Spirulina platensis improves growth, oil content, and antioxidant activitiy of rosemary plant under cadmium and lead stress

In the present study, a pot experiment was conducted to investigate the response of rosemary (Rosmarinus officinalis L.) plants to foliar application of Spirulina platensis at 0.0, 0.1, 0.2, and 0.4%; soil irrigation with heavy metals (Cd nitrate, Pb acetate, and Cd + Pb, each at 100 ppm), and Spirulina platensis at 0.1% + heavy metals. Spirulina platensis significantly improved growth parameters, oil yield/fed, photosynthetic pigments, and activity of superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT), and polyphenol oxidase (PPO) with a maximum promoting effect at 0.2% algal extract. On the other hand, heavy metal stress reduced growth criteria, photosynthetic pigments, and oil yield, while, significantly increased levels of antioxidant enzymes (SOD, CAT, GR) and corresponding non-enzymatic antioxidants (ascorbic acid, total antioxidant capacity, phenolics and flavonoids). Bioaccumulation factor (BF) and translocation factor (TF) indicated that Cd and Pb accumulated largely in the roots, with little transfer to the shoots. Nevertheless, compared with heavy metal treatments, S. platensis at 0.1% significantly increasing growth parameters, oil content, photosynthetic pigments, and the activity of non-enzymatic and enzymatic antioxidants, while, slightly reduced TF of Cd and Pb, alleviated membrane lipid peroxidation, and significantly lowered the content of malondialdehyde, hydrogen peroxide, and indole acetic acid oxidase (IAAO) activity in heavy metal (Cd, Pb, and Cd + Pb)-treated rosemary plants.

www.nature.com/scientificreports/ Atomic Emission Spectroscopy (Agilent Technologies 4210 MP-AES). According to the manufacturer's user manual, the instrument settings and operational instructions were adjusted. The soil adhering to rosemary roots, the shoots and roots samples were taken individually from various pots and dried at 110 °C for 24 h. Samples were crushed and sieved through a 2 mm sieve after cooling. The shoots and roots of rosmary plants (0.5 g) were digested in a 15-mL acid mixture of HNO 3 : HCl (1:1, v/v) and heated on a hot plate until the digest turned clear. The digest was cooled, filtered, and diluted with twice-de-ionized water to a volume of 25 mL. Pb and Cd concentrations were measured and expressed in mg kg −1 on a dry matter basis.
Moreover, the bioaccumulation factor (BF), which assesses the ability of rosemary plants to accumulate Pb and Cd with respect to its concentration in the substrate 17 , was calculated in the following way: BF = C rr /C s , where C rr and C s represent the concentrations of Cd and Pb in rosemary root and soil, respectively. In addition, the translocation factor (TF), which determines the relative translocation of Pb and Cd metals from root to shoot of the plant species 17 , was estimated as: TF = C rs /C rr , where C rs and C rr represent Cd and Pb concentrations in rosemary shoots and roots, respectively.
Oxidative stress markers. Lipid peroxidation (malondialdhyde content). As described by 18 , lipid peroxidation was measured in leaf tissue by the thiobarbituric acid (TBA) test, which determines malondialdhyde (MDA) as a product of lipid peroxidation. Leaf material (0.5 g) was homogenised in 10 ml of TBA reagent (18% Tri-chloroacetic acid (TCA) mixed with 0.45% TBA in a 1:2 ratio). After 15 min of incubation in a boiling water bath, the hot mixture was filtered through Whatman No. 42 filter paper, and the reaction was terminated by placing the reaction tubes in an ice bath. The samples were then centrifuged for 10 min at 6000 rpm, and the absorbance of the supernatant was measured at 532 nm. Non-specific absorbance at 600 nm was removed from the data at 532 nm. The concentration of MDA was measured in µmol g −1 d. wt. equivalent.
