ORIGINAL ARTICLE
https://doi.org/10.1007/s10341-019-00445-1
SNP Mitigates Malignant Salt Effects on Apple Plants
Servet Aras1· Hakan Keles1· Ahmet Eşitken2Received: 23 January 2018 / Accepted: 21 June 2019 / Published online: 8 July 2019 © Springer-Verlag GmbH Deutschland, ein Teil von Springer Nature 2019
Abstract
Sodium nitroprusside (SNP) a nitric oxide donor is utilized as an antioxidant under stress conditions in order to mitigate stress damages. To probe into the potential relieving salinity malignant effects, we investigated the protective roles of SNP. An apple plant (Malus domestica Borkh.) cv. Fuji grafted on MM106 and M9 clonal rootstocks was chosen for the experiment and imposed to salinity stress for 4 months with 35 mM NaCl. SNP with different three doses (1, 2 and 4 mM) was applied to the roots of the salt-stressed apple plants except control. SNP applications inhibited apple plants growth depression through increasing stomatal conductivity, chlorophyll and protein content and decreasing electrolyte leakage and lipid peroxidation. Beside that, SNP triggered chlorophyll biosynthesis and maintained better cell membrane stability compared to control. In cv. Fuji/MM106, 1 mM SNP application had the highest SPAD value (48.6) even more than control plants (44.8). 4 mM SNP showed the highest stomatal conductivity (313 mmol m–2s–1) and the lowest value was obtained
from salt plant (141 mmol m–2s–1). In cv. Fuji/M9, 4 mM SNP elevated the protein content by 73% compared to control.
Information from current experiment SNP can be utilized to improve soil management practises under salt stress condition. Moreover, SNP affected apple plants through antioxidant mechanism, but did not have impact on osmotical adjustment.
Keywords Chlorophyll contents · Enzyme activity · Growth · Malus · Tolerance indices
SNP mindert die gefährlichen Auswirkungen von Salzbelastungen bei Apfelbäumen
Schlüsselwörter Chlorophyllgehalte · Enzym-Aktivität · Wachstum · Toleranzindizes
Introduction
Salinity is one of the major environmental stresses in arid, semi-arid and seaside regions due to the poor drainage, excessive fertilization and coastal areas. Many malignant effects of salt stress have been showed in many plants such as decrease in the photosynthesis, metabolisms, water sta-tus and plant growth, increase in reactive oxygen species (ROS) leading cellular damage through oxidation of lipids and protein (Niu et al.1995; Foyer and Noctor2000; Yin et al.2010). Furthermore, higher accumulation of sodium
Servet Aras
servet.aras@bozok.edu.tr
1 Faculty of Agriculture, Department of Horticulture, Bozok
University, 66200 Yozgat, Turkey
2 Faculty of Agriculture, Department of Horticulture, Selcuk
University, 42030 Konya, Turkey
(Na) ion possess the detrimental effects on cell membrane affecting metabolic activities (Zhu2001).
The plants produce many physiological and biochemi-cal responses to cope with the detrimental effects of salin-ity, such as accumulation of proline, glycine betaine, syn-thesizing of antioxidants against ROS (Khan et al. 2012). Therefore, increase of defense mechanisms in plants is nec-essary for improving the tolerance to salt stress. In recent years, many experiments showed that the nitric oxide (NO) and its donors such as sodium nitroprusside (SNP), S-nitro-Nacetylpenicillamine (SNAP) help plant growth and de-velopment under stress conditions (Siddiqui et al. 2011; Bellin et al. 2013; Jian et al.2016). In previous studies, it has been demonstrated that NO is involved in many processes of plants such as induction in cell death (Pe-droso and Durzan2000), stomatal movement (García-Mata and Lamattina2007), photosynthesis regulation (Takahashi and Yamasaki2002) and floral regulation (He et al.2004). Among the NO donors, SNP is one of the most utilized one (Filippou et al. 2012). Many studies revealed
exoge-nous SNP could mitigate the damage caused by salt stress in cucumber (Shi et al.2007), cotton (Liu et al.2014) and medicago (Jian et al.2016). Moreover, SNP application re-markably mitigated the oxidative damage of salt stress to lupin (Kopyra and Gwozdz2003) and cucumber (Fan et al.
2007). However, no attempts have to date been made to study effects of SNP on the morphological, physiological and biochemical properties in apple plants under salinity stress.
