Alleviation of Lead Toxicity by 5-Aminolevulinic Acid Is Related to Elevated Growth, Photosynthesis, and Suppressed Ultrastructural Damages in Oilseed Rape

Lead (Pb) is a widely spread pollutant and leads to diverse morphological and structural changes in the plants. In this study, alleviating role of 5-aminolevulinic acid (ALA) in oilseed rape (Brassica napus L.) was investigated with or without foliar application of ALA (25 mg L−1) in hydroponic environment under different Pb levels (0, 100, and 400 µM). Outcomes stated that plant morphology and photosynthetic attributes were reduced under the application of Pb alone. However, ALA application significantly increased the plant growth and photosynthetic parameters under Pb toxicity. Moreover, ALA also lowered the Pb concentration in shoots and roots under Pb toxicity. The microscopic studies depicted that exogenously applied ALA ameliorated the Pb stress and significantly improved the cell ultrastructures. After application of ALA under Pb stress, mesophyll cell had well-developed nucleus and chloroplast having a number of starch granules. Moreover, micrographs illustrated that root tip cell contained well-developed nucleus, a number of mitochondria, and golgi bodies. These results proposed that under 15-day Pb-induced stress, ALA improved the plant growth, chlorophyll content, photosynthetic parameters, and ultrastructural modifications in leaf mesophyll and root tip cells of the B. napus plants.


Introduction
Environmental pollutants like heavy metals are toxic even at low concentration. Lead (Pb) is amongst one of the most toxic pollutants which is not an essential nutrient for plants [1]. The contamination of Pb in environment is of major ecological alarm due to its long persistent nature that causes risk effects on human health through the food chain [2]. Lead accumulation is recognized to cause highly deleterious effects on growth and yield of plants [3]. It was reported that Pb significantly suppressed the root elongation in mesquite (Prosopis sp.) [4]. Plant growth retardation under Pb stress may be due to nutrient imbalance, disturbed photosynthesis, obstacle in electron transport system, and inadequate levels of carbon dioxide due to stomatal closure [5,6]. It is documented that Pb prohibits the nutrient uptake, net photosynthetic rate, and cellular respiration and causes damage to cell membrane [7]. It has been stated that photosynthesis inhibition is a wellrecognized symptom of Pb toxicity [8]. Lately, Jiang and Liu [9] proposed that exposure of Pb for 72 hr to Allium sativum roots induced ultrastructural changes, that is, loss of cristae, mitochondrial swelling, dictyosomes, endoplasmic reticulum vacuolization, impairment into lamellar organization of the chloroplast, and cell division [10]. Thus, it is very important to exploit the potential of oilseed rape plant against Pb stress.
The plant growth regulators (PGRs) are mostly used to alleviate abiotic stress in plants. The 5-aminolevulinic acid (ALA), one of the most vital plant growth regulators, is an essential precursor for the formation of tetrapyrroles like protochlorophyllide (which is converted into chlorophyll when exposed to light) [11]. In our previous research, ALA had been found to promote growth of B. napus under cadmium (Cd) stress [12,13]. Similarly, Zhang et al. [14] applied the ALA in oilseed rape plants and suggested that it alleviates the toxicity of herbicides. Wang et al. [15] delineated that treatment of B. campestris L. with ALA yielded a positive effect on the seedlings growth. Furthermore, ALA improved the plant growth in terms of biomass of different plant parts under salinity-stress conditions [16] and ALA is the major photosynthetic light harvesting pigment [17]. Recently, it has been documented that root morphology was exalted under the combined treatment of ALA and Cd in B. napus [18].
It is evident from a number of previous reports that ALA, like other known PGRs, is effective in counteracting the injurious effects of various abiotic stresses on plants, but the mechanisms how it regulates growth under stressful cues are not fully elucidated yet. The present research was undertaken to investigate the changes in the level of plant growth, photosynthetic activity, and Pb uptake due to ALA application on B. napus under Pb stress. It is hypothesized that ALA regulates the growth, photosynthetic attributes, chlorophyll contents, and ultrastructural changes under Pb stress conditions in B. napus L.

Plant Material and Treatments.
For the experiment, B. napus L. cv. ZS 758 seeds were obtained from Zhejiang University. The seeds were grown in plastic pots (170 mm × 220 mm) which were filled with peat moss. At five-leaf stage, uniform seedlings were plugged into plate holes on plastic pots (five plants per pot) containing Hoagland nutrient solution [19], in which concentration of KH 2 PO 4 was kept as minimum as 0.01 mM in order to avoid precipitation of lead (Pb). The composition of Hoagland nutrient solution was as follows (in mol L −1 ): 3000 KNO  After two weeks, solutions were supplied to required Pb as Pb(NO 3 ) 2 concentration (0, 100, and 400 M) and simultaneously plants were sprayed with an aqueous solution of ALA (Cosmo Oil Co. Ltd., Japan) at concentrations of 0 and 25 mg L −1 ALA. The spray was done on the lower as well as the upper leaf surfaces with a hand-held atomizer [20]. ALA was applied as a foliar spray at a rate of 20 mL of formulated solution/plant. After the five days of first spray, subsequent application was followed. The control plants were sprayed with distilled water. Fifteen days after treatment, all morphological data and photosynthetic gas exchange parameters were recorded. Samples for chlorophyll contents and microscopic studies of leaf mesophyll and root tip cell were collected as described next.

