Melatonin mediates gamma-aminobutyric acid induced mitigation of cadmium toxicity on growth, PSII photochemistry, and oxidative stress in Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120

Melatonin mediates gamma-aminobutyric acid induced mitigation of cadmium toxicity on growth, PSII photochemistry, and oxidative stress in Nostoc muscorum ATCC 27893 and Anabaena sp. 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PCC 7120 Sakshi Pandey, Varunendra Kumar Singh, Shobhit Raj Vimal, Sheo Mohan Prasad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7857714/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cyanobacteria, being nitrogen fixers most often beneficially associated with paddy crops and support the productivity. However, the growth of these symbionts is relatively affected by abiotic stresses. In the present study, the impact of Cd toxicity was explored in cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120, in the presence of signalling molecules melatonin (MT; 90 nM) and gamma-aminobutyric acid (GABA; 40 nM). Cadmium at 5 µM disturbed light-harvesting pigments and photosynthesis (whole cell oxygen evolution and PS II photochemistry), and finally diminished the growth of cyanobacteria. Excessive intracellular accumulation of Cd raised the level of oxidative biomarkers i.e., superoxide radical, hydrogen peroxide, and malondialdehyde equivalents content despite of accelerated activity of enzymatic antioxidants i.e., superoxide dismutase, peroxidase, catalase, and glutathione- S -transferase. Furthermore, exogenous application of MT and GABA individually supported the growth by improving light-harvesting pigments and photosynthetic activity, and it was more pronounced with GABA. This could occur due to considerable decline in Cd accumulation as a result, the declining trend in the oxidative stress biomarkers was taken place by further augmentation of antioxidant activity. The endogenous MT inhibitor CPA (90 µM) suggested the interactive role of MT and GABA in Cd toxicity alleviation. Hence, the study concludes that GABA, being the prime player in Cd toxicity alleviation, appears to act downstream of MT. Further, more insight is needed by involving molecular techniques to enrich the cyanobacteria with these signalling molecules to use them as a biofertilizer for sustainable agriculture, particularly paddy, even under prevailing stress conditions. Abiotic stress cyanobacteria heavy metal stress mitigation signaling molecules Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The current decade has seen an exponential rise in several environmental issues because of industrialization, primarily due to the quick human population growth and rising demand for numerous domestic materials that have increased pollutants in the environment (Pimentel et al. 2025 ). Heavy metal (HM) contamination in the environment has become an inevitable fact. They are discharged into the environment by industries and other anthropogenic activities. Cadmium (Cd) causes damage to living beings at more than 0.2 ppm due to its high toxicity and a significant degree of bioaccumulation (WHO 1996; Biswal et al. 2025 ). Cd is mainly used in Cd-Ni batteries, phosphate fertilizers, paints, dyes, cement, and other products, and frequently released into the environment. Besides strict rules in developing countries, toxic metals-containing effluents are being released directly into the rivers without proper treatment (Singh and Prasad 2024 ). Furthermore, due to the canal irrigation system, the polluted water from rivers reaches far away and heavily contaminates soils, including paddy fields. Rice is the most significant cereal among the worldwide crops, and serves as the primary food source for millions of households (Nawaz et al. 2024 ). Cd is structurally similar to calcium therefore, it uses calcium channel to pass through a cell membrane and get accumulated in the cytosol. Cadmium disturbs the flow of electron transport related to photosynthesis and respiration, inhibits the activities of enzymes and the functioning of nucleic acids, hence declines the growth of cyanobacteria and plants (Ahad and Syiem 2019 ; Zulfiqar et al. 2022 ). Furthermore, overproduction/ accumulation of ROS eventually damages the antioxidant system and membrane integrity (Ahad and Syiem 2019 ). Cd contamination in the paddy field directly reduces rice yield by affecting the beneficial microflora, i.e., cyanobacteria, which fix atmospheric nitrogen into ammonia (Verma and Prasad 2021a ). Cyanobacteria are the first species on Earth to evolve free oxygen and are important microorganisms for the development of sustainable agriculture (Nawaz et al. 2024 ). They are essential components of the ecosystem and substantial contributors to the global nitrogen cycle due to their special ability to fix nitrogen, which makes them a natural biofertilizer (Chittora et al. 2020 ; Singh and Prasad 2024 ). Most efficient nitrogen-fixing cyanobacteria, such as Aulosira fertilisima, Anabaena sp., Tolypothrix sp., Calothrix sp., Nostoc sp., Nostoc linkia , and Scytonema sp., commonly occur in paddy fields and support the growth and development (Singh et al. 2016 ). It is noticed that cyanobacteria can fix upto 20–25 kg/ha of atmospheric nitrogen, and some species of Anabaena may fix 60 kg of nitrogen per hectare in each season and improve soil quality with organic matter as well (Chittora et al. 2020 ). Since agriculture is crucial for both economic growth and food security, it is supposed to increase food production up to 50% by 2050 to feed the global population (FAO 2022 ). In recent years, several approaches are being applied to maintain the crop productivity, and one such method may be the application of signaling molecules (Chittora et al. 2020 ). Cyanobacteria have been shown to synthesize several bioactive compounds, including GABA, a non-protein four carbon containing amino acid (Mahawar et al. 2022 ). Pandey et al. ( 2022 ) have noticed that GABA alleviated UV-B-induced toxicity in N. muscorum and Anabaena sp. by involving nitric oxide. Under stress, GABA potentially regulates carbon: nitrogen metabolism in bacteria, cyanobacteria, and in eukaryotic organisms, including plants (Mahawar et al. 2022 ). Likewise, another signaling molecule MT, directly scavenges oxidative radicals and plays a significant role in mitigating the toxicity under stress in some microbes (Yuan et al. 2022 ) and in plants (Jahan et al. 2023 ). The presence of MT is reported in all living beings, nonetheless, its function varies in bacteria, cyanobacteria, fungi, plants, and animals (Zhao et al. 2019 ). It has been reported that MT treatment induces endogenous MT synthesis and regulates the GABA shunt pathway in tomato (Sharafi et al. 2019 ) and in peach (Wu et al. 2023 ) under cold stress. However, very little information has been shown to how these two signalling molecules act in collaboration in abiotic stress alleviation. As far as we know that in cyanobacteria, the alleviatory role of GABA and MT works, either separately or in interaction against HM toxicity, particularly Cd, remains elusive. Therefore, the present study is aimed to explore the ameliorating role of GABA and MT in conjunction on various physiological and biochemical attributes, such as growth, photosynthetic pigments contents, PS II photochemistry, oxidative stress, and the antioxidant system of two cyanobacteria N. muscorum and Anabaena sp. Further, we have also attempted to understand whether endogenous MT regulates GABA-induced allevitory effect against Cd toxicity in these microbes. Materials and Methods Experimental organisms and culture conditions The cultures of filamentous, heterocystous, and homogenous cyanobacteria, Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 were maintained in BG-11 medium (25±2°C) at pH 7.5 under photosynthetic active irradiance of 75 µmol photons m −2 s −1 (PAR) under 14:10 h (light–dark) conditions. These cyanobacteria were selected for the present study, and for each experiment, exponentially growing cells were harvested and used in each experiment. Purity of cultures was often inspected through a light microscope. Experimental design The concentration of cadmium (Cd), i.e., 5 µM, which corresponds to LC 22 for N. muscorum and LC 25 for Anabaena sp., was selected for the current study, after screening experiments with various doses (1, 2, 3, 4, 5, 6, 7, and 8 µM) of Cd. Likewise, after screening experiments 90 nM MT and 40 nM GABA were chosen, at which maximum alleviation against Cd toxicity on growth was recorded. Further, to understand the signaling relationship between the MT and GABA, 90 µM CPA (4-Chloro DL-phenylalanine) was also selected for the present study. For the experimental setup, the early exponentially growing healthy cultures of both the selected cyanobacteria were harvested (4,000 ×g for 10 min) separately, and pellets were rinsed thrice with sterile DDW and finally resuspended in fresh BG-11 medium and used as inoculum. In each experiment, 100 mL of cultures (A 750 nm, 0.1) were poured in conical flask having different chemicals as required and placed under defined experimental conditions. The experimental setups were consisted of: control, Cd, Cd+MT, Cd+GABA, and Cd+GABA+CPA. All the experimental setups were carried out under aseptic conditions. After 72 h of experiment, the cells from each setup were harvested, and various physiological and biochemical parameters were determined. Determination of growth attributes Measurement of dry weight The growth of each sample was determined in terms of dry weight. The cells of both the cyanobacteria were harvested from each sample after 72 h of the experiment. For this, the cells from 100 mL culture from each sample were harvested by centrifugation at 4,000 ×g for 10 min, and pellets were rinsed thrice with sterile double-distilled water. Then, the pellets were carefully transferred onto the filter papers and placed in an oven, and dried at 80 °C for 48 h till the weight became constant. Thereafter, the weight of each sample was determined by the single pan electronic balance (Mettler-Toledo AG CH-8608, Switzerland). Determination of light-harvesting pigments The photosynthetic pigments chlorophyll a (Chl a ), carotenoids (Cars), and phycobillins [phycocyanin (PC), phycoerythrin (PE), and allophycocyanin (APC)] were quantified by using the protocols of Porra et al. (1989), Goodwin (1954), and Bennett and Bogorad (1973), respectively. For Chl a and Cars, cells were harvested, and pellets were washed carefully with distilled water prior to mixing with the required amount of pure methanol. To extract the pigment completely, cells suspended in methanol were kept in a refrigerator at 4°C overnight. Further, each sample was centrifuged at 10,000 ×g for 15 min, and the absorbance of the clear solution for Chl a and Cars was measured at 665 nm and 450 nm, respectively, by using a UV-visible spectrophotometer (Shimadzu, Japan). Thereafter, the amounts of Chl a and Cars were calculated by using the equation given by Porra et al. (1989) and Godwin (1954), respectively. The amount of Chl a and Cars is expressed as µg (mg dry weight) -1 . For the determination