Impaginato 11 Adv. Hort. Sci., 2020 34(1): 11­24 DOI: 10.13128/ahsc­8252 Effect of foliar spray of calcium lactate on the growth, yield and biochemical attribute of lettuce (Lactuca sativa L.) under water deficit stress A. Khani 1, T. Barzegar 1 (*), J. Nikbakht 2, Z. Ghahremani 1 1 Department of Horticultural Sciences, Faculty of Agriculture, University of Zanjan, Zanjan, Iran. 2 Department of Water Engineering, Faculty of Agriculture, University of Zanjan, Zanjan, Iran. Key words: anthocyanin, antioxidant enzymes, leaf water status, nutrient uptake. Abstract: The field experiment was conducted to evaluate the effect of foliar spray of calcium lactate (Ca) on fresh yield and biochemical attribute of lettuce (Lactuca sativa L.) under water deficit stress, in a split plot form based on a ran­ domized complete block design with three Irrigation regimes (70, 85 and 100% ETc) and three calcium lactate treatment levels (0, 0.75 and 1.5 g L­1) in three replicates. Results revealed that water deficit stress significantly reduced the growth and yield of plant, leaf relative water contents, excised leaf water retention and N, P and Mg absorption while led to increase anthocyanin, phe­ nol and flavonoids contents, antioxidant activity, peroxidase and catalase activ­ ity and water use efficiency. The results of our research indicated that the application of CaL 1.5 g L­1 is capable of increasing lettuce yield, under field con­ ditions with 30% less than optimal irrigation. CaL treatment showed a clearly protective effect in stressed plants, enhancing their leaf water status, antioxi­ dant capacity and N and Ca contents in comparison to untreated plants. Therefore, feeding leaves by CaL with increasing antioxidant activity and nutri­ ents content especially N led to increase growth and fresh yield of lettuce under normal irrigation and water deficit conditions. 1. Introduction Abiotic stresses such as high temperature, drought, salinity and chemi­ cal toxicity, are the most important limiting factors to crop productivity. Drought is undoubtedly one of the most important stresses that have huge impact on growth and productivity of the crops (Fahad et al., 2017; Hussain et al., 2018). Water stress is the most prominent abiotic stress limiting agri­ cultural crop growth and productivity (Gholipoor et al., 2013; Ihsan et al., 2016). Deficit irrigation stress as a consequence of the progressive decrease in water availability has been a hot topic regarding food security during the last two decades (UNESCO, 2012). Growth and development of plants is influenced by reduction in turgor that result in decreased nutrient acquisi­ (*) Corresponding author: tbarzegar@znu.ac.ir Citation: KHANI A., BARZEGAR T., NIKBAKHT J., GHAHRE­ MANI Z., 2020 ­ Effect of foliar spray of calcium lactate on the growth, yield and biochemical attribute of lettuce (Lactuca sativa L.) under water deficit stress ­ Adv. Hort. Sci., 34(1): 11­24. Copyright: © 2020 Khani A., Barzegar T., Nikbakht J., Ghahremani Z. 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 31 October 2019 Accepted for publication 14 May 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(1): 11­24 12 tion from dry soil (Luo et al., 2011). Due to the threat of climate change, there is a need to limit the use of water resources in arid and semi­arid climates. It is therefore important to find new approaches to avoid crop productivity losses in ‘limited fresh­water’ areas. Lettuce (Lactuca sativa L.), an annual plant of Asteraceae family, is considered as one of the most important salad vegetables as a cool season crop. Lettuce leaves contain vitamins C and E, carotenoids, phenolic acids with anti­free radical activity, and min­ erals with a lot of fiber, which are an important part of the human diet. Moreover, lettuce contains lac­ tocin and lactucopicrin which improve the quality of sleep (Chakraborti et al., 2002; FAOSTAT, 2016). Since most of vegetable species are shallow­root­ ed, they are sensitive to mild water stress. In lettuce production, it is particularly important to preserve optimal growth through a well­scheduled irrigation program, where the harvested part of the plant is the photosynthetic leaf area, (Ahmed et al., 2000; Casanova et al., 2009). Its leaves have high water content and it is sensitive to mild water deficit stress due to its shallow root system (Kizil et al., 2012). Therefore, in lettuce, new strategies will become crit­ ical to enhance productivity under deficit irrigation (Malcom et al., 2012). Foliar application of agro­chemicals has widely been used in agriculture as a rapid, low­cost and effective way for enhancing growth and productivity of many vegetable crops under water deficit stress especially green leafy vegetables like lettuce. Calcium lactate is considered as one of important agro­chemi­ cal which can be spray and play important roles in physiological and biochemical