Comparative analyses of plant responses to drought and salt stress in related taxa: A useful approach to study stress tolerance mechanisms.

Introduction Salinity and drought are the most important environmental stress conditions reducing crop yields worldwide and limiting the distribution of wild plants in nature. Soil salinity, especially secondary salinity caused by anthropogenic practices, such as prolonged irrigation, lead to substantial agricultural yield losses, especially in arid and semiarid regions. Drought, caused by reduced water content in the soil, occurs due to disorders in nature´s water cycle, chiefly when evapotranspiration exceeds precipitation in a certain area, to the point where soil water reserves can no longer support plant growth. Drought and salt stress trigger the activation of a series of basic stress mechanisms that includes among others, the control of ion transport, exclusion and compartmentalization, as well as the accumulation of compatible solutes (‘osmolytes’), and the activation of antioxidant systems. These mechanisms are conserved in all plants, stress tolerant and sensitive alike, and don’t necessarily confer tolerance. To decipher those mechanisms and have a better understanding on the contribution of different stress responses to the stress tolerance of a given species, we have carried out comparative studies on the responses to drought and salinity in a number of genetically related taxa with different tolerance potentials. Methodology The work concentrated on studying the responses to salt and drought stress in genetically related plants with different tolerance to abiotic stress. The studied taxa included salt-tolerant (halophytes) and salt-sensitive (glycophytes) wild species of two different genera: Juncus (monocotyledonous) and Plantago (dicotyledonous), as well as plants of crop species: Solanum lycopersicum var. cerasiforme (cherry tomatoes) and different Phaseolus cultivars, one of P. coccineus and three of P. vulgaris. The experimental approach was mostly based on i) establishing the relative tolerance to water and salt stress in the studied species from their distribution in nature (in the case of wild species) and through the relative inhibition of growth in the presence of stress, and ii) correlating changes in the levels of biochemical ‘stress markers’ associated to specific response pathways (ion transport, osmolyte accumulation…) upon stress treatments, with the already established relative tolerance to stress. This strategy proved to be appropriate to distinguish mere general responses to stress from those mechanisms relevant for stress tolerance of the investigated species and cultivars. The work also sheds light on other aspects affected by salt stress, specifically regarding germination and reproductive success or anatomical changes in salt-stressed plants. The expression patterns of the gene NHX1, encoding a vacuolar Na+/H+ antiporter were also studied in the Plantago taxa, as a first step in the full characterisation of this ion transporter, that appears to play an important role in the mechanisms of salt tolerance in this genus. Results and discussion Through the results attained from this work, we have been able to establish which general stress responses are relevant for tolerance in the investigated species or cultivars, and which are not. Thus, we provide clear evidence that, although all plants seem to activate the same mechanisms of defense in response to abiotic stress, their relative contribution to stress tolerance differs widely in different genera and species. Moreover, in general, the relative tolerance of the investigated species and cultivars were the same, when referring to salt stress and to water stress, and the same mechanisms – except some related to ion transport and homeostasis – were relevant for tolerance to both stresses. In the studied Phaseolus cultivars, P. vulgaris cv. 'Maxidor' showed the smallest growth inhibition under salt and water stress conditions, and therefore was defined as the most tolerant. 'Maxidor' accumulated lower levels of toxic ions and proline, and recorded higher levels of myo-inositol than the other cultivars. We concluded that blocking ion transport from the roots to the leaves and myo-inositol accumulation, were the mechanisms most relevant for stress tolerance in Phaseolus. Proline is a reliable stress biomarker in this genus, indicating the degree of stress affecting the plants, but is not directly involved in tolerance mechanisms. In the studied Plantago species it was found that the more tolerant taxa transported Na+ and Cl- to the leaves more efficiently than the most sensitive P. major, and tended to accumulate large amounts of proline, albeit only under extreme stress conditions; these responses appear to be the most relevant for tolerance in Plantago. I Toxic ions transported to the leaves are presumably accumulated in vacuoles, which gave incentive to isolating, sequencing and studying the expression of the Na+/H+ vacuolar antiporter gene NHX1 in the studied species. Upon short-term treatments with high NaCl concentrations, the more tolerant species showed higher salt -induced expression of the aforementioned gene, supporting the contribution of the NHX1 antiporter to salt tolerance in Plantago. Meanwhile, the tolerant Juncus species were able to partly inhibit ion transport from the roots to the plants aerial parts and recorded a much larger increment (about 60- fold over the controls) in proline contents, as compared to the stress-sensitive congener. Therefore, blocking accumulation of toxic ions and inducing accumulation of proline in the culms appear to be the most important mechanisms of tolerance in Juncus. On the other hand, we did not detect significant stress-induced anatomical differences when comparing salt tolerant and sensitive Juncus taxa. Conclusion The results obtained in this work contribute to a better understanding of general stress tolerance mechanisms in plants, and provides clear insights into the mechanisms conferring tolerance, specifically, to drought and salt stress in some wild species and crops. This work also shed more light on the highly efficient responses to stress in halophytes, plants that could be viewed as nature´s answer to the aforementioned adverse environmental conditions via evolution and adaptation. Halophytes can therefore be considered as a suitable source – underutilized at present, in our opinion – of knowledge, genetic resources and biotechnological tools for the needed improvement of stress tolerance in crops. This work has yielded eight scientific manuscripts (published, under review, or in preparation), that are considered as subchapters of the results section of this thesis and are listed below: 1) Al Hassan, M. , Pacurar, A. , Gaspar, A. , Vicente, O. , Boscaiu, M. (2014). Growth and reproductive success under saline conditions of three Plantago species with different levels of stress tolerance. Notulae Botanicae Horti Agrobotanici Cluj- Napoca 42(1): 180-186. 2) Al Hassan, M. , Fuertes, M. , Sanchez, F. , Vicente, O. , Boscaiu, M. (2015). Effects of Salt and Water Stress on Plant Growth and on Accumulation of Osmolytes and Antioxidant Compounds in Cherry Tomato. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 43(1): 1-11. 3) Al Hassan, M. , Gohari, G. , Boscaiu, M. , Vicente, O. , Grigore, M. (2015). Anatomical modifications under salt stress in two ecologically distinct Juncus species. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 43(2): 501-506. 4) Al Hassan, M. , Morosan, M. , Lopez-Gresa, M.P., Boscaiu, M. , Vicente, O. (2015). Selection and characterisation of salt and drought-resistant Phaseolus cultivars: a ‘proof-of-concept’ study. (Under revision). 5) Al Hassan, M. , Pacurar, A. , Lopez-Gresa, M.P., Llinares, J., Boscaiu, M. , Vicente, O. (2015). Effects of Salt and Water Stress on Three Ecologically Distinct Plantago Species. (Under revision). 6) Al Hassan, M. , Lopez-Gresa, M.P., Boscaiu, M. , Vicente, O. (2015). Stress tolerance mechanisms in Juncus: Responses to salinity and drought in three Juncus species adapted to different natural environments. (Under revision). 7) Al Hassan, M. , Cortes, J., Gaspar, A. , Boscaiu, M. , Vicente, O. (2015). Differential anti-oxidative responses under salinity and drought challenges in two halophytes and one glycophyte of the genus Juncus. (In preparation). 8) Al Hassan, M. , Daniso, E. , Martinelli, F. , Boscaiu, M. , Vicente, O. (2015). Expression of the vacuolar Na+/H+ antiporter gene (NHX1) in three Plantago species differing in salt tolerance (In preparation). Other manuscripts, published or submitted during the period of work, more or less related with its topic, but not included in the Thesis, are listed in the appendix at the end of this document.