of PC, PE, and APC, the cultures from each sample were centrifuged, and pellets were thoroughly mixed with few drops of toluene and kept in refrigerator at 4 o C for 24 h. Afterward, the permeabilized cells were suspended in the required amount of phosphate buffer (2.5 mM, pH 7.0). At the end, the samples were centrifuged at 10,000 ×g at 4 o C for 10 min using mini cooling centrifuge. The transparent supernatant was used to record the absorbance at 562 nm, 615 nm, and 652 nm, and thereafter, the following equations were used to quantify the contents of PC, PE, and APC. PC=A 615 -(0.474*A 652 )/5.34 APC =A 652 -0.208 (A 615 )/5.09 PE=A 562 * 2.41 (PC)-0.849(APC)/9.62 The amounts of PC, APC, and PE are expressed as µg [(mg dry weight) -1 ]. Determination of intracellular cadmium accumulation The intracellular Cd was determined in cells of each sample by using the method of Allen et al. (1976). For this, the required amount of cultures from each sample at the end of each experiment was centrifuged, and pellets were rinsed thrice with 1 mM EDTA to remove extracellular Cd. Further, the pellets were kept in oven at 60–70 o C for 3 days. The dried samples were subjected to digestion by mixing with HCl: HNO 3 in a 3:1 ratio, at 120 o C till the solution became transparent. Digested samples were allowed to cool, and the solution was diluted with DDW and filtered. Further, the filtrate from each sample was used to determine the intracellular Cd by using ICP-MS inductively coupled plasma mass spectrometry (Model-Agilent 5800 ICP-OES). The amount of Cd in each sample is expressed as µg Cd (mg dry weight) -1 . Measurement of photosynthetic O 2 yield, PSII photochemistry and respiratory oxygen uptake (i) Determination of photosynthetic O 2 yield and respiratory oxygen uptake The photosynthesis rate of each sample was recorded in terms of oxygen evolved. The required vol. of cells from each sample was poured into air-tight vessels of Clark type oxygen electrode (Digital Oxygen System, Model-10, Rank Brothers, UK), and cells were exposed under the light intensity of 360 µmol photons m -2 s -1 (PAR), and oxygen evolution was measured for 5 min at 25 o C. Similarly, the respiratory oxygen consumption of the same samples was recorded under darkness. The photosynthetic oxygen yield and respiratory oxygen uptake are expressed as µmol O 2 evolved (mg Chl a ) -1 h −1 and µmol O 2 consumed (mg Chl a ) -1 h −1 respectively. (ii) Determination of chlorophyll a fluorescence kinetics (JIP-test) The chlorophyll a fluorescence kinetics of both the test cyanobacteria were evaluated in 30 min dark-adapted cells by using hand-held fluorometer (AquaPen AP 100, Photon System Instruments, Czech Republic). Prior to measurement of chlorophyll a fluorescence, the absorbance (A 750 nm) of cells of each sample was maintained, and transients were recorded accordingly as per the method described by Strasser et al. (2000). Measurement of oxidative stress biomarkers (i)Biochemical analysis To evaluate oxidative stress biomarkers, superoxide radical (SOR; O 2 .- ), hydrogen peroxide (H 2 O 2 ), and MDA equivalents content were analysed biochemically as per the method described by Elstner and Heupel (1976), Velikova et al. (2000), and Heath and Packer (1968), respectively. At the end of the experiment, cells of each sample were crushed in the required amount of phosphate buffer. The SOR content in each sample was measured by the formation by NO 2 as a result of the reaction between hydroxylamine and superoxide, and the amount of superoxide was calculated by comparing the standard curve prepared by a graded solution of NaNO 2. The SOR content in each sample is represented as nmol SOR radical (mg dry weight) -1 . For the estimation of H 2 O 2, the cells of each sample were crushed in the required amount of 0.1 % of TCA solution, and the H 2 O 2 content was estimated by recording the amount of product formed due to the reaction between H 2 O 2 and potassium iodide. Finally, the amount of H 2 O 2 was calculated with the help of a standard curve prepared by the graded solution of H 2 O 2, and the value of H 2 O 2 is expressed as nmol H 2 O 2 (mg dry weight) -1 . The lipid peroxidation products, MDA equivalent, were determined in each sample by the TBA adduct reactive substance. For this, cells from each sample, were crushed in the required amount of 5% TCA solution. Supernatant was added to the TBA-TCA solution. Finally, the amount of MDA equivalent content in each sample was calculated by using an extinction coefficient of 155 mM −1 cm −1 . The amount of lipid peroxidation product in the sample is expressed as nmol MDA equivalent content (mg dry weight) -1. (ii) Histochemical analysis Superoxide radical (SOR; O 2 •- ) and hydrogen peroxide (H 2 O 2 ) were visualized by the formation of blue chromophores with NBT and brown with DAB, respectively, as per the protocol of Forster et al. (2005). Similarly, to visualize the oxidative damage, MDA equivalent compounds were allowed to react with Schiff's reagent, exhibiting pink colour in cyanobacterial cells as described by Pompella et al. (1987). Further, an image-capturing light microscope (Model: DM 2500) was employed. Assay of enzymatic antioxidant The treated and untreated cells of cyanobacteria at the end of the experiment were harvested by centrifugation (4,000 ×g ; 10 min) and the pellets were crushed separately in the required amount of phosphate buffer. For SOD, POD, CAT, and GST estimation methods of Giannopolitis and Reis (1977), Aebi (1984), Gahagan et al. (1968), and Habig et al. (1974), respectively, were followed. The quantity of enzyme that inhibits the reduction of NBT by 50% is considered as one unit of SOD activity, and enzyme activity is expressed as U mg -1 protein. For the measurement of CAT activity, the dissociation of H 2 O 2 was recorded at 240 nm, and CAT activity was calculated by employing the extinction coefficient of 39.4 mM -1 cm -1 . One unit is defined as the dissociation of 1 nmol hydrogen peroxide per min, and the activity is expressed as U mg -1 protein. To measure the POD activity, the oxidation of pyrogallol was quantified, and the absorbance was recorded at 430 nm. One unit of enzyme activity is defined as one nmol oxidation of pyrogallol per min, and POD activity is expressed as U mg -1 protein. GST activity was estimated by measuring the formation of conjugates between CDNB and GSH, and the absorbance was read at 340 nm. One unit of enzyme activity is considered as the formation of CDNB-GSH conjugates and the activity is presented as U mg -1 protein. Statistical analysis The DATA were treated with one-way analysis of variance (ANOVA) by using Duncan's multiple range test (DMRT) SPSS-26. The mean separation for significant differences was determined among the treatments at P <0.05 significance levels. The data include means ± standard errors of three replicates in three independent experiments, each with n=3. Results Effects of melatonin and gamma-aminobutyric acid on the growth under cadmium stress The growth of both test cyanobacteria was determined by estimating the dry weight, and the results are displayed in Fig . 1a .The test metal Cd at 5 µM diminished the growth significantly ( P <0.05) by 22% and 25% in N. muscorum and Anabaena sp., respectively, over the respective controls values. The exogenous supplementation of MT in the growth medium significantly alleviated Cd toxicity on growth, exhibiting only 11% and 15% reduction, while GABA treatment more efficiently recovered the growth, as there was only 5% and 9% declining effect on the growth of N. muscorum and Anabaena sp., respectively. Further, with the addition of 4 Chloro DL-phenylalanine (CPA), biosynthetic inhibitor of MT, the recovery in growth under Cd stress by GABA was slowed down, as there was 8% and 12% reduction in N. muscorum , and Anabaena sp., respectively. Fig.1 Impacts of melatonin and gamma-aminobutyric acid on intracellular cadmium accumulation The intracellular Cd accumulation in both the cyanobacteria is presented in Fig.1b. The results reveal that cyanobacteria N. muscorum and Anabaena sp., when treated with 5 µM Cd, accumulated significant amount of test metal i.e., 7.625±0.132 and 9.438±0.163 µg Cd mg -1 dry weight, respectively. After supplementation of MT (90 nM) to the growth medium N. muscorum and Anabaena sp. exhibited less Cd accumulation, as it was 4.538±0.079 and 5.662±0.098 µg Cd mg -1 dry weight, respectively. In another set, when GABA was administered, the declining trend on Cd accumulation was more prominent, hence intracellular Cd was 2.648±0.045 and 3.493±0.057 µg Cd mg -1 dry weight in N. muscorum and Anabaena sp., respectively. Unlike this, the efficiency of GABA in restricting Cd accumulation declined when applied together with the endogenous MT inhibitor, thereby showing greater amount of Cd accumulation i.e., 3.662±0.063 and 4.624±0.080 µg Cd mg -1 dry weight in N. muscorum and Anabaena sp., respectively, as compared to GABA alone. Effects of melatonin and gamma-aminobutyric acid on light-harvesting pigments under cadmium stress The results pertaining to the effects of signaling molecules MT and GABA on Chl a , Cars, and phycobilins (PC, PE, and APC) contents in Cd-stressed and unstressed cultures of both the strains are displayed in Table 1. Cadmium at 5 μM caused substantial ( P <0.05) reduction in photosynthetic pigments of both cyanobacteria i.e. a reduction of 21% and 24% for Chl a and 17% and 20% for Cars in N. muscorum, and Anabaena sp., respectively. Similarly, Cd caused damaging effect on phycobilproteins as the reduction was 25% and 27% for PC, 26% and 29% for PE, and 23% and 26% for APC in N. muscorum and Anabaena sp., respectively. When MT was subjected to the cultures, a significant recovery in the reduction of pigment under Cd stress was noticed. Under this condition, the percentage decrease was 12% and 17% for Chl a , 9% and 14% for Cars, and 15% and 20% for PC, 17% and 22% for PE, and 13% and 18% for APC in N. muscorum, and Anabaena sp., respectively. The other signaling molecule GABA, induced more pronounced ameliorating effects on the toxicity of Cd on pigment contents in both strains. The supplementation of GABA to the growth medium resulted in comparatively less decline in Chl a (7% and 12%), Cars (5%, and 10%), PC (8% and 12%), PE (10% and 16%), and APC (6% and 10%) in N. muscorum , and Anabaena sp., respectively than that with MT. Furthermore, GABA without endogenous MT (in the presence of biosynthetic inhibitor, CPA) reversed Cd-induced damaging effect, but it was lesser extent as observed with GABA alone in both the strains. Table 1 Effects of melatonin and gamma-aminobutyric acid on photosynthetic oxygen yield, PS II photochemistry, and respiratory activity under cadmium stress The photosynthetic oxygen yield in treated and untreated cells of both the cyanobacteria is displayed in Fig. 1c. The 5 μM Cd caused significant ( P <0.05) inhibition in photosynthetic activity, showing 26% and 28% reduction in N. muscorum and Anabaena sp., respectively, over the value of the respective controls. In contrast to this, exogenous MT resulted in significant amelioration in the Cd-induced