processes. Calcium (Ca) is the mineral nutrient most commonly decrease absorption under water deficit condition, so increas­ ing the calcium content in the leafy vegetables could further improve Ca concentrations in plant tissues (Grusak, 2002). Ca is an essential macronutrient for plant growth and development, and is considered as an important intracellular messenger, mediating responses to hor­ mones, stress signals and a variety of developmental processes. Furthermore, Ca is an important compo­ nent in the structure of cell walls and cell membranes (Hepler and Winship, 2010). Ca plays a role in the regulation of various mechanisms of plants under environmental conditions such as water stress, heat, cold and salinity. In addition, calcium signaling is required for acquisition of tolerance or resistance to the stress (Cousson, 2009). Positive effect of calcium in improving stress tolerance can be attributed to regulate of water status, antioxidant systems activity, osmolytes accumulation, improving photosynthetic pigment content, and nutritional balances (Kurtyka et al., 2008). Ca plays an important role in oxidative stress signaling, linking H2O2 perception and induc­ tion of antioxidant genes in plants (Rentel and Knight, 2004). Ca participates in most cellular signaling processes (Sanders et al., 2002) and interacts strong­ ly with reactive oxygen species (Evans et al., 2005). Since the combined effects of Ca and water deficit stress have hardly been reported, the current study was, therefore, designed to evaluate the influence of foliar application of calcium lactate on the growth, yield and biochemical attribute of lettuce cv. New Red Fire under water deficit stress. 2. Materials and Methods Experimental design The field experiment was carried out at the Research farm of the Agriculture faculty, University of Zanjan, Iran, during 2017. The experiment was performed using a split plot based on a randomized complete block design with three Irrigation regimes (70, 85 and 100% ETc) as the main plot and three cal­ cium lactate (CaL) treatment levels (0, 0.75 and 1.5 g L­1) as the sub­plot in three replicates. The soil prop­ erties of experimental filed as well as average daily climatic data during the growing seasons was shown in Tables 1 and 2, respectively. Plant material Seeds of lettuce (Lactuca sativa L.) cv. New Red Fire was obtained from a “Takii seed” company. Lettuce seeds were sown in the nursery on the 2nd of August. Seedlings were transplanted at the 3­4 leaf stage when the seedlings were four weeks old with 2 5 c m s p a c i n g w i t h i n r o w a n d 3 5 c m s p a c i n g between rows that there were about 11.5 plants per square meters (plants m­2). Table 1 ­ Soil physical and chemical properties on the site of experimental field Soil texture Organic matter (%) pH EC (dS m­1) N (%) Ca (g kg­1) Na (g kg­1) K (g kg­1) Loam clay 0.94 7.4 1.49 0.07 0.12 0.13 0.20 Khani et al. ‐ Protective effect of calcium lactate on lettuce 13 Irrigation treatments and calcium lactate applications After plant establishment, lettuce plants were sprayed with different concentration calcium lactate at 6­7th leaf stage, 10 and 20 days after first spraying for 3 times, during the plant growth. Irrigation treat­ ments were applied one week after the first spraying. All foliar sprayings time were the same and distilled water was used for control treatment. The three irri­ gation levels were calculated based on actual evapo­ transpiration (ETc): (1) control, irrigated 100% crop water requirement (I100), (2) deficit irrigation 85% ETc (I85), and (3) deficit irrigation 50% ETc (I50). The Water requirement of the plant for control treatment was estimated using long­term average daily data of meteorological parameters recorded at Zanjan Meteorological Station and following relation. ETC = ET0 × KC ETC: Water requirement of lettuce (mm/day), ET0: E v a p o t r a n s p i r a ti o n o f g r a s s r e f e r e n c e p l a n t (mm/day) and KC: Vegetable coefficient of lettuce (no unit). It is necessary to explain that ET0 values were estimated based on the standard FAO­Penman­ Monteith method. Table 2 shows the long­term average of meteorological parameters of Zanjan synoptic station during the period of plant growth which was used to calculate ET0 and ETC values. After calculating the ETC values, the net and gross i r r i g a ti o n w a t e r r e q u i r e m e n t s o f l e tt u c e w e r e estimated based on cropping intervals, type of irrigation system and irrigation interval and then give the plant at each irrigation time. Based on the calculations, the amount of irrigation water given to the control plants was estimated to be 895.7 m3.ha­1. Water requirement of other treatments was estimat­ ed and distributed based on the water requirement of control treatment and water stress (Allen et al., 1998). All necessary management practices such as weeds