In cold-tolerant plants; proline is the predominant amino acid acting as a protective compound against cold and the elevated proline concentration in tissues is the mechanisms by which, to withstand cold stress. Moreover, the accumulation of this amino acid has been reported in many plants under cold stress due to the increased biosynthesis or a decreased degradation rate [2, 4]. Protecting the plant from osmotic changes, keeping the integrity of biological membranes, maintaining pH, protecting cellular enzymes, as well as storing energy for post-frost recovery are the major functions of proline in face of cold stress [18]. During cold stress, the total protein content increases which goes on to a certain extent and then decreases. These fluctuations can be interpreted that in the onset of stress, the plant begins to increase the expression of genes involved in the biosynthesis of defense enzymes to protect cellular structures to keep their normal activities. Therefore, by producing a sufficient amount of defense enzymes by the cells, it is not necessary to further increase the number of enzymes as a subset of total protein; and behind a sufficient period from the onset of stress, the conditions are under control by the plant cells [2–4]. Also, among the three cultivars studied, the lowest increase in malondialdehyde content compared to the control treatment was devoted to ‘Perllete’. Malondialdehyde is one of the end products of membrane lipids peroxidation resulting from the reactive oxygen species activity. In other words, malondialdehyde levels are often used as an indicator of oxidative stress damage [19]. In the present study, cold stress increased the peroxidation of membrane lipids and as a result, increased the amount of malondialdehyde in the tissues of three grape cultivars. Commonly, the level of malondialdehyde in plant tissue is an indicator of stress-induced damage. Similar results on the effect of cold stress on malondialdehyde content were obtained in the previous studies with different grape cultivars [20]. Based on the results obtained in the present study, ‘Perllette’ can be introduced as the most tolerant among the tested cultivars, and ‘Ghiziluzum’ can be placed in the next rank in terms of cold tolerance. Plants show variety of morphological, biochemical, and physiological adaptations in response to stresses, including changes in the activities of certain enzymes such as ascorbate peroxidase. According to the literature, ascorbate peroxidase, as the most important antioxidant enzyme in plants; regenerates many free radicals especially hydrogen peroxide. The importance and role of this enzyme have been emphasized in many other plants especially in tangerine [21]. Higher APX activity was observed in Jatropha macrocarpa as a response to high H2O2, which improved cold stress tolerance, whereas reduced APX activity in J. curcas was linked with the increased sensitivity under cold stress conditions [22]. The results of the study conducted by Karimi Alvije et al., [1] also showed an increase in the content of guaiacol peroxidase in 7 different grapevine cultivars due to cold. They stated that placing grape seedlings at 4 ºC initially and significantly increased the content of this enzyme, but then stress caused decreasing pattern in its content. In cold-tolerant plants; the more efficient mechanisms enable them to protect themselves against the destructive effects of ROSs [4, 6]. This group of plants uses enzymes such as Superoxide Dismutase (SOD), Catalase (CAT), Ascorbate Peroxidase (APX) and Glutathione Reductase (GR) as well as non-enzymatic compounds including Ascorbate, Tocopherol, Carotenoids and other compounds (including flavonoids, polyanols, mannitol) to gain the ability to reduce reactive oxygen species damage [1, 3, 6]. In plant cell, the AsA-GSH cycle is the major antioxidant defense pathway to detoxify H2O2, and redox homeostasis [21, 23]. Hydrogen peroxide and reactive oxygen radicals are produced under natural conditions in small amounts during the common metabolism in diverse organelles, including chloroplasts, mitochondria, and peroxisomes and in any places where there is an electron transport chain [5, 19]. ‘Ghiziluzum’ with its high H2O2 production was more sensitive to cold stress than ‘Khalili’ and ‘Perllete’ and had low cold tolerance. Numerous studies, including research on apples and pears, showed an increase in H2O2 accumulation under stress conditions [24]. Cell membrane is the first site of damage by the cold stress and the change in membrane state as a result of cold stress causes the membrane malfunction. So, measuring the electrolyte leakage of tissues is an acceptable criterion for evaluating plant resistance to cold stress [21, 22]. In the present study, ‘Perllete’ was more tolerant to the cold stress in terms of electrolyte leakage than ‘Khalili’ and ‘Ghiziluzum’. The results obtained in this study were consistent with the findings on different grape cultivars [20], which reported the increased electrolyte leakage in response to the cold stress. Plant survival under environmental stresses, especially cold stress, requires the control of harmful compounds inside plant cells, which ensure plant survival. These harmful compounds can be ROS and compounds resulting from the oxidation of biological substances in cell metabolism. These compounds cause metabolic disorders due to intense electron demand. Antioxidant enzymes reduce cell damage by scavenging, ROS, and antioxidants are involved in reducing the oxidation of biomolecules, such as lipid peroxidation, by supplying the electrons needed [4, 6, 20].