inhibitory effect on photosynthetic oxygen yield, as the decrease was only 13% and 16% in Cd-stressed N. muscorum and Anabaena sp. cells, respectively, compared to the value of the respective controls. The GABA treatment more potentially recovered photosynthetic oxygen yield under Cd stress as decrease was only 6% and 9% in N. muscorum and Anabaena sp., respectively. Further, when biosynthetic inhibitor of MT i.e. CPA, was applied, the GABA-induced recovery was less pronounced in N. muscorum (10%) and in Anabaena sp. (13%) under Cd stress. Photosynthetic activity of both the cyanobacteria was further analysed by recording Chl a fluorescence (JIP Test), and the results are presented in Fig. 2a,b. The findings regarding to this vividly demonstrates that Cd caused negative impact on fluorescence kinetics parameters by decreaseing the values of photosynthetic quantum yield (Fv/F M or Phi_Po), quantum yield of electron transport flux (Phi_Eo), efficiency of electron transport for each trapped exciton (Psi_ 0 ), size and quantity of active photosynthetic reaction centers (Fv/F 0 ) and performance index (PI ABS ). Reverse to this effect under similar stress conditions, the values of energy flux parameters, such as absorption flux for each reaction center (ABS/RC), trapping flux per reaction center (TR 0 /RC), dissipation flux per reaction center (DI 0 /RC), and electron transport flux per reaction center (ET 0 /RC) were increased. Exogenously subjected MT to the N. muscorum and Anabaena sp., cultures reversed the damaging effect of Cd stress on PSII photochemistry by regulating fluorescence kinetics and energy fluxes, and this effect was more prominent with exogenous GABA treatment. Notwithstanding this, GABA-induced recovery on these parameters under Cd stress became less pronounced when cultures were grown with biosynthetic inhibitor (CPA) of MT than that of GABA alone. The respiratory activities measured as oxygen uptake in both the strains exposed to Cd stress, together with and without exogenous signalling molecules, are displayed in Fig. 1d. The tested dose of Cd accelerated the respiratory rate in N. muscorum by 25% and in Anabaena sp. by 27% as compared to the values of their respective controls. The application of signaling molecule MT tended to show a decreasing trend toward normalization however, it was still higher in N. muscorum by 15% and in Anabaena sp. by 17% over the values of the respective controls. Further, exogenous GABA continued declining trends, but it was still remained greater in N. muscorum (by 7%) and Anabaena sp. (by 10%) than the values of controls. When the impact of endogenous MT was abolished by treating the cultures with its biosynthetic inhibitor i.e, CPA, the role of GABA towards the normalization of respiratory rate was further hampered, showing a rise of 11% and 14% in the rate of respiration in N. muscorum, and Anabaena sp., respectively, over the values of controls. Fig.2 Effects of melatonin and gamma-aminobutyric acid on oxidative stress biomarkers under cadmium stress Cadmium at 5 μM exacerbated the oxidative stress, expressing the sudden rise in oxidative stress biomarkers i.e, superoxide (by 27% and 31%), H 2 O 2, (by 30% and 33%) and MDA equivalent contents (by 36% and 39%) in N. muscorum (by 27%, 30%, and 36%) and Anabaena sp. respectively Table 2 . Furthermore, exogenous MT produced a normomilizing effect on oxidative biomarkers (SOR, H 2 O 2, MDA equivalents content) however, it was still higher in Anabaena sp. (by 17%, 18%, and 24%) and in N. muscorum (by 14%, 16%, and 21%), respectively, over the value of controls. It was further noticed that exogenous GABA caused more reducing effect however, oxidative biomarkers were still greater in Anabaena sp. (by 9%, 11%, and 13%) in N. muscorum (by 6%, 7%, and 10%), respectively. In addition to this, when endogenous MT biosynthetic inhibitor CPA was used, the contents of oxidative biomarkers ( SOR, H 2 O 2, and MDA equivalents content) were further increased in both the cyanobacteria as compared to values recorded in the presence of exogenous GABA without CPA. The results related to the oxidative biomarkers (SOR, H 2 O 2, and MDA equivalents content) were also expressed by histochemical analysis under different treatments in the cells of both the strains and represented in Fig. 3. The intensity of colour for superoxide (SOR; O 2 .- ), (blue), H 2 O 2 (brown) and MDA equivalents (pink) was more intense when cells are treated with Cd and then decreasing trend was followed by Cd+MT, Cd+GABA+CPA, Cd+GABA, controls in both the strains. Table 2 Fig.3 Effects of melatonin and gamma-aminobutyric acid on the activity of enzymatic antioxidants under cadmium stress The results shown in Table 3 depict the enzymatic antioxidant activities (SOD, POD, CAT, and GST) under Cd stress in both the test cyanobacteria. Under Cd stress the data reveal that the activity of SOD, POD, CAT, and GST was accelerated by 37%, 30%, 33%, and 26% in N. muscorum , and by 35%, 26%, 29%, and 22 % in Anabaena sp ., respectively, over the values of controls. The MT addition to Cd stressed cultures further augmented the enzymatic antioxidant activity SOD (by 50% and 46%), POD (by 43% and 40%), CAT (by 55% and 51%), and GST (by 59% and 54%) in N. muscorum, and Anabaena sp., respectively. Likewise, GABA treatment to Cd-stressed cultures caused a more profound enhancing effect in SOD, POD, CAT, and GST activity as it was shown by 62%, 57%, 75% , and 81% in N. muscorum, and 59%, 53%, 72%, and 78% in Anabaena sp. , respectively. When the effect of endogenous MT was masked by CPA, GABA also maintained the enhancing effect on these enzymatic antioxidant activities in both strains however, the rise in the activity over the values of controls was comparatively less than that recorded in only with GABA. Table 3 Discussion Melatonin and gamma-aminobutyric acid upregulate the growth performance under cadmium stress Melatonin (MT) and gamma-aminobutyric acid (GABA) are the signaling molecules that are endogenously produced in every living being including cyanobacteria. The alleviating effect of the signaling molecules MT and GABA in cyanobacteria is recorded howover, this is the first report in cyanobacteria under abiotic stress, especially in Cd stress. The results revealed that Cd (5 μM) significantly reduced the growth performance of N. muscorum and Anabaena sp. Fig 1a. This could be due to (i) excessive intracellular Cd accumulation (Fig. 1b), (ii) decreased light-harvesting pigment contents (Table 1 ) , photosynthesis [whole cell oxygen evolution (Fig. 1c) , and PS II photochemistry (Fig. 2a , b)] , (iii) increased oxidative stress, (Table 2) , despite of accelerated activity of the enzymatic antioxidant system (Table 3) . The current results are incongruent with earlier findings, where the growth of Anabaena sp. PCC7120, A. L31, and A. doliolum at 10 μM Cd (Singh et al. 2018), and N. muscorum ATCC 27893 and Anabaena sp. PCC7120 at 6 μM Cd (Verma and Prasad 2021a;b) were found to be diminished. The two signalling molecules MT and GABA, which were exogenously applied separately, induced significant recovery in growth of Cd toxicity in both strains however, GABA appeared to be more prominent (Fig. 1a). This was primarily due to a significant reduction in intracellular Cd content (Fig. 1b), upregulation of photosynthetic pigments (Table 1), photosynthesis (Fig. 1c and Fig. 2 a,b) and lowering in oxidative stress (Table 2) by more strengthening antioxidant system (Table 3). A significant reversal in the improvement of growth performance of both the cyanobacteria treated with CPA (a biosynthetic inhibitor of MT) points towards the requisite for better functioning of GABA under Cd stress. In the previous study of Aghdam and Fard (2017), it was investigated that the addition of MT mitigated postharvest decay and improved nutritional quality in strawberry fruits by enhancing the GABA shunt pathway, resulting in higher energy levels. Furthermore, Sharafi et al. (2019) demonstrated that exogenous supplementation of MT promoted endogenous MT accumulation, thereby triggering GABA pathway activity in tomato fruits during cold storage. As a result, the quality of fruit was maintained under postharvesting conditions. It is worthy to mention that there is no such study which demonstrates the functioning of MT and GABA in interaction to deal with toxicity amelioration, especially in cyanobacteria. Melatonin and gamma-aminobutyric acid regulate light-harvesting components, photosynthetic oxygen yield, PS II photochemistry, and respiration under Cd stress Growth performance of photosynthetic organisms is regulated by their light-harvesting component, photosynthesis, and respiration, and they are considered as stress indicators. HMs have been shown to cause negative impact on light-harvesting components such as Chl a , Cars, and phycobiliproteins (PC, PE, and APC, a main light-harvesting complex of PS II) in cyanobacteria. Cd at 5 μM caused significant negative impact on Chl a , Cars, and phycobiliprotein in both the test cyanobacteria, and the effect was greater in Anabaena sp. (Table 1) . These results are in consonance with the earlier findings of Singh et al. (2018) in different Anabaena sp. and also in the previous study of Ahad and Syiem (2019) in Nostoc muscorum Meg 1 under Cd stress. The damaging effect of Cd on Chl a may be correlated with the (i) negative impact on Chl a biosynthesis, including δ-aminolevulinic acid dehydrogenase and protochlorophyllide reductase (Parmar et al. 2013), (ii) activation of Chl a degrading enzyme, i.e., chlorophyllase, and (iii) substitution of Mg and Fe, crucial elements for the synthesis of chlorophyll (Aziz et al. 2015). Further, the impact of Cd may destabilize the thylakoid membrane in which the light-harvesting pigments are integrated (Verma and Prasad 2021b). The light-harvesting pigment, i.e, carotenoids, are primarily responsible for the protection of the photosynthetic machinery however, due to higher doses of Cd, their content declines as a result of downregulation in biosynthesis and upregulation in the damaging process (Chen et al. 2022). The greater impact on phycobiliprotein (PE, PC, and APC) could be explained on the basis of their localization on the outer surface of the thalakoid membrane as well as their strong affinity with Cd due to their proteinaceous nature. Similar results were also reported by Goswami et al. (2015) in Anabaena doliolum Ind1 where Cd was shown to induce the damaging effect on Chl a , Cars, PC, PE, and APC content. The addition of MT and GABA individually recovered the photosynthetic pigments, which could be correlated with the possible positive effect of these signaling molecules on the biosynthesis of pigment and stabilization of the thylakoid membrane. Furthermore, both the signalling molecules caused substantial reduction in Cd accumulation (Fig. 1b), thereby significantly lowering of ROS (Table 2), which contributed to the recovery in photosynthetic pigments (Table 1). In the presence of a biosynthetic inhibitor of endogenous MT (CPA), GABA appears to be less efficient in the restoration of photosynthetic pigment under stress. This might have occurred