Salinity Depression in Vitro Impress the Growth Potential and, Mediates the Antioxidants Pool and the Tolerance Genes Expression of Grapevine

Undoubtedly, salinity is the major environmental stress affecting the crops growth, development and yield. Grapevine is a dominant horticulture crop and mildly sensitive to the salinity. Salinity triggers variations at the cellular and molecular levels and hence induces some specic genes expression. The responses of two grapevine cultivars (Fakhri and Sultanin) were evaluated for its tolerance to the salinity stress in vitro. The results for the explants exposed to the salinity revealed that the viability, fresh weight of the regenerated explant and the proliferation rate were declined compared to the control ones. The activity of SOD enzyme and MDA content were increased with salinity. However, protein content declined. There was no signicant difference in CAT and APX activities with the salinity treatment. With salinity adding up, the DREB/CBFs genes expression pattern was signicantly increased in both cultivars. ‘Fakhri’ was more responsive in growth parameters and the activity of antioxidant enzymes and higher expression rate of DREB/CBFs under salinity compared to the variety ‘Sultanin’. in grapevine 8,22 an acceptable recommended procedure there are techniques for the ecient screening in mulberry 23 citrus and cherry rootstocks The present study planned to assay the in vitro response of some grapevine cultivars to the by the tracing of morphological and as well as the dened genes expression pattern variations.

To have the extract of CAT, GPX, SOD and soluble proteins; 0.2 g of plant material (shoots after 2 month inoculation) was digested in liquid nitrogen. 2 ml of phosphate buffer (pH= 7.5) containing EDTA (0.5 mol) was added. Then after, the extracts were centrifuged at -4°C for 15 min at 15000 rpm, then collected the supernatant and stored it for assay 26 .
Due to the short half-life of APX under ex-vitro condition and for keeping the cell structure and components; PVP (5%) and 2 ml ascorbate was added to the enzymatic solution. APX was assayed as; the reaction mixture was containing 250 μL phosphate buffer (pH=7) along with EDTA, 10 μL H 2 O 2 (1 mmol), 250 μL sodium ascorbate (0.25 mmol) and 50 μL enzyme solution. The absorbance was measured at 290 nm 27 .
For the activity measurement of GPX the reaction mixture was contained 1 ml phosphate buffer (100 mmol, pH=7) along with EDTA (0.1 mmol), 1 mL guaiacol (15 mmol), 1 ml H 2 O 2 (3 mmol) and 50 μL of the extracted enzyme solution. The reaction response was measured at 470 nm for 1 min 28 . For CAT, the reaction mixture was containing 1.5 ml phosphate buffer (100 mmol, pH=7), 0.5 ml H 2 O 2 (7.5 mmol) and 50 μL of extracted enzyme solution. The absorbance at 240 nm during 1 minute was measured 29 . SOD activity was evaluated in a reaction mixture having 1.6 ml phosphate buffer (100 mmol) containing 0.1 ml EDTA, and sodium carbonate (1.5 M), 0.2 ml of L-methionine (0.2 M), 1 ml of distilled water, 0.1 ml of NBT (2.25 mM) and 50 μL of enzyme solution. The enzyme reaction and activity was initiated with the addition of 0.1 ml ribo avin (60 μL) and the samples absorbance was recorded at 560 nm 30 .
Malondialdehyde assay. 0.2 g of plant sample was homogenized in 2 ml of TCA (20%) containing 0.05% TBA. The samples were incubated at 95°C for 30 minutes and then were transferred on ice. The samples were centrifuged at 10000 rpm for ten minute and the absorbance was recorded at 532 and 600 nm 27 .
Soluble protein assay.
To prepare the solution, 100 µl of enzymatic solution, 200 µl of Bradford reagent and 700 µl of deionized water were mixed. 2 min after the complex formation of Bradford reagent (interaction with the amino acids), the absorbance was recorded at 523 nm. Proteins content of the samples was calculated based on cow albumin-serum calibration curve 31 .
Data were analyzed by SPSS (v. 21) and mean comparisons were accomplished by Duncan's multiple range test.
RNA extraction and DNA synthesis.
Total RNA was extracted from the in vitro derived shoots and the RNA extract was puri ed by the method of Tatterall et al. 32 .
The quanti cation and quali cation of the extracted RNAs was carried out by the A260/A280 ratio (1.8-2) and A260/A230. The extracted RNAs were identi ed in 2% agarose gel by the golden red staining method. The reaction mixtures (Table 1) were prepared in microtubes on ice and reached 20 µl by RNase-free water.
Primer design and RT-qPCR analysis.
RNA sequences of genes of VvCBF 1,2,3,4 and MDH (Reference gene) were acquired from NCBI (www.ncbinlm.nih.gov) and the forward/reverse primers were designed by oligo 7 software (Table 2). RT-qPCR analysis was done by ABI StepOne (Applied biosystems) and were detected by SYBR Green PCR Master Mix (TakaRa, Toyo to, Japan). Reaction mixture (Table 3) of 20 µl was prepared for each sample.
Initially, denaturation was done at 5°C for 5 min. 40 cycles of 95°C for 5 sec and 60°C for one minute. After calculation of the ampli cation e ciency of the primers; the reaction was done by the method of 2 -∆∆CT . The analysis of Real-time PCR data was accomplished based on the threshold cycle acquired for the target and reference genes. The difference between ct reference genes from the mean of ct target gene was considered as ∆ct index for both the test and control groups. ct s difference of test and control groups were employed for the calculation of ∆∆ct.