There are two sources of the soil salinization: natural or primary salinity results from the accumulation of solu-ble salt in soils or groundwater over long geological periods and secondary salinization grow out of different agricultural practices, mainly irrigation and fertilization. Moreover, oc-currence of secondary salinization is becoming ever more visible (Hasanuzzaman et al.2013). High fertilizer applica-tion, irrigation rates and intensive continuous cropping are main reasons for aggravating soil secondary salinization. Because apple growing often requires a greater degree of management and involves much larger inputs of nutrient and irrigation, high irrigation and excessive fertilizer have been used in apple growing. Therefore, the plant growth and development are negatively affected under saline con-ditions. Apple (Malus domestica Borkh.) is sensitive to salt, and as a perennial woody plant the mechanism of salt stress adaption and response to SNP application could be different from that of annual plants, such as Arabidopsis, cucumber, cotton and medicago, and could be various at rootstocks and/or varieties.
Considering the pivotal role of NO in plants, the current study was undertaken to evaluate the effects of SNP in apple plants in order to mitigate the salt stress damages and to probe into role of SNP in regulation of the defense system as physiological and biochemical under saline conditions.
Material and Method
Pot Trials and Experimental Design
The study was conducted in 2014 in a heated greenhouse of Department of Horticulture at Selcuk University in Turkey. An apple plant (Malus domestica Borkh.) cv Fuji grafted on MM106 and M9 clonal rootstocks was chosen for the experiment with following a randomized plot design in-volving three replications, with three plants per replication and was planted in pots in March. Up until the start of the experiment, all plants were irrigated with tap water and 1 month later (in April) plants were watered with 35 mM NaCl solution. Two months after the salt stress, three differ-ent SNP doses (1, 2 and 4 mM) were treated twice a month interval (in June and July) to plant rhizosphere as solution except control. Control and salt plants were not applied
with SNP, salt plants were watered with NaCl solution com-pared to control. Four months after the salinity (in August), many morphological, physiological and biochemical prop-erties were evaluated. Three leaves per plant were used for the measurements.
Growth Measurements and Physiological Determinations
The growth promoting effects of SNP treatments were eval-uated by determination of rootstock and scion diameter and shoot length. Tolerance indices (TI) of rootstock and scion diameter and shoot height were determined according to Shetty et al. (1995) with some modifications and calculated as follows: TI = (rootstock and scion diameter and shoot length of SNP + salt applied plant/rootstock and scion di-ameter and shoot length of control plant) × 100. Relative chlorophyll (SPAD) value was measured with a Minolta SPAD-502 chlorophyll meter (Minolta Camera Co, Ltd, Osaka, Japan). Stomatal conductivity was conducted on the youngest fully expanded leaves on upper branches of the plants with leaf porometer.
The procedure of membrane permeability (electrolyte leakage) based on Lutts et al. (1996) was used to assess membrane permeability. Electrolyte leakage was measured using an electrical conductivity (EC) meter. Mature leaves per plant were taken and cut into 1 cm segments. The leaf samples were then placed in individual stoppered vials containing 10 mL of distilled water after three washes with distilled water for removing surface contamination. These samples were incubated at room temperature (25 °C) on a shaker (100 rpm) for 24 h. Electrical conductivity of bathing solution (EC1) was measured after incubation. The same samples were then placed in an autoclave at 120 °C for 20 min and the second measurement (EC2) was taken after cooling solution to room temperature. The elec-trolyte leakage was calculated as EC1/EC2 and expressed as percent.
In order to determine leaf relative water content (LRWC), the leaves were collected from the young fully expanded leaves of three plants per replicate. The individual leaves first detached from the stem and then weighted to de-termine fresh weight (FW). In order to dede-termine turgid weight (TW), the leaves were floated in the distilled water inside a closed petri dish. The leaf samples were weighted periodically, after gently wiping the water from the leaf sur-face with the tissue paper until a steady state was achieved. At the end of imbibition period, the leaf samples were placed in a pre-heated oven at 72 °C for 48 h, in order to determine dry weight (DW). Values of FW, TW, and DW
were used to calculate LRWC using the equation given below (Smart and Bingham1974):
LRWC.%/ = Œ.FW − DW/=.TW − DW/ 100
Biochemical Determinations
Mature leaves of the plants were used for the protein con-tent determination. The leaf segments were ground in cold phosphate buffer (pH: 6.5) and then filtered. The filtrate was centrifuged at 4000 g for 20 min at 4 °C. The super-natant was decanted and added Bradford Protein Kit. The mixture was vigorously shaken with vortex. The sample absorbance was read at 595 nm. The protein levels were estimated by the method of Bradford (1976) using bovine serum albumin as standard and expressed at mg protein g–1
fresh weight (fw).