due to further rise in ROS content following more intracellular Cd accumulation. The growth of photoautotrophs is mainly depends on the leading anabolic process, such as photosynthesis. Hence, in order to understand the impact of test metal on growth was explored by analyzing photosynthetic oxygen evolution and PS II photochemistry. Cadmium caused substantial inhibition to the photosynthetic oxygen evolution rate in both the test cyanobacteria, and the damage was more pronounced in Anabaena sp. (Fig. 1c). In consonance to our study, Verma and Prasad (2021b) also reported significant decline in oxygen evolution rate in N. muscorum and Anabaena sp. and explained that Cd impaired the functioning of the oxygen-evolving complex by replacing the calcium ion. Further, it was also correlated with the Cd-induced damage to the photosynthetic electron transport system, light-harvesting complex, and downregulation in the gene expression responsible for D1 and D2 proteins synthesis (Song et al. 2025). Exogenous application of MT and GABA majorly recovered photosynthetic oxygen evolution rate under Cd stress, and this effect was greater with GABA. The similar normalization in photosynthetic activity was also observed after MT exposure under Cd and Al stress, as reported in the study of Sami et al. (2020) in Brassica napus L., and with GABA exposure in cyanobacteria under UV-B stress (Pandey et al. 2022). Wherein, it was suggested that this recovery occurred as a result of improvement in the functioning of the oxygen-evolving complex, PSII assembly, and photosynthetic electron transport system (Jahan et al. 2021; Li et al. 2021; Jahan et al. 2023). Further, after masking the role of endogenous MT (due to the addition of CPA, an endogenous inhibitor), GABA induced reversal in the inhibitory effect of Cd in photosynthetic activity (oxygen yield) was declined. This effect might have occurred due to less positive effect on the oxygen-evolving complex, D1 and D2 protein, light-harvesting complex, and the photosynthetic electron transport system in both the cyanobacteria under Cd stress. In order to pinpoint the negative impact on PSII-mediated oxygen evolution rate under Cd stress was further elaborated by observing PS II photochemistry (Chl a fluorescence kinetics) in both the cyanobacteria with the presence and absence of MT/GABA (Fig. 2a,b). Cd treatment damaged PS II photochemistry as indicated by lowering in maximum quantum yield (Fv/Fm or Phi Po), the size and number of active reaction centers (Fv/F 0 ), yield of electron transport per trapped exciton (Psi_ 0 ), quantum yield of electron transport (Phi_Eo), and overall performance index (PI ABS ) in both the cyanobacteria. The damaging effect was noticed because of direct impact on the oxygen-evolving complex, PS II assembly, and thylakoid membrane, as reported in earlier findings (Verma and Prasad 2021b). The Cd-induced damage to the reaction centre and thylakoid membrane was also revealed by increased energy fluxes, i.e., absorption flux per reaction center (ABS/RC), dissipation energy per reaction center (DI 0 /RC), electron transport flux per reaction center (ET 0 /RC), and trapped energy flux per reaction center (TR 0 /RC). The significant improvement in PS II photochemistry following MT/GABA treatments, which occurred as a consequence of reversal in inhibitory/damaging effect on PS II reaction centre, antenna pigment, and thylakoid membrane. GABA appears to be more efficient in normalizing the PS II efficiency under Cd stress. These results are also in consonance with the earlier findings, where PS II photochemistry was found to be improved by GABA in cyanobacteria under UV-B stress (Pandey et al. 2022) while MT strengthened PS II in wheat seedlings exposed to salt stress (Ke et al. 2018). The study based on the endogenous MT inhibitor (CPA) revealed that the efficiency of GABA in recovering the PS II photochemistry under Cd stress was more profound with the conjugation of MT. Under stress conditions, respiratory rate is found to increase to maintain the energy loss due to decline in photosynthetic activity in cyanobacteria as some components of electron transport are shared in photosynthesis and respiration (Singh and Prasad 2024). In the current study, it was also noticed that the respiratory rate in both tested cyanobacteria exposed to Cd stress significantly increased (Fig. 1d). The findings of Verma and Prasad (2021b) in N. muscorum and Anabaena sp. under Cd stress are also in consonance with the current study. Carfagna et al. (2013) repoted that in Chlorella sorokiniana treated with Pb and Cd substantial increase in respiratory oxygen consumption was the result of an adaptation approach to make up for the energy loss due to decreased photosynthetic activity. Hence, they have suggested that C . sorokiniana cells use respiration to produce extra energy to support cell metabolism. Nonetheless, in the current study, after exogenous supplementation of MT, oxygen consumption showed a declining trend due to improved photosynthetic rate (Fig. 1c). In exogenously supplied GABA, the declining trend further continued, but it was still higher than that of the controls. Similar result was also reported in cyanobacteria with exogenous GABA by Pandey et al. (2022) under UV-B stress. The interactive role of MT and GABA towards the normalization of respiratory rate even under Cd stress was clearly demonstrated by masking the effect of endogenous MT. Melatonin and gamma-aminobutyric acid downregulate oxidative stress by stimulating antioxidant enzymes Under aerobic conditions, all living organisms, including cyanobacteria and plants, generate reactive oxygen species [superoxide radical (SOR; O 2 .- ), hydroxyl radical (OH . ), H 2 O 2 etc.] during the respiratory, photosynthetic process, and membrane electron transport even under normal conditions. Hence, causes oxidation to protein, lipid, and nucleic acid (Sabahi et al. 2018; Smirnoff and Arnaud 2019). These organisms are equipped with enzymatic (superoxide dismutase, peroxidase, catalase, glutathione S transferases, etc.) and non-enzymatic (ascorbate, glutathione, flavonoid, proline, cysteine, etc.) antioxidants, thereby scavenging ROS efficiently to maintain homeostasis under normal conditions (Verma and Prasad 2021a; Zhu et al. 2023). However, under stress, ROS production was excessively increased, which could not be controlled by the antioxidant system, thereby cell homeostasis was disturbed. Under Cd stress, oxidative biomarkers [superoxide radical (SOR; O 2 .- ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde equivalents content (MDA)] were increased despite of accelerated activity of SOD, POD, CAT, and GST in both the test cyanobacteria (Table 3). As a consequence of this, the rate of lipid peroxidation was also enhanced as evidenced by higher levels of MDA equivalent content (Table 2). Similar results were also reported by Verma and Prasad (2021a) under Cd stress in cyanobacteria and by Zulfiqar et al. (2024) in Matthiola incana L. where increased oxidative stress caused cellular damage, thereby declined vital metabolic pathways and finally reducing the growth. Under similar stress conditions, when cyanobacterial cells were treated with exogenous MT, the activity of enzymatic antioxidants i.e. SOD, POD, CAT, and GST, were further augmented (Table 3), and concurrently, the ROS level tended to bring down significantly (Table 2). As a result of this, damage to cyanobacteria was lessened. Hence, MT fostered as an alleviating role in reducing the Cd toxicity on photosynthesis and growth in both cyanobacteria. It has also been reported that MT directly scavenges ROS and also by improving the antioxidant system in soybean under osmotic stress (Jahan et al. 2023). Another signaling molecule, GABA, induced more profound effect on alleviating Cd toxicity on lipid peroxidation by keeping the ROS level under limits. This could occur as a result of further enhancement in the activity of the enzymatic antioxidant system (Table 3). It has also been demonstrated that exogenous GABA application enhanced the gene expression related with sod and cat in green alga Haematococcus pluvialis exposed to salinity and high-light stress conditions (Li et al. 2021). Further, rise in ROS production/accumulation in the presence of endogenous MT inhibitor (CPA) suggests the role of MT for the significant functioning of GABA in Cd toxicity alleviation in test cyanobacteria. The study reveals that GABA, being the prime player, acts downstream to MT in upregulating growth, and PS II photochemistry by attenuating oxidative stress in N. muscorum and Anabaena sp. under Cd stress. Fig.4 Conclusion In conclusion, the growth of both cyanobacteria was significantly hampered due to excessive intracellular accumulated Cd induced toxicity on light-harvesting pigments and PS II photochemistry. Under this condition, the accelerated activity of enzymatic antioxidants could not keep oxidative stress biomarkers under control. The signaling molecules MT as well as GABA tended to restrict the entry of Cd significantly inside the cell, thereby strengthening effects on pigments and PS II photochemistry supported the growth of cyanobacteria (Fig. 4 ). The finding further reveals that GABA is a primary player and acts downstream to MT in relieving the cyanobacteria from Cd-induced toxicity. The study pinpoints towards the engineering of cyanobacteria by involving molecular approaches so that MT and GABA-enriched cyanobacteria can support the paddy crops in Cd stress, in particular, and stresses in general. Abbreviations ABS/RC : Absorption flux per reaction center ANOVA : Analysis of variance APC : Allophycocyanin Cars : Carotenoids CAT : Catalase Cd : Cadmium CDNB : 1-chloro-2, 4-dinitrobenzene Chl a : Chlorophyll a CPA : 4-Chloro DL-phenylalanine DAB : 3, 3' diaminobenzidine DDW : Double-distilled water DIo/RC : Dissipation energy per reaction center DW : Dry weight ETo/RC : Electron transport per reaction center Fv/Fo : Size and number of active reaction centers) GABA : Gamma-aminobutyric acid GST : Glutathione- S -transferase H 2 O 2 : Hydrogen peroxide HM : Heavy metal ICP-MS : Inductively coupled plasma mass spectrometry MDA : Malondialdehyde MT : Melatonin O 2 •− : Superoxide radical OH − : Hydroxyl radicals PC : Phycocyanin PE : Phycoerythrin Phi_Eo : Quantum yield of electron transport Phi_Po : Quantum yield of primary photochemistry PI ABS : Overall performance index POD : Peroxidases Psi_o : Yield of electron transport per trapped exciton ROS : Reactive oxygen species SOD : Superoxide dismutase TRo/RC : Trapping flux per reaction center Declarations All authors read and approvd this manuscript. Competing interests Authors have no competing intrests. Funding Sheo Mohan Prasad is thankful to CSIR, New Delhi, India, for providing financial support through the Emeritus Scientist Scheme, File No. 21/1166/24/EMR-II. Author Contribution SMP designed the experiments. SP,VKS and SRV performed the experiments. SP analyzed the data and wrote the manuscript. SP, SRV and SMP finalized the draft. Acknowledgement The authors are thankful to Head, Department of Botany, University of Allahabad, Prayagraj, India for providing the necessary facilities. SP and VKS are thankful as AU research scholars to the University Grant Commission, New Delhi. SMP is thankful to CSIR, New Delhi, India, for providing financial support through the Emeritus Scientist Scheme, File No. 21/1166/24/EMR-II. Availability of data and material Data will be provide on reasonable request. References Aebi II (1984) Catalase in vitro. Method Enzymol 105:121-126. 