Efficacy of a multi-dentate Schiff base and its vanadyl complex on various morphological and biochemical parameters of Vigna radiata L.

Abstract A N2O2 donor Schiff base polydentate ligand was synthesized by the condensation of benzidine and benzil in ethanolic medium. The formed ligand was condensed further with VOSO4.xH2O to get the corresponding vanadyl complex. Both the ligand and the complex were characterized by spectroscopic and elemental analyses. Mung bean (Vigna radiata L.) was selected as a plant material. Various morphological and biochemical parameters, e.g. leaf senescence assay, chlorophyll content, and different reactive oxygen species (ROS) were estimated and were compared to those with ammonium vanadate. Outcomes of the experiments revealed that the Schiff base complex has less toxic effects than ammonium vanadate on mung bean seedlings and provide better tolerance to vanadium toxicity. Though different stress marker and ROS accumulation were less and minimum pigment damage was noticed in the Schiff base complex-treated seedlings but the optimum positive impact largely depends on the dose. Beyond certain concentration, the complex may show inhibitory effects on the plants. Therefore, the present study revealed that heavy metal Schiff base complexes can be used as potential supplement to meet up micronutrient deficiency and at the same time such complexes can minimize the toxicity generated by application of different heavy metals.


Introduction
Vanadium, one of the important trace elements for plants is found scattered in the environment through the leaching of rocks, combustion of coal or petroleum products, and residual slag from the steel industry (Baken et al. 2012). Although Vanadium (V) was discovered in early eighteenth century, very little work was done on V before the 1950s (Bertrand 1950). Initial studies showed that V is toxic for most of the plant species and that is why there was very little interest in evaluating its effect on plants (Witz and Osmond 1886). Interest intensified when Arnon and Wessel (1953) concluded that V is essential for some plants (Trejo-T ellez G omez-Merino, and Alcantar-Gonz alez 2016). Subsequent studies showed that V is generally toxic to terrestrial plants when applied in amounts greater than pico-molar levels (Davis, Beckett, and Wollan 1978). However, it is found to be beneficial for plant growth and development when applied in trace amount (Prarr 1966;Welch 1973). Further studies indicated that V due to its various oxidation states (-I to þ V), the toxicity varies. It was also found that the pentavalent state (V þ5 ) is more toxic than the corresponding tetravalent state (Tian, Yang, and Huang 2015). Moreover V þ4 , predominantly found in the soil, is responsible for the development of plants (Larsson et al. 2013). Study revealed that V acts as constituent of the cofactors in vanadate-dependent haloperoxidases and vanadium nitroginase (Hu, Lee, and Ribbe 2012). Many important electron transfer processes and plasma membrane hydrogen (H þ )-translocation ATPase are largely dependent upon V (Vara and Serrano 1982). The monomeric form of vanadate is both structurally and electronically identical with phosphate. This facilitates vanadate to inhibit or to activate the enzymes that interact with phosphorylated species (Akabayov and Akabayov 2014). Generally, vanadium-containing fertilizers (e.g. NH 4 VO 3 ) are used to provide V to plants in optimum level. These types of fertilizers, being ionic in nature, are responsible for the alteration of pH of soil (Thind and Rowell 1999). So nowadays, more emphasis is given to metal chelates that are less reactive and can solve the V deficiency as well as V toxicity for longer period of time without hampering the medium (Khoshgoftarmanesh et al. 2010;Wallace and Wallace 1982). Inspired by these facts, a polydentate (N 2 O 2 donor type) Schiff base ligand and its oxo vanadium complex have been synthesized and their effects on Vigna radiata L. were thoroughly monitored. Mung bean (V. radiata L.) is chosen as plant material because of its global importance as pulse. It is grown in South, East, and Southeast Asia where 90% of global production currently occurs (Tomooka et al. 1992). Mung bean provides significant amounts of protein, carbohydrates, and a range of micronutrients in diets. Its cultivation is also important as it maintains the soil fertility through nitrogen fixation. In this study, the responses of vigna seedlings to vanadium toxicity when exposed to ammonium vanadate and vanadium Schiff base complex are thoroughly monitored in terms of relative water content, biochemical components, oxidative stress markers, and overall tolerance level in vigna plants.