The proline content was estimated by the method of Bates et al. (1973). The plant material was homogenised in 3% aqueous sulfosalicylic acid and the homogenate was centrifuged at 10,000 rpm. The supernatant was used for estimation of the proline content. The reaction mixture con-sisted of 2 ml supernatant, 2 ml acid ninhydrin and 2 ml of glacial acetic acid, which was boiled at 100 °C for 1 h. After termination of the reaction in ice bath, the reaction mixture was extracted with 4 ml of toluene and the absorbance was read at 520 nm.
For the determination of chlorophyll (a, b and a + b) content, the fine powder (0.1 g) of the leaves was homog-enized in 10 mL of 80% acetone, and then centrifuged at 12,000 g for 10 min. The chlorophyll (a, b, and a + b) content was spectrophotometrically determined by mea-suring absorbance at 663 and at 646 nm. The chlorophyll (a, b and a + b) content was calculated using the equa-tions of Porra et al. (1989), as follows: (1) chlorophyll a (µg mL–1) = 12.25 A 663– 2.55 A646; (2) chlorophyll b (µg mL–1) = 20.31 A 646– 4.91 A663; and (3) chlorophyll a + b (µg mL–1) = 17.76 A 646+ 7.34 A663. Chlorophyll stability
index (CSI) was calculated as follows (Sairam et al.1997): CSI = (total chlorophyll under stress/total chlorophyll under control) × 100.
For the quantification of total phenols, methanol was used for extraction. Total phenolic content was assayed by A765with Folin-Cocalteau reagent (Singleton and Rossi 1965). The results were expressed as µg of p-hydroxycin-namic acid (g fresh weight).
The lipid peroxidation was determined by estimating the malondialdehyde (MDA) content in 1 g leaf fresh weight according to Madhava Rao and Sresty (2000). MDA is a product of the lipid peroxidation by thiobarbituric acid reaction. The concentration of MDA was calculated from
the absorbance at 532 nm by using extinction coefficient of 155 mM–1cm–1.
The ascorbate peroxidase (APX) activity was estimated according to the method of Nakano and Asada (1981). The enzyme activity was determined by the decline in absorbance of ascorbate at 290 nm. The reaction mixture consisted of enzymatic extract, 50 mM sodium phosphate buffer, pH 7, 0.5 mM ascorbate, 0.5 mM H2O2and 0.1 mM
EDTA, in a 0.3 ml final volume. The reaction started af-ter the hydrogen peroxide addition. The molar extinction coefficient 2.8 mM–1cm–1 was used to calculate ascorbate
peroxidase activity. Enzyme activity was expressed at unit’s mg–1protein. One unit of enzyme was the amount necessary
to decompose 1μmol of the substrate per minute at 25°C. The statistical analyses were performed with the sta-tistical software package SPSS, version 20.0. Data were subjected to two-way ANOVA and were seperated by the Duncan’s test at a significance level of P < 0.05.
Results
The apple plants were exposed to moderate salinity at 35 mM for 4 months at the vegetative stage and relative damage to the indices of stress and the role of exogenously supplied SNP were evaluated.