control were done according to recommend­ ed practices during the crop growth. Measurements Anthocyanin content. Anthocyanin content in leaf tissue was determined according to the method of Mita et al. (2000). Fresh weight of leaves (0.1 g) was homogenized in methanol containing 1% (v/v) HCl and then was filtrated. The filtration was stored at 4°C for 24 hours in dark conditions. The absorbance of filtration was recorded at 550 nm using UV­vis spectrophotometer (Specorp 250 Jena­History) and the anthocyanin was expressed as µmol g­1 FW. Total phenols and flavonoids contents The fresh leaf tissue (2.0 g) was washed with deionized water, and homogenized in 80% cold methanol (20:80, V/V). The homogenate was cen­ trifuged at 10,000 rpm for 10 min, and the super­ natant was collected for the measurement of, total phenolic and flavonoid content. Total phenolics assay was carried out according to the procedure described in the literature (Meda et al., 2005). The results were expressed as mg of gallic acid equivalents (GAE) per 100 g of fresh weight based on a standard curve using gallic acid as standard. Total flavonoids were determined by the colorimetric method (Kim et al., 2002). Quercetin was used as a reference standard, and the results were expressed as mg quercetin equivalents per 100 g fresh weight of leaf. Antioxidant activity As mentioned in the previous paragraph, 2.0 g of leaves were homogenated in methanol and then was centrifuged. The filtration was used to determine free radical scavenging activity using the 2, 2,­ diphenyl­2­picryl­hydrazyl (DPPH) method at optical density 517 nm (Sun et al., 2007). Antioxidant activity (%) was calculated using the following equation: Antioxidant activity = A DPPH ­ A sample (517 nm)/A DPPH × 100 Catalase (CAT) and peroxidase (POX) enzymes activity Samples were taken from the fully expanded leaf and transferred to the laboratory in the ice. Leaf sample (0.5 g) was frozen in liquid nitrogen and ground using a porcelain mortar and pestle. Catalase (CAT) activity was measured by following the decomposition of H2O2 at 240 nm with a UV spec­ trophotometer (Cakmak and Horst, 1991). Samples Table 2 ­ Average daily climatic parameters of Zanjan Synoptic station during the growth seasons (2017) of lettuce Meteorological parameter May June July August September Rainfall (mm) 0.01 1.11 5.00 0.00 0.02 Average temperature (˚C) 22.94 25.71 27.68 24.79 15.73 Minimum temperature (˚C) 11.29 16.8 17.61 14.68 7.89 Maximum temperature (˚C) 32.47 33.96 36.82 35.12 25.05 Adv. Hort. Sci., 2020 34(1): 11­24 14 without H2O2 were used as blank. The activity of CAT was calculated by the differences obtained at OD240 values at 30 second interval for 2 min after the initial biochemical reaction. Peroxidase (POX) activity was measured using modified method of the Tuna et al. (2008) with guaiacol at 470 nm. A change of 0.01 units per minute in absorbance was considered to be equal to one unit POX activity, which was expressed as unit g­1 FW min­1. Leaf water status (RWC, ELWR) T h e f r e s h w e i g h t o f y o u n g l e a v e s ( F W ) w a s recorded and then was kept in Petri dishes for 24 hours immersed in distilled water. The turgid weight (TW) was measured after saturation of leaves with water. The leaves were dried at 70˚C to constant weight and then weighted (DW). Leaf relative water contents (RWC) were calculated according to the fol­ lowing formula reported by Hanson and Hitz (1982). (%) RWC= (FW­DW) / (TW­DW) × 100 For the determination of excised leaf water reten­ tion (ELWR), The youngest leaves collected for each treatment were weighed to record fresh weight (FW), kept at room temperature (25˚C) for 6 hours and reweighed (WL). ELWR was calculated using the following formula suggested by Lonbani and Arzani (2011). ELWR= [1­(FW­WL)/FW] ×100 Nutrient contents The lettuce leaf samples from each treatment were collected at the end of the experiment. For mineral analysis, leaf samples were taken and oven­ dried at 70°C until constant weight. Then 0.3 g of the dry samples was taken and digested using a mixture of sulphuric acid (H2SO4) and hydrogen peroxide (H2O2) as described by Allen et al. (1974). All the stud­ ied elements were assayed in the digest of the con­ cerned plant samples. Total nitrogen was determined using Kjeldahl method as described by Piper (1950). Phosphorus determination was done by complexing it with ammonium molybdate, which on reduction with ascorbic acid gives stable blue colour, the con­ tent of P was measured by spectrophotometer at 882 n m a c c o r d i n g t o W a t a n a b e a n d O l s e n ( 1 9 6 5 ) . Potassium, calcium and magnesium content were analyzed by flame photometer (Chapman and Pratt, 1961). Yield and water use efficiency (WUE) Lettuce plants were weighed after harvest with a digital gravimetric scale. The average weight of single plant was calculated in grams and total yield was estimated in kg/m2. Also water use efficiency (WUE) was obtained from the ratio of the amount of yield of each treatment to the amount of water consumed by the same treatment in kg m­3. Statistical analyses of the data The analysis of variance (two­way ANOVA) and least significant difference (LSD) test (P≤0.05 and P≤0.01) used to compare means within each sam­ pling date. The Statistical analysis and standard error calculation were carried out using SAS software (v. 9.1). 