The results of the present study show that the application of sodium nitroprusside in two stages (pre and post-harvest) leads to less reduction of weight of flowers as compared to control. Normally, RFW is also strongly associated with the vase life of cut flowers. Maintaining the fresh weight of cut flowers during vase life period is an important parameter to evaluate the vase life and the postharvest quality of flowers and it has been proven that the heavier the cut flowers, the longer the vase life 28. Prior studies have noted the importance ofnitric oxide in increasing water absorption, as a result, increasing the relative fresh weight of cut flowers by influencing the regulation of ion channels of guard cells, closing stomata 10, decreasing osmotic pressure, transpiration and an effect on abscisic acid 14. In this study, sodium nitroprusside was also used in the pre-harvest stage, it is possible that sodium nitroprusside, as a nitric oxide-releasing compound, increased endogenous β-gluconase activity. The enzyme β-gluconase is involved in the hydrolysis of glycosidic bonds between glucose units within the cell wall, thereby leading to cell wall relaxation and increased cell development. In a study, it was stated that nitric oxide deficiency in mutant Arabidopsis plants reduces the activity of β-gluconase enzyme, so plant growth remains limited. Since the growth process is associated with an increase in turpentine pressure and consequently an increase in the relative water content, nitric oxide deficiency leads to a decrease in turpentine and fresh weight in plants 29. The effect of nitric oxide on many other processes, including stem elongation, meristem development, and relationship between organs, has been reported by the vascular system. The presence of nitric oxide in vascular tissues and epidermal cells of roots, stems and leaves has been proven 30, Therefore, it seems that the application of sodium nitroprusside in the pre-harvest stage in this study could be effective through the development and improvement of the vascular system and also the positive effect on plant water relations on the fresh weight of the plant. In confirmation of the results of the present study in Abdi’s 31 study on roses of ‘Avalanch’, they reported the effect of nitric oxide on increasing the relative fresh weight of flowers in the post-harvest stage. In accordance with the present results, previous studies have demonstrated the positive effects of sodium nitroprusside or other nitric oxide releasing compounds on the improvement of relative fresh weight. For example, Mittal and Shalini 32 reported the effect of sodium nitroprusside on gladiolus, Rahimian Booger and Salehi 33 on gladiolus, Mirzaei Esgandian and Jabbarzadeh 34 on rose ‘Utopia’ and ‘Dolce Vita’, Karamian et al. 35 on gerbera flowers and Dwivedi et al. 16 reported on gladiolus.
In our study, the Alstroemeria cut flowers absorbed more solution and performed better when sprayed with SNP and then pulse treated with SNP than control (Figure 2). Vase life of cut flowers is dependent upon the water status in flowers i.e., turgidity. The flowers are acceptable only when petals are turgid. When a flower is harvested, the uptake of water from parent plant ceases but the transpiration loss continues that results in water deficit followed by petal wilting 36 . Flowers with wilted petals become unacceptable and their vase life is over. So, water status plays an important role in determining the vase life of flowers 37, 38, 39, 40. Uptake of water by the plant is one of the important factors to increase the vase life of cut flowers 18. Many studies have reported that nitric oxide causes the stomata to close, which in turn leads to the production of the abscisic acid, and as we know, this hormone plays a key role in responding to water stress and increases water absorption 41. Nitric oxide has a lead role in xylem differentiation which could be accounted for a higher amount of solution/water uptake by flowers with the application of SNP, NO donor, 42. SNP responses are dose dependent so at low concentration, it resulted in better uptake through stem whereas at high concentration, it interfered with translocation channel. The decrease in the ability to absorb solution at a high concentration of SNP could also be ascribed to microbial growth in vase solution as well as on the stem surface and air embolism of xylem tissues 43. In addition, nitric oxide leads to improved quality through stem strength and chlorophyll retention 44. It is indirectly effective in water uptake.