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Tables Table 1 Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid on the photosynthetic pigments: chlorophyll a (Chl a ) and carotenoids (Car), and phycobiliproteins: phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) in Nostoc muscorum and Anabaena sp. after cadmium treatment. Data signifies the mean±standard error of three replicates, each with three independent experiments ( n = 3). Values within same column followed by different superscripts have significant difference at P<0.05 level according to Duncan’s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine. Photosynthetic pigments [µg (mg dry weight) -1 ] Treatments Nostoc muscorum ATCC 27893 Anabaena sp. PCC 7120 Chl a Cars PC APC PE Chl a Cars PC APC PE Control 15.74±0.27 a 7.23±0.12 a 54.05±0.93 a 8.72±0.15 a 8.90±0.15 a 15.01±0.26 A 6.74±0.11 A 49.72±0.86 A 7.90±0.13 A 8.10±0.14 A Cd 12.38±0.21 c 5.98±0.09 d 40.52±0.70 e 6.68±0.11 d 6.55±0.11 d 11.35±0.19 D 5.40±0.09 D 36.10±0.62 D 5.81±0.10 D 5.72±0.09 D Cd+ MT 13.81±0.23 b 6.58±0.11 c 45.83±0.77 d 7.55±0.13 c 7.38±0.12 c 12.44±0.21 C 5.79±0.10 C 39.76±0.68 C 6.44±0.11 C 6.30±0.10 C Cd + GABA 14.61±0.25 b 6.85±0.12 b 49.70±0.86 b 8.16±0.14 b 7.99±0.13 b 13.23±0.23 B 6.04±0.10 B 43.70±0.75 B 7.09±0.12 B 6.79±0.11 B Cd+GABA+ CPA 14.15±0.24 b 6.72±0.11 bc 46.85±0.81 c 7.83±0.13 bc 7.62±0.13 bc 12.88±0.22 BC 5.91±0.10 BC 41.52±0.72 BC 6.71±0.11 BC 6.56±0.11 BC Table 2 Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid on the contents of SOR, H 2 O 2 and MDA equivalents contents in Nostoc muscorum and Anabaena sp. after cadmium treatment. Data signifies the mean±standard error of three replicates, each with three independent experiments ( n = 3). Values within same column followed by different superscripts have significant difference at P<0.05 level according to Duncan’s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine SOR, H 2 O 2, and MDA equivalents contents Treatments Nostoc muscorum ATCC 27893 Anabaena sp. PCC 7120 SOR H 2 O 2 MDA SOR H 2 O 2 MDA [nmol (mg dry weight) -1 ] [nmol (mg dry weight) -1 ] Control 4.7±0.08 d 7.72±0.13 d 0.89±0.01 d 5.20±0.09 D 8.20±0.14 D 0.94±0.01 D Cd 5.97±0.10 a 10.06±0.17 a 1.21±0.02 a 6.80±0.11 A 10.90±0.18 A 1.31±0.02 A Cd+ MT 5.38±0.09 b 8.98±0.15 b 1.08±0.01 b 6.09±0.10 B 9.71±0.16 B 1.17±0.02 B Cd + GABA 5.00±0.08 c 8.29±0.14 c 0.98±0.01 c 5.68±0.09 C 9.11±0.15 C 1.06±0.01 C Cd+GABA+ CPA 5.19±0.09 bc 8.66±0.15 bc 1.03±0.01 bc 5.89±0.10 BC 9.45±0.16 BC 1.11 ±0.02 BC Table 3 Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid on the activity of enzymatic antioxidants SOD, POD, CAT, and GST in Nostoc muscorum and Anabaena sp. after cadmium treatment. Data signifies the mean±standard error of three replicates, each with three independent experiments ( n = 3). Values within same column followed by different superscripts have significant difference at P<0.05 level according to Duncan’s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine Treatments Nostoc muscorum ATCC 27893 Anabaena sp. PCC 7120 SOD POD CAT GST SOD POD CAT GST (U mg -1 protein) (U mg -1 protein) Control 11.5±0.19 d 25.20±0.43 d 12.66±0.21 e 46.00±0.79 e 9.93±0.17 D 23.50±0.40 D 11.83±0.20 E 44.50±0.77 E Cd 15.81±0.27 c 32.80±0.56 c 16.85±0.29 d 58.05±1.00 d 13.43±0.23 C 29.70±0.51 C 15.31±0.26 D 54.35±0.94 D Cd+ MT 17.26±0.29 b 36.11±0.62 b 19.63±0.34 c 73.03±1.26 c 14.53±0.25 B 32.98±0.57 B 17.88±0.30 C 68.59±1.18 C Cd + GABA 18.68±0.56 a 39.61±0.68 a 22.16±0.38 a 83.32±1.44 a 15.80±0.27 A 35.97±0.62 A 20.36±0.35 A 79.04±1.36 A Cd+GABA+ CPA 17.98±0.31 ab 37.90±0.65 ab 20.92±0.36 b 78.36±1.35 b 15.21±0.26 AB 34.41±0.59 AB 19.10±0.33 B 73.91±1.28 B Additional Declarations No competing interests reported. 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16:33:31","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167972,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/8aeb0ee4ccf33a9aea4c3cff.html"},{"id":95196508,"identity":"ea7a093c-bb8b-405f-8d15-c5fc7b1b314a","added_by":"auto","created_at":"2025-11-05 11:35:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85143,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of exogenous supplementation of melatonin and gamma-aminobutyric acid on dry mass (a), Intracellular cadmium accumulation (b), photosynthetic oxygen evolution (c), and respiratory oxygen consumption (d) in \u003cem\u003eNostoc muscorum \u003c/em\u003eATCC 27893\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Anabaena \u003c/em\u003esp. PCC 7120 exposed to cadmium stress. Data are means ± standard error of three independent biological replicates (\u003cem\u003en \u003c/em\u003e= 3). The bars followed by different letters show significant difference at \u003cem\u003eP\u0026lt;\u003c/em\u003e0.05 significance level according to the Duncan’s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/9dcc9ed797e40eae0fc8235a.jpg"},{"id":95196509,"identity":"ca68be18-8edd-4074-b07e-13f98b78cc89","added_by":"auto","created_at":"2025-11-05 11:35:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205785,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of exogenous supplementation of melatonin and gamma-aminobutyric acid on Chl \u003cem\u003ea \u003c/em\u003efluorescence transient (JIP-test) of \u003cem\u003eAnabaena \u003c/em\u003esp. PCC 7120 (a) and\u003cem\u003e Nostoc muscorum \u003c/em\u003eATCC 27893\u003cem\u003e \u003c/em\u003e(b) exposed to cadmium stress. Each parameter is represented as normalized values of the control which is represented by 1 (100%). Each value is average of 3 independent biological replicates (\u003cem\u003en \u003c/em\u003e= 3). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/251c42dd922a34fc123e6cf4.jpg"},{"id":95196539,"identity":"9b9bfe3a-a0e7-4ff7-a34e-aeb9e316e1ad","added_by":"auto","created_at":"2025-11-05 11:35:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60490,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of exogenous supplementation of melatonin and gamma-aminobutyric acid on \u003cem\u003ein-vivo \u003c/em\u003evisualization of superoxide radical (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e• -\u003c/sup\u003e ), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and MDA equivalents content of \u003cem\u003eNostoc muscorum \u003c/em\u003eATCC 27893\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Anabaena \u003c/em\u003esp. PCC 7120 exposed to cadmium stress. Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/c6be71f540003112f7ca64de.jpg"},{"id":95196512,"identity":"360dc0d6-8b30-453c-85e3-9cfa4fcc5cc8","added_by":"auto","created_at":"2025-11-05 11:35:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46972,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation exhibiting melatonin and gamma-aminobutyric acid \u0026nbsp;signaling in cadmium stress-exposed cyanobacterial cells. Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/7e435420b0a2bd8efa4ffe64.jpg"},{"id":96708293,"identity":"d3aa4076-9259-47b2-b5d8-426cba61be88","added_by":"auto","created_at":"2025-11-25 10:00:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873444,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7857714/v1/890ea1a2-5c1c-4cb0-a2f4-2304832b8249.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMelatonin mediates gamma-aminobutyric acid induced mitigation of cadmium toxicity on growth, PSII photochemistry, and oxidative stress in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNostoc muscorum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC 27893 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAnabaena \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. PCC 7120\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe current decade has seen an exponential rise in several environmental issues because of industrialization, primarily due to the quick human population growth and rising demand for numerous domestic materials that have increased pollutants in the environment (Pimentel et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Heavy metal (HM) contamination in the environment has become an inevitable fact. They are discharged into the environment by industries and other anthropogenic activities. Cadmium (Cd) causes damage to living beings at more than 0.2 ppm due to its high toxicity and a significant degree of bioaccumulation (WHO 1996; Biswal et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cd is mainly used in Cd-Ni batteries, phosphate fertilizers, paints, dyes, cement, and other products, and frequently released into the environment. Besides strict rules in developing countries, toxic metals-containing effluents are being released directly into the rivers without proper treatment (Singh and Prasad \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, due to the canal irrigation system, the polluted water from rivers reaches far away and heavily contaminates soils, including paddy fields. Rice is the most significant cereal among the worldwide crops, and serves as the primary food source for millions of households (Nawaz et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cd is structurally similar to calcium therefore, it uses calcium channel to pass through a cell membrane and get accumulated in the cytosol. Cadmium disturbs the flow of electron transport related to photosynthesis and respiration, inhibits the activities of enzymes and the functioning of nucleic acids, hence declines the growth of cyanobacteria and plants (Ahad and Syiem \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zulfiqar et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, overproduction/ accumulation of ROS eventually damages the antioxidant system and membrane integrity (Ahad and Syiem \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cd contamination in the paddy field directly reduces rice yield by affecting the beneficial microflora, i.e., cyanobacteria, which fix atmospheric nitrogen into ammonia (Verma and Prasad \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyanobacteria are the first species on Earth to evolve free oxygen and are important microorganisms for the development of sustainable agriculture (Nawaz et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They are essential components of the ecosystem and substantial contributors to the global nitrogen cycle due to their special ability to fix nitrogen, which makes them a natural biofertilizer (Chittora et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Singh and Prasad \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Most efficient nitrogen-fixing cyanobacteria, such as \u003cem\u003eAulosira fertilisima, Anabaena\u003c/em\u003e sp., \u003cem\u003eTolypothrix\u003c/em\u003e sp., \u003cem\u003eCalothrix\u003c/em\u003e