Membrane permeability. The permeability of cell membranes was measured using electrolyte leakage as described by 19 . Fresh leaves (2.5 g) were cut into 2-cm segments and placed in individual glass vials holding 25 mL de-ionized water after being washed with distilled water. After 30 min of soaking in water, the bathing solution's electrolytic conductivity (EC1) was measured with a conductivity metre (Model Ohm-419). The leaves were then brought to a boil, the bathing solution was cooled to room temperature, and the electrolytic conductivity was measured once more (EC2). The plasma membrane's relative permeability was determined as follows: Hydrogen peroxide content. The level of hydrogen peroxide (H 2 O 2 ) was measured according to 20 . In an ice bath 0.5 g leaf tissue was homogenised in 5 mL of 0.1 percent (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged for 30 min at 6000 rpm and 4 °C. Non-enzymatic antioxidants. Ascorbic acid. Estimation of ascorbic acid in rosemary was done using colorimetric assay kits acquired from Biodiagnostic Co., Cairo, Egypt, as described by 21 . One gram of fresh leaf tissue was extracted with 5 mL of TCA (10%). After centrifugation at 2000 rpm and 4 °C for 10 min, the supernatant obtained was used for estimation. The reaction mixture contained 200 μL of enzyme extract, 1.0 mL of buffer R1 (100 mM), and 2.0 mL 2,6-dichlorophenolindopheno R2 (DCPIP) at 1.0 mM. The assay mixture of the blank contained 200 μL of dist H 2 O instead of the enzyme extract. This mixture was well mixed and added to a clean quartz cuvette, and the absorbance of samples and blank were read in a spectrophotometer against dist water at 520 nm. The content of ascorbate in the samples was measured and expressed as (mg L −1 ).
Total antioxidant capacity. The total antioxidant capacity (TAC) of fresh rosmary leaf extract was measured according to 22 using colorimetric assay kits purchased from Biodiagnostic Co., Cairo, Egypt. In brief, 0.02 mL of extract solution (1 mg mL −1 ) was well mixed with 0.05 mL R1 (substrate, H 2 O 2 ) and incubated at 37 °C for 10 min, then added 0.5 mL working reagent (containing equal volumes of R2 (Chromogen) and R3 (Enzyme-Buffer)). The reaction mixture was mixed and incubated at 37 °C for 5 min. The assay of the blank contained 0.02 mL of dist. H 2 O instead of the enzyme extract solution. Then the absorbance of the blank and sample solution was read at 505 nm against dist. water. The antioxidant capacity of the extract was calculated and expressed as mM L −1 extract.
Total phenolic content. Air-dried powdered rosemary leaves (0.5 g) were extracted by stirring 25 mL of ethanol 70% at room temperature until the extraction solvent became colorless. As described by 23 , total phenolic content (TPC) was evaluated using Folin-Ciocalteu reagent and gallic acid as a standard. 0.5 mL filtered extracts were combined with 2.5 mL of Folin-Ciocalteu's reagent (diluted with ethanol 1:10) and 2 mL of Na 2 CO 3 (7.5%) and thoroughly mixed. After 15 min of incubation at room temperature, the absorbance of the resulting blue-colored solution was measured against a blank reagent at 765 nm using a Jenway 6405 UV-Vis spectrophotometer. The total phenolic content of the extract was measured in mg of gallic acid equivalents (GAE) per g of extract (mg GAE g −1 d. wt.).
Relative permeability(%) = (EC1/EC2) × 100 www.nature.com/scientificreports/ Total flavonoids. The total flavonoid concentration (TFC) of the rosemary extract was determined using an aluminium chloride colorimetric assay 24 . 0.5 mL of sample solutions (0.5 g in 25 mL of ethanol 70%) were combined with 2 mL of dis. H 2 O, followed by 0.15 mL of 5% NaNO 2 solution. After a 6-min incubation period, 0.15 mL of 10% AlCl 3 solution was added and left to stand for 6 min before adding 2 mL of 4% NaOH. The mixture was diluted to 5 mL with methanol and thoroughly mixed. The absorbance was measured spectrophotometerically against a blank at 510 nm after a 15-min incubation period. The total flavonoid concentration was measured in mg of quercetin equivalents (QE) per gram of dry weight (mg QE g −1 d. wt.). Total flavonoids were calculated using a quercetin standard curve.
Antioxidants enzymes. Superoxide dismutase (EC 1.15.1.1). The superoxide dismutase (SOD) enzyme was assayed as U g −1 of fresh leaf tissue as described by 25 , using a superoxide dismutase Biodiagnostic ready kit purchased from Biodiagnostic Co., Cairo, Egypt. For extraction, 0.25 g of fresh leaf tissue was homogenised in 5 mL of cold 100 mM potassium buffer (pH 7.0), then centrifuged for 15 min at 4000 rpm and 4 °C. In a glass tube, add 0.5 mL of cold absolute ethanol/chloroform (60/40, v/v) to 1.0 mL of supernatant. Mix thoroughly for at least 30 s, then centrifuge for 10 min at 4000 rpm and 4 °C. The supernatant obtained was used for an enzyme assay. Before use, 2 mL of 50 mM potassium phosphate buffer (pH 8.5), 1 mM nitrobluetetazolium, and 1 mM NADH were mixed in a 10:1:1 ratio. Then, 0.2 mL of enzyme extract and 0.2 mL of distilled water were added to the assaying mixture. The assay mixture of the control contained the previous mixture without enzyme extract. This mixture was combined and placed in a clean quartz cuvette, and the reaction was started by adding 0.2 mL of 0.1 mM phenazine methosulphate (PMS) (diluted 1000 times before use) into the cuvette and mixing it immediately. The cuvette was inserted in the spectrophotometer for 5 min at 25 °C to record the increase in absorbance at 560 nm for control (∆ control) and the leaf tissue sample (∆ sample). SOD activity is expressed as (U g −1 ).