Morphological traits.
The results for the explants exposed to the salinity during 2 months with one sub-culture showed that the viability statistically declined with salinity intensity especially in 'Sultanin' cultivar. In 'Fakhri', 0.25 and 0.5 gL -1 of salinity had no meaningful effect on the viability of explants. Shoot growth in both cultivars correspondingly declined in response to the salinity. The fresh weight of the regenerated explants was impacted by the salinity compared to the control. So that, at 1 gL -1 salinity, the fresh weight was statistically lower than control and other salinity levels. Shoot number was not affected by the salinity treatment at any levels (Table 4).
Proliferation rate was declined with the salinity; however, there was no noticeable difference between the salinity levels. Leaf number per shoot in'Fakhri' was not in uenced by the salinity of up to 0.75 gL -1 . But, in 'Sultanin', there were signi cant differences between the control and saline treatments considering the number of leaves. The number of three nodal shoots per explant was drastically impacted by 1 gL -1 salinity in both cultivars.

Biochemical parameters.
For the APX activity and for both cultivars, there were no differences between control and NaCl treatments. But the APX activity of cultivar 'Fakhri' was signi cantly higher than that of the other cultivar. GPX attained the top-activity with 'Fakhri' (Fig. 1).
The highest recorded activity of CAT was at 0.5% and 1 gL -1 for 'Fakhri' and 0.25 gL -1 NaCl for 'Sultanin'. The least data for CAT was belonged to control plants of cultivar 'Sultanin'. SOD activity in 'Fakhri' was correspondingly increased with salinity from 0.25, 0.75 gL -1 . However, in 'Sultanin', there was no signi cant difference in SOD activity between control and the saline treatments up to 0.75 gL -1 . Signi cant SOD activity differences were observed between the cultivars (Fig. 2).
MDA content as the biomarker of membrane lipids peroxidation was uniformly responded to the salinity levels in both cultivars. Soluble proteins content in both cultivars was diminished. However, the reduction was more highlighted in 'Fakhri' (Fig. 3).

Correlation coe cient.
As shown in table 5, the positive or negative response were observed between the traits in response to salinity stress. Considering the signi cant positive relationship was recorded between the shoot lengths with the fresh weight of proliferated explants, variability, shoot number, proliferation rate and leaves number. Furthermore, positive and signi cant correlation was calculated in between the APX activity and the activity of GPX and CAT. Moreover, the relationship was observed between GPX with CAT and SOD. Alongside, negative correlations were recorded between the activity of SOD and GPX with MDA.
Overall, considering all the results, the traits Y1 to Y7 and the activity of enzymes; APX, GPX and SOD would be reliable criteria in assessing the grapevine genotypes against the salinity stress and to select the promising salt tolerant grapevine cultivars.
Factor and bi-plot analysis.
Using the rst three main factors, the proportional effect of the traits on the main factors were depicted by a bi-plot chart (Fig. 4). The results of the bi-plot analysis revealed the traits; stem length, the fresh weight of proliferated explants, viability, shoot number, proliferation rate and leaves number had reasonable relationships. Furthermore, factor analysis showed that the two major factor with the top proportional rate 60.395% of total variations were explained by the variables. The rst main factors with the highest positive impact on the observed variation were Y1 to Y7. For the second main factor, the traits Y8-Y11 were the most in uential positive ones. But, Y12 had the most negative and bi-plot analysis is in agreement with correlation results that Y1-Y7 signi cantly correlated to each other (Table 6 and 7).

Gene expression.
Expression pattern of DREB/CBFs genes in response to the in vitro salinity was also concentration dependent and, showed an ascending pattern with increase in salinity. Expression of CBF 2,3 and 4 were increased with salinity of 0.25 to 1 gL -1 and compared to control in both cultivars. CBFs expression in 'Fakhri' was more than 'Sultanin' in response to the salinity. For CBF 4 , with salinity up to 1 gL -1 ; the expression was 5.66 and 4.25 fold more than control in 'Fakhri' and 'Sultanin', respectively. CBF 3 expression was 3.95 and 2.94 fold higher than control for 'Fakhri' and 'Sultanin' again with the highest salinity level tested. At 0.25 gL -1 salinity level, CBF3 expression did not show signi cant differences between the treatments. The same trend of expression pattern was followed for CBF 2 with up to 1 gL -1 salinity (2.45 and 2.14 fold increase for 'Fakhri' and 'Sultanin', respectively). However, there were no differences between cultivars with regards to salinity levels ( Fig. 5). The most recorded CBF 1 expression for both cultivars was belonged to 0.75 gL -1 salinity level. Meanwhile, the trend was decreasing with the higher level of salinity (1 gL -1 ).

