Impaginato 165 Adv. Hort. Sci., 2021 35(2): 165­174 DOI: 10.36253/ahsc­8343 Growth response nitrogen metabolism of grafted cucumber fertilized with different ratios of nitrate: ammonium fertilizer M. Haghighi 1 (*), O. Zamani 1, L. Abbey 2 1 Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran. 2 Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, PO Box 550, Truro, B2N 5E3, Nova Scotia, Canada. Key words: Endemic accession, nutrient absorption, N metabolism, photosynthe­ sis. Abstract: The use of an endemic plant as rootstock has many merits but its application for cucurbits production has not been extensively investigated. The present study determined the growth responses of grafted cucumber using two endemic rootstocks from Cucurbita pepo L. fertilized with different ratios of nitrate (NO3 ­): ammonium (NH4 +) fertilizer. A greenhouse study was carried out using cucumber (C. sativus ‘Dominos’) grafted on two accessions of Cucurbita pepo L. collected from Babol and Isfahan with the control being ungrafted C. sativus ‘Dominos’. Different ratios of NO3 ­/NH4 + as follows: 100:0 (NO3 − alone), 25/75, 50/50, 75/25 and 0/100 (NH4 + alone) were applied. It was found that dif­ ferent rootstock has the same physiology but different growth attributes. The growth of the ungrafted cucumber was lower than the grafted ones, and Babol showed better or equal growth compared to the Isfahan rootstock. The NO3 ­ /NH4 + effect on growth of the cucumber shoot and root fresh and dry weights, root and shoot lengths, nodes, and number of leaves were increased in the 75/25 ratio compared to the other treatments. Grafting on the Isfahan and Babol showed the same effect of N metabolism i.e., grafting increased nitrate reductase activity and NO3 ­ concentration in the 75/25 and the 100/0. Protein content and amino acids content of leaves increased in the grafted cucumber treated with 50/50 NO3 ­/NH4 +. The same response of photosynthesis parame­ ters was observed in the different rootstocks. In conclusion, the result suggest­ ed that the grafted ‘Dominos’ on Babol endemic rootstock at 50/50 NO3 ­/NH4 + ratio gave the high growth. 1. Introduction Grafting is a horticultural technique performed by joining two plants i.e., a scion and a rootstock. Grafting of plants can minimize the detrimen­ tal effects of biotic and abiotic stress if the proper rootstock is used (Lee (*) Corresponding author: mhaghighi@iut.ac.ir Citation: HAGHIGHI M., ZAMANI O., ABBEY L., 2021 ­ Growth response nitrogen metabolism of grafted cucumber fertilized with different ratios of nitra‐ te: ammonium fertilizer. ­ Adv. Hort. Sci., 35(2): 165­174 Copyright: © 2021 Haghighi M., Zamani O., Abbey L. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 16 April 2020 Accepted for publication 24 Dcember 2020 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-8343 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2021 35(2): 165­174 166 and Oda, 2003; Rouphael et al., 2008; Keshavarzi and Shekafandeh, 2019). Many studies have shown that grafting promotes root and shoot growth, increase plant resistance against diseases, increase plant tol­ erance to temperature extremities and soil salinity, increase nutrient and water absorption, and plant productivity (Colla et al., 2010; Lee et al., 2010). Nutrient uptake like nitrogen (N) and phosphorus (P) absorptions improved by grafting of cucumber to fig leaf gourd (Cucurbita ficifolia L.) (Pogonyi et al., 2005). Cucumber ‘Adrian’ was grafted onto three root­ stocks of Lagenaria siceraria, Cucurbita maxima × C. moschata, and zucchini. Results showed that grafting improved total yield,leaves number, total soluble solids and titratable acidity. The effect of grafting is highly dependent on the choice of rootstock (Goreta et al., 2014 ; Dadashpour et al., 2017) to improve growth and fruit production. The benefits of grafting on vegetative growth have been reported for cucurbit­type crops (Goreta et al., 2014) but using the endemic cucurbits as a rootstock