sp., \u003cem\u003eNostoc\u003c/em\u003e sp., \u003cem\u003eNostoc linkia\u003c/em\u003e, and \u003cem\u003eScytonema\u003c/em\u003e sp., commonly occur in paddy fields and support the growth and development (Singh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is noticed that cyanobacteria can fix upto 20\u0026ndash;25 kg/ha of atmospheric nitrogen, and some species of \u003cem\u003eAnabaena\u003c/em\u003e may fix 60 kg of nitrogen per hectare in each season and improve soil quality with organic matter as well (Chittora et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Since agriculture is crucial for both economic growth and food security, it is supposed to increase food production up to 50% by 2050 to feed the global population (FAO \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, several approaches are being applied to maintain the crop productivity, and one such method may be the application of signaling molecules (Chittora et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cyanobacteria have been shown to synthesize several bioactive compounds, including GABA, a non-protein four carbon containing amino acid (Mahawar et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Pandey et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have noticed that GABA alleviated UV-B-induced toxicity in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. by involving nitric oxide. Under stress, GABA potentially regulates carbon: nitrogen metabolism in bacteria, cyanobacteria, and in eukaryotic organisms, including plants (Mahawar et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Likewise, another signaling molecule MT, directly scavenges oxidative radicals and plays a significant role in mitigating the toxicity under stress in some microbes (Yuan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and in plants (Jahan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The presence of MT is reported in all living beings, nonetheless, its function varies in bacteria, cyanobacteria, fungi, plants, and animals (Zhao et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that MT treatment induces endogenous MT synthesis and regulates the GABA shunt pathway in tomato (Sharafi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and in peach (Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) under cold stress. However, very little information has been shown to how these two signalling molecules act in collaboration in abiotic stress alleviation. As far as we know that in cyanobacteria, the alleviatory role of GABA and MT works, either separately or in interaction against HM toxicity, particularly Cd, remains elusive. Therefore, the present study is aimed to explore the ameliorating role of GABA and MT in conjunction on various physiological and biochemical attributes, such as growth, photosynthetic pigments contents, PS II photochemistry, oxidative stress, and the antioxidant system of two cyanobacteria \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. Further, we have also attempted to understand whether endogenous MT regulates GABA-induced allevitory effect against Cd toxicity in these microbes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental organisms and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cultures of filamentous, heterocystous, and homogenous cyanobacteria, \u003cem\u003eNostoc muscorum\u003c/em\u003e ATCC 27893 and \u003cem\u003eAnabaena\u003c/em\u003e sp. PCC 7120 were maintained in BG-11 medium (25\u0026plusmn;2\u0026deg;C) at pH 7.5 under photosynthetic active irradiance of 75 \u0026micro;mol photons m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;1\u003c/sup\u003e (PAR) under 14:10 h (light\u0026ndash;dark) conditions. These cyanobacteria were selected for the present study, and for each experiment, exponentially growing cells were harvested and used in each experiment. Purity of cultures was often inspected through a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of cadmium (Cd), i.e., 5 \u0026micro;M, which corresponds to LC\u003csub\u003e22\u0026nbsp;\u003c/sub\u003e for \u003cem\u003eN.\u003c/em\u003e \u003cem\u003emuscorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eLC\u003csub\u003e25\u003c/sub\u003e for \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp., was selected for the current study, after screening experiments with various doses (1, 2, 3, 4, 5, 6, 7, and 8 \u0026micro;M) of Cd. Likewise, after screening experiments 90 nM MT and 40 nM GABA were chosen, at which maximum alleviation against Cd toxicity on growth was recorded. Further, to understand the signaling relationship between the MT and GABA, 90 \u0026micro;M CPA (4-Chloro DL-phenylalanine) was also selected for the present study. For the experimental setup, the early exponentially growing healthy cultures of both the selected cyanobacteria were harvested (4,000\u003cem\u003e\u0026times;g\u0026nbsp;\u003c/em\u003efor 10 min) separately, and pellets were rinsed thrice with sterile DDW and finally resuspended in fresh BG-11 medium and used as inoculum. In each experiment, 100 mL of cultures (A\u003csub\u003e750\u0026nbsp;\u003c/sub\u003enm, 0.1) were poured in conical flask having different chemicals as required and placed under defined experimental conditions. The experimental setups were consisted of: control, Cd, Cd+MT, Cd+GABA, and Cd+GABA+CPA. All the experimental setups were carried out under aseptic conditions. After 72 h of experiment, the cells from each setup were harvested, and various physiological and biochemical parameters were determined. \u003cstrong\u003e\u003cs\u003e\u0026nbsp;\u003c/s\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of growth attributes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of dry weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth of each sample was determined in terms of dry weight. The cells of both the cyanobacteria were harvested from each sample after 72 h of the experiment. For this, the cells from 100 mL culture from each sample were harvested by centrifugation at 4,000\u003cem\u003e\u0026times;g\u0026nbsp;\u003c/em\u003efor 10 min, and pellets were rinsed thrice with sterile double-distilled water. Then, the pellets were carefully transferred onto the filter papers and placed in an oven, and dried at 80 \u0026deg;C for 48 h till the weight became constant. Thereafter, the weight of each sample was determined by the single pan electronic balance (Mettler-Toledo AG CH-8608, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of light-harvesting pigments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photosynthetic pigments chlorophyll \u003cem\u003ea\u003c/em\u003e (Chl \u003cem\u003ea\u003c/em\u003e), carotenoids (Cars), and phycobillins [phycocyanin (PC), phycoerythrin (PE), and allophycocyanin (APC)] were quantified by using the protocols of Porra et al. (1989), Goodwin (1954), and Bennett and Bogorad (1973), respectively. For Chl \u003cem\u003ea\u003c/em\u003e and Cars, cells were harvested, and pellets were washed carefully with distilled water prior to mixing with the required amount of pure methanol. To extract the pigment completely, cells suspended in methanol were kept in a refrigerator at 4\u0026deg;C overnight. Further, each sample was centrifuged at 10,000\u003cem\u003e\u0026times;g\u003c/em\u003e for 15 min, and the absorbance of the clear solution for Chl \u003cem\u003ea\u003c/em\u003e and Cars was measured at 665 nm and 450 nm, respectively, by using a UV-visible spectrophotometer (Shimadzu, Japan). Thereafter, the amounts of Chl \u003cem\u003ea\u0026nbsp;\u003c/em\u003eand Cars were calculated by using the equation given by Porra et al. (1989) and Godwin (1954), respectively. The amount of Chl \u003cem\u003ea\u003c/em\u003e and Cars is expressed as \u0026micro;g (mg dry weight) \u003csup\u003e-1\u003c/sup\u003e. For the determination of PC, PE, and APC, the cultures from each sample were centrifuged, and pellets were thoroughly mixed with few drops of toluene and kept in refrigerator at 4 \u003csup\u003eo\u003c/sup\u003eC for 24 h. Afterward, the permeabilized cells were suspended in the required amount of phosphate buffer (2.5 mM, pH 7.0). At the end, the samples were centrifuged at 10,000\u003cem\u003e\u0026times;g\u003c/em\u003e at 4 \u003csup\u003eo\u003c/sup\u003eC for 10 min using mini cooling centrifuge. The transparent supernatant was used to record the absorbance at 562 nm, 615 nm, and 652 nm, and thereafter, the following equations were used to quantify the contents of PC, PE, and APC.\u003c/p\u003e\n\u003cp\u003ePC=A\u003csub\u003e615\u003c/sub\u003e-(0.474*A\u003csub\u003e652\u003c/sub\u003e)/5.34\u003c/p\u003e\n\u003cp\u003eAPC =A\u003csub\u003e652\u003c/sub\u003e-0.208 (A\u003csub\u003e615\u003c/sub\u003e)/5.09\u003c/p\u003e\n\u003cp\u003ePE=A\u003csub\u003e562\u003c/sub\u003e* 2.41 (PC)-0.849(APC)/9.62\u003c/p\u003e\n\u003cp\u003eThe amounts of PC, APC, and PE are expressed as \u0026micro;g [(mg dry weight) \u003csup\u003e-1\u003c/sup\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of intracellular cadmium accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intracellular Cd was determined in cells of each sample by using the method of Allen et al. (1976). For this, the required amount of cultures from each sample at the end of each experiment was centrifuged, and pellets were rinsed thrice with 1 mM EDTA to remove extracellular Cd. Further, the pellets were kept in oven at 60\u0026ndash;70 \u003csup\u003eo\u003c/sup\u003eC for 3 days. The dried samples were subjected to digestion by mixing with HCl: HNO\u003csub\u003e3\u003c/sub\u003e in a 3:1 ratio, at 120 \u003csup\u003eo\u003c/sup\u003eC till the solution became transparent. Digested samples were allowed to cool, and the solution was diluted with DDW and filtered. Further, the filtrate from each sample was used to determine the intracellular Cd by using ICP-MS inductively coupled plasma mass spectrometry (Model-Agilent 5800 ICP-OES). The amount of Cd in each sample is expressed as \u0026micro;g Cd (mg dry weight) \u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of photosynthetic O\u003csub\u003e2\u003c/sub\u003e yield, PSII photochemistry and respiratory oxygen uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i) Determination of photosynthetic O\u003csub\u003e2\u003c/sub\u003e yield and respiratory oxygen uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photosynthesis rate of each sample was recorded in terms of oxygen evolved. The required vol. of cells from each sample was poured into air-tight vessels of Clark type oxygen electrode (Digital Oxygen System, Model-10, Rank Brothers, UK), and cells were exposed under the light intensity of 360 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e (PAR), and oxygen evolution was measured for 5 min at 25 \u003csup\u003eo\u003c/sup\u003eC. Similarly, the respiratory oxygen consumption of the same samples was recorded under darkness. The photosynthetic oxygen yield and respiratory oxygen uptake are expressed as \u0026micro;mol O\u003csub\u003e2\u003c/sub\u003e evolved (mg Chl \u003cem\u003ea\u003c/em\u003e)\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e\u0026minus;1\u003c/sup\u003e