Glutathione reductase (EC. 1.6.4.2). A glutathione reductase (GR) enzyme was measured in leaf tissue as described by 26 using a glutathione reductase Biodiagnostic ready kit for research reagents purchased from Biodiagnostic Co., Cairo, Egypt. 0.25 g of fresh leaf tissue was homogenised in 5 mL of cold potassium buffer (pH 7.5) containing 1 mM EDTA and centrifuged for 20 min at 4000 rpm and 4 °C. For the enzyme assay, the supernatant was employed, 2 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA, 0.2 mL of oxidized glutathione (GSSG), and 0.2 ml of 2 mM nicotinamide adenine dinucleotide phosphate (NADPH) were added to the assaying mixture. The reaction is started by adding 0.1 mL of enzyme extract to the assaying liquid in a clean cuvette. The decrease in absorbance min −1 was recorded for 5 min at 340 nm. The activity of GR is expressed as unit L −1 .
Catalase (EC 1.11.1.6). According to 27 , fresh rosemary leaves were used for making enzyme extracts. In brief, 0.25 g of fresh leaf was homogenised in 5.0 mL of cold phosphate buffer (Na/K phosphate 0.1 M, pH 6.8), centrifuged for 10 min at 6000 rpm and 4 °C, and the supernatants were completed to a known volume and used for assaying the activity of catalase (CAT), polyphenol oxidase (PPO), and indole acetic acid (IAA) oxidase. Catalase was measured using a modified version of 28 29 . The assay mixture contained 3 mL of buffered catechol solution (0.01 M catechol, freshly prepared in 0.1 M phosphate buffer, PH 6.0) and 1 mL of crude enzyme extract. Immediately, the colour intensity was read at 495 nm. Follow the change in absorbance for 30 s intervals up to 5 min. The enzyme activity was expressed as optical density g −1 d. wt. equiv. h −1 .
Indole acetic acid oxidase (EC 1.2.3.7). The activity of IAA oxidase was assessed using a modified approach of 30 . The reaction mixture (3 mL) contained 0.1 mL of 1 mM 2,6 dichlorophenol, 0.1 mL of 1 mM MnCl 2 , 0.4 mL of 0.1 mM potassium phosphate buffer pH 6, and 0.2 mL of 10 mM IAA. The reaction was initiated by adding 0.2 mL of enzyme extract, followed by 1 mL of p-dimethyl aminocinnamaldhyde (0.5% in 1 N HCl) as a control. The remaining reaction mixture was incubated in the dark. 1 ml of p-dimethyl aminocinnamaldhyde was added after 2 h and left in the dark for 1 h to develop the colour. The developed colour was spectrophotometrically monitored at 562 nm. The activity of an enzyme was expressed as optical density g −1 d. wt. equiv. h −1 .

Statistical analysis.
For growth criteria, the data was expressed as the mean of six replicates, and for chemical analysis, the data was expressed as the mean of three replicates. Statistical analysis was carried out using oneway ANOVA followed by Duncan's Multiple Comparison Test in IBM Statistical Product and Service Solutions, SPSS Statistics for Windows, Version 21 at P 0.05, which was denoted as statistically significant for the means compared, using the least significant difference (LSD at 5 percent level).

Results
Growth parameters. Resultsshowed that foliar application of Spirulina platensis at 0.1, 0.2, and 0.4% on rosemary plants enhanced all growth criteria (i.e., plant height, root length, branch number per plant, fresh and dry leaf, stem, and root weights (g plant −1 ) compared to the untreated control plants. The increase in growth characteristics was significantly greatest with 0.2% of S. platensis during the two cuttings ( Fig. 1).