3. Results Anthocyanin content The data in Table 3 and figure 1, displays the anthocyanin contents of lettuce leaf applied with dif­ ferent concentrations of CaL under water deficit. Mean comparisons of data showed that deficit irriga­ tion led to a significant increase in antioxidant activi­ ty compared to control. However, the effect of CaL on the anthocyanin contents was depended to the ** and * represent significance at the 1 and 5% probability levels, respectively, and NS represents non­significance at p<0.05. Table 3 ­ Variance analysis (ANOVA) of effect of calcium lactate on physiological characteristics in lettuce under deficit irrigation S.O.V df Mean of squares Anthocyanin Total phenols Flavonoids CAT activity POX activity Antioxidant activity RWC ELWR Replication 2 1.003 0.002 0.308 0.057 0.003 16.725 8.614 23.677 Irrigation 2 2.162 ** 6.187 ** 11.932 ** 1.953 ** 0.851 ** 132.47 ** 42.799 * 238.260 ** Error (a) 4 5.648 0.135 0.330 0.009 0.001 23.524 9.036 14.106 Calcium lactate 2 4.292 * 1.057 ** 24.265 ** 0.702 ** 0.052 * 318.416 ** 186.691 ** 420.124 ** Calcium lactate × irrigation 4 2.770 * 0.475 * 1.149 * 0.075 * 0.030 * 43.004 * 1.302 NS 29.661 * Error (b) 12 6.388 0.121 0.338 0.023 0.008 9.686 9.323 6.176 Coefficient of Variation (%) ­ 11. 18 2. 31 4.19 8.87 16.93 3.73 3.9 3.4 Khani et al. ‐ Protective effect of calcium lactate on lettuce 15 Total phenols and flavonoids contents The exposure to water deficit stress significantly (P<0.05) increased total phenols and flavonoids con­ tents (Table 3, Figs. 2A, B). Besides, the results of the present study also showed that foliar application of CaL increased total phenols and flavonoids contents under normal and deficit irrigation, however, the effects of CaL was dependent to the irrigation levels. The maximum value of phenols and flavonoids con­ tents was recorded in plant treated with 0.75 g L­1 CaL under irrigation 70% ETc. In all levels of irrigation, application of 0.75 g L­1 CaL had the greatest effect on total flavonoid content, although did not show significant difference with CaL 1.5 g L­1 under irriga­ tion 70% ETc. Phenolic compounds include many secondary metabolites in plants that display antioxidant proper­ ties (Barbagallo et al., 2012). Some of the phenolic compounds, such as phenolic acids or flavonoids, are widely recognized in most of the plant species (Jwa et a l . , 2 0 0 6 ) . P h e n o l i c c o m p o u n d s a r e i m p o r t a n t because of their contribution to the nutritional quali­ ty attributes of fruits and vegetables such as color, astringency, bitterness and flavor (Vinson et al., 2001). The role of phenols as antioxidant is support­ ed by several researches and the recovery methods h a v e a g r e a t i m p o r t a n c e f o r i n d u s t r i a l u s e (Barbagallo et al., 2012). Environmental stress can cause an increase in the content of phenolic com­ pounds of cell (Weidner et al., 2009). Aghdam et al. (2013) reported that the total phe­ nols and flavonoids contents increased in the cor­ nelian cherry fruit with CaCl2 treated. Their results suggested that CaCl2 treatment may stimulate the accumulation of phenols and flavonoids fruits by acti­ vating their biosynthetic pathways. Biosynthesis of phenols such as flavonoids in plants carried out via the shikimate­phenylpropanoid pathways. Ca2+ plays irrigation regime treatment. The highest value of anthocyanin content was obtained from plant treat­ ed with 1.5 g L ­1 CaL under irrigation 85% ETc. According to the results, there was no significant dif­ ference between different levels of CaL under irriga­ tion 100% ETc. However, anthocyanin content at 85% ETc was significantly increased by using CaL while under irrigation of 70% ETc with the increase of anthocyanin content did not show any significant dif­ ference between different levels of CaL. Secondary metabolic products, which are inten­ sively biosynthesized under drought, are antioxidants (Do Nascimento and Fett­Neto, 2010). Anthocyanin pigments as one of secondary metabolites and antioxidative systems play many important eco­phys­ iological roles in plants, including roles in stress pro­ tection (Winkel­Shirley, 2002). Increased anthocyanin contents are thought to mask chlorophyll and/or act as a filter for preventing high light absorption by leaves and thus minimize photoinhibition (Farrant, 2000). Therefore anthocyanin accumulation in drought­stressed leaves confirms a possible protec­ tive role of anthocyanins as sun­screens and reactive oxygen species (ROS) scavengers in stressed plants (Merzlyak and Chivkunova, 2000), that similar results have also been reported by Jazizadeh and Mortezaei Nejad (2016) in chicory. The obtained results indicated that CaL was effec­ tive in preserving and increasing anthocyanin con­ tent. These findings were in agreement with Abd­ Elhady (2014) findings who also observed that CaL pretreatments proved to be effective for increasing the retention of anthocyanin in frozen strawberry. Fig. 2 ­ Effects of CaL treatments on total phenol and flavonoids contents of lettuce under deficit irrigation. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are statistically different P<0.05. Fig. 1 ­ Effects of CaL treatments on anthocyanin content of let­ tuce under deficit irrigation. Values are means with stan­ dard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are stati­ stically different P<0.05. http://https://www.sciencedirect.com/science/article/pii/S0570178314000116" /l "! http://https://www.sciencedirect.com/science/article/pii/S0570178314000116" /l "! 16 Adv. Hort. Sci., 2020 34(1): 11­24 a d i r e c t r o l e i n t h e b i o s y n t h e s i s o f p h e n o l s (Castañeda and Perez, 1996). CaL might be a poten­ ti a l m o l e c u l e f o r a c ti v a ti n g p h e n y l p r o p a n o i d ­ flavonoids pathways of fruits by increasing the PAL activity (Jacobo­Velazquez et al., 2011; Aghdam et al., 2013). Catalase (CAT) and peroxidase (POX) enzymes activity Significant differences among irrigation treat­ ments were observed for CAT and POX enzyme activi­ ty (Table 3, Figs. 3A, 3B). The antioxidant enzyme activates increased with the decrease of irrigation water applied. As the results showed CAT and POX enzymes activity increased with increasing CaL con­ centration under deficit irrigation, although no signif­ icantly differences was observed in normal irrigation. The highest CAT and POX enzymes activity were recorded in plant treated with 1.5 g L­1 CaL under irri­ gation 70% ETc, which had no significant difference with 0.75 g L­1. The production of ROS (Vurukonda et al., 2016) is another major factor that impairs plant growth under water deficit (Liting et al., 2015). Plants employ a number of mechanisms, at molecular, cellular and p h y s i o l o g i c a l l e v e l s t o p e r s i s t s t r e s s c o n d i ti o n (Shinozaki and Yamaguchi­Shinozaki, 2000). The acti­ vation of antioxidant enzymes is one of the major types of these mechanisms which enable plants to control ROS (Shahid et al., 2014). CAT, ascorbate per­ oxidase (APX), superoxide dismutase (SOD) and POX are the key antioxidant enzymes involved in detoxifi­ c a ti o n o f s u p e r o x i d e a n d h y d r o g e n p e r o x i d e (Kadkhodaie et al., 2014). A relationship between antioxidant enzymes activity and water stress or salinity tolerance was confirmed by comparison of a tolerant cultivar with a sensitive cultivar in several plant species, such as tomato (Mittova et al., 2002). Calcium is known to regulate different metabo­ lisms in plants mediating signaling pathways, which modulate gene expression in response to stress and its adaptation (Upadhyaya et al., 2011). Upadhyaya et al. (2011), observed POX activity was increased in the stressed plant as compared to controls, but recovering plants showed POX activity increasing after rehydration, which was enhanced by CaCl2 and reported that CaCl2 treatment resulted in increased non enzymatic antioxidant and enhanced activities of enzymatic antioxidant, including SOD, POX and CAT, and thus reduced ROS accumulation and lipid peroxi­ dation ultimately leading to improved post­drought recovery potential in Camellia sinensis. Calcium applied alleviation of drought­induced damage has been clarified in numerous plants e.g. Zoysia japonica (Xu et al., 2013), and Phaseolus vulgaris (Abou El­ Yazied, 2011). Antioxidant activity (AA) AA was affected significantly by the irrigation treatments, and water deficit stress increased AA, w h i c h n o s i g n i fi c a n t d i ff e r e n c e w a s o b s e r v e d between irrigation 100 and 85%ETc (Table 3, Fig. 4). In present study, the exogenous application of CaL significantly (P<0.05) increased AA of lettuce under different irrigation regimes compared to control plant. The highest AA (93.03%) was observed in CaL 0.75 g L−1 under irrigation 85% ETc, however had no significant difference with CaL 1.5 g L­1 treatment under irrigation 70% ETc (Fig. 4). The antioxidant activity in lettuce arises from phe­ nolic compounds, secondary plant products, such as Fig. 3 ­ Effects of CaL treatments on catalase (CAT) and peroxida­ se (POX) enzymes activity of lettuce under deficit irriga­ tion. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are statistically different P<0.05. Fig. 4 ­ Effect of CaL treatments on antioxidant activity of lettuce under deficit irrigation. Values are means with standard errors (n = 3). Data were subjected to two­way ANOVA and different letters mean that values are statistically dif­ ferent P<0.05. Khani et al. ‐ Protective effect of calcium lactate on lettuce 17 flavonoids and phenols, and also anthocyanin. Also, the antioxidant activity strongly correlated to the presence of efficient oxygen radical scavengers, such as vitamin C and phenolic compounds (Tulipani et al., 2008). In current study a significant correlation was found between antioxidant activity and antho­ cyanins, phenols and flavonoids contents; which the anthocyanin, phenolic and flavonoids content and CAT and POX enzymes activity, as well as the total antioxidant activity also increased with increasing water deficit stress and Cal concentration. This find­ ing described that phenolic compounds and antho­ cyanin, and antioxidant enzyme activity makes an important contribution to the antioxidant capacity in lettuce leaf. Velioglu et al. (1998) reported a strong relationship between total phenolic content and antioxidant activity in fresh fruits and vegetables. Leaf water status (RWC, ELWR) Based on the findings (Table 3, Figs. 5A, 6), deficit irrigation caused a significant reduction in RWC and ELWR contents. The application of CaL significantly ameliorated relative water content (RWC) and excised leaf water retention (ELWR) contents (Figs. 5B, 6). Mean comparisons of data, displayed that pre­ treatment with CaL markedly reduced the effects of water deficit stress and also improved ELWR under control irrigation and water deficit stress. The highest value of ELWR content was obtained in plant treated with CaL 1.5 g L­1 under irrigation of 85 and 100% ETc. RWC and ELWR are among the main physiological criteria that influence plant water relations and have been used for assessing drought tolerance (Xing et al., 2004). Under drought stress, leaf RWC plays an important role in the tolerance of plants to stress by inducing osmotic adjustments due to the accumula­ tion of osmoprotectants (Barnabás et al., 2008; Zhang et al., 2012). The maintenance of a high plant water status during stress is a significant defensive mechanism to maintain enough water by minimizing water loss (e.g. caused by stomatal closure, tri­ chomes, reduced leaf area, senescence of older leaves, etc.) and maximizing water uptake (e.g. by increased root growth) (Barnabás et al., 2008). Because of the decrease in leaf area, the accumula­ tion of chlorophyll has increased, but due to high transpiration, the plant loses more water and as a result, the RWC of leaf and consequently photosyn­ thesis decreases (Farooq et al., 2012). Farooqi et al. (2000) indicated that RWC of lemongrass leaves decreased in all the cultivars due to drought but after rehydration, RWC gradually increased to pre­stress level, which has also been reported in several crop species such as melon (Mani, 2014). As well as drought stress significantly decreased RWC and ELWR in spring safflower (Balian et al., 2015). Ruiz­Lozano and Azcon (1997) reported that calci­ um application significantly increased RWC in lettuce. The results of this research showed that the RWC of leaves increased with calcium application. Increasing relative water content means increasing water hold­ ing capacity, which can prevent water loss in leaves in a dry environment (Ma et al., 2005). Nutrient contents According to the results (Table 4, Fig. 7), N con­ tent in lettuce leaves increased with increasing CaL concentration, indeed the highest value of N was obtained at CaL 1.5 g L­1 under irrigation 100 %ETc that had significant difference with deficit irrigation treatments (85 and 70% ETc), whereas in other treat­ ments there were not any significant differences. Mean comparisons of data, showed that deficit irri­ Fig. 5 ­ Effects of irrigation (A) and CaL (B) treatments on leaf relative water content (RWC) of lettuce. Data were subjected to two­way ANOVA and different letters mean that values are statistically different P<0.05. Fig. 6 ­ Effects of CaL treatment on excised leaf water retention (ELWR) content of lettuce under deficit irrigation. Values are means with standard errors (n= 3). Data were subjec­ ted to two­way ANOVA and different letters mean that Adv. Hort. Sci., 2019 33(3): 11­24 18 gation significantly increased P content in lettuce leaves and decreased K content compared to control irrigation (Table 4, Figs. 8A, 8B). The Ca content increased with the deficit irriga­ tion treatments, in particular with moderate deficit irrigation (85% ETc). Ca content in lettuce leaves increased in response to higher CaL concentration ( F i g . 