and \u0026micro;mol O\u003csub\u003e2\u003c/sub\u003e consumed (mg Chl \u003cem\u003ea\u003c/em\u003e)\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e\u0026minus;1\u003c/sup\u003e respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(ii) Determination of chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence kinetics (JIP-test)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chlorophyll \u003cem\u003ea\u0026nbsp;\u003c/em\u003efluorescence kinetics of both the test cyanobacteria were evaluated in 30 min dark-adapted cells by using hand-held fluorometer (AquaPen AP 100, Photon System Instruments, Czech Republic). Prior to measurement of chlorophyll \u003cem\u003ea\u0026nbsp;\u003c/em\u003efluorescence, the absorbance (A\u003csub\u003e750\u003c/sub\u003e nm) of cells of each sample was maintained, and transients were recorded accordingly as per the method described by Strasser et al. (2000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of oxidative stress biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i)Biochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate oxidative stress biomarkers, superoxide radical (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and MDA equivalents content were analysed biochemically as per the method described by Elstner and Heupel (1976), Velikova et al. (2000), and Heath and Packer (1968), respectively. At the end of the experiment, cells of each sample were crushed in the required amount of phosphate buffer. The SOR content in each sample was measured by the formation by NO\u003csub\u003e2\u003c/sub\u003e as a result of the reaction between hydroxylamine and superoxide, and the amount of superoxide was calculated by comparing the standard curve prepared by a graded solution of NaNO\u003csub\u003e2.\u0026nbsp;\u003c/sub\u003eThe SOR content in each sample is represented as nmol SOR radical (mg dry weight)\u003csup\u003e-1\u003c/sup\u003e. For the estimation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e the cells of each sample were crushed in the required amount of 0.1 % of TCA solution, and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was estimated by recording the amount of product formed due to the reaction between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and potassium iodide. Finally, the amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was calculated with the help of a standard curve prepared by the graded solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and the value of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is expressed as nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (mg dry weight)\u003csup\u003e-1\u003c/sup\u003e. The lipid peroxidation products, MDA equivalent, were determined in each sample by the TBA adduct reactive substance. For this, cells from each sample, were crushed in the required amount of 5% TCA solution. Supernatant was added to the TBA-TCA solution. Finally, the amount of MDA equivalent content in each sample was calculated by using an extinction coefficient of 155 mM\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;1\u003c/sup\u003e. The amount of lipid peroxidation product in the sample is expressed as nmol MDA equivalent content (mg dry weight)\u003csup\u003e-1.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(ii) Histochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide radical (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;-\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003ewere visualized by the formation of blue chromophores with NBT and brown with DAB, respectively, as per the protocol of Forster et al. (2005). Similarly, to visualize the oxidative damage, MDA equivalent compounds were allowed to react with Schiff\u0026apos;s reagent, exhibiting pink colour in cyanobacterial cells as described by Pompella et al. (1987). Further, an image-capturing light microscope (Model: DM 2500) was employed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay of enzymatic antioxidant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treated and untreated cells of cyanobacteria at the end of the experiment were harvested by centrifugation (4,000\u003cem\u003e\u0026times;g\u003c/em\u003e; 10 min) and the pellets were crushed separately in the required amount of phosphate buffer. For SOD, POD, CAT, and GST estimation methods of Giannopolitis and Reis (1977), Aebi (1984), Gahagan et al. (1968), and Habig et al. (1974), respectively, were followed. The quantity of enzyme that inhibits the reduction of NBT by 50% is considered as one unit of SOD activity, and enzyme activity is expressed as U mg\u003csup\u003e-1\u003c/sup\u003eprotein. For the measurement of CAT activity, the dissociation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was recorded at 240 nm, and CAT activity was calculated by employing the extinction coefficient of 39.4 mM\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eOne\u003csup\u003e\u0026nbsp;\u003c/sup\u003eunit is defined as the dissociation of 1 nmol hydrogen peroxide per min, and the activity is expressed as U mg\u003csup\u003e-1\u003c/sup\u003eprotein. To measure the POD activity, the oxidation of pyrogallol was quantified, and the absorbance was recorded at 430 nm. One unit of enzyme activity is defined as one nmol oxidation of pyrogallol per min, and POD activity is expressed as U mg\u003csup\u003e-1\u003c/sup\u003eprotein. GST activity was estimated by measuring the formation of conjugates between CDNB and GSH, and the absorbance was read at 340 nm. One unit of enzyme activity is considered as the formation of CDNB-GSH conjugates and the activity is presented as U mg\u003csup\u003e-1\u003c/sup\u003eprotein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DATA were treated with one-way analysis of variance (ANOVA) by using Duncan\u0026apos;s multiple range test (DMRT) SPSS-26. The mean separation for significant differences was determined among the treatments at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 significance levels. The data include means \u0026plusmn; standard errors of three replicates in three independent experiments, each with n=3.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on the growth under cadmium stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth of both test cyanobacteria was determined by estimating the dry weight, and the results are displayed in Fig\u003cstrong\u003e.\u003c/strong\u003e1a .The test metal Cd at\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e5 \u0026micro;M diminished the growth significantly (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) by 22% and 25% in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively, over the respective controls values. The exogenous supplementation of MT in the growth medium significantly alleviated Cd toxicity on growth, exhibiting only 11% and 15% reduction, while GABA treatment more efficiently recovered the growth, as there was only 5% and 9% declining effect on the growth of \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively. Further, with the addition of 4 Chloro DL-phenylalanine (CPA), biosynthetic inhibitor of MT, the recovery in growth under Cd stress by GABA was slowed down, as there was 8% and 12% reduction in \u0026nbsp;\u003cem\u003eN. muscorum\u003c/em\u003e, and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpacts of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on intracellular cadmium accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intracellular Cd accumulation in both the cyanobacteria is presented in Fig.1b. The results reveal that cyanobacteria \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., when treated with 5 \u0026micro;M Cd, accumulated significant amount of test metal i.e., 7.625\u0026plusmn;0.132 and 9.438\u0026plusmn;0.163 \u0026micro;g Cd mg\u003csup\u003e-1\u003c/sup\u003edry weight, respectively. After supplementation of MT (90 nM) to the growth medium \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. exhibited less Cd accumulation, as it was 4.538\u0026plusmn;0.079 and 5.662\u0026plusmn;0.098 \u0026micro;g Cd mg\u003csup\u003e-1\u003c/sup\u003edry weight, respectively. In another set, when GABA was administered, the declining trend on Cd accumulation was more prominent, hence intracellular Cd was 2.648\u0026plusmn;0.045 and 3.493\u0026plusmn;0.057 \u0026micro;g Cd mg\u003csup\u003e-1\u003c/sup\u003edry weight in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively. Unlike this, the efficiency of GABA in restricting Cd accumulation declined when applied together with the endogenous MT inhibitor, thereby showing greater amount of Cd accumulation i.e., 3.662\u0026plusmn;0.063 and 4.624\u0026plusmn;0.080 \u0026micro;g Cd mg\u003csup\u003e-1\u003c/sup\u003edry weight in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively, as compared to GABA alone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; on light-harvesting \u0026nbsp;pigments under cadmium stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results pertaining to the effects of signaling molecules MT and GABA on Chl \u003cem\u003ea\u003c/em\u003e, Cars, and phycobilins (PC, PE, and APC) contents in Cd-stressed and unstressed cultures of both the strains are displayed in Table\u0026nbsp;1. Cadmium at 5 \u0026mu;M caused substantial (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) reduction in photosynthetic pigments of both cyanobacteria i.e. a reduction of 21% and 24% for Chl \u003cem\u003ea\u003c/em\u003e and 17% and 20% for Cars in \u003cem\u003eN. muscorum,\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively. \u0026nbsp;Similarly, Cd caused damaging effect on phycobilproteins as the reduction was \u0026nbsp;25% and 27% for PC, 26% and 29% for PE, and 23% and 26% for APC in \u003cem\u003eN. muscorum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively. When MT was subjected to the cultures, a significant recovery in the reduction of pigment under Cd stress was noticed. Under this condition, the percentage decrease was 12% and 17% for Chl \u003cem\u003ea\u003c/em\u003e, 9% and 14% for Cars, and 15% and 20% for PC, 17% and 22% for PE, and 13% and 18% for APC in \u003cem\u003eN. muscorum,\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively. The other signaling molecule GABA, induced more pronounced ameliorating effects on the toxicity of Cd on pigment contents in both strains. The supplementation of GABA to the growth medium resulted in comparatively less decline in Chl \u003cem\u003ea\u0026nbsp;\u003c/em\u003e(7% and 12%), Cars (5%, and 10%), PC (8% and 12%), PE (10% and 16%), and APC (6% and 10%) in \u003cem\u003eN. muscorum\u003c/em\u003e, and \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp., respectively than that with MT. Furthermore, GABA without endogenous MT (in the presence of biosynthetic inhibitor, CPA) reversed Cd-induced damaging effect, but it was lesser extent as observed with GABA alone in both the strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; on photosynthetic oxygen yield, PS II photochemistry, and respiratory activity under cadmium stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photosynthetic oxygen yield in treated and untreated cells of both the cyanobacteria is displayed in Fig.