for this family has not been extensively investigated. Ammonium, nitrate and urea are the three forms of inorganic nitrogen that increase plant growth (Niu et al., 2011). Most plants prefer nitrate to ammoni­ um or a combination of them. The optimal NO3 ­/NH4 + ratio depends on many factors such as plant species, plant growth and maturity stages, and environment condition (Marschner, 2012). Yet, little is known about how the NO3 ­/NH4 + ratio may affect grafted plant and their nitrogen metabolism. Higher NO3 ­/NH4 + ratio produce higher biomass than other ratios in mesquite (Prosopis velutina) (Hahne and Schuch, 2006). Conversely, higher NO3 ­ improves growth and flowering in Phalaenopsis orchid (Phalaenopsis) (Wang et al., 2008). In some plants, N form has no significant effects on growth. For instance, the dry weight of shoots and roots, and root/shoot ratio were not affected by NO3 ­/NH4 + ratio in Sophora secundiflora (Niu et al., 2011). However, using high NO3 ­ in plant nutrition is a risk for the envi­ ronment and human health. Replacing NO3 ­ in plant nutrition with an appropriate amount of NH4+ may alleviate these concerns. Plants usually grow on NO3 ­ and NH4 + as nitrogen sources, which eventually influ­ ence the synthesizing of amino acids and proteins (Xing et al., 2015). The optimum level of N and the best ratio of NO3 ­ /NH4 + are required by each species according to their respective growth and productivity (Fernandez­Nava et al., 2010). On the other hand, the process of up taking NO3 ­ or NH4 + has a substantial impact on the uptake of other cations and anions and rhizosphere­ pH. When roots take up NO3 ­ with a negative charge and NH4 + with a positive charge, they release a specif­ ic charged molecule to keep a balanced pH inside the plant cells. NH4 + reduces rhizosphere pH while NO3 ­ increases pH (Marschner, 2012). High levels of NH4 + can also inhibit the uptake of cations such as calcium and magnesium (Siddiqi et al., 2002). Ammonium application reduces the rate of iron deficiency but increases phosphate and sulfate uptake due to changing substrate pH. In contrast, nitrate reduces the absorption of those essential anions. Thus, most of the time, supplying the proper NO3 ­/NH4 + ratio results in the highest growth rates and plant yield by balancing nutrient absorption (Marschner, 2012). Cucumber has a shallow root system and could not uptake nutrient well (Causin et al., 2004). Information on the influence of endemic accession as a rootstock on cucumber nutrient absorption and growth is limited. To better understand the effect of NO3 ­/NH4 + ratio for grafted cucumber, the present study was designed. The present study determined plant growth response of grafted cucumber using two endemic rootstocks from Cucurbita pepo L. fertil­ ized with different ratios of NO3 ­/NH4 + under green­ house conditions. 2. Materials and Methods Production of rafte seedlings and experimenta design The greenhouse experiment was carried out at the Isfahan University of Technology, Isfahan, Iran. Cucurbita pepo L. accession as the rootstocks were collected from the Babol (Babol), Isfahan region (Isfahan), and C. sativus ‘Dominos’ is a common culti­ var used as scion. The ungrafted Dominos used as control. Total nitrogen in the nutrient solution was equal and comprised NO3 ­/NH4 + ratio 100:0, 25:75, 50:50, 75:25, or 0:100. Rootstock and scion seeds were sown in cocopeat and perlite (50/50 v/v). Scion seeds had been cultured 10 days before the root­ stock seeds in cocopeat: perlite ratio of 1:1. Scion plants and rootstocks were cut beneath and above the first true leaves, respectively. The hole insertion grafting method as described by Lee (1994) was adopted. Firstly, true leaves and meristem tissue were removed at the growing tip of the rootstock Haghighi et al. ‐ Nitrogen metabolism of grafted cucumber 167 before making a slit across the growing point from the bottom of one cotyledon to the other side of the hypocotyl. The newly grafted cucumber