9 A ) . I n f a c t , t h e h i g h e s t v a l u e o f C a w a s observed in treatments with application of 1.5 g L­1 CaL under irrigation 70 and 85% ETc. The overall effect of deficit irrigation on Mg content was nega­ tive, with a decrease of 0.79% (Fig. 9B) under deficit i r r i g a ti o n 7 0 % E T c . M g c o n t e n t i n t h e l e a v e s decreased when the concentration of CaL applied was increased. The lowest value of Mg content was obtained in plant applied with 1.5 g L­1 CaL under deficit irrigation 70% ETc. Drought stress and associated reduction in soil moisture can decrease plant nutrient uptake by reducing nutrient supply through mineralization (Sanaullah et al., 2012), and nutrient diffusion in the soil (Chapin III, 1991; Lambers et al., 2008). Drought can depress plant growth by reducing N and P uptake, transport and redistribution (Rouphael et al., 2012). A majority of studies have indicated that plants decrease N and P uptake with a decline in soil moisture (Sardans and Peñuelas, 2012). N uptake was reduced in maize under stress conditions, which indicates that the absorption of nutrients is limited in conditions of water deficit stress, which may be reduced due to reduced transpiration rate, active transfer and membrane permeability (Naeem et al., 2017). Owing to a reduction in stomatal conduc­ tance, photosynthesis and transpiration rates also Table 4 ­ Variance analysis (ANOVA) of effects of calcium lactate on nutrient contents and fresh yield in lettuce under deficit irrigation ** and * represent significance at the 1 and 5% probability levels, respectively, and NS represents non­significance at p<0.05. Source of variations df Mean of Squares N content P content K content Ca content Mg content Fresh yield WUE Replication 2 0.030 0.008 0.008 0.002 0.004 206838.82 0.380 Irrigation 2 0.208 ** 0.111 ** 0.153 ** 0.621 ** 2.157 ** 21349043.15 ** 3.293 ** Error (a) 4 0.008 0.008 0.003 0.005 0.004 83934.71 0.183 Calcium lactate 2 1.158 ** 0.024 NS 0.025 NS 0.291 ** 0.095 ** 6105376.18 ** 10.144 ** Calcium lactate × Irrigation 4 0.116 ** 0.006 NS 0.010 NS 0.047 ** 0.018 * 413961.66 * 0.317 NS Error (b) 12 0.012 0.006 0.008 0.004 0.005 101407.46 0.170 Coefficient of Variation (%) ‐ 1.47 3.48 1.43 2.09 3.4 2.82 2.78 Fig. 7 ­ Effects of CaL treatments on nitrogen content of lettuce leaves under deficit irrigation. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are stati­ stically different P<0.05. Fig. 8 ­ Effect of irrigation treatments on phosphorus (A) and potassium (B) contents of lettuce leaves. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are statistically different P<0.05. Fig. 9 ­ Effect of CaL treatments on calcium (A) and magnesium (B) contents of lettuce leaves under deficit irrigation. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and different letters mean that values are statistically different P<0.05. Khani et al. ‐ Protective effect of calcium lactate on lettuce 19 decrease, and CO2 assimilation rates progressively decline in response to drought (Farooq et al., 2012). Therefore, drought effects on plant may depend on the reduction in N and P uptake relative to the decrease in CO2 assimilation (He and Dijkstra, 2014). Based on the current findings, increasing K and Ca contents and decreasing Mg content of lettuce leaves under water deficit stress as compared to well­ watered conditions that also reported by Tadayyon et al. (2018) in castor plants. Potassium has a positive correlation with the physiological effects of plants, such as water use efficiency, stomatal control, air and underground body biomass, and is likely to play an important role in photosynthesis (Sardans et al., 2012). Increasing K content of leaves with decreasing irrigation rate maybe due to role of this cation in the regulation of osmotic pressure and stomatal control, (Zhao et al., 2000). Nahar and Gretzmacher (2002) reported that with increasing deficit irrigation, Mg concentration in tomato tissues decreased, which is similar to results of the present study. The same results were reported from other authors, that high concentrations of Ca often result in increased leaf Ca along with a marked reduction in leaf Mg (Nassery et al., 1979; Borghesi et al., 2011). As well as, Naeem et al. (2018) revealed that concen­ tration of macronutrients (N, K, Ca) in maize grains was markedly improved by foliar supply of calcium which indicates its synergistic effect on uptake and translocation of these nutrients. Tuna et al. (2007) also observed leaf N, K and Ca content increased in tomato plants supplemented with calcium under stress conditions. In safflower, by decreasing soil moisture K and Mg content decreased. Following this reduction, there was a significant increase in calcium concentration, which is justified by the antagonistic relationship between Ca and Mg (Vafaie et al., 2013). Morard et al. (1996) reported an intense antagonistic relation­ ship between Ca and Mg, that Mg transfer to leaves was affected by calcium. Fresh yield Lettuce plants grown under control and deficit irrigation conditions exhibited