The Plant Growth
After 4 months of the treatment, our data confirmed that applying SNP with different doses to the roots significantly mitigated the growth reduction of apple plants. The dif-ferences between rootstocks were not significant while the effects of applications on rootstock diameter was signifi-cant. Salt treatment limited increase in rootstock diameter and SNP applied salted plants overcame salinity damages and provided rootstock diameter growth similar with con-trol (Table1). The differences in both applications and root-stocks were significant for scion diameter. SNP application ameliorated salinity damages in salt plants while salt treat-ment decreased scion diameter compared to control. 1 and 2 mM SNP application provided shoot length similar with control plant, while salt treatment caused decrease in shoot length similar with rootstock and scion diameter. Rootstock TI was affected significantly as well as rootstock diame-ter. SNP applications increased salinity tolerance of plants for both rootstocks (Table 2). For shoot length TI, it is shown that 1 and 2 mM SNP increased salinity tolerance of plants. SNP applications did not have significant effects on scion diameter TI. In Fuji/MM106, 4 mM SNP showed the highest rootstock diameter (22.2 mm) while the salt plants possessed the lowest one (18.6 mm) (Table1). The control plants had the highest scion diameter (19.2 mm). This
effec-Table 1 Effect of SNP on rootstock and scion diameters and shoot length
Treatments Rootstock diameter (mm) Scion diameter (mm) Shoot length (cm)
M9 MM106 Means M9 MM106 Means M9 MM106 Means
Control 21.0 a 20.8 ab 20.9 A 17.5 a 19.2 a 18.4 A 44.3 a 46.8 bc 45.6 A Salt 18.9 b 18.6 b 18.8 B 13.2 b 17.3 b 15.3 B 32.7 b 43.2 c 38.0 B 1 mM SNP + Salt 21.1 a 19.8 ab 20.5 A 15.6 ab 17.3 b 16.5 B 45.3 a 51.0 b 48.2 A 2 mM SNP + Salt 20.0 ab 21.4 a 20.7 A 14.3 b 17.5 b 15.9 B 42.0 a 57.3 a 49.7 A 4 mM SNP + Salt 19.9 ab 22.2 a 21.1 A 14.0 b 19.0 a 16.5 B 33.9 b 44.9 bc 39.4 B Means 20.2NS 20.6 – 14.9 B 18.1 A – 39.6 B 48.6 A – Rootstocks × Treatments NS NS NS
Means separation within column and line by Duncan’s multiple range test (P < 0.05) NS not significant
Table 2 Effect of SNP on tolerance indices (TI) of rootstock and scion diameters and shoot length and chlorophyll stability index (CSI) Treatments Rootstock diameter TI Scion diameter TI Shoot length TI Chl a + b CSI
M9 MM106 Means M9 MM106 Means M9 MM106 Means M9 MM106 Means Salt 90 b 90 b 90 B 75 b 90 b 83NS 74 b 93 c 84 B 89 d 95 c 92 C 1 mM SNP + Salt 100 a 95 ab 98 A 89 a 90 b 90 102 a 109 b 106 A 149 b 136 a 143 A 2 mM SNP + Salt 95 ab 103 ab 99 A 82 ab 91 b 87 95 a 123 a 109 A 141 c 131 a 136 B 4 mM SNP + Salt 95 ab 107 a 101 A 80 ab 99 a 90 77 b 96 c 87 B 157 a 106 b 132 B Means 95NS 99 – 81.5 B 92.5 A – 87 B 105 A – 134 A 117 B – Rootstocks × Treatments NS * NS ***
Means separation within column and line by Duncan’s multiple range test (P < 0.05) NS not significant
*- pÄ 0.05, ***- pÄ 0.001
tive relief was also reflected by SNP in our data for the shoot length (Table1). 2 mM SNP increased the shoot length by 18.4 and 32.6% compared to the control and salt treated plants, respectively. Moreover, 4 mM SNP had the highest values of the tolerance indices of the rootstock and scion diameters (107 and 99, respectively) (Table2). The highest shoot length tolerance index was determined in 2 mM SNP (123).
In cv. Fuji/M9, 1 mM SNP had the highest rootstock diameter (21.1 mm) while the salt plant possessed the low-est one (18.9 mm) (Table 1). The highest scion diameter was observed in the control plants (17.5 mm) and salt de-creased scion diameter by 32.6% compared to the control. 1 mM SNP treated plants possessed the highest shoot length (45.3 cm). Furthermore, 1 mM SNP showed the highest tol-erance indices of the rootstock and scion diameters, and the shoot length (100, 89 and 102, respectively) (Table2). Ex-ogenous SNP significantly offset the declines in the plant growth of Fuji cv grafted onto both rootstocks.