plant was kept in a grafting chamber with approximately 65% relative humidity and exposed at 16 h fluorescent light at 25­30°C for two weeks. The grafted plants were moved to the greenhouse and maintained at 30­35°C and 60­65% relative humidity for one week to gradually adapt to the greenhouse conditions (Kashi et al., 2008). Fertilization with a half­modified Johnson nutrient solution was applied to the grafted plants including (mM): MgSO4 (2), KH2PO4 (1), H3BO3 (50), MnCl2 (10), CaCl2 (1), MnSO4 (10), CuSO4 (1.5), ZnSO4 (0.8), Na2MoO4 (0.4), Co(NO3)2 (0.1), KNO3 (10), FeCl3 (0.1), EDTA (0.3) and H3BO3 (50.5) mM (Jones, 2005). Ten (10) days after adaptation of the grafted plants, they were treated with the NO3 ­/NH4 + ratios on a daily basis. The control treatment was irrigated with a complete Johnson nutrient solution. The plants were kept in the nutrient solution at varying NO3 ­/NH4 + ratios with 5 min airing in every 15 min in 2liter container. The EC and pH of the nutrient solu­ tion were kept at 2.0±0.2 dS m­1 and 6.0±0.3, respec­ tively, by adding HNO3 and H3PO4 into the nutrient solution. Plants were maintained for six more weeks before final harvest. Data collection Greenness. Chlorophyll index was measured with portable SPAD (SPAD­502 plus, Minolta, Japan). Growth trait assay. All the leaf and nod number were counted. The shoots of seedlings were separat­ ed from the roots and after recording the fresh weight, they were oven dried at 70°C to constant weight. Root volume was measured by change in water volume in a graduated container (Haghighi et al., 2012). Shoot and root length measured by ruler and the stem thickness was measured using a pair of caliper. Photosynthesis traits assay. Gas exchange para­ meters including photosynthesis rate, transpiration, stomata conductivity, and intercellular CO2 of stoma­ ta were measured by a portable photosynthesis meter (Li­Cor Li­3000, USA) on a sunny day. Photosynthetically active radiation (PAR) intensity was 1000 μmol m­2 ꞏs­1 and CO2 concentration was 350 μmolꞏmol. The same leaves of each plant was used for chlorophyll measurement using chlorophyll meter (SPAD­502, Minolta Corp., NJ, USA). Mesophyll conductance (mmol CO2 m­2 s­1) was measured according by using the formula: Photosynthetic rate/sub­stomatal CO2 concentration (Ahmadi and Siosemardeh, 2005). Phenolic content. The Folin­Ciocalteu method was used for measuring the total phenolic content of the root exudate. The absorbance was measured at 725 nm with a spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan) (Motamedi et al., 2019). Prolin. Plant leaves were homogenized in 3% sul­ fosalicylic acid at 4°C. Then the solution incubated and centrifuged at 5000 rpm for 20 min. The super­ natant was mixed with 2.5% ninhydrin, 60% phos­ phoric acid (v/v) and 1 ml of glacial acetic acid (100%). The absorbance was measured at 518 nm by spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan) (Bates et al., 1973). Total amino acid. Total amino acids measured by high­performance liquid chromatography (HPLC). Samples were hydrolyzed with 6 M HCl and 10 mg phenol (for protection of tyrosine) at 110 °C for 24 h. HPLC system was equipped with MD­1510 diode­ array detector and set to 263 nm (λmax). The sam­ ples were injected with a 20 μL loop using a 7125 valve (Rheodyne, Cotati, CA) onto a Purospher RP­18 column and operated at 25°C with a flow rate of 1.0 mL/min using 50 mM acetate buffer (pH 4.2) as elu­ ent A and acetonitrile as eluent B (El­Abagy et al., 2014). The level of amino acids present in 100 g of leaves. Protein. Na­phosphate buffer (pH 7.2) was used to homogenized 1 g fresh leaf samples then centrifuged at 4°C. The absorbance of the supernatants and dye measured using a spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan) at 595 nm. Bovine serum albumin (BSA) used as protein standard (Bradford, 1976). Nitrate reductase enzyme. The activity of nitrate reductase enzyme was measured according to the method proposed by (Cazetta and Villela, 2004). The amount of 400 mg of leaf samples was placed in a phosphate solution (100 mg, pH=7.5) containing 4% propanol and potassium nitrate, and stored in dark­ ness for an hour at a temperature of 30°C. Then, 1 mL of the solution containing sulfanilic acid was dis­ solved in 2 ml of chloride acid and 1 mL of naph­ thylethylene diamide solution (0.02%) and after 20 min, the absorbance was measured at 540 nm wave­ length. Sodium nitrite (NaNO3) was used to prepare the standard solution and the enzyme activity was calcu­ lated based on µmol nitrite/gr FW h. Adv. Hort. Sci., 2021 35(2): 165­174 168 Nitrate concentration. The leaf nitrate content was determined following the procedures described by Atanasova (2008). Nutrient concentrations. The amount of calcium was measured by an atomic absorption device (model: Perkin Elmer, AA200) (Sharifi et al., 2016). zK concentration was determined by atomic absorption after digestion with HCl (Murillo­Amador et al., 2007). Nitrogen concentration of leaves measured by Kjeldahl method. Phosphorus was estimated by the vanadomolybdo phosphoric acid colorimetric method at 460 nm (Estan et al., 2005). Ca concentrations. Twelve fruits for each treat­ ment when reached market ripe harvested at the end of the experiment for measurements. Four leaf samples, consisting of young, fully expanded leaves were collected, washed thoroughly with tap water, gave a final rinsing with deionized water, dried at 65°C to constant weight. The extraction of Ca from the plant tissue material was performed using 1 N HCl after dry ashing at 550°C for five h. The amount of calcium was measured by an atomic absorption device (model: Perkin Elmer, AA200) (Sharifi et al., 2014). K, Mg, and P concentrations. The concentrations of K, Mg, and P were measured (Shield Torch System, Agilent 7500a). Meanwhile, phosphorus was estimat­ ed by the vanadomolybdo phosphoric acid colorimet­ ric method at 460 nm (Sharifi et al., 2014). P was col­ orimetrically determined using a spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan). Statistical analysis The factorial experiment was arranged in a com­ pletely randomized design with 10 replications. Data were analyzed with Statestix 8 (Tallahassee FL, USA) and treatment means were separated using the least significant difference (LSD) test at the 5% level of sig­ nificance when the analysis of variance indicated sig­ nificant difference between treatments at P≤0.05. 3. Results The ANOVA in Table 1 showed that all growth and development characteristics were affected by NO3 ­ /NH4 + ratio and rootstock. The interaction of NO3 ­/NH4 + ratio and rootstock significantly affected all measured variables except rootstock stem thickness and root volume. Except for leaf greenness, all physiological parameters were affected by NO3 ­/NH4 + ratio and rootstock and their interaction (Table 2). Table 2 ­ Analysis of variance effect of NO3:NH4 ratio on physiological characteristics of cucumber NS, *, ** not significant or significant at 5% or 1%. Table 1 ­ Analysis of variance effect of NO3:NH4 ratio a rootstock on growth characteristics of cucumber NS, *, ** not significant, significant at 5% or 1%, respectively. Source df Shoot length Rootstock stem thickness Number scion leaves Number scion nodes Shoot fresh weight Root fresh weight Root volume Root length Shoot dry weight NO3:NH4 ratio (N) 4 59.92 ** 3.03 ** 7.23 ** 12.88 ** 24.69 ** 1.08 ** 2.44 ** 100.49 ** 0.06 ** Rootstock (R) 2 290.70 ** 6.51 ** 36.87 ** 24.01 ** 14.03 * 1.16 ** 2.94 ** 92.88 ** 0.26 ** NxR 8 37.40 ** 1.67 ns 6.19 ** 4.03 * 24.73 ** 0.64 ** 2.04 ns 44.01 ** 0.09 ** Error 30 26.78 0.40 3.44 1.60 3.40 0.05 0.11 5.07 0.01 CV 20.31 14.22 28.98 20.89 31.31 38.97 39.76 18.63 24.10 Source df Greeness Photo­ synthesis Tran­ spiration Inter­ cellular CO2 concentra­ tion of stomata Stomata conductivity Meso­ philic conductivity Nitrate reductase activity Proline Root phsenol Protein Nitrate Amino acid NO3:NH4 ratio (N) 4 14.34 