significant differences between CaL treatments in fresh yield (Table 4, Fig. 10). Water deficit stress caused significant reductions in yield. In fact, deficit irrigated plants showed a 6.8 and 15.8% decrease in fresh yield, respectively. As the results showed, with increasing CaL concentra­ ti o n s , l e tt u c e y i e l d s i g n i fi c a n t l y i n c r e a s e d a n d reached to highest value (1.37 kg.m­2) at CaL 1.5 g L­1 under irrigation 100% ETc (Fig. 10). Lettuce is one of the leaf­edible vegetables that it is extremely sensitive to water deficit stress due to shallow root system (Sabedze and Wahome, 2010). Our results are in agreement with many open­field studies on lettuce (Jiménez­Arias, 2019) and lettuce (Sayyari et al., 2013). Deficit irrigation defined as a practice that applies water below full crop­water requirements, deliberately exposes plants to a cer­ tain level of moisture stress. It is well known that drought stress results in dehydration of the cell and osmotic imbalance that impairs numerous metabolic and physiological processes in plants (Mahajan and Tuteja, 2005). Reduction in fresh yield of lettuce with deficit irrigation might be attributed to the suppres­ sion of cell division and expansion, and growth due to the low turgor pressure and also closure of stomata leaf and more leaf senescence under drought stress (Sayyari et al., 2013). Foliar application of CaL enhanced fresh yield of lettuce. Naeem et al. (2013) reported that crop pro­ ductivity and photosynthetic efficiency in Senna occi‐ dentalis was improved under Ca application. With low calcium availability, a reduction in bean plant height, leaf area and shoot and root growth has been reported (Leal and Prado, 2008). Foliar applied of chelated calcium enhanced the seed yield and relat­ ed attributes in common bean under water­deficit conditions (Abou El­Yazied, 2011). Water use efficiency (WUE) According to the results (Table 4, Figs. 11A, 11B), irrigation and CaL treatments significantly affected WUE, but their interaction showed no significant dif­ ferences. Water deficit stress significantly increased WUE and the highest WUE was recorded in 70% ETc deficit­irrigated plants that had no significant differ­ Fig. 10 ­ Effect of CaL treatments on fresh yield of lettuce under deficit irrigation. Values are means with standard errors (n= 3). Data were subjected to two­way ANOVA and dif­ ferent letters mean that values are statistically different P<0.05. Adv. Hort. Sci., 2019 33(3): 11­24 20 ence with deficit irrigation 85% ETc (Fig 8A). WUE increased with increasing CaL concentration and the highest WUE (15.8 kg m­3) was obtained at 1.5 g L­1 CaL (Fig. 8B). W U E , t h e p h y s i o l o g i c a l p a r a m e t e r o f c r o p , describes the relationship between plant water use a n d d r y m a tt e r p r o d u c ti o n ( C a i e t a l . , 2 0 1 2 ) . Regarding water resource constraints, it is essential to find ways to preserve water and increase water use efficiency in plants (Topcu et al., 2007; Alenazi et al., 2015). The highest WUE value was determined in 70% ETc irrigation. It was calculated that WUE values increased with the decrease in the amount of water. These results are similar to the previous finding of Şimşek et al. (2004), who reported that the maximum WUE for watermelon was obtained with low irriga­ tion. With increased WUE, there is a greater biomass production per amount of water transpired, and less w a t e r i s n e e d e d f o r g r o w t h a n d d e v e l o p m e n t (Nemali and van Iersel, 2008). WUE is strongly related to photosynthetic activity and transpiration efficiency, and can be affected by irrigation (Monneveux et al., 2006). Ca is directly involved in photosynthesis processes, and its deficit reduces the plant’s biomass by reducing the efficien­ cy of carboxylation and photosynthesis (Alarcon et al., 1999). The results of the current experiment showed that N content was increased with Ca appli­ cation, which increasing N content leads to increase dry matter production as well as the WUE. Therefore, Ca maybe increased the WUE by increasing the amount of N and Ca in lettuce plants. 4. Conclusions The results obtained in this investigation pro­ posed that lettuce is sensitive to water deficit stress during their entire growing period. Hence, it could be concluded that under water deficit, decrease in the relative water content in the leaves is related to the decrease markedly in the fresh yield. Application of CaL showed a clearly protective effect on yield of plants under water deficit stress. The result also revealed that treating the plants with calcium lactate led to increase N and Ca accumulation. CaL appears to promote water deficit tolerance by acting at differ­ ent levels: leaf water status and antioxidant defens­ es, without evidence of toxic effects on the soil. 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