Physiological Responses
SNP treatments significantly influenced the physiology of salinity exposed apple plants (Table3). Salt treatment
caused sigfinicant decrease in SPAD value. Moreover, SNP applications increased SPAD value even more than con-trol plants. Similar with SPAD, salt and SNP applications significantly affected membrane permeability. SNP appli-cations leaded decrease in membrane permeability similar with control value, while salt treatment caused increase in membrane permeability. Salt stress caused significant decrease in stomatal conductivity and that leaded stom-ata closure in response to salinity. Furthermore, 2 and 4 mM SNP increased stomatal conductance through open-ing stomata. LRWC were not significantly affected by applications. The salinity adversely affected SPAD value. In cv. Fuji/MM106, 1 mM SNP application had the highest SPAD value (48.6) even more than control plants (44.8). The highest LRWC value was determined in the control plant (78.9%). 1 mM SNP possessed the lowest membrane permeability (21.2%), while the salt plant had the highest one (41.6%) and 1 mM SNP application showed better membrane permeability compared to non-salted plants. Moreover, the stomatal conductance recorded a steep de-cline by salt treatment. 4 mM SNP showed the highest stomatal conductivity (313 mmol m–2s–1) and the lowest
Table 3 Effect of SNP on physiological parameters
Treatments SPAD LRWC (%) Membrane permeability (%) Stomatal conductance (mmol m–2s–1)
M9 MM106 Means M9 MM106 Means M9 MM106 Means M9 MM106 Means Control 48.7 b 44.8 b 46.8 B 71.6NS 78.9 a 75.2NS 28.1 b 27.6 bc 27.8 B 346 a 308 a 327 A Salt 44.5 c 35.4 c 40.0 C 66.0 67.5 b 66.7 48.4 a 41.6 a 45.0 A 175 b 141 b 158 C 1 mM SNP + Salt 49.5 ab 48.6 a 49.0 A 67.5 67.3 b 67.4 30.6 b 21.2 c 25.9 B 225 b 146 b 186 BC 2 mM SNP + Salt 50.0 ab 48.4 a 49.2 A 75.7 62.7 b 69.2 26.8 b 31.4 b 29.1 B 187 b 294 ab 241 ABC 4 mM SNP + Salt 53.0 a 47.9 a 50.4 A 68.0 61.8 b 64.9 25.6 b 34.2 ab 29.9 B 232 b 313 a 273 AB Means 49.1 A 45.0 B – 69.7NS 67.6 – 31.9NS 31.2 – 233NS 240 – Rootstocks × Treatments ** NS NS NS
Means separation within column and line by Duncan’s multiple range test (P < 0.05) NS not significant
**- pÄ 0.01
In cv. Fuji/M9, the highest SPAD value was seen in 4 mM SNP application (53.0), while the salt plant had the lowest value (44.5) (Table3). LRWC value was not significantly affected in the study. 4 mM SNP demonstrated the lowest membrane permeability (25.6%), while the salt plant had the highest one (48.4%).
Biochemical Responses
In cv. Fuji/MM106, the SNP treatment did not significantly affect total protein and proline content (Table4). Salt and SNP applications significantly affected chlorophyll contents (Table5). SNP applications had higher Chl a, b and a + b contents than control, while salt stress decreased the values compared to control. Chl SI significantly increased by all SNP doses. Thus, it is seen that SNP helps plants to protect chlorophyll content. 2 and 4 mM SNP applications had the highest protein content, while control, salt and 1 mM SNP
Table 4 Effect of SNP on protein, proline, MDA and APX content
Treatments Protein (µg g–1fw) Proline (µmol g–1fw) MDA (µmol g–1fw) APX (µmol g–1fw min–1)
M9 MM106 Means M9 MM106 Means M9 MM106 Means M9 MM106 Means Control 0.011 c 0.015NS 0.013 B 0.011NS 0.015NS 0.013NS 0.0021 a 0.0022 c 0.0022 B 227 b 238 b 233 B Salt 0.013 c 0.012 0.013 B 0.016 0.037 0.027 0.0022 a 0.0025 b 0.0024 A 282 a 305 a 294 A 1 mM SNP + Salt 0.014 bc 0.012 0.013 B 0.013 0.012 0.013 0.0015 b 0.0017 d 0.0016 D 223 b 250 b 237 B 2 mM SNP + Salt 0.017 ab 0.020 0.019 A 0.012 0.012 0.012 0.0012 c 0.0027 a 0.0020 C 150 c 263 ab 207 B 4 mM SNP + Salt 0.019 a 0.018 0.019 A 0.013 0.012 0.013 0.0005 d 0.0017 d 0.0011 E 197 bc 253 b 225 B Means 0.015NS 0,015 – 0.010NS 0.018 – 0.0015 B 0.0022 A – 216 B 262 A – Rootstocks × Treatments NS NS *** *
Means separation within column and line by Duncan’s multiple range test (P < 0.05) NS not significant
*- pÄ 0.05, ***- pÄ 0.001
