APPROACHES TO SOLVE THE PROBLEM OF SALINITY
The use of good land management practice and the association of plants with plant growth-promoting bacteria (PGPB) or breeding and molecular biology have been considered veiy promising approaches to solve the problems related to salinity.
In saline land, the adequate maintenance of the physical/chemical characteristics of soil is a fundamental goal that can be reached by rational use of good quality water, even though not always applicable, and the wise use of fertilizers together with appropriate cultural practices. Lakhdar et al. (2009) emphasize the effectiveness of the use of composted municipal solid waste in salt-affected soil, suggesting this practice as a valid alternative to counteract the negative effects of salt exposure on plants. However, compost quality needs to be very carefully checked in order to do not enhance the risk due to the presence of either pollutants or pathogenic organisms in the compost.
Considering the achievement of salt tolerance in plants, a note of caution is necessary since most of the results, obtained so far, come from laboratory and greenhouse experimental conditions. Thus, we do not know yet the limitations and challenges that may be encountered when these approaches will be transferred to the field. Hydroponics is the most popular growth condition applied in these studies with respect to the utilization of soil mixture (reviewed by Shavrukov et al. 2012; Shavrukov, 2013) but plant response to salinity in hydroponics and in soil may be different (Tavakkoli et al., 2010) and a soil-based system is needed to simulate the field responses. Saline soils usually contain different salts, beside NaCl, that can be also harmful. Moreover, most of the studies are focused on the plant response to single stress, even though in field conditions the plants may be challenged by concurrent different stresses.
To obtain a better crop performance, plant acclimation can be considered, as also reported above, both gradual exposure and low-salt level can activate processes leading to an enhancement of tolerance to salinity in different species, providing an improvement in survival, growth, and yield (Pandolfi et al., 2016).
Exogenous applications of hormones or polyamine or the establishment of association with bacteria/mycorrhizal fungi may ameliorate plant tolerance to salinity. Some of the adverse effects of salt can be diminished by exogenous application of hormones (Afzal et al., 2005; Liu et al., 2015). For example, the positive effect of exogenous application of CK has been described and ascribed to its antioxidant activity related to purine breakdown protection (Javid et al., 2011). According to Ghanem et al. (2011), the enhanced synthesis of CK in the root offers the possibility to improve both shoot hormonal and ion status, thus, decreasing the effects of salinity on growth. However, controversy still exists in the literature about the positive role of CK, since CK-deficient mutants showed improved salt and drought tolerance (Nishiyama et al., 2011; Nishiyama et al., 2012).
Exogenous application of polyamines has been suggested (Gill and Tuteja, 2010), basing on the possible relation between the enhanced amount of polyamine concentration and improvement of salt tolerance (Zapata et al., 2004; Alcazar et al., 2010). However, plant response can be highly variable depending on the organ developmental stage that influences the transport, cytoplasmic accumulation, metabolization, and functional expression (Pandoffi et al., 2010).
Associations of crops with rhizospheric/endophytic bacteria and mycor- rhizal fungi have also been suggested as an alternative and environmentally sustainable strategy to increase crop yields in salt-affected field (Dodd and Perez-Alfocea, 2012; Egamberdieva, 2009; Fomi et al., 2017; Gamalero et al., 2010). The production of different plant hormones by PGPB improves the growth of the plants (Glide et al., 2007; Fomi et al., 2017). Moreover, PGPB can alleviate the symptoms and protect the plants from stress by several mechanisms that confer salt tolerance, for example, through the modulation of the level of the plant hormones, involved in stress response, or osmolyte production (Glick et al., 2007; Glick, 2012; Forni et al., 2017; Mayak et al., 2004).
Screening for tolerant cultivars is important to select genotypes to be used in breeding programs. The existence of wide variability in the responsiveness of crop plants to abiotic stresses and in the control of the response by gene networks with epistatic interactions is well-known (Dolferus, 2014). Salt tolerance is a quantitative trait that is quite difficult to study and requires efficient means to evaluate the level of tolerance. Nevertheless, the identification of germplasm, helps to maintain the biomass production under different stress conditions, needs to be foreseen (Dolferus, 2014).
Because of the long-time required in obtaining salt-tolerant cultivars by conventional breeding programs, alternative approaches have been considered in parallel. Today, the available techniques and tools allow the study of gene expression in plants exposed to different stress, including salinity (Takahashi et al., 2004). Up- and downregulated genes responding to stress have been identified (reviewed by Xiong and Zhou, 2002; Jamil et al., 2011; Shavrukov, 2013; Deinlein et al., 2014; Cabello et al., 2014). Based on the results of these studies, the biotechnology seeks the possibility of manipulation of stress-responsive genes (Munns, 2005; Denlein et al., 2014;
Cabello et al., 2014). In this perspective, the identification of candidate genes for plant salt tolerance improvement can become easier by quantitative trait locus (QTL) analyses coupled with marker-assisted selection (Ashraf and Foolad, 2013).