On the other hand, Cd, Pb, and Cd + Pb stress significantly lowered the growth parameters of rosemary plants during the two cuttings compared to their relative controls and other treatments (Fig. 1). Cadmium stress significantly reduced the fresh (19.27, 43.45 g plant −1 ) and dry (9.32, 15.98 g plant −1 ) weights of leaves at the first and second cuttings, respectively, when compared to their related controls (20.26, 65.06 g plant −1 ) for f.wt and (10.81, 20.82 g plant −1 ) for d. wt. www.nature.com/scientificreports/ Morover, S. platensis at 0.1% combined with heavy metals (Cd, Pb, Cd + Pb), reduced the harmful effect of heavy metals and improved growth parameters under stress conditions as compared to heavy metal treatments during the two cuttings. The most significant increases in growth metrics were seen in S. platensis at 0.1% + Pb, followed by S. platensis + Cd + Pb.
Oil content. Figure 2 shows that the treatment of S. platensis alone enhanced essential oil (EO) percent and yield per feddan (L fed −1 ) on a dry weight equivalent basis of rosemary plants at both the 1st and 2nd cuts. When the two cuts were pooled together, the maximum increase in EO percent (40.58 and 34.27%) and oil yield (101. 46 and 69.76% more than their corresponding controls) were obtained at 0.2 and 0.4% S. platensis, respectively. However, Cd, followed by Cd + Pb, then Pb stress, recorded a decrease of 27.57, 19.40 and 3.61% in oil percentage, respectively, during the two cuts, as compared to the control. A more or less comparable trend was found with regard to the oil yield per feddan.
Considering the combined effect of S. platensis at 0.1% with heavy metals, application of S. platensis + Pb, followed by S. platensis + Cd + Pb, then S. platensis + Cd increased oil percent of the two cuts (increased 22.61, 12.57, and 7.74%) and oil yield (increased 35.61, 27.32, and 20.00% more than their controls), respectively. In most cases, as compared to untreated controls, the increase in oil content and yield were frequently significant (Fig. 2). Figure 3 demonstrates that when S. platensis was applied at any concentration or in combination with heavy metals, total photosynthetic pigments (TPP) in rosemary leaves were enhanced more than controls at the first cut. S. platensis, at 0.2 percent, was the most effective, followed by 0.4 percent, although heavy metals alone drastically reduced photosynthetic pigments.

Photosynthetic pigments.
Foliar application of S. platensis at 0.1, 0.2, and 0.4% significantly enhanced Chl a and b, carotenoids, and, as a result, TPP in the leaves of rosemary plants. The highest values of carotenoids and TPP (0.76 and Figure 2. Essential oil percentage (mL 100 g −1 ) and oil yield (L fed −1 ) on a dry weight basis of rosemary (R. officinalis) plants as influenced by foliar spray with Spirulina platensis at 0.0, 0.1, 0.2, and 0.4%, irrigation with heavy metal (Cd as Cd nitrate; Pb as lead acetate; Cd + Pb each at 100 ppm), either alone or in combination with 0.1% S. platensis, at the 1st and the 2nd cuts. Statistical analysis was carried out using Duncan test. Vertical bars represent LSD at 0.05 level and different letters show significant variation at 0.05 P. Bioaccumulation and translocation factors. The data of the bioaccumulation factor (BF) indicated that rosemary plants had a BF for Cd greater than one and higher than Pb in rosemary treated with heavy metals (Cd, and Cd + Pb) or S. platensis 1% + heavy metals-treated soils (Fig. 4). The highest BF (1.753) for Cd and Pb (1.111) were attained in Cd + Pb and Pd-treated soils, respectively.
As shown in Fig. 4, the translocation factor (TF) of Cd and Pb varied among all the treatments. The translocation factor of Cd was higher in rosemary when treated with Cd, Cd + Pb, singly or combined with S. platensis at 0.1% concentration. On the other hand, Pb and Cd + Pb, singly or combined with S. platensis 1% foliar application, increased the TF of Pb in rosemary, with the highest TF (0.393) for Cd and Pb (0.158) were recorded at Cd and Cd + Pb, irrigated soils, respectively.  www.nature.com/scientificreports/ Generally, the TFs were less than 1 for Cd and Pb treated with different treatments, suggesting a slight ability of rosemary to translocate Cd and Pb. Also, the TFs of Cd and Pb were reduced with the application of S. platensis 0.1% + heavy metals compared with heavy metals (pb, and Cd + Pb), singly.
