The salinity effects on the growth characteristics were evident. Salinity stand-up in the growing media restricted the growth potential of explants with regard to the proliferation rate, fresh weight, viability, shoot length and three nodal shoots number.
The in vitro and in vivo effects of salinity treatments on ‘Sultanin’7,33,34 Black Corinth and Emperor35 Perlette, Beauty seedless and Delight36 grapevines have been previously reported.
The previous data shows that the salinity treatments up to 1 gL− 1 in ‘Sultanin’ grape drastically reduced the explants viability7. High Na+ concentrations in the growing media antagonistically prevent the absorption of other cations. Researchers demonstrated that the more sodium availability limited Mg2+, Ca2+ and K+ intake in grapevines. Salinity restrains the overall growth and development of plants2. The others reported the same idea emphasizing that the shoots growth in grapevine was greatly inhibited by the action of Cl− ions3,37.
Salinity damage is dependent upon the NaCl concentration, treatment time-course, and cultivar type. Several researchers have verified the mentioned side effects of salinity on plant35,38. Salinity impedes the vegetative and reproductive growth by the direct or indirect action on the physiological dynamics of cells and whole plants. The damages on tissues come from not only the osmotic pressure but also, more intensified by the toxic effects of Na+ and Cl− ions2,3.
In the present study, APX enzyme activity in ‘Fakhri’ under the diverse salinity levels had no significant differences with the control ones. However, in ‘Sultanin’ grape, the APX activity was increased with salinity. In another study on apple and pear cultivars in MS medium, the highest APX activity was devoted to 150 µm of NaCl25,39.
For GPX, the highest data were recorded with 0.5 gL− 1 of NaCl. The increased levels of GPX have been frequently reported in salt tolerant species. Some documents say that this enzyme activity has been increased in both sensitive and tolerant cultivars. However, the increasing pattern and rate are different in diverse species40,41.
CAT activity was increased in ‘Sultanin’ grape in line with salinity levels. On the contrary, in ‘Fakhri’, there was no significant difference in CAT activity between shoots under various salinity treatments. Sweet cherry rootstocks showed broad diversity in CAT activity in vitro exposed to salinity treatments25. The similar results are available for apple39. So, all the antioxidant enzymes do not follow a liner increase in the activity in response to the salinity. Since, the salinity stress treated feedback of plants is dependent on species, growth stage, salts concentration and type, treatment exposure times and absolutely, the activity would be different for the diverse enzymes11,42.
SOD activity was initially increased for all the salinity treatments; but, later declined. Under the high salinity concentrations, the biochemical responses of plants are not correspondingly related to the salinity treatments, although the growth and yield attributes are negatively affected. With increasing the salinity levels, the antioxidant system of plants is activated and hence with the production and activity of SOD as the forefront barrier against ROS radicals; the tissues and whole body of the plants get ready to face with the salinity stress39. Furthermore, with high salinity levels and the age of the plants, the SOD activity was considerably increased43. The elevated SOD activities under the salinity stress have been reported for mulberry44, citrus crops45 and maize46.
Salinity stress triggers oxidative membranes deterioration. Plants evolved some mechanisms to denature and decline the activity of ROS molecules by motivating the antioxidants potential to control the stress damages. The first step in this passage is the scavenging of H2O2 to protect the damages caused at cellular, and tissue level and to mitigate the oxidative stress47,48.
With salinity, MDA levels were increased. Lipids peroxidation coincides with the increased antioxidant enzymes activity in the salt tolerant plants. MDA ameliorates the oxidative stress and intensifies the scavenging of ROS radicals46,49.
Many findings have noted that the consequence of ROS molecules over-production is the membrane lipids peroxidation leading to the production of aldehydes and specially MDA and some other products like ethylene50. The production of these compounds can be annotated as the lipids peroxidation and fatty acids oxidation and their structural denaturation. The high MDA content under salinity conditions is an outcome of the reduced content and activity of SOD, GR and CAT39,44,48. Salinity apart from the lipids deterioration denatures membrane-anchored proteins and hence reduces the total protein content of plants. ROS radicals' over-activity under the salinity environments oxidase the amino acid chains and finally denatures proteins and alters their conformational moiety46.
The same trend was followed for CBF4 and the expression rate in cultivar ‘Fakhri’ was higher than that in cultivar ‘Sultanin’. CBF1, and in ‘Fakhri’, the expression was additive up to 0.75 gL− 1 of NaCl. But, then after decreased. In ‘Sultanin’, the CBF1 expression rate with salinity of up to 0.75 gL− 1 declined but, was higher again at 1 gL− 1.
CBF/DREB proteins are fundamental compounds in the expression of specific genes involved or responsive in abiotic stresses tolerance. The mentioned proteins are target compounds of the breeding programs for the environmental stressors. The majority of CBF genes are characterized to be influential in the induction of tolerance responses to the low temperature stress51,52,53, Otherwise, several studies have claimed that DREB/CBF genes are induced by the several stressors and, ABA activates a bunch of DREB/CBF genes51,54,55. CBF4 is the major gene respondent to the drought stress. CBF4 expression is more activated by the exogenous application of ABA in Arabidopsis and grapevine. CBF1 − 3 expression was also enhanced with the ABA external applications in main part due to the increased activity of CBF promoters in reply to ABA54,56,57. The differences in CBF proteins in grapevine reflect the positioning of CBF4 in the separate phylogenetic groups compared to CBF1,2,319,58,59. This possibly justifies that CBF4 in grapevine behaves in different manner than CBF1,2 and 3. CBF1,2,3 are more expressed in young tissues60. However, CBF4 is typically under the active expression in the young and mature leaves and in the nodes. Seemingly, CBF/DREB genes act as connector or interaction point of several biosynthetic pathways and simultaneously regulate the tolerance to the stressors like drought, cold and salinity. Transgenic Arabidopsis lines of DREB1B/CBF1 or DREB1A/CBF3 exhibited reasonable salinity tolerance. It seems that DREBs/CBFs target several genes61.
CBFs genes play inimitable role in the growth and development of grapevine plants. So, may be employed as biomarkers in the screening of salt tolerant cultivars in the breeding programs of grapevines.