Synthesis of tetradentate N 2 O 2 donor Schiff base ligand and its vanadium(IV) complex
Ethanolic solution of benzidine (purity > 99%, procured from Sigma-Aldrich, Germany) was refluxed with benzil (purity l > 98%, procured from Sigma-Aldrich) in a round-bottom flask in 1:2 molar ratio for $4 hr at temperature of 40-60 C with constant stirring to get the olive green colored ligand L1 [C 40 H 28 N 2 O 2 . 2H 2 O]. The ligand was filtered off, washed, and recrystallized from ethanol. Then it was dried in a vacuum desiccator. To synthesize vanadium(IV) complex, the ligand was further refluxed with an ethanolic solution of VOSO 4 .xH 2 O (procured from Sigma-Aldrich) for $8 hr at 40-60 C in a molar ratio of 1:1 with constant stirring. The complex C1 obtained with a molecular formula of C 80 H 56 N 4 O 6 V 2 .5H 2 O was washed with ethanol and dried over anhydrous CaCl 2 . Both the synthesized ligand and the complex were characterized by elemental microanalysis, infrared, and electronic spectroscopic spectra. These results were similar to those reported earlier in the literature (Ahmed and BenGujji 2009).

Maintenance of plants
Vigna seeds were surface sterilized using 1% (wt/vol) sodium hypochlorite solution and rinsed with double distilled water. Seeds were then transferred to plastic pots (diameter 11 cm) containing sterile soil. Each pot contained five seeds and the pots were kept in temperature of 25 ± 2 C for a photoperiod of 8 hr with 65%-70% relative humidity regime. Seedlings were watered regularly every alternate day and after one month plants were utilized for further experiments. After a growth period of 30 days, the plants in their vegetative phase were taken, roots were gently washed with sterile H 2 O and transferred to 20% Steiner nutrient solution (1.8 mM Ca(NO 3 ) 2 . 4H 2 O, 0.8 mM MgSO 4 .7H 2 O, 0.2 mM KH 2 PO 4 , 0.6 mM KNO 3 , 0.6 mM K 2 SO 4 , 89.31 lM Fe, 42.37 lM Mn, 7.12 lM Zn, 39.98 lM B, 2.93 lM Cu, 1.80 lM Mo). The plants were then allowed to acclimatize in this solution for 48 h. After 48 h of acclimation, this nutrient solution was entirely replaced and treatments were applied in the renewed nutrient solution with different concentrations (5, 10, and 20 lM V) of schiff base ligand (L1), Schiff base vanadyl complex (C1), and NH4VO 3 (AV) along with a control (no treatment of ligand, Schiff base complex, NH4VO 3 in nutrient solution) separately for 7 days. Each treatment had three replicate sets and experiment was conducted in randomized design method. After 7 days, leaf samples were collected, frozen in liquid nitrogen, and subsequently used for biochemical tests. The fresh weight of seedlings was taken immediately after sampling to avoid any water loss from leaf samples.

Growth parameters
Plants from different treatment sets were harvested and weighed to get fresh biomass (F.W.). The samples were then absorbed to full turgidity for 6 hr at 25 C to get turgid weight (T.W.). Subsequently, the samples were parched in a hot air oven at 70 C for 48 h to get dry biomass (D.W.) of each sample. Relative water content (RWC) was calculated by using the method of Farooqui et al. (2000).

Cell viability
Briefly, 10-mm leaf disc from the control and the treated plants were kept in glass vials with 1% MTT (i.e. 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution in dark for 12 hr. Leaf samples were placed in 5% alcohol and kept for boiling till all the alcohol evaporated off. Thereafter, the absorbance of the purple colored extract was measured at 485 nm.

Electrolyte leakage and membrane injury
Electrolyte leakage was measured as per Yan et al. (1996). Leaves from each treatment were washed carefully with deionized water, kept in culture tubes containing 10 mL of deionized water and incubated at 25 C on a rotary shaker for 24 h. The electrical conductivity of the solution (C 1 ) was measured with a conductivity meter. Then the samples were autoclaved at 120 C for 20 min and cooled to room temperature prior to assessing the final electrical conductivity (C 2 ). Electrolyte leakage was measured as follows: Membrane lipid peroxidation was assessed in terms of malondialdehyde (MDA) content according to Heath and Packer (1968). Fresh leaves (0.5 g) were ground in precooled 0.1% (wt/ vol) trichloroacetic acid (TCA) followed by centrifugation at 10,000 rpm for 15 min at 4 C. About 0.5 mL of the slurry was mixed with 2 mL of 0.5% Thiobarbituric acid (TBA) in 20% TCA, followed by heating for 30 min at 95 C and subsequent cooling on ice. The absorbance of the reaction mixture was measured at 532 and 600 nm and the MDA content was calculated using an extinction coefficient of 155 mM À1 cm À1 .

