving crop adaptation. Here we review the present situation and propose the development of crops tolerant to drought and salt stress for addressing the challenge of dramatically increasing food production in the near future. The success in the development of crops adapted to drought and salt depends on the efficient and combined use of genetic engineering and traditional breeding tools. Moreover, we propose the domestication of new halophilic crops to create a ‘saline agriculture’ which will not compete in terms of resources with conventional agriculture.

Environmentally induced changes in antioxidant phenolic compounds levels in wild plants

Different adverse environmental conditions cause oxidative stress in plants by generation of reactive oxygen species (ROS). Accordingly, a general response to abiotic stress is the activation of enzymatic and non-enzymatic antioxidant systems. Many phenolic compounds, especially flavonoids, are known antioxidants and efficient ROS scavengers in vitro, but their exact role in plant stress responses in nature is still under debate. The aim of our work is to investigate this role by correlating the degree of environmental stress with phenolic and flavonoid levels in stress-tolerant plants. Total phenolic and antioxidant flavonoid contents were determined in 19 wild species. Meteorological data and plant and soil samples were collected in three successive seasons from four Mediterranean ecosystems: salt marsh, dune, semiarid and gypsum habitats. Changes in phenolic and flavonoid levels were correlated with the environmental conditions of the plants and were found to depend on both the taxonomy and ecology of the investigated species. Despite species-specific differences, principal component analyses of the results established a positive correlation between plant phenolics and several environmental parameters, such as altitude, and those related to water stress: temperature, evapotranspiration, and soil water deficit. The correlation with salt stress was, however, very weak. The joint analysis of all the species showed the lowest phenolic and flavonoid levels in the halophytes from the salt marsh. This finding supports previous data indicating that the halophytes analysed here do not undergo oxidative stress in their natural habitat and therefore do not need to activate antioxidant systems as a defence against salinity.

Responses to salt stress in Juncus acutus and J. maritimus during seed germination and vegetative plant growth

Abstract Responses to increasing salinity, during seed germination and vegetative plant growth, were studied in two related species of Juncus, J. maritimus and J. acutus. In both species, germination was optimal in the absence of salt, reduced by about 50% in the presence of 200 mM NaCl, and completely inhibited by NaCl concentrations above 300 mM. Previous exposure of the seeds to salt, up to 500 mM NaCl, did not affect the germination capacity in J. acutus, and clearly enhanced it in J. maritimus. A concentration-dependent inhibition of plant growth was observed in the presence of NaCl for both species, together with the parallel accumulation of sodium ions in the leaves, as determined by cation exchange HPLC. Regarding the levels of divalent cations, in J. acutus Ca2+ and Mg2+ increased up to about two-fold in plants treated with 500 mM NaCl, as compared to control plants, whereas in J. maritimus they were three- to four-fold higher than in J. acutus in the absence of salt, and did not change significantly with increasing NaCl concentrations. These results suggest that Ca2+ and Mg2+ participate in defence mechanisms against salt stress, which would be constitutive in J. maritimus and salt-inducible in J.acutus.

Effects of salt stress on the reproductive biology of the halophyte Plantago crassifolia

Floral phenology, pollen quality and seed set of Plantago crassifolia plants, grown in the presence of increasing NaCl concentrations, were studied to test how this Mediterranean halophyte responded to salt stress during the reproductive phase of its life cycle. “Reproductive success” was maximal in plants grown in non-saline conditions, or in the presence of 100 mM NaCl, but it was negatively affected by higher salinities, due to a progressive reduction of pollen fertility, seed set, and seed viability.

Native-Invasive Plants vs. Halophytes in Mediterranean Salt Marshes: Stress Tolerance Mechanisms in Two Related Species

Dittrichia viscosa is a Mediterranean ruderal species that over the last decades has expanded into new habitats, including coastal salt marshes, ecosystems that are per se fragile and threatened by human activities. To assess the potential risk that this native-invasive species represents for the genuine salt marsh vegetation, we compared its distribution with that of Inula crithmoides, a taxonomically related halophyte, in three salt marshes located in “La Albufera” Natural Park, near the city of Valencia (East Spain). The presence of D. viscosa was restricted to areas of low and moderate salinity, while I. crithmoides was also present in the most saline zones of the salt marshes. Analyses of the responses of the two species to salt and water stress treatments in controlled experiments revealed that both activate the same physiological stress tolerance mechanisms, based essentially on the transport of toxic ions to the leaves—where they are presumably compartmentalized in vacuoles—and the accumulation of specific osmolytes for osmotic adjustment. The two species differ in the efficiency of those mechanisms: salt-induced increases in Na+ and Cl− contents were higher in I. crithmoides than in D. viscosa, and the osmolytes (especially glycine betaine, but also arabinose, fructose and glucose) accumulated at higher levels in the former species. This explains the (slightly) higher stress tolerance of I. crithmoides, as compared to D. viscosa, established from growth inhibition measurements and their distribution in nature. The possible activation of K+ transport to the leaves under high salinity conditions may also contribute to salt tolerance in I. crithmoides. Oxidative stress level—estimated from malondialdehyde accumulation—was higher in the less tolerant D. viscosa, which consequently activated antioxidant responses as a defense mechanism against stress; these responses were weaker or absent in the more tolerant I. crithmoides. Based on these results, we concluded that although D. viscosa cannot directly compete with true halophytes in highly saline environments, it is nevertheless quite stress tolerant and therefore represents a threat for the vegetation located on the salt marshes borders, where several endemic and threatened species are found in the area of study.