NS 9.59 NS 3.01 NS 19146.7 NS 0.0041 NS 0.0051 NS 0.006 NS 2.33 NS 1.61 NS 3307 NS 0.0000013 NS 35460 NS Rootstock ( R) 2 3.19 NS 11.17 NS 1.97 NS 34138.4 NS 0.0043 NS 0.0033 NS 0.013 NS 1.77 NS 1.56 NS 29073 NS 0.000075 NS 251790 NS NxR 8 23.42 ** 19.16 ** 4.11 ** 13750.9 ** 0.005 ** 0.005 ** 0.003 ** 2.39 ** 2.05 ** 59005 ** 0.000015 ** 236095 ** Error 22 0.92 9.17 9.17 9.17 9.17 9.19 9.17 9.18 9.17 30518 0.000043 72919 CV 38 12.52 4.69 7.41 3.23 0.16 0.15 0.02 0.65 0.08 17.18 11.08 24.17 Haghighi et al. ‐ Nitrogen metabolism of grafted cucumber 169 The main effect of nutrient absorbtion was not affected by NO3 ­/NH4 + ratio and rootstock, but the interaction showed significant changes (Table 3). The main result of the N source indicated that the thickest rootstock stem was at 75/25 NO3 ­/NH4 + ratio and the most significant root volume was influenced by treatment 50/50 NO3 ­/NH4 + ratio. The ungrafted plants had the least root volume and Babol has the most root volume (Table 4). Shoot length increased when the ratio of NO3 ­ /NH4 + increased, which led to higher NO3 ­ in shoot length compared to the root length (Fig. 1A and B). The shoot length was highest in the 0/100 and then in the 75/25 of NO3 ­/NH4 + ratio in all rootstock. It seemed the root had the best growth in the 50/50 NO3 ­/NH4 + ratio, and when the NO3 ­/NH4 + ratio was increased by more than 75% of total­N, the root length decreased. Shoot and root fresh and dry weights were not Fig. 1 ­ The interaction effect of different rootstocks and NO3:NH4 ratio on A) shoot length and B) root length. improved with only NO3 ­ or NH4 + (Fig. 2A­D). The Babol rootstock caused the best shoot and root growth in the 50/50 NO3 ­/NH4 + ratio. Ungrafted plant showed the lower growth in all the treatment N ratios. It seemed that the different NO3 ­/NH4 + ratio did not affect the growth in ungrafted plants. Isfahan rootstock increased both shoot and root growth in the 75/25 NO3 ­/NH4 + ratio. The highest root and shoot growth were seen in the Babol and the 50/50 NO3 ­/NH4 + ratio compared to all the other treat­ ments. The numbers nodes and leaves increased by increasing the NO3 ­ portion of the nutrient solution for the grafted plants, but there were no significant changes in the ungrafted plants in the different NO3 ­ /NH4 + ratios (Fig. 3A and B). The lowest number of Table 4 ­ Effects of NO3:NH4 ratio and rootstocks on root volume and cucumber rootstock stem NS, *, ** not significant or significant at 5% or 1%. Table 3 ­ Analysis of variance effect of NO3:NH4 ratio on nutrient concentration of cucumber NS, *, ** not significant, significant at 5% or 1%, respectively. Source df N concentration P concentration K concentration Ca concentration Mg concentration NO3:NH4 ratio (N) 4 230. 9 NS 244.56 NS 43.12 NS 78.12 NS 6.12 NS Rootstock (R) 2 23.45 NS 150.01 NS 35.93 NS 602.45 NS 13.08 NS N×R 8 26.54 ** 111.77 ** 4.65 ** 81.12 ** 4.20 * Error 22 5.27 67.38 3.23 76.32 1.35 CV 38 25.62 13.04 15.50 23.97 44.38 Treatments Rootstock stem thickness (cm) Root volume (mm3) NO3:NH4 ratio 100/0 4.51 b 0.35 c 25/75 3.62 c 0.47 bc 50/50 4.50 b 1.85 a 75/25 5.46 a 0.80 b 0/100 4.40 b 0.78 b Rootstock Un­grafted 3.71 b 0.49 b Babol 4.66 a 1.40 a Isfahan 5.13 a 0.67 b 170 Adv. Hort. Sci., 2021 35(2): 165­174 leaves and nodes was seen in the ungrafted plants. These findings were in line with the increase in shoot length, which was higher in treatments with increased NO3 ­/NH4 + ratio for grafted plants. Photosynthesis was more significant in both graft­ ed cucumbers in all the NO3 ­/NH4 + ratio compared with the ungrafted plant, and it seemed that it is not related to the changes of the greenness index (Fig. 4A). The greenness index did not change between treatments significantly, except for the high increase in Babol from the50/50 NO3 ­/NH4 + ratio. Furthermore, the photosynthesis rate increased in the 50/50 NO3 ­/NH4 + ratio in the ungrafted cucumber (Fig. 4B). Transpiration in all the rootstock was increased by