Oxidative stress markers. The data indicates that foliar application of S. platensis, singly or combined with heavy metal stress reduced levels of malondialdhyde (MDA) as a product of lipid peroxidation damage by free radicals, electrolyte leakage (EL) as a measure of cell membrane permeability, and hydrogen peroxide content, one of the most damaging forms of reactive oxygen species in the leaves of rosemary plants at first cut, relative to heavy metal treatments and controls (Fig. 5). The lowest values of MDA (95.0 µmol g −1 d. wt. equiv), relative permeability (14.86%), and H 2 O 2 levels (0.049 µmol g −1 d. wt. equiv.) were recorded at 0.2% S. platensis compared with (117.0 µmol g −1 d. wt. equiv., 19.58%, and 0.069 µmol g −1 d. wt. equiv.) for their respective controls (Fig. 5).
Non-enzymatic antioxidants. The results presented in Fig. 6  Moreover, heavy metal (Cd, Pb, and Cd + Pb) stress increased the above measured non-enzymatic antioxidants as compared with untreated control plants (Fig. 6).
Furthermore, the combination of S. platensis with heavy metals enhanced the content of total antioxidant capacity, ascorbic acid, total phenols, and flavonoids in the leaves of rosmary plants relative to their corresponding controls and heavy metals applied singly. The most promising increase in non-enzymatic antioxidants was obtained by foliar application of S. platensis at 0.1% + Cd, followed by S. platensis at 0.1% + Cd + Pb. Figure 7 shows that foliar application of S. platensis at 0.1, 0.2, and 0.4% significantly increased antioxidant (superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT)) activity as well as oxidative enzyme (polyphenol oxidase (PPO)) in rosemary leaves compared to controls at first cut. This stimulating effect was maximal for four measured enzymes at 0.2% S. platensis and thereafter declined; this is in contrast to the activity level of the oxidative enzyme indoleacetic acid oxidase (IAAO), which was reduced in response to the application of S. platensis, notably at 0.2 percent.

Antioxidants and oxidative enzymes.
On the other hand, heavy metals significantly improved the activity of antioxidants (SD, GR, and CAT) and oxidative enzymes (PPO and IAAO) in rosemary leaves relative to untreated controls (Fig. 7).
Under heavy metal (Cd, Pb, and Cd + Pb) stress, an enhancing effect was observed in the activity of antioxidants (GR, SOD, and CAT) and oxidative enzymes (PPO) by foliar spray with S. platensis at 0.1%, compared with their corresponding controls, except for IAAO, where the activity decreased.   34 .This also agree with 35 who reported that Low uptake of N, P, and K in bitter gourd under Cd toxicity might be a major factor for reduction in yield, fruit length, and fresh weight. In this respect, cadmium promoted a significant reduction in biomass production, height, root parameters, and inhibited the production and expansion of leaves in potato (solanum tuberosum) plants at 50 µM 11 , and inhibited growth of wheat plants by accumulation of Cd in plant tissue 36 . Pb significantly reduced shoot height, root length, and dry mass in Leucaena leucocephala seedlings at 300-700 µM Pb (NO 3 ) 2 37 . Intuitively, application of S. platensis under heavy metals stress revealed a much greater ability to promote the growth of rosemary plants compared with untreated plants. In this connection, the association of bacteria or mycorrhizae with plants showed a reduced deleterious effect of Cd, indicating other key biochemical and molecular mechanisms involved in Cd-stress tolerance 13 . The reduction of the negative effect of abiotic stresses observed in plants because of the interaction with cyanobacteria has been verified both directly, through its action in the soil, and indirectly, because of the activation of specific responses in plants 38 . Chen and Pan 39 stated that living Spirulina cells had a high tolerance to lead and are good adsorbing agents. This may play arole in decreasing plant absorbance to Pb and Cd from soil. Indeed, besides its www.nature.com/scientificreports/ naturally fertilizing potential, Spirulina is able to release various bioactive molecules (polysaccharides, amino acids, phytohormones, etc.) that enhances plant growth and increase tolerance to biotic and abiotic stresses 40 . The essential oil yield was generally higher during the second cut, compared to the first one, and the application of S. platensis, particularly at 0.2%, significantly increased the EO content and yield (L fed −1 ) of rosemary during the two applied cuttings compared to the corresponding control plants under normal conditions and in plants affected by heavy metals stress. This was accomplished by enhancing photosynthetic activity, fresh matter production plant −1 , and EO biosynthesis. This was also proved by Gharib et al. 41 on rosemary plants by foliar application of Sargassum latifolium extract at 0.2, and 0.4 percent which improved essential oil percentage and oil output per plant on a dry matter basis.The results obtained indicated that Cd and Cd + Pb stress, significantly reduced oil content on a dry weight basis for the two cuts due to reduction in photosynthesis, fresh aerial parts plant −1 , essential oil biosynthesis, as the synthesis of essential oil in epidermal glands depends on providing photosynthetic carbon. In accordance with the present study, high toxic doses of Cd and Pb, disturb carbon translocations and consequently reduce essential oil percent of peppermint 42 and significantly reduced oil yield of vetiver (Vetiveria zizanoides) grass in metal-contaminated soils 43 . In our investigation, Pb at 100 ppm had no effect on the essential oil output fed −1 in rosemary. Similarly, peppermint oil yield was not affected by the application of Pb (50-500 mg L −1 ) and Cd (2-10 mg −1 ) 42 . However, 44 reported an enhancement in the essential oil percentage of certain aromatic plants under heavy metal stress.