applied plants had the same value. Phenolic content sig-nificantly increased by salt treatment compared to control. Moreover, 1 and 2 mM SNP significantly increased phenolic content compared to control. SNP applications leaded de-crease in MDA content even lower than control, while salt treatment significantly increased MDA content. Although APX activity significantly increased by salt treatment com-pared to control, SNP applications maintained APX activity as low as control. However, there is a considerable increase in the chlorophyll content. The highest chlorophyll a, b and a + b were obtained from the 1 mM SNP treatment (5.24, 2.45 and 7.77 µg g–1fw, respectively) while the lowest ones
were in the salt plants (3.56, 1.88 and 5.43 µg g–1fw,
re-spectively). Moreover, the SNP applications considerably increased the chlorophyll content under the salinity com-pared to control. In addition the chlorophyll content, 1 mM SNP increased the chlorophyll stability index by 30.1% compared to the salt plant. The 2 mM SNP showed the
high-Table 5 Effect of SNP on chlorophyll and phenolic content
Treatments Chl a (µg g–1fw) Chl b (µg g–1fw) Chl a + b (µg g–1fw) Phenolic (µg GAE 100 g–1fw)
M9 MM106 Means M9 MM106 Means M9 MM106 Means M9 MM106 Means Control 3.20 d 3.61 c 3.40 D 1.82 c 2.04 bc 1.93 C 5.07 d 5.73 bc 5.40 D 0.091 c 0.103 c 0.097 C Salt 3.11 d 3.56 c 3.33 D 1.95 bc 1.88 c 1.91 C 4.53 e 5.43 c 4.98 E 0.105 b 0.106 c 0.106 B 1 mM SNP + Salt 5.14 b 5.24 a 5.20 A 2.37 a 2.45 a 2.41 A 7.54 b 7.77 a 7.65 A 0.123 a 0.113 b 0.118 A 2 mM SNP + Salt 4.84 c 5.19 a 5.01 B 2.23 ab 2.27 ab 2.25 AB 7.14 c 7.47 a 7.30 B 0.096 c 0.120 a 0.108 B 4 mM SNP + Salt 5.49 a 4.21 b 4.85 C 2.40 a 1.90 c 2.15 B 7.96 a 6.07 b 7.01 C 0.065 d 0.104 c 0.084 D Means 4.35NS 4.36 – 2.15NS 2.11 – 6.45NS 6.50 – 0.096 B 0.109 A – Rootstocks × Treatments *** ** *** ***
Means separation within column and line by Duncan’s multiple range test (P < 0.05) NS not significant
**- pÄ 0.01, ***- pÄ 0.001
est value of the phenolic content (0.120 µg GAE 100g–1fw).
1 mM SNP and 4 mM SNP had the lowest value of MDA (0.0017 µmol g–1fw) and the control plant had the lowest
APX (238 µmol g–1fw min–1).
In cv. Fuji/M9, 4 mM SNP elevated protein content by 73% compared to control. There was no statistically signif-icant change in proline content. Chlorophyll a, b and a + b were significantly affected by the interaction of rootstocks and SNP applications (p 0.001, p 0.01 and p 0.001, respectively). 4 mM SNP treatment exhibited the highest chlorophyll a, b and a + b content (5.49, 2.40 and 7.96 µg g–1 fw, respectively) (Table5). Moreover, 4 mM SNP
ele-vated chlorophyll stability index by 76% compared to salt plant. The highest phenolic content was seen in 1 mM SNP (0.123 µg GAE 100g–1fw). Concerning the MDA content,
it is showed a significant effect of SNP on MDA accu-mulation by the leaves (p 0.001). 4mM SNP showed the lowest value of MDA (0.0005 µmol g–1fw) while the
high-est one was in salt plant (0.0022 µmol g–1fw). Moreover, it
is indicated a significant interaction (p 0.05) on the APX activity in plant leaves. 2 mM SNP possessed the lowest APX (150 µmol g–1fw min–1) while salt plant had the
low-est value (282 µmol g–1fw min–1).
Discussion
Apple is an important temperate zone fruit tree grown in salted-areas due to poor drainage, excessive fertilization and coastal areas. Salt stress has many deleterious influences on plants may cause dysfunction in photosynthetic process (Munns and Tester2008). In our experiment, we applied 35 mM NaCl solution to Malus plants at moderate salinity stress. In addition that, we applied the SNP with different three doses in order to cope with the adverse effects of salt stress. SNP was applied to salt stressed apple plants,
because we wanted to reveal the effects of SNP on apple plants grown under salinity conditions. Thus, in the current study it was also studied the effects of SNP on the plants exposed salt stress. Nitric oxide has been identified as a cy-toprotectant in plants when applied at low concentrations promotes the plant growth (Leshem and Haramaty 1996; Beligni and Lamattina1999,2001). SNP is a donor of NO acts as its behaviour (Jian et al.2016).
