Physiological, epigenetic and genetic regulation in some olive cultivars under salt stress

Abstract

Cultivated olive, a typical fruit crop species of the semi-arid regions, could successfully face the new scenarios driven by the climate change through the selection of tolerant varieties to salt and drought stresses. In the present work, multidisciplinary approaches, including physiological, epigenetic and genetic studies, have been applied to clarify the salt tolerance mechanisms in olive. Four varieties (Koroneiki, Royal de Cazorla, Arbequina and Picual) and a related form (O. europaea subsp. cuspidata) were grown in a hydroponic system under different salt concentrations from zero to 200 mM. In order to verify the plant response under salt stress, photosynthesis, gas exchange and relative water content were measured at different time points, whereas chlorophyll and leaf concentration of Na⁺, K⁺ and Ca²⁺ ions, were quantified at 43 and 60 days after treatment, when stress symptoms became prominent. Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effects of salt stress on plant DNA methylation. Several fragments resulted differentially methylated among genotypes, treatments and time points. Real time quantitative PCR (RT-qPCR) analysis revealed significant expression changes related to plant response to salinity. Four genes (OePIP1.1, OePetD, OePI4Kg4 and OeXyla) were identified, as well as multiple retrotransposon elements usually targeted by methylation under stress conditions.

Figure 1

The plants growing condition in control and 200 mM of NaCl treatment. (A) Plants of subspecies cuspidata (after 14 DAT), (B) Plants of cv. Royal and (C) Plants of cv. Koroneiki (both after 43 DAT).

Figure 2

Physiological parameters measured for plants of cultivars Koroneiki and Royal under 0-100-200 mM NaCl at 15, 36 and 43 DAT (except for chlorophyll content, data showed only for 43 DAT) of treatment. (A) Net photosynthesis (Pn), (B) Stomatal conductance (gs), (C) Sub-stomatal CO₂ concentration (Ci), (D) Transpiration rate (E), (E) Leaf Relative water content (RWC), and (F) Chlorophyll content. ANOVA tests were performed inside each cultivar for three treatments (0-100-200 mM NaCl) and separately for each time point. Different letters correspond to significantly different values at P ≤ 0.05.

Figure 3

Content of Na⁺, K⁺ and Ca²⁺ ions in leaves, shoots and roots of cv. Koroneiki and Royal plants at zero and 200 mM NaCl, after 60 DAT of treatment. To different letters correspond significantly different values at P ≤ 0.05.

Figure 4

Relative mRNA levels of differentially methylated genes under salt stress, as determined by RT-qPCR. Leaves of plants under control conditions or treated at 200 mM NaCl were analyzed at 8, 15 and 43 DAT of treatment. Transcripts analyzed: (A,B) Alleles (A,B) of an aquaporin - subfamily membrane intrinsic protein (OePIP1.1); (C) a cytochrome b6 (OePetD); (D) a phosphatidylinositol 4-kinase (OePI4Kg4); (E) a xylose isomerase (OeXylA); (F) an unknown mitochondrial region (OeMit); and (G,H,I) Three reverse transcriptases Ty3-gypsy (OeRT1-2-3 Ty3-gypsy). Values are means of three biological replicates and three technical replicates. To different letters correspond significantly different values at P ≤ 0.01.

Figure 5

Olive plants of four olive cultivars (Koroneiki, Royal, Picual and Arbequina) and a sample of O. europaea subsp. cuspidata kept in hydroponics under different levels of salt stress (0-100-200 mM NaCl).