Photosynthetic pigments in rosemary leaves were enhanced by the application of S. platensis, singly and under heavy metal stress (Fig. 3). Spirulina platensis might improve cell metabolic rate, delay senescence and Chl destruction and/or increase Chl biosynthesis by increasing N and Mg intake (structural component of Chl), resulting in increased Chl accumulation and a faster rate of photosynthesis. In this connection, Spirulina improved absorption of NPK and resulted in a significant increase in chlorophyll content in Portulaca grandiflora 4 . Also, the highest levels of chlorophylls and carotenoids were obtained in onion plants treated with S. platensis + cow dung, Chlorella vulgaris + cow dung, followed by C. vulgaris, S. platensis, cow dung, and then control plants, respectively 45 . In rosemary, treatment with heavy metals (Cd, Pb, and Cd + Pb) makedly decreased Chl a and b, carotenoids, and TPP in the leaves, which is a visual indicator of impaired growth more than control plants (Fig. 3). In this connection, Cd-negatively affected plant photosynthetic enzyme activity, pigment content, leaf stomatal conductance, induced chlorophyll degradation, and disorder in grana and thylakoids arrangements, resulting in decreased photosynthesis rate, and chlorophyll content in potato plants 11 . According to 37 chlorophyll decreases in Leucaena leucocephala seedlings have been attributed to Pb (NO 3 ) 2 at 300-700 µM induced chlorophyll synthesis inhibition. In another research, stress of cadmium (10 and 40 mg kg −1 ) and lead (60 and 180 mg kg −1 ) significantly decreased chl a, chl b, chl a + b and carotenoid in Matricaria chamomilla 46 .
Our results indicate that S. platensis at 0.1% might concomitantly alleviate the adverse effects of heavy metals, promoting plant biomass and enhancing the content of photosynthetic pigments in leaves. This is due to the fact that S. platensis can provide rosemary plants with some nutrients, vitamins, and hormones and also reduce Cd and Pb absorption from the soil, thereby improving plant growth and photosynthesis when compared to plants exposed to Cd and/or Pb stress. In this regard, providing soil amendments can provide plants with nutrients while minimising soil absorption of Cd and Pb, thereby promoting plant growth and improving photosynthesis 47 . Also, foliar application of S. platensis (100 mg L −1 ) extract significantly improved pigment content, photosynthetic activity, and transpiration rate under salinity stress in Vicia faba plants 48 .
In this study, Cd and Pb concentrations increased in both the shoots and roots of R. officinalis under Cd and/ or Pb stress singly rather than when combined with S. platensis. The accumulations of Cd and Pb in the roots of rosemary plants were larger than those in the shoots. In this respect, more accumulation of Cd and Pb was observed in roots than in shoots of Salix mucronata 49 . However, the toxic effect of Cd was reduced by using a bacterium-containing biofertilizer in maize and sunflower plants 50 . The bioaccumulation factor (BF) generally indicates the transport of heavy metals from soil to the plant root, indicating the effectiveness of bio-available metal intake from the environment. According to our current investigation, the BF revealed the transfer and availability of Cd and Pb in rosemary, for soil treated with heavy metals (Cd, Pb, and Cd + Pb), singly and when combined with foliar spray with S. platensis. Rosemary plants can accumulate a higher Cd concentration in the roots than in the leaves, and the mobility of Cd in plants is higher than Pb, since it has an electronic configuration and a zinc-like valence state. The BF showed low transfer of Cd (1.316) and Pb (0.697) in rosemary treated with S. platensis + Cd + Pb compared with their respective values (1.753 and 0.944) for Cd + Pb treatment. Similar, low BF for Cd and Pb were obtained in wheat treated with four soil amendments (sepiolite, single superphosphate, triple superphosphate, and calcium magnesium phosphate) compared to the control. Also, the application of soil amendments is an effective phytostabilization for remediating Cd-and Pb-contaminated soil in ramie plants 51 . On the other hand, the translocation factor (TF) of Cd was greater than that of Pb in rosemary. due to its similar properties to Zn, but, Pb translocation was low because Pb was early recognised as a toxic molecule by plant roots and sequestered in the vacuole or in the cell walls of the root, resulting in low translocation in the shoots. In our research, the translocation factor (TF) was less than one for Cd and Pb in all treatments, indicating the potential of the rosemary plant for their phytostabilization in the root. Similar findings were reported by 45 on Mentha species and 46 on Matricaria chamomilla L.