Two mechanisms of SNP were described according to many previous studies conducted on many plants for the plant stresses. First is behaving as an antioxidant scaveng-ing the reactive oxygen species (ROS) (Radi et al. 1991; Wink et al. 1993; Caro and Puntarulo 1998; Gould et al.
2003). Second one belongs to signal molecule behaviour of SNP under stress conditions helps plants indirectly (Foiss-ner et al.2000; Klessig et al.2000; Delledonne et al.2001; Wendehenne et al.2001).
In our study, the responses of the rootstocks to SNP ap-plications were different with regard to the plant growth. In general, higher concentrations of the SNP were effec-tive in MM106 while lower concentrations were effeceffec-tively observed in M9. Reduction in the plant growth is a conce-quence of the salt stress (Nazar et al.2011; Ruiz-Carrasco et al. 2011). Decline in the growth is a result of the ma-lignant effects of salt stress related with dysfunctions in the photosynthesis and water relations (Banuls and Primo-Millo 1992). In our study, the SNP applications deceler-ated the depression of the apple plants rootstock and scion diameters and shoot length and even increased the plant rootstock diameter and shoot length compared to the con-trol and salt treated plants. In MM106, the highest rootstock and scion diameter were obtained with 4 mM SNP, while the longest shoots were in 2 mM SNP. Related with that, the highest TI of rootstock and scion diameter were seen in 4 mM SNP and the highest TI of shoot length was ob-served in 2 mM SNP. Beside that 2 mM SNP application
increased the shoot length of cv. Fuji/MM106 plants by 18.36% compared to control. Therefore, in MM106 it was observed 2 and 4 mM SNP were more effective against salt stress. By contrast with, 1 mM SNP were the most effective among the applications and had the highest TI values in M9. Thus that reflects 1 mM SNP has better contributions to M9 rootstock against salinity. The protective effect of SNP on the plant growth under salinity was also reported by Khan et al. (2012). Many publications showed SNP has amelio-rative effects on the plant growth under salt stress (Hai-Hua et al. 2004; Shi et al.2007). In addition that, the chloro-phyll and stomata play pivotal roles in the plant growth and development. Under salt stress condition decline in the stomatal conductance (Flexas et al.2004) and chlorophyll SPAD value (Murkute et al.2006) retards the photosynthe-sis following by the plant growth depression. The stomata regulates stomatal conductance, leaf transpiration and net CO2 assimilation rate (Arbona et al. 2005). SNP treated
plants had better growth may be attributed more SPAD and stomatal conductance compared to control.
Salinity influences membrane functioning and leads ex-tensive lipid peroxidation as a result of the oxidative dam-age in membrane (Hernández and Almansa2002; Karabal et al. 2003). Salt stress makes the membranes leaky and peroxidazes lipids that lead electrolyte leakage (Verma and Mishra 2005), thus the membrane permeability increases. Similar with these findings, we observed that the SNP ap-plications showed lower level of membrane permeability and MDA content. The lesser degree of membrane damage (lower MDA content and membrane permeability) observed in the SNP + NaCl-treated apple plants indicated that SNP applications possess a protective role against the cellular damage. We suggest that the SNP decreases the permeabil-ity of plasma membranes and membrane lipid peroxidation and maintains the membrane integrity and functions un-der salinity, thus alleviating salt damages. Furthermore, the low lipid peroxidation results in elevated biomass produc-tion, thus increases the plant growth. It was reported that the MDA accumulation causes reduction in the plant growth under salinity (Li2009). Therefore, the increase in mem-brane permeability and lipid peroxidation may be a reason of the plant growth reduction similar with our findings. De-creasing MDA and inDe-creasing membrane integrity by SNP application in our study reinforce the previous studies re-garding chickpea, cucumber and barley plants (Shi et al.
2007; Li et al.2008; Sheokand et al.2008).
Phenolics are important compounds in plants due to be-having as antioxidant (Rice-Evans et al.1997). Plants sub-jected to salt stress accumulate phenolics as a defense re-sponse and that may contribute directly to antioxidative be-haviour (Awika et al.2003; Parida et al.2004). Similar with phenolics the APX activity could be indicative of elevated ROS content and establishes the protective mechanism to
decrease the oxidative damage (Meloni et al. 2003). Al-though phenolics and APX activity increase as a defense mechanism, elevated level of phenolics and APX activity may exhibit the level of stress damage, thus, it can be inter-preted low contents of them show low stress damage. How-ever, we assume that the SNP acted as antioxidant in order to decrease the MDA and prevent membrane permeability and had a high capacity for the scavenging of ROS gen-erated by salinity. Similar with our APX result, decreasing trend in the activity of the APX activity by SNP application was reported by Khan et al. (2012).