When light is at a moderate level; its majority is employed in photochemical activities for photosynthesis and a small part of its energy is emitted as fluorescence known as basal or minimal fluorescence (F0) [25]. In the present study, the amount of F0 in all studied cultivars increased due to cold application. An increase in F0 indicates a damage to the photosystem II electron transfer chain due to a decrease in the capacity of quinone A (QA) and lack of its complete oxidation to a slow flow of electrons along the photosystem II pathway and the inactivation of photosystem II. A rise in F0 is associated with photoinhibitory damage but not with zeaxanthin retention [26]. The increased F0, due to cold stress, has been observed in plants such as basil (Ocimum basilicum L.) and lettuce (Lactuca sativa) [27]. The stress conditions causes structural changes in the pigments in photosystem II and the fluorescence function such as maximal fluorescence is changed, making it possible to use these factors as an indicator for estimating stress-induced damage to the plant photosynthetic system [25, 26]. The results showed that cold reduced the maximal fluorescence in all studied cultivars and this drop of Fm occurred at the maximum with applying 4 hours of cold stress. Researchers suggested that a decrease in Fm may be related to a decrease in the activity of the water-degrading enzyme complex as well as the electron transfer cycle in or around photosystem II [26, 27]. Research on tomatoes (Solanum lycopersicum) [27], also showed a decrease in Fm due to cold stress. Therefore, the measurement of Fv/Fm can be used as a successful method to determine the status of the photosynthetic apparatus and identify the degree of cold tolerance in plants [25]. By slowing down the insertion of protein D1 into the center of photosystem II, cold stress slows down the plants recover and causes membrane degradation and chlorophyll oxidation, thereby reduces the Fv/Fm ratio (maximum photochemical quantum yield of photosystem II under adaptive conditions to light). It is an estimate of the maximum photochemical quantum yield of photosystem II [28]. In many plant species, when the Fv/Fm ratio is about 0.7 to 0.8, it means that no stress has been applied on the plant. Therefore, values less than this amount indicate the effects of stress on plants [25–27]. In fact, chlorophyll fluorescence indicates a decrease in the initial health of the plant before the signs of deterioration appear, so that this trait indirectly indicates health (fluidity, stability, and cohesion) of photosynthetic membranes [25, 28]. Considering that high Fv/Fm indicates high cold resistance; among the three studied grapevine cultivars, ‘Perllete’ with the highest maximum photochemical quantum yield of photosystem II facing cold was more cold tolerant than the other two cultivars. Cold stresses increase F0 and decrease Fv/Fm, which indicates the discontinuity of light-absorbing pigments from the photosystem II complex, leading to a decrease in the quantum performance of photosystem II. The activity of the photosystem II is severely reduced or even stopped under cold conditions, and chloroplasts, stromal carbon metabolism, and photochemical reactions in the thylakoid lamella have been cited as primary sites of cold stress injury [25–28].
A study showed that drought, salinity and exogenous abscisic acid in plants caused VaCBF4 expression. Overexpression of VaCBF4 in transgenic Arabidopsis increased the tolerance to cold, salinity and drought compared to control plants [29]. The expression of VrCBF4 gene under stress conditions in different plant tissues continued for several days, which indicates the role of this gene in acclimation to stress conditions. Biological age did not affect CBF4 expression, and transcription of this gene was observed in young and old leaves of V. vinifera and V. riparia for several days [10]. There are similar sequences in the promoter of two CBF1 and CBF4 genes, that protein product of ICEr1 gene binds to that and induce this gene not only by cold but also by the other stresses [9]. In the present study, similar to some previous researches, CBF4 gene expression accelerated immediately after cold exposure and decreased after 8 hours, but, is in contrast to the findings of other researchers who reported long-term accumulation of CBF genes [7, 10]. VvNAC26 showed the greatest expression change in response to abiotic stress in the whole NAC family and acts as a COR NAC gene. VvNAC18 induces freezing tolerance by increasing DE NAC genes, so NAC genes are expressed in response to cold stress and increase plant tolerance [11, 12]. VvNAC1 may involve several signals, including developmental processes and responses to biotic and abiotic stresses, and can act as a novel node between different signaling pathways. Functional analysis has shown that VvNAC1 has a positive role in abiotic stress tolerance [30]. NAC transcription factors increase stress tolerance in plants by modulating transcript levels of CBFs and their putative downstream such as COR / ERD / RD genes. One study showed that the function of VaNAC17 in a CBF-dependent signaling pathway induced drought stress tolerance by modulating the expression of stress-responsive genes [14]. NAC transcription factors participate in various signaling pathways to against with cold stress, so the study of NAC transcription factors is a prerequisite for improving the effects of stress on plants. NAC1 gene expression increases under cold stress by tolerant cultivars with producing signaling and increasing the expression of ROS inhibitory genes and also increases the response of CBFs genes, especially CBF4. CBFs cold-response proteins encode DNA-binding domains. These proteins bind to the DRE/CRT sequence and regulate cold-induced promoters.