Decrease in MDA production and relative permeability by application of S. platensis under normal and heavy metal stress suggests greater protection of rosemary plants from oxidative damage as compared to Cd and Pb treatments. Reduced lipid peroxidation (due to the inhibitory effect of S. platensis) favoured vegetative growth and the accumulation of essential oil (Figs. 1, 2)  www.nature.com/scientificreports/ suggests that the efficiency of redox processes has increased in the presence of S. platensis. In this regard, 50% hydrogen-rich water (HRW) + Cd(NO 3 ) 2 at1 μM significantly reduced the content of malondialdehyde, hydrogen peroxide, superoxide radical (O 2 − ), relative electrical conductivity, lipoxygenase activity, plasma membrane permeability, and protect the membrane lipid stability in cucumber (Cucumis sativus L.) explants compared to Cd treatment 52 . Moreover, maximum production of H 2 O 2 , induced damage to MDA content and membrane integrity, was recorded under single Cd followed by Pb stress, which showed a 55.11 and 39.99% increase in electrolyte leakage, respectively, relative to control. Cd and/or Pb, treatments, cause a rise in malondialdehyde concentration, which indicates the production of free oxygen radicals and may result in changes in membrane fluidity, alterations in phospholipids, and an increase in electrolyte leakage (Fig. 5). In this connection, Cadmium stress induces overproduction of ROS and subsequent oxidative stress, with increase in the content of H 2 O 2 and TBARS in cucumber explants 53 and rice seedlings under Cd, lead (Pb) or Cd + Pb stress 54 . ROS can speed up lipid peroxidation, affecting cell membrane fluidity and permeability due to changes in membrane lipid composition 55 . On the other hand, any rise in H 2 O 2 has severe effects on the damaged cell. In our investigation, Cd stress resulted in a 33.33% increase in H 2 O 2 levels, while Pb stress resulted in a 27.54% increase over the control value (Fig. 5). A larger accumulation of H 2 O 2 under stress indicates that H 2 O 2 metabolising enzymes are not working properly. Cd poisoning cause electrolyte leakage, overproduction of hydrogen peroxide, and malondialdehyde content in wheat (Triticum aestivum L.) plants 56 .
Foliar spray with S. platensis, especially at 0.2%, significantly increased the content of non-enzymatic antioxidants (total antioxidant capacity, ascorbic acid, total phenols, and flavonoids), relative to untreated control rosemary plants. Similarly, a significant increase in total phenols and enhanced growth were obtained by S. platensis in onion plants 45 . Furthermore, heavy metal stress increased the measured non-enzymatic antioxidants as compared with untreated control rosemary plants (Fig. 6). During Cd stress, the rise in the production of nonenzymatic antioxidants, such as ascorbate and glutathione (GSH), as well as vitamins, flavonoids, and carotenoids, may have a valuable role in detoxify ROS produced under stress conditions 57 . According to 57 , total phenol level, ascorbic acid (AsA), α-tocopherol and carotenoids contents were all significantly higher in cadmium-exposed plant species. The rise in phenolics attributed to an increase in the activity of enzymes involved in phenolic metabolism, implying de novo phenolic synthesis in the presence of metal stress. However, 54 found that Cd, Pb, and Cd + Pb treatments enhanced dehydroascorbate (DHA) level while decreasing AsA level and the AsA/DHA ratio in rice seedlings. Our results indicated that the most promising increase in non-enzymatic antioxidants (total antioxidant capacity, ascorbic acid, total phenols and flavonoids) was obtained by foliar application of S. platensis at 0.1% + Cd, followed by S. platensis + Cd + Pb in the leaves of rosmary plants, relative to their corresponding controls and single heavy metal stress. In this respect, treatments with Spirulina spp. extract increased the antioxidant capacity in Vicia faba under salinity stress 46 . Also, hydrogen-rich water (50%) alleviated oxidative stress caused by Cd (NO 3 ) 2 in cucumber (Cucumis sativus L.) explants and induced increases in a nonenzymatic protection system, such as AsA/DHA and GSH/GSSG ratio 53 .