Several genes can be the possible candidate for genetic engineering as suggested by numerous authors (Munns, 2005; Ji et al., 2013; Cabello et al., 2014; Wang et al., 2016). Results of the experiments, related to these topics, are summarized and reviewed by Munns (2005), Denlein et al. (2014), and Cabello et al. (2014). In the perspective of genetic engineering, the expression of inducible promoters should be preferred to the constitutive ones, since they do not affect growth, while in the meantime they can increase tolerance when stress is applied (Munns and Tester, 2008).
CASE REPORT: BRASSICA SPP.
The Brassicaceae family includes a wide range of horticultural crops with more than 30 species and several varieties and hybrids (Rakow, 2004). Some of them have economic significance in agriculture as a source of food, vegetable oil (Wanasundara, 2011), medicinal products (Pagliaro et al., 2015), and possible utilization in phytoremediation projects (Szczyglowska et al., 2011; Mourato et al., 2015). There are six economically important species:
B. juncea L. (Indian mustard, target of several phytoremediation works) (Mourato et al., 2015), B. oleracea L. (varieties of this species include vegetables used as food), B. rapa L. and B. nigra (L.) W. D. J. Koch (used for the oil content of the seeds) (Kopsell et al., 2007), B. napits L. (oilseed rape, canola, a globally important oil crop worldwide, used in human nutrition and industry as lubricant and biodiesel) (Hua et al., 2012). The seeds of the latter species are a fundamental source of oil for human consumption and have a protein content between 20 and 35% of dry weight (Wanasundara, 2011).
Abiotic stresses, such as drought, cold, and high salinity, can frequently affect the yield and quality of these important crops. Overall, the data collected on tolerance to salinity in different Brassica species show a significant interspecific and intraspecific variation in the level of salt tolerance within the genus (Kumar et al., 2009).
Chakraborty et al. (2016), by evaluating and comparing the role of plasma membrane transporters in salt tolerance in B. juncea, B. oleracea and B. napns, considered the latter as the most tolerant species.
In B. juncea, the experiments by Fatma et al. (2016) showed that in the presence of salt stress the combined application of NO and S (100 pM NO and 200 mg S/ kg soil, respectively) decreased the negative effects of salt on stomatal behavior, photosynthetic activity, and growth. The enzymatic activities of ATP-sulfurylase, catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) and optimized NO generation reduced the oxidative stress caused by salt exposure.
The effectiveness of the activity of some antioxidant enzymes, polyphenol oxidase, APX and CAT, in minimizing oxidative stress is also confirmed by the data obtained in our work (unpublished results), where an enhancement of the activities was observed in B. napus cv Edirnax Cl plants, exposed either gradually or directly to salt for 28 and 35 days.
Proteomic analyses have been also applied to determine differentially expressed proteins in plants of Brassica exposed to salt. Jia et al. (2015) reported changes in protein patterns in leaves of B. napus exposed to 200 mM NaCl for 24 h, 48 h, and 72 h. The proteins differentially expressed were those associated with protein metabolism, damage repair, and defense response, which contribute to alleviate the salt detrimental effects on chlorophyll biosynthesis, photosynthesis, energy synthesis, and respiration in oilseed rape leaves.
Seedlings of the same species, pretreated with 245 mM NaCl (salt treatment) or 25% polyethylene glycol 6000 (drought treatment) (Luo et al., 2015), showed significant decreases in water content and photosynthetic rate, whereas compatible osmolytes were accumulated; oxidative damage was also observed. The proteomic profiles of family proteins, related to stress response, were changed in treated plants (Luo et al., 2015).
A study on the transcriptome profiles was performed on canola roots at the germination stage, that is, up to 24 h after H,0 (control) and NaCl treatments. Changes in the expression of genes involved in the metabolism of proline, inositol and carbohydrates, and in oxidation-reduction processes were detected, thus, evidencing the importance of these genes in stress response at this growth stage (Long et al., 2015).
To ameliorate salt tolerance in Brassica, several studies have been focused on the identification of candidate genes to be used in molecular breeding for salt tolerance (Kumar et al., 2015; Yong et al. 2015; Lang et al., 2017). The data provided by the QTL analyses represent valuable information for studying the genetic control of salt tolerance in this genus, and they will be very useful in marker-based breeding. Progress has been made in this field, even though few salt-tolerance cultivars and lines have been obtained so far because of the difficulties of transferring stress-tolerant traits from interspecific and intergeneric sources (reviewed by Zhang et al., 2014).
Plant transformation with gene of bacterial origin may be also helpful to obtain enhancement of salinity tolerance. The canola cv. Westar was transformed to express a bacterial ACC deaminase (EC 18.104.22.168) gene under the control of different promoters (Sergeeva et al., 2006). Transformed and nontransformed plants were treated with 0-200 mM NaCl, and the fresh and dry weights of plants, leaf proteins, and chlorophyll concentrations were determined. In the transgenic canola lines, the activity of ACC deaminase lowered the synthesis of stress ET and improved salt tolerance.