Plant Morphological Parameters.
After 15 days of treatment, harvested plants were separated into leaf, stem, and root. The plant height, stem length, root length, and leaf area were recorded simultaneously. Root surface area, volume, diameter, and number of root tips of randomly selected plants were determined using root automatism scan apparatus (MIN MAC, STD1600 + ), equipped with WinRHIZO software offered by Regent Instruments Inc.

Photosynthetic Attributes.
Photosynthetic parameters were analyzed 15 days after treatment by LiCor-6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). All the photosynthetic attributes were measured on the intact topmost fully expanded leaf after 2 hr of acclimatization in a growth cabinet, at a temperature of 18 ∘ C under a light intensity of 1,000 mol m −2 s −1 , relative humidity of 60%, and took at least eight readings per treatment [21]. Chlorophyll contents in B. napus leaves were estimated according to Porra et al. [22] and Pei et al. [23].

Analysis of Pb Concentration.
Pb contents in different plant parts were determined with the method of Hsu and Kao [24]. The tissue samples were dried at 65 ∘ C for 24 hr and then ashed in Muffle furnace at 550 ∘ C for 20 hr. Then the ash incubated was with 31% HNO 3 and 17.5% H 2 O 2 at 70 ∘ C for about 2 hr, and then this was dissolved in distilled water. Pb concentration was determined by using spectrophotometer (AA-6800, Shimadzu Co. Ltd., Japan).

Microscopic Study.
After 15 days of treatment, topmost leaf fragments without veins and root tips were collected from randomly selected plants and then fixed overnight in 4% glutaraldehyde (v/v) in 0.1 M PBS (sodium phosphate buffer, pH 7.4) followed by three times washing with the same PBS. Samples were postfixed in 1% OsO 4 (osmium (VIII) oxide) for 1 hr, washed three times in 0.1 M PBS (pH 7.4) with 10 min interval between each washing. Then with 15-20 min interval, the samples were dehydrated in a graded series of ethanol (50%, 60%, 70%, 80%, 90%, 95%, and 100%) and at the end washed by absolute acetone for 20 min. The samples were then infiltrated and embedded in Spurr's resin overnight. After heating at 70 ∘ C for 9 hr, ultrathin sections (80 nm) of specimens were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOL TEM-1230EX) at an accelerating voltage of 60.0 kV.

Statistical
Analysis. The SPSS version 16.0 (SPSS, Chicago, IL, USA) was used to analyze the data. A two-way variance analysis (ANOVA) was carried out, followed by the Duncan's multiple range test.

ALA Ameliorated Pb-Induced Inhibition of Plant Growth.
The data regarding plant height, stem length, root length, and number of leaves per plant under different treatments of ALA and Pb is given in Table 1. The results showed that Pb toxicity significantly inhibited the plant growth as compared  Foliar applied ALA did not show any impact on Chl contents under Pb stress conditions; however, it significantly increased the total Chl contents by 11.88% under higher concentration of Pb (400 M), as compared to its respective control. Moreover, the higher chlorophyll values were found under ALA alone conditions. The effects of different treatments of ALA and Pb on photosynthetic parameters have been presented in Figure 1. The data depicted that Pb toxicity significantly inhibited all the photosynthetic attributes, that is, net photosynthetic rate, stomatal conductance, internal CO 2 concentration, and transpiration rate as compared to control, and this inhibition was recognized as dosedependent. Exogenous application of ALA alone exhibited a significant positive effect on stomatal conductance and transpiration rate of the rapeseed plants.  The TEM of root cells of ZS 758 at control treatment with low and high magnifications have been shown in Figures  4(a) and 4(b). The micrograph showed a clear nucleus with nuclear membrane and nucleolus. Moreover, clear endoplasmic reticulum and mitochondria were found in root tip cell. The cell wall and cell membrane were smooth and continuous, and golgi bodies can also be observed in the micrograph. A big and round shape nucleus was observed with nucleolus and nuclear membrane at the level of 25 mg L −1 ALA alone (Figures 4(c) and 4(d)). A well-developed mitochondria and endoplasmic reticulum were found in