Antioxidant responses under salinity and drought in three closely related wild monocots with different ecological optima

We studied the level of oxidative stress and the activation of antioxidant responses in three rush species - sea rush (Juncus maritimus), spiny rush (J. acutus) and jointleaf rush (J. articulatus) - subjected to salt and water stress treatments. The halophytes J. maritimus and J. acutus were the most tolerant taxa; they were less affected by oxidative stress than the salt-sensitive J. articulatus under both conditions, due to more efficient activation of enzymatic and non-enzymatic antioxidant systems. Our results show the relative importance of different antioxidant responses for stress tolerance in species with distinct ecological requirements.

Identification of Salt Stress Biomarkers in Romanian Carpathian Populations of Picea abies (L.) Karst.

The Norway spruce (Picea abies), the most important tree species in European forests, is relatively sensitive to salt and does not grow in natural saline environments. Yet many trees are actually exposed to salt stress due to the common practice of de-icing of mountain roads in winter, using large amounts of NaCl. To help develop strategies for an appropriate use of reproductive seed material on reforestation sites, ensuring better chances of seedling survival in salt-affected areas, we have studied the responses of young spruce seedlings to salt treatments. The specific aim of the work was to identify the optimal salt stress biomarkers in Picea abies, using as experimental material seedlings obtained by germination of seeds with origin in seven populations from the Romanian Carpathian Mountains. These responses included general, conserved reactions such as the accumulation of ions and different osmolytes in the seedlings needles, reduction in photosynthetic pigments levels, or activation of antioxidant systems. Although changes in the contents of different compounds involved in these reactions can be associated to the degree of stress affecting the plants, we propose that the (decreasing) levels of total phenolics or total carotenoids and the (increasing) levels of Na+ or K+ ions in Picea abies needles, should be considered as the most reliable and useful biomarkers for salt stress in this species. They all show very high correlation with the intensity of salt stress, independently of the genetic background of the seeds parental population, and relatively easy, quantitative assays are available to determine their concentrations, requiring simple equipment and little amount of plant material.

Proline and glycine betaine accumulation in two succulent halophytes under natural and experimental conditions

Proline (Pro) and glycine betaine (GB) contents were determined in two Mediterranean halophytes, Plantago crassifolia and Inula crithmoides, to assess their possible role in salt tolerance of both taxa. Plant material was collected in a littoral salt marsh under different environmental conditions, and from plants subjected to salt treatments in a growth chamber. Relative growth inhibition by NaCl indicated that I. crithmoides is more salt-tolerant than P. crassifolia, in agreement with the distribution of the two species in nature. Field and laboratory data confirmed GB as the major osmolyte responsible for osmotic adjustment in I. crithmoides, but with only a minor role – if any – as “osmoprotectant” in the salt tolerance of P. crassifolia. Under natural conditions, Pro contents were very low in both taxa, but increased to levels high enough to contribute significantly to osmotic balance when plants were artificially treated with 450–600 mM NaCl – higher salt concentrations than those they would normally encounter in their natural habitats. These data suggest that halophytes possess built-in mechanisms, such as accumulation of additional osmolytes, to rapidly adapt to increasing salinity levels in their natural ecosystems; for example, those expected to be caused by climate change in salt marshes in the Mediterranean region.

Effects of Salt Stress on Three Ecologically Distinct Plantago Species.

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.

Effects of salinity and drought on growth, ionic relations, compatible solutes and activation of antioxidant systems in oleander (Nerium oleander L.)

Nerium oleander is an ornamental species of high aesthetic value, grown in arid and semi-arid regions because of its drought tolerance, which is also considered as relatively resistant to salt; yet the biochemical and molecular mechanisms underlying oleander’s stress tolerance remain largely unknown. To investigate these mechanisms, one-year-old oleander seedlings were exposed to 15 and 30 days of treatment with increasing salt concentrations, up to 800 mM NaCl, and to complete withholding of irrigation; growth parameters and biochemical markers characteristic of conserved stress-response pathways were then determined in stressed and control plants. Strong water deficit and salt stress both caused inhibition of growth, degradation of photosynthetic pigments, a slight (but statistically significant) increase in the leaf levels of specific osmolytes, and induction of oxidative stress—as indicated by the accumulation of malondialdehyde (MDA), a reliable oxidative stress marker—accompanied by increases in the levels of total phenolic compounds and antioxidant flavonoids and in the specific activities of ascorbate peroxidase (APX) and glutathione reductase (GR). High salinity, in addition, induced accumulation of Na+ and Cl- in roots and leaves and the activation of superoxide dismutase (SOD) and catalase (CAT) activities. Apart from anatomical adaptations that protect oleander from leaf dehydration at moderate levels of stress, our results indicate that tolerance of this species to salinity and water deficit is based on the constitutive accumulation in leaves of high concentrations of soluble carbohydrates and, to a lesser extent, of glycine betaine, and in the activation of the aforementioned antioxidant systems. Moreover, regarding specifically salt stress, mechanisms efficiently blocking transport of toxic ions from the roots to the aerial parts of the plant appear to contribute to a large extent to tolerance in Nerium oleander.