Determination of free amino acids and total sugar
From the ethanol extraction of leaves, 500 lL were mixed with 500 lL of ascorbic acid and sodium citrate buffer mix (0.2% wt/vol), at a pH of 5.2 and 1000 lL of ninhydrin (1% wt/vol) in ethanol at 70% (vol/vol) was added. The samples were kept in a water bath at 95 C for 30 min and followed by cooling at room temperature. The free amino acids were measured in a spectrophotometer using leucine (10 mM in ethanol 70%) as standard at 570 nm.
To measure total soluble sugar, leaves (0.5) were homogenized in 10 mL of 95% ethanol (Harborne 1973). Then 1 mL of extract and 4 mL of anthrone reagent (0.2%) were kept in boiling water bath for 10 min followed by quick cooling. Then absorbance was taken at 620 nm and quantified using glucose as standard (Plummer 1978).
H 2 O 2 levels in the leaves were estimated according to Jana and Choudhuri (1982) with minor modifications. H 2 O 2 levels were calculated using extinction coefficient 0.28 mmol À1 cm À1 .

Chlorophyll content
Chlorophyll was extracted from the leaves using 80% (vol/vol) acetone according to Lichtenthaler (1987). Absorbance was taken spectrophotometrially using at 480 nm, 645 nm, and 663 nm. Total chlorophyll, chlorophyll a, and chlorophyll b were calculated using following formula (Arnon 1949

Leaf disc bioassay
The fully expanded and fresh leaves from the plants were gently washed in deionized water and 1-cm diameter leaf discs were then floated in a 5 mL solution of schiff base ligand (L1), Schiff base VO(II) complex (C1), and NH 4 VO 3 (AV) for 6 days. Leaf discs floated in sterile distilled H 2 O served as the experimental control The effects of different complexes on leaf discs were assessed on the basis of the phenotypic alteration especially leaf color (Fan, Zheng, and Wang 1997).

Statistical analysis
Data were analyzed by using standard error and Least Significant Difference (LSD) tests at p 0.05 probability level using IBM SPSS statistics 21 software.

Results and discussion
Characterization of the prepared ligand and its vanadyl complex The analytical and spectral data recorded for the Schiff base (L1) and its vanadyl complex (C1) was found to be almost as same as reported in the literature (Ahmed and BenGujji 2009). Some of the characteristic analytical and spectral data and scheme of reaction are listed in Tables 1 and 2 and in Supplementary Material. Characteristic Infrared (IR) bands at 1620, 1731, and 3375 cm À1 appeared due to C¼N , C¼O , and OÀH vibrations, respectively for the ligand (L1). After complexation the C¼N band shifted to 1624 cm À1 due to coordination bond formation. For the complex, two new bands appeared at 982 and 491 cm À1 due to VÀO and VÀN vibrations, respectively. The electronic spectra were measured with 5 Â 10 À4 molar solutions in dimethylformamide for both the compounds. The ligand (L1) has a characteristic k max at 263.1 nm due to p-p Ã transition and its vanadyl complex (C1) manifested three peaks at 844.7, 445.1, and 367.8 nm, respectively due to characteristic transitions as reported earlier in the literature (Ahmed and BenGujji 2009).

Effects of the ligand and its vanadyl complex on vigna seedlings
Leaf disc bioassay V sensitivity of mung leaf was determined by leaf disc senescence bioassay. It is represented in terms of degree of leaf decoloration and percentage (%) decrease of the chlorophyll content of the detached leaves at the concentration range of 5 mM, 10 mM, and 20 mM of L1, C1, and NH 4 VO 3 in comparison to the detached leaves kept in sterile distilled water. For control and L1treated Vigna seedlings, the leaf color and chlorophyll content were almost alike after 7 days of treatment indicating that L1 ligand do not have negative impact on seedlings (Figures 1 and 2). The leaf discs turned slightly blackish when kept at 20 mM concentration of NH 4 VO 3 for 7 days. On the contrary, leaf decoloration was found to be least for C1-treated leaf discs indicating less negative impact of the Schiff base complex (C1) on seedlings chlorophyll.