Discussion
Lead (Pb) is a highly phytotoxic heavy metal in the environment causing alteration in various physiological processes and ultrastructural changes in plants [7]. Generally, the sensitivity to heavy metal of a given plant depends on metal concentration, dose duration, plant variety and type, age of plant, and plant tissue being analyzed [25]. The present study was carried to analyze the beneficial role of 5-aminolevulinic   Table 1). The reduction in plant growth would be the adverse effect of Pb on physiological processes and disturbance in plant mineral nutritional status. Moreover, reduction in root growth might be due to Pbinduced inhibition of cell division in root cell [7]. On the other hand, plant growth was improved under the combined treatment of ALA and Pb stress (Table 1). This increase in growth with ALA might be due to the fact that ALA has a promotive role in regulating different metabolic processes, thereby improving growth and yield of most plants under abiotic stresses [26]. Recently, it has been recognized that ALA enhanced the plant growth by alleviating cadmium effects in B. napus [12]. The data showed that Pb toxicity significantly reduced the root morphology of plants ( Table 2). The possible reason behind is that Pb toxicity leads to the imbalance of water status and disturbed nutrient uptake and inhibits the cell division in the root tip cell [7]. Previously, it was observed that Pb toxicity demoted the root morphology in two ecotypes of Elsholtzia argyi [3]. Schwarz and Grosch [27] also conducted similar studies to discover the effects of Pb on the relationship between yield and root morphology. Meanwhile, when plant sustained foliar application of ALA, it significantly improved the root morphology of B. napus plants under the Pb stress ( Table 2). This improvement in root morphology under the application of ALA might be due to the fact that ALA activates the heme-based antioxidant systems (APX, POD, and CAT) to scavenge the reactive oxygen species like H 2 O 2 [18,28]. Moreover, this study showed that Pb stress alone reduced the chlorophyll contents (Table 3) and photosynthetic parameter as compared to control (Figure 1). This reduction in photosynthetic attributes might be due to the destruction of protein complex, chloroplast, and photosynthetic apparatus under metal stress [29], and at the same time heavy metal stress might inhibit the photosynthetic electron transport chain [30]. Moreover, decomposition of chlorophyll might be due to increase in chlorophyllase activity under heavy metal stress [31]. It has been well documented in the literature that heavy metals can affect stomatal conductance, gas exchange, and chlorophyll contents, thereby affecting the net photosynthetic rate [32]. At the same time, increase in chlorophyll and net photosynthesis rate with foliar application of ALA confirms that ALA performs as growth regulator and enhances the parameters involved in photosynthesis. The role of ALA in improving photosynthetic attributes can therefore be due to the significant improvement in Chl content [33] and subsequently increasing light harvesting capacities in the treated plants. Ali et al. [12] found that foliar application of ALA significantly elevated the photosynthetic parameters in B. napus plants under cadmium stress conditions.
The results showed that Pb contents were increased in different plant parts with increasing Pb concentration in the medium (Table 4). These results about more uptake of Pb contents in roots than shoots express the capability of plants to avoid metal-induced changes [34]. In this study, it was found that foliar application of ALA reduced the uptake of Pb in different parts of plants under Pb stress. It may be due to the application of ALA which strengthens the plant defense system by activating antioxidant machinery, thus prevents the uptake or movement of heavy metal in plant tissues [12,13]. So, it can be suggested that exogenous ALA could be applied for the purpose of phytoextraction.
Microscopic examination was performed as it helped to study the changes at cellular level [35]. In the present study, plant mesophyll cell and root tip cells were significantly damaged under the Pb toxicity alone conditions. It was advocated that chloroplasts are highly susceptible to oxidative stress [36]. In mesophyll cell, Pb-induced stress brought about the disappearance of starch granules inside the chloroplast and dissolved thylakoids membranes (Figures 3(a) and 3(b)). Under Pb stress, metal deposition was present in vacuoles and cell walls and all the organelles were totally damaged in root tip cells (Figures 5(a) and 5(b)). Previously, similar ultrastructural disorders in plant cells were noticed under heavy metal stress [37]. However, foliar application of ALA improved these structures under Pb toxicity in both mesophyll cell (Figures 3(c) and 3(d)) and root tip cell (Figures 5(c) and 5(d)). Improved cell structures with foliar application of ALA under Pb-treated plants could be the indication of less oxidative stress and might be due to that ALA helped in inducing the antioxidant system in the thylakoids and cell membranes. The higher activity of ascorbic peroxidases catalyzed the breakdown of H 2 O 2 in Halliwell-Asada pathway originally described in the chloroplast [38]. So, it can be suggested that activity of ascorbic peroxidases can potentially protect thylakoids from activated oxygen species under the Cd stress. Naeem et al. [39] and Ali et al. [13,18] also observed that ALA improved the cell structures in B. napus under the salinity and cadmium stress, respectively. Previously, Castelfranco and Jones [40] suggested that ALA showed alleviating role to trigger the heme-based molecules. It can be concluded that ALA may alleviate oxidative stress induced by Pb toxicity. From the present findings, it could be suggested that ALA ameliorated the Pb-induced inhibition of plant growth, photosynthesis, and ultrastructural changes in plant cells.

Conclusions
According to the present study, we observed that Pb reduced the plant growth and root morphology as well as damaged the cell ultrastructures. Meanwhile, exogenously applied ALA increased the plant growth and improved cell ultrastructures under the Pb toxicity. Overall, results from present research reveal that ALA has the ability to get rid of the adverse effects of Pb on B. napus by increasing plant growth and net photosynthesis rate and improving the ultrastructural changes induced by Pb stress. To examine the precise ameliorative role of ALA under Pb stress conditions a soil environment-based approach is needed. Values are the means of four replications ± SD. Variants are significant at ≤ 0.05.