The current work clearly reported the ability of SNP to significantly prolong the flowers longevity and maintain the postharvest quality of them. The mechanisms thereby SNP treatments exhibited these effects most probably ascribed to water relation maintenance, antimicrobial effect, alleviating the oxidative stress, membrane integrity and chlorophyll maintenance of cut flowers. These mechanisms altogether regulated and delayed the senescence of Alstroemeria cut flowers. SNP treated flowers showed greater ability to maintain water as indicated by higher RWC and RFW of the flowers. Maintaining water relations during postharvest has shown to be very important to extend the vase life while disturbed water balance leads to senescence of flowers 45, 46, 47, also reported that maintaining the fresh weight of flowers may be ascribed to increased water uptake and/or transpiration reduction, thus preventing the fresh weight loss. In accordance with our data, previous publications indicated that SNP treatment improved water relations and increased the RWC during vase life period 48, 49, 50.
The application of sodium nitroprusside in both pre and post-harvest stages reduced the amount of malondialdehyde, although over time, the amount of malondialdehyde showed an increasing trend. Malondialdehyde is a product that is produced by peroxidation of membrane lipids and its amount inside the cell can indicate the amount of oxidative stress in plants as well as cell membrane homeostasis. So, the higher membrane stability plays a key role in inhibiting leakage of electrolytes, sugars, pigment, solute leakage, and also lipid peroxidation as well as in delay senescence during cut flowers postharvest 51. Our results showed that the change in lipid peroxidation occurrence resulting from the MDA production were alleviated by SNP concentrations (50, 100 μM). These results are in agreement with those reported earlier by Mansouri 52Kazemzadeh-Beneh et al. 53, Mohasseli and Sadeghi 54; who suggested that SNP prolonged the vase life of flowers. Cut flower senescence is linked to a sequence of highly regulated physiological and biochemical processes such as degradation of proteins, DNA content, peroxidation of lipids and membrane leakage, degradation of macromolecules, cellular decompartmentalization, floral abscission, color change, leaf yellowing, and weight loss 55. These changes are due to the increase in the production of reactive oxygen species, which is due to the imbalance of reactive oxygen radicals and antioxidant enzymes, leading to secondary oxidative damage due to the loss of permeability of the membrane 56. Lipid peroxidation is a complex process that begins with the removal of hydrogen atoms from unsaturated fatty acids, which in turn leads to the production of lipid radicals. The increase in the lipid peroxidation process is determined by a change in the amount of malondialdehyde. Sodium nitroprusside, as a nitric oxide-releasing compound, can reduce lipid peroxidation by three mechanisms. A) In the first mechanism, nitric oxide reacts rapidly with alloxy lipids and peroxide radicals, thereby stopping the peroxidation chain and thereby reducing the amount of malondialdehyde. B) In the process of lipid peroxidation, the enzyme lipoxygenase is one of the key oxidizing enzymes in this process, and its mechanism is also mentioned by reducing Fe3 + to Fe2 + in the active site of the lipoxygenase enzyme 57. C) The third mechanism is through the effect of nitric oxide on the increase of antioxidant enzymes, which leads to the removal of activated oxygen radicals, thereby reducing cell membrane damage and thus reducing the amount of malondialdehyde 54.
Considering that it has been reported in various studies that the application of high concentrations of nitric oxide releasing compounds depending on the plant genotype can lead to toxicity in plants 16, so considering that, at a concentration of 200 μM, malondialdehyde levels showed an increasing trend. It can be assumed that this concentration is high for Alstroemeria and leads to toxicity thus increased MDA.