Discussion
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 Emperor 35 Perlette, Beauty seedless and Delight 36 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 viability 7 . High Na + concentrations in the growing media antagonistically prevent the absorption of other cations. Researchers demonstrated that the more sodium availability limited Mg 2+ , Ca 2+ and K + intake in grapevines. Salinity restrains the overall growth and development of plants 2 . The others reported the same idea emphasizing that the shoots growth in grapevine was greatly inhibited by the action of Cl − ions 3,37 .
Salinity damage is dependent upon the NaCl concentration, treatment time-course, and cultivar type. Several researchers have veri ed the mentioned side effects of salinity on plant 35,38 . 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 intensi ed by the toxic effects of Na + and Cl − ions 2,3 .
In the present study, APX enzyme activity in 'Fakhri' under the diverse salinity levels had no signi cant 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

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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 species 40,41 .
CAT activity was increased in 'Sultanin' grape in line with salinity levels. On the contrary, in 'Fakhri', there was no signi cant difference in CAT activity between shoots under various salinity treatments. Sweet cherry rootstocks showed broad diversity in CAT activity in vitro exposed to salinity treatments 25 . The similar results are available for apple 39 . 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 enzymes 11,42 . 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 stress 39 . Furthermore, with high salinity levels and the age of the plants, the SOD activity was considerably increased 43 . The elevated SOD activities under the salinity stress have been reported for mulberry 44 , citrus crops 45 and maize 46 .
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 rst step in this passage is the scavenging of H 2 O 2 to protect the damages caused at cellular, and tissue level and to mitigate the oxidative stress 47,48 .
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 intensi es the scavenging of ROS radicals 46,49 .
Many ndings 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 ethylene 50 . 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 CAT 39,44,48 . 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 nally denatures proteins and alters their conformational moiety 46 .
The same trend was followed for CBF 4 and the expression rate in cultivar 'Fakhri' was higher than that in cultivar 'Sultanin'. CBF 1 , and in 'Fakhri', the expression was additive up to 0.75 gL − 1 of NaCl. But, then after decreased. In 'Sultanin', the CBF 1 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 speci c 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 in uential in the induction of tolerance responses to the low temperature stress 51,52,53 , Otherwise, several studies have claimed that DREB/CBF genes are induced by the several stressors and, ABA activates a bunch of DREB/CBF genes 51,54,55 . CBF 4 is the major gene respondent to the drought stress. CBF 4 expression is more activated by the exogenous application of ABA in Arabidopsis and grapevine. CBF 1 − 3 expression was also enhanced with the ABA external applications in main part due to the increased activity of CBF promoters in reply to ABA 54,56,57 . The differences in CBF proteins in grapevine re ect the positioning of CBF 4 in the separate phylogenetic groups compared to CBF 1,2, 3 19,58,59 . This possibly justi es that CBF 4 in grapevine behaves in different manner than CBF 1,2 and 3.

Conclusions
The growth parameters were evaluated in two-grapevine cultivars in vitro with four salinity treatments. Shoot length, explant weight, viability, number of shoots, proliferation ratio and the number of 3 nodal shoots were signi cantly decreased by salinity. The explants viability in 'Sultanin' signi cantly decreased with increasing salinity levels. The growth parameters in 'Sultanin' showed signi cant decrease compared to 'Fakhri' under different levels of salinity. However, there was no signi cant difference response of explants to 0.75 and 1 g/l of salinity except for the viability trait. The activity of antioxidant enzymes APX, GPX, CAT and SOD in 'Fakhri' cultivar was higher than that of 'Sultanin' with the diverse salinity levels. 1 g/l salinity signi cantly reduced the activity of SOD. As salinity levels increased, malondialdehyde content was increased in both cultivars. The expression rate of CBF 2,3,4 genes showed a signi cant raise with salinity. CBF 1 expression rate was increased with the salinity of up to 0.75 g/l in both cultivars. 'Fakhri' cultivar was more tolerant to the salinity more possibly due to its high antioxidant capacity and the high expression of CBFs genes.       The effects of NaCl treatment on APX and GPX activity of two grapevine cultivars; 'Fakhri' and 'Sultanin' in vitro. Means followed by the same letter on columns are not signi cantly different at 0.05 level, according to the Duncan's multiple range test. Data are mean±SD (n=4 replicates).

Figure 2
The effects of NaCl treatment on CAT and SOD activity of two grapevine cultivars; 'Fakhri' and 'Sultanin', in vitro. Means followed by the same letter on columns are not signi cantly different at 0.05 level, according to the Duncan's multiple range test. Data are mean±SD (n=4 replicates).

Figure 3
The effects of NaCl treatment on MDA and protein content of two grapevine cultivars; 'Fakhri' and 'Sultanin', in vitro. Means followed by the same letter on columns are not signi cantly different at 0.05 level, according to the Duncan's multiple range test. Data are mean±SD (n=4 replicates).