the 25/75 NO3 ­/NH4 + and decreased with increasing NO3 ­/NH4 + ratio in the nutrient solution. It seemed Fig. 2 ­ The interaction effect of different rootstocks and NO3:NH4 ratio on A) shoot fresh weight, B) shoot dry wei­ ght, C) root fresh weight and D) root dry weight. Fig. 3 ­ The interaction effect of different rootstocks and NO3:NH4 ratio on A) number of leaves and B) number of nodes. Fig. 4 ­ The interaction effect of different rootstocks and NO3:NH4 ratio on A) Greenness (SPAD), B) photosynthe­ sis, C) transpiration D) stomata conductivity, and E) inter­ cellular CO2 concentration of stomata. Haghighi et al. ‐ Nitrogen metabolism of grafted cucumber 171 that by increasing transpiration in all plants stomata conductivity was increased by the25/75 NO3 ­/NH4 + treatment (Fig. 4C). Stomata conductivity was highly raised in the Isfahan rootstock and the ungrafted plants (Fig. 4D). The stomata conductivity was enhanced in the ungrafted cucumber, especially at 25/75 NO3 ­/NH4 +. Conversely, the CO2 concentration in the stomata reduced in the 25/75 NO3 ­/NH4 + and increased with NO3 ­ increment in the nutrient solution (Fig. 4E). Phenol exudate of the root was highest in 25/75 NO3 ­/NH4 + and decreased by increasing the NO3 ­ por­ tion (Fig. 5A). It was highest in the ungrafted com­ pared to the grafted cucumber plants. The highest proline content was seen in the 100/0 and the 25/75 NO3 ­/NH4 + and decreased by increasing the NO3 ­ con­ centration (Fig. 5B). Nitrate reductase activity was highest in the 25/75 NO3 ­/NH4 + and was reduced by increasing the NO3 ­ portion. The lowest NO3 ­ concentration was recorded by the 0/100 and the 25/75 NO3 ­/NH4 +. Nitrate con­ centration was higher in the 0/100, and 100/0 NO3/NH4 and was the same in between grafting plants in 25/75 and 50/50 NO3/NH4 (Fig. 6B). The amino acid was higher in grafted cucumber, especial­ ly in 5/75 and 50/50 NO3/NH4. Protein content was higher in 50/50 NO3/NH4 in all rootstock and un­ grafted (Fig. 6A). It seemed that the most nutrient absorption was recorded by the Babol and the Isfahan rootstock, especially in the 100/0, 75/25 followed by the 50/50 NO3 ­/NH4+ for N and K (Fig. 7). Conversely, the high­ est Ca absorption was recorded by the 50/50 NO3 ­ /NH4 + and to lesser extent by the 75/25 NO3 ­/NH4 +. Fig. 5 ­ The interaction effect of different rootstocks and NO3:NH4 ratio on A) proline and B) root phenol. Fig. 6 ­ The interaction effects of different rootstocks and NO3:NH4 ratio on A) protein, B) nitrate and C) amino acid D) nitrate reductase (NR) activity. Adv. Hort. Sci., 2021 35(2): 165­174 172 The Mg and P absorption were less absorbed, but highest in the Babol rootstock. 4. Discussions and Conclusions The effect of NO3 ­/NH4 + and grafting on the cucumber growth A wide range of morphological and physiological characteristics are influenced by scions, rootstocks and their interactions (He et al., 2009). In this study, it was observed that growth in terms of shoot and root fresh and dry weights, root and shoot lengths, numbers of node and leaves were increased by the 75/25 treatment. However, some parameters were not significantly affected. All the growth parameters were improved by the 75/25 treatment in Isfahan rootstock. On the other hand, the best result in plant growth improvement was seen in the 50/50 treat­ ment for Babol. These results revealed that different rootstock act differently in different NO3 ­/NH4 + ratio to promote growth. In all the growth parameters, the ungrafted cucumber was lower than the grafted ones and Babol showed a better or similar increase than Isfahan rootstock. When using NO3 ­ or NH4 + alone, plant growth was not significantly different except for the numbers of node and leaves and root length in the grafted cucumber following application of NH4 + alone. It noted that the use of both sources of N indi­ vidually was not economical. Less plant growth was seen in the ungrafted cucumber by using NO3 ­ or NH4 + alone compared to the grafted