Decline in the chlorophyll (Chl) a content reduction was accompanied by Chl b and Chl a + b by SNP applications, similarly to what has been demonstrated in a previous ex-periment under salt stress (Tavallali et al. 2008). The re-duction of Chl content could be due to an elevation of the Chl degradation and/or decreasing in mineral acquisi-tion needed for the Chl synthesis (El-Desouky and Atawia
1998). In current study, there are considerable inhibitions of the Chl reductions by SNP compared to control. Moreover, inhibition of the Chl loss by SNP attributed the plant vege-tative growth through prevention of the photosynthesis re-duction. Sheokand et al. (2008) also reported SNP applica-tion prevented the Chl loss in chickpea plants under salinity condition. Numerous studies have shown the improvement in the chlorophyll content in response to the root applica-tion by SNP under salinity (Beligini and Lamattina1999; Sheokand et al. 2008; Khan et al.2012). Similar with the Chl content, the chlorophyll stability index (CSI) was in-creased by SNP applications. CSI can be utilized as a rapid method estimating resistance to the salt stress. Beside that, the SNP applications increased the Chl content even com-pared to control under salt stress condition. That may reflect SNP triggers the chlorophyll biosynthesis and/or possesses chlorophyll repair potential. In addition to Chl, the SNP application significantly affected the protein content of the apple leaves. Many studies demonstrated that the salt stress leads the protein oxidation, thus decrease in the protein con-tent (Remorini et al.2009; Tanou et al.2009a). Tanou et al. (2009b) proposed that NO mitigates the protein oxidation that helps to inhibition of protein loss. Similar with that the SNP applications considerably protected the protein content and inhibited protein reduction in our study.
SNP applications did not have significant effect on the proline content for both rootstocks. Thus, it is seen SNP establishes the salt-tolerance in apple plants on the basis of enzymatic antioxidants. Proline, an amino acid, is synthe-sized as osmoprotectant by plants against stresses. However, for cv. Fuji grafted onto both rootstocks the proline content was not affected by SNP applications. It can be considered SNP did not have effects on mechanisms which is not en-zymatical. On the other hand, for both rootstocks decrease in lipid peroxidation and membrane permeability, increase
in APX activity and SPAD, chlorophyll a, b and a + b con-tents and also elevation of phenolic and protein content and stomatal conductance show SNP is effective especially with regards to enzymatical activites on providing salt-tolerance. Moreover, this is the first publication indicated that the ef-fects of SNP on salt stress in the perennial woody plant. The previous studies have shown the effects in herbaceous plants, but the plant responses against stresses are distinct between the woody and herbaceous plants. Therefore, cur-rent study reveals meliorative effects of SNP on the woody plants compared to the herbaceous plants such as acting an-tioxidant and possessing enzymatical defence mechanisms.
Conclusion
The data of our study may strongly support the conclu-sion that the SNP application plays a fundamental role in adaptive response of apple plants to salt stress. The vi-sual symptoms were accompanied by diminishing of leave burns of the salinity in SNP applied plants. The current study exhibited relations between morphological, physio-logical and biochemical properties of apple plants in order to cope with the salinity damages. We anticipated that re-ductions in the plant growth could correspond to increases in the membrane permeability and lipid peroxidation and declines in chlorophyll through alteration in the photosyn-thesis. Moreover, decrease in the stomatal conductance by salinity could cause a reduction in the carbon assimilation that limits the plant growth. Beside that, SNP consider-ably triggered the chlorophyll biosynthesis and/or inhibited chlorophyll degradation, and maintained better cell mem-brane stability and the plant growth compared to control. The mitigation effects of SNP on the plant growth, phys-iological and biochemical responses of salt-stressed apple plants could be explained by behaving as antioxidant. SNP exhibited some abilities including membrane stabilizing and antioxidant properties. Thus, we suggest that application of SNP to roots could be utilized as a method to maintain better growth in apple plants under salinity conditions. We consider 1 mM SNP dose is suit for apply to maintain better growth in apple plants under salinity.
Conflict of interest S. Aras, H. Keles and A. E¸sitken declare that they
have no competing interests.
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