As shown in figure 5, the application of sodium nitroprusside in both preand post-harvest stages increases the activity of catalase, antioxidant enzyme. In the post-harvest stage of flowers and plants, the production of reactive oxygen species is one of the main causes of damage that changes the structure of membrane compounds and antioxidants. Under natural conditions, reactive oxygen species are constantly produce at low concentration in different parts of the plant, including chloroplasts and mitochondria, plasma membranesand etc., which increase in stress conditions and senescence. Earlier studies have been confirmed that reactive oxygen species ultimately cause damage to proteins, RNA, DNA, and cell membranes, and cause to lipid peroxidation 58. The action of nitric oxide as an antioxidant and its cytotoxic role due to the reaction with free radicals have been considered 16. In addition, nitric oxide induces the production of antioxidant enzymes, thereby suppressing free radicals and delaying senescence 14. Also nitric oxide could delay leaf senescence by enhancing CAT and SOD activities and inducing the production of other enzymes, such as glutathione 59.Various reports indicate that nitric oxide has a high affinity for iron-containing enzymes and improves the activity of these protective enzymes. The application of nitric oxide varies depending on the species, cultivar and concentration used, but it has been shown that the application of nitric oxide in low concentrations leads to the expression of genes involved in the synthesis of protective enzymes 60. Earlier studies have shown that nitric oxide, after reacting with reactive oxygen species, leads to the production of peroxynitrite and reduces the production of these reactive oxygen species. When the pH is in the physiological range, the peroxynitrite produced can be decomposed into a nitrate and proton anion or react with a radical of hydrogen peroxide, thereby reducing reactive oxygen species 60, which may be the reason for the increase in antioxidant enzymes activity in the present study. The results were in accordance with the findings of Naing et al. 55 that reported a positive effect of nitric oxide on the activity of antioxidant enzymes on gerbera cut flowers and Mittal and Shalini 32 that reported the application of SNP on gladiolus flowers increases the activity of antioxidant enzymes such as catalase and ascorbate peroxidase. In the present study, it was shown that the application of high concentrations (200 μM) of SNP cause a decrease in the activity of antioxidant enzymes and antioxidant capacity. Negative effects induced by high concentrations of SNP in CAT activity were dose- dependent 14, therefore, in the present study, preharvest foliar application of 100 μM SNP jointly with pulse application of 50 μM of it were selected as optimal concentrations for CAT activity.
In the present study, the application of sodium nitroprusside increased the vase life of Alstroemeria flowers. In cut flowers, one of the limitations of their maintenance in the postharvest stage is senescence. In senescence process, the formation of reactive oxygen species leads to oxidative damage, which indicates the inability of the plant to antioxidant defense at this stage 61. In recent years, nitric oxide has been recognized as an antioxidant molecule in plants 62.It should be noted that the effect of nitric oxide depends on the concentration and species of plants, and high concentrations of nitric oxide, through nitrosative stress in cooperation with oxidative stress, cause more destructive damage 63. Nitric oxide, in high concentration as a free radical, has the ability to damage membranes, proteins and nucleic acids in plant cells, but in low concentrations is necessary for plant growth and acts as a defense system in plants, therefore, NO has both protective and toxic effects 64. In this study, preharvest application of 200 μM SNP led to a reduction in vase life, but at a concentration of 50 μM, the highest vase life was observed, which shows the positive effect of low concentrations of sodium nitroprusside on vase life. Sensitivity to ethylene and water stress is the main factor limiting the postharvest life of cut flowers 21. In the present study, the application of sodium nitroprusside at a concentration of 100 μM in the preharvest stage and a concentration of 50 μM in the postharvest stage, in addition to increasing vase life, led to a reduction in water loss. In Alstroemeria, one of the most important problems in the postharvest stage is yellowing of leaves and reduced vase life. Therefore, SNP application for exogenous NO could act as a promising tool to enhance the postharvest performance of Alstroemeria cut flowers. It is believed that NO protects plants against oxidative stress and early senescence by two mechanisms, i.e., the antioxidant effect and changing the expression of defensive genes in plant 65. The results were in accordance with the findings of Salachna and Byczynska 64on cut Eucomis, Ghaei et al. 50 on Gerbera, Mirzaei Esgandian and Jabbarzadeh 34 on two cultivars of rose, Dwivedi et al. 16 on cut gladiolus and Kazemzadeh-Beneh et al. 53 (2018) on gladiolus.