Spirulina platensis foliar treatment, at any dose significantly raised the activity levels of superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT) and PPO in the leaves of rosemary plants, while, the opposite result was achieved with IAAO activity levels that dropped in response to S. platensis treatment, particularly at 0.2 percent compared with controls. In this connection, application of Ascophyllum nodosum extract elevated antioxidant levels in spinach leaves 58 . Under single Cd, Pb, and Cd + Pb stress, the activities of SOD, GR, CAT, and PPO were significantly enhanced in rosemary, which indicates the scavenging mechanism of the plants against the ROS produced in response to the metal stress. According to 59 , Cd induces several ROS, including the superoxide radical (O2•−), hydrogen peroxide, and hydroxyl radical (OH•), systems capable of preventing uncontrolled oxidation are induced or stimulated, including antioxidant enzymes such as SOD, GR, CAT, glutathione peroxidase (GPX), ascorbate peroxidase (APX), and other peroxidases, which detoxify H 2 O 2 60 . Superoxide dismutase is the principal scavenger in active oxygen species detoxification in plants, converting superoxide to hydrogen peroxide and oxygen, protecting cells from superoxide-induced oxidative stress. In addition, CAT detoxifies H 2 O 2 into H 2 O and O 2 . High cadmium stress (100 μmol L −1 ) increased GR 337.7%, SOD 688.6%, POD 72.2%, CAT 229.0%, and APX 226.5% activity in Arachis hypogaea L. leaves 59 , significantly increased activity of POD, PPO, and CAT in pea seedlings at 25-50 µM cadmium acetate concentrations 57 and increased the activities of enzymatic antioxidant defense system (ADS) to some extent, while non-enzymatic ADS dramaticly decreased in Capsicum annuum under Cd and/or Pb stress 60 . However, the rise in PPO activity was also observed in pea seedlings under Cd-stress, and in both Ceratophyllum demersum L. and Hydrilla verticillata L. plants under Pb (5-80 µM) stress 56 . Our results indicated that under heavy metal (Cd, Pb, and Cd + Pb) stress, S. platensis treatment at 0.1% increased SOD, CAT, GR, and PPO activity, while significantly decreasing IAAO activity, as compared to control rosemary plants, and this may be related to differences in antioxidant enzyme expression to get rid of reactive oxygen species and increase plant tolerance in response to heavy metal stress. Avoiding oxidative stress and reestablishing redox homeostasis are critical in alleviating metal stress 54 . Under salinity stress, S. platensis decreased the large levels of SOD, POD, CAT, and GPX in Vicia faba as compared to salt-stressed plants 48 . Furthermore, under Cd stress, hydrogen-rich water treatment significantly reduced IAAO activity, ascorbic acid content, and reduced glutathione (GSH) in cucumber explants while significantly increasing the activity and related gene expression of ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and GR in comparison to Cd treatment. It also reduced oxidative damage by increasing the activities of PPO and POD 55 . Improvement in plant antioxidant capacity by application of Spirulina algae is agood indication of increase in rosemary plant tolerance toward heavy metal stress and overcome the damage may be caused by reactive oxygen species produced under stress.

Conclusion
Environmental pollution by heavy metals increases by time due to development in industrial and agricultural aspects. Spirulina platensis (biostimulant) promoted rosemary (R. officinalis L) growth, physiological performance in terms of photosynthetic production, essential oil content, and was efficient in mitigating Pb and/or Cd toxicity. Spirulina platensis alleviated membrane lipid peroxidation, inhibited the production and accumulation of ROS through modulating the antioxidant defence system and promoted the reduction of Cd and Pb absorption and accumulation in rosemary. The TFs were less than 1 for Cd and Pb in rosemary irrigated with heavy metals (Cd, Pb, and Cd + Pb), and sligtly reduced with the application of S. platensis at 0.1%, which indicates low translocation. Therefore, this study indicates that foliar spray with S. platensis may be a viable novel strategy for agricultural improvement of medicinal and aromatic plants under normal and heavy metal (Cd, Pb, or Cd + Pb) stress.

Data availability
All data generated or analyzed during this study are included in this article. In addition to provided supplementary materials.