cucumber. Different rootstocks have different root size and different absorbance abilities which can affect vege­ tative growth rates. Rootstocks improve photosyn­ thetic ability and increase yield in grafted plants (Massai et al., 2004). Rootstocks improve growth in plant by improving nutrient uptake, hormonal status and root growth (Lee et al., 2010). Grafting, especial­ ly Babol rootstocks, contributed to better vegetative growth of ‘Dominos’ due to higher root distribution, perhaps resulting in more nutrient uptake. It should be considered that increasing number of nodes is a sign for more yield because the flower initiate in nodes so the more node means more flowers and fruits as obtained with grafted cucumber. The effect of NO3 ­/NH4 + and grafting on the metabo‐ lism of cucumber Results revealed that grafting on the Isfahan and Babol showed the same effect on N metabolism, i.e., NR activity decreased and NO3 ­ concentration increased in plants treated with the 75/25 and the 100/0 NO3 ­/NH4 +.Protein and amino acids contents of leaves was increased at the 50/50 treatment in graft­ ed cucumber and protein showed the same trend like amino acid. It can be concluded that the healthiest cucumber plant was obtained from the 50/50 treat­ ment in the grafted cucumber although the most nitrate metabolism was achieved by treatment 75/25 NO3 ­/NH4 +. Despite the effect of rootstock on growth, it was seen that the rootstock has no difference in the metabolism N in the cucumber plants. The other reason for the promotion of plant growth by chang­ ing the NO3 ­/NH4 + ratio could be that after NR reduced NO3 ­ to nitrite, it was changed to ammoni­ um, and amino acids were produced, which can later combine to produce proteins (Haghighi et al., 2012). On the other hand, nitrate through producing active forms of cytokinins, as an osmolyte in vacuoles, stim­ ulates leaf function and growth, causing cell exten­ sion and improved growth (Wang et al., 2008). Increasing root length and cytokinin production helped the plant to absorb more water and nutrients to improve vegetative growth (Haghighi et al., 2016 a). The effect of NO3 ­/NH4 + and grafting on the stress photosynthesis traits of cucumber Photosynthesis was not affected by N ratio but increased by grafting. Stomata conductance, internal CO2 of stomata and transpiration increased with increasing NO3 ­/NH4 + ratio and was reduced by graft­ ed cucumber. It seemed changes in photosynthesis traits were more related to stomata status, which can be associated with the rootstock. Photosynthesis was improved with grafting due to an efficient root system of the rootstock with regards to nutrient uptake compared to the ungrafted plants Fig. 7 ­ The effects of different rootstocks on nutrients (N, P, K, Ca and Mg) absorption in different NO3:NH4 ratio. Haghighi et al. ‐ Nitrogen metabolism of grafted cucumber 173 (Lee et al., 2010). Furthermore, more vigorous root­ stocks could absorb more water and nutrients. Consequently, photosynthesis improved when these rootstocks were used compared to non­grafted plants as reported previously by Haghighi et al. (2016 b). In all NO3 ­/NH4 + ratios, grafting reduced stomata conductivity, which resulted in lower transpiration and improved water use efficiency so plants could deal with challenged conditions more efficiently (Duan et al., 2001). In conclusion, the performance of the different cucumber plant accessions used as rootstocks i.e. nutrient absorption and growth parameters varied. The two accessions used in this study i.e. Isfahan and Babol responded similarly to N metabolism and pho­ tosynthesis traits. Therefore, it seems that different rootstock has the same physiology but different growth pattern due to their pre­existing genetic dif­ ferences. More noticeably, we found that, shoot and root fresh and dry weights, root and shoot lengths, nodes, and number of leaves were increased by the 75/25 ratio of NO3 ­/NH4 +. 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