Fresh biomass, dry biomass and relative water content
Plants from different treatment sets were harvested and weighed to get fresh biomass. Subsequently, to get dry biomass the samples were parched in a hot air oven at 70 ᭺ C for 48 h. The outcomes reveal that plants treated with the complex (C1) were able to retain higher percentage of fresh mass and dry mass over the period of time than NH 4 VO 3 -treated plants with increasing concentration suggesting less toxicity of the Schiff base complex (C1) (Figures 3 and 4). Same trend has been observed for relative water content (RWC). Drastic decrease in relative water content at higher concentration for NH 4 VO 3 -treated plants signified higher stress in cells (Table 3).

Oxidative stress
Plants facing adverse conditions produce reactive oxygen species (ROS) at vital cell organelles like chloroplast, mitochondria, and peroxisomes. These ROS are formed as a byproduct of plant aerobic metabolism (Huang et al. 2016;Dietz, Turkan, and Krieger-Liszkay 2016;Sandalio and Romero-Puertas 2015). Amongst a variety of ROS, H 2 O 2 is highly stable and can remain in cell causing damage to cell viability and induce senescence. ROS also increases lipid peroxidation in both cellular and organelle membranes and thus further induce membrane injury, protein degradation, and thereby affects photosynthesis (Sarkar, Chakraborty, and Chakraborty 2018;Huang et al. 2019). Lipid peroxidation produces malonaldehyde as the end product in a chain of reactions with membrane phospholipid molecules (Huang et al. 2019). Application of beneficial   In all the plants, electrolyte seepage from the membranes increased gradually with the increasing concentration of the Schiff base complex (C1) and NH 4 VO 3 (AV) whereas in L1 there were no such changes in these parameters in relation to control. At 5 lM level, EL remained similar for both C1 and AV whereas 10 lM and 20 lM of AV caused considerable membrane leakage as compared to C1-treated plants. H 2 O 2 accumulation was pretty similar in the Schiff base complex   (C1) and AV-treated plant leaves up to 10 lM whereas at 20 lM, there was greater H 2 O 2 accumulation in AV-treated plants as compared to those treated with C1. Similar trends were also noticed for membrane lipids peroxidation (MDA). Greater impact of the Schiff base complex (C1) treatment on MDA accumulation was observed at 20 lM whereas in AV-treated plants significant induction was observed from 20 lM. Survival prospect of plants was measured in terms of cell viability. There were no significant changes in cell viability across all concentrations in L1treated plants and even in C1-and AV-treated plants when given at 5 lM. But beyond that there was gradual drop of cell viability in both the Schiff base complex (C1) and AV-treated plants and this drop was found be slightly lesser in Schiff base complex-treated plants. Findings of the present study suggest that the ligand (L1) has neither any of positive or negative impact on the accumulation of stress indicators. So far the Schiff base complex (C1) and AV both elicited certain level of membrane injury and ROS accumulation. While at low concentrations (5 lM) both C1 and AV have similar impacts but at higher concentrations harmful effects of the Schiff base complex (C1) was comparatively lesser compared to those with AV regarding membrane injury and oxidative stress, i.e. it can impart comparatively better cell survival. Total free amino acid and total soluble sugar Amino acids play as key player in metal chelation by which plant detoxify or alleviate heavy metal stress. Therefore, it can be suggested that plants experiencing higher amount of vanadiuminduced stress can accumulate more amount of free amino acid. Results revealed that although both the C1 and AV are responsible for alleviating free amino level in vigna seedlings but the accumulation is much higher in AV-treated plants. This signifies the more toxicity of AV than C1 for vigna plants ( Figure 6). Total soluble sugar content was also estimated. Higher sugar content in cell symbolizes less stress. Results show that both the C1 and AV are responsible for the increase of soluble sugar content at low concentrations (5 lM). But at higher concentration (20 lM), the soluble sugar content decreases drastically for AV than C1-treated plants (Figure 7). This justifies that both the C1 and AV are beneficial for vigna plants at low concentrations but at higher concentrations AV become more toxic than C1.

Conclusions
Outcomes of the present experiment reveal that the Schiff base complex (C1) has less toxic effects than NH 4 VO 3 on mung bean seedlings and it also provide better tolerance to vanadium toxicity. Though different stress marker and reactive oxygen species (ROS) accumulation were less and minimum pigment damage was noticed in the Schiff base complex (C1)-treated seedlings, the optimum positive impact largely depends on the dose. Beyond certain concentration, the complex (C1) may show inhibitory effects on the plants. Therefore, the present study revealed that heavy metal Schiff base complexes can be used as potential supplement to meet up micronutrient deficiency and at the same time such complexes can minimize the toxicity generated by application of different heavy metals.