\u0026nbsp;1c. The 5 \u0026mu;M Cd caused significant (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) inhibition in photosynthetic activity, showing 26% and 28% reduction in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp., respectively, over the value of the respective controls. In contrast to this, exogenous MT resulted in significant amelioration in the Cd-induced inhibitory effect on photosynthetic oxygen yield, as the decrease was only 13% and 16% in Cd-stressed \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. cells, respectively, compared to the value of the respective controls. The GABA treatment more potentially recovered photosynthetic oxygen yield under Cd stress as decrease was only 6% and 9% in \u003cem\u003eN. muscorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Anabaena\u003c/em\u003e sp., respectively. Further, when biosynthetic inhibitor of MT i.e. CPA, was applied, the GABA-induced recovery was less pronounced in \u003cem\u003eN. muscorum\u003c/em\u003e\u0026nbsp; (10%) and in \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. (13%) under Cd stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhotosynthetic activity of both the cyanobacteria was further analysed by recording Chl \u003cem\u003ea\u003c/em\u003e fluorescence (JIP Test), and the results are presented in Fig. 2a,b. The findings regarding to this vividly demonstrates that Cd caused negative impact on fluorescence kinetics parameters by decreaseing the values of photosynthetic quantum yield (Fv/F\u003csub\u003eM\u003c/sub\u003e or Phi_Po), quantum yield of electron transport flux (Phi_Eo), efficiency of electron transport for each trapped exciton (Psi_\u003csub\u003e0\u003c/sub\u003e), size and quantity of active photosynthetic reaction centers (Fv/F\u003csub\u003e0\u003c/sub\u003e) and performance index (PI\u003csub\u003eABS\u003c/sub\u003e). Reverse to this effect under similar stress conditions, the values of energy flux parameters, such as absorption flux for each reaction center (ABS/RC), trapping flux per reaction center (TR\u003csub\u003e0\u003c/sub\u003e/RC), dissipation flux per reaction center (DI\u003csub\u003e0\u003c/sub\u003e/RC), and electron transport flux per reaction center (ET\u003csub\u003e0\u003c/sub\u003e/RC) were increased. Exogenously subjected MT to the \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., cultures reversed the damaging effect of Cd stress on PSII photochemistry by regulating fluorescence kinetics and energy fluxes, and this effect was more prominent with exogenous GABA treatment. Notwithstanding this, GABA-induced recovery on these parameters under Cd stress became less pronounced when cultures were grown with biosynthetic inhibitor (CPA) of MT than that of GABA alone.\u003c/p\u003e\n\u003cp\u003eThe respiratory activities measured as oxygen uptake in both the strains exposed to Cd stress, together with and without exogenous signalling molecules, are displayed in Fig. 1d. The tested dose of Cd accelerated the respiratory rate in \u003cem\u003eN. muscorum\u003c/em\u003e by 25% and in \u003cem\u003eAnabaena\u003c/em\u003e sp. by 27% as compared to the values of their respective controls. The application of signaling molecule MT tended to show a decreasing trend toward normalization however, it was still higher in \u003cem\u003eN. muscorum\u0026nbsp;\u003c/em\u003eby 15% and in \u003cem\u003eAnabaena\u003c/em\u003e sp. by 17% over the values of the respective controls. Further, exogenous GABA continued declining trends, but it was still remained greater in \u003cem\u003eN. muscorum\u003c/em\u003e (by 7%) and \u003cem\u003eAnabaena\u003c/em\u003e sp. (by 10%) than the values of controls. When the impact of endogenous MT was abolished by treating the cultures with its biosynthetic inhibitor i.e, CPA, the role of GABA towards the normalization of respiratory rate was further hampered, showing a rise of 11% and 14% in the rate of respiration in \u003cem\u003eN. muscorum,\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp., respectively, over the values of controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on oxidative stress biomarkers under cadmium stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCadmium at 5 \u0026mu;M exacerbated the oxidative stress, expressing the sudden rise in oxidative stress biomarkers i.e, superoxide (by 27% and 31%), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e (by 30% and 33%) and MDA equivalent contents (by 36% and 39%) in \u003cem\u003eN. muscorum\u003c/em\u003e (by 27%, 30%, and 36%) and \u003cem\u003eAnabaena\u003c/em\u003e sp. respectively Table 2\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eFurthermore, exogenous MT produced a normomilizing effect on oxidative biomarkers (SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e MDA equivalents content) however, it was still higher in \u003cem\u003eAnabaena\u003c/em\u003e sp. (by 17%, 18%, and 24%) and in \u003cem\u003eN. muscorum\u003c/em\u003e (by 14%, 16%, and 21%), respectively, over the value of controls. It was further noticed that exogenous GABA caused more reducing effect however, oxidative biomarkers were still greater in \u003cem\u003eAnabaena\u003c/em\u003e sp. (by 9%, 11%, and 13%) in \u003cem\u003eN. muscorum\u003c/em\u003e (by 6%, 7%, and 10%), respectively. In addition to this, when endogenous MT \u0026nbsp;biosynthetic inhibitor CPA was used, the contents of oxidative biomarkers ( SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and MDA equivalents content) were further increased in both the cyanobacteria as compared to values recorded in the presence of exogenous GABA without CPA. The results related to the oxidative biomarkers (SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and MDA equivalents content) were also expressed by histochemical analysis under different treatments in the cells of both the strains and represented in Fig. 3. The intensity of colour for superoxide (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e), \u0026nbsp;(blue), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (brown) and MDA equivalents (pink) was more intense when cells are treated with Cd and then decreasing trend was followed by Cd+MT, Cd+GABA+CPA, Cd+GABA, controls in both the strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of melatonin and gamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on the activity of enzymatic antioxidants under cadmium stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results\u0026nbsp;shown in Table 3\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edepict the enzymatic antioxidant activities (SOD, POD, CAT, and GST) under Cd stress in both the test cyanobacteria. Under Cd stress the data reveal that the activity of SOD, POD, CAT, and GST was accelerated by 37%, 30%, 33%, and 26% in \u003cem\u003eN. muscorum\u003c/em\u003e, and by 35%, 26%, 29%, and 22 % in \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.,\u0026nbsp;\u003c/em\u003erespectively, over the values of controls. The MT addition to Cd stressed cultures further augmented the enzymatic antioxidant activity SOD (by 50% and 46%), POD (by 43% and 40%), CAT (by 55% and 51%), and GST (by 59% and 54%) in \u003cem\u003eN. muscorum,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Anabaena\u003c/em\u003e sp., respectively. Likewise, GABA treatment to Cd-stressed cultures caused a more profound enhancing effect in SOD, POD, CAT, and GST activity as it was shown by 62%, 57%, 75%\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand 81% in\u003cem\u003e\u0026nbsp;N. muscorum,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003e59%, 53%, 72%, and 78% in \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp.\u003cem\u003e,\u0026nbsp;\u003c/em\u003erespectively. When the effect of endogenous MT was masked by CPA, GABA also maintained the enhancing effect on these enzymatic antioxidant activities in both strains however, the rise in the activity over the values of controls was comparatively less than that recorded in only with GABA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eMelatonin and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;upregulate the growth performance under cadmium stress\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMelatonin (MT) and gamma-aminobutyric acid (GABA) are the signaling molecules that are endogenously produced in every living being including cyanobacteria. The alleviating effect of the signaling molecules MT and GABA in cyanobacteria is recorded howover, this is the first report in cyanobacteria under abiotic stress, especially in Cd stress. The results revealed that Cd (5 \u0026mu;M) significantly reduced the growth performance of \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. Fig 1a. This could be due to (i) excessive intracellular Cd accumulation (Fig. 1b), (ii) decreased light-harvesting pigment contents (Table\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e1\u003cstrong\u003e)\u003c/strong\u003e, photosynthesis [whole cell oxygen evolution (Fig. 1c)\u003cstrong\u003e,\u003c/strong\u003e and \u0026nbsp;PS II photochemistry (Fig. 2a\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eb)]\u003cstrong\u003e,\u003c/strong\u003e (iii) increased oxidative stress, (Table 2)\u003cstrong\u003e,\u003c/strong\u003e despite of accelerated activity of the enzymatic antioxidant system (Table 3)\u003cstrong\u003e.\u003c/strong\u003e The current results are incongruent with earlier findings, where the growth of \u003cem\u003eAnabaena\u003c/em\u003e sp. PCC7120, \u003cem\u003eA.\u0026nbsp;\u003c/em\u003eL31, and \u003cem\u003eA. doliolum\u003c/em\u003e at 10 \u0026mu;M Cd (Singh et al. 2018), and \u003cem\u003eN. muscorum\u003c/em\u003e ATCC 27893 and \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. PCC7120 at 6 \u0026mu;M Cd (Verma and Prasad 2021a;b) were found to be diminished. The two signalling molecules MT and GABA, which were exogenously applied separately, induced significant recovery in growth of Cd toxicity in both strains however, GABA appeared to be more prominent (Fig. 1a). This was primarily due to a significant reduction in intracellular Cd content (Fig. 1b), upregulation of photosynthetic pigments (Table 1), photosynthesis (Fig. 1c and Fig. 2 a,b) and lowering in oxidative stress (Table 2) by more strengthening antioxidant system (Table 3). A significant reversal in the improvement of growth performance of both the cyanobacteria treated with CPA (a biosynthetic inhibitor of MT) points towards the requisite for better functioning of GABA under Cd stress. In the previous study of Aghdam and Fard (2017), it was investigated that the addition of MT mitigated postharvest decay and improved nutritional quality in strawberry fruits by enhancing the GABA shunt pathway, resulting in higher energy levels. Furthermore, Sharafi et al. (2019) demonstrated that exogenous supplementation of MT promoted endogenous MT accumulation, thereby triggering GABA pathway activity in tomato fruits during cold storage. As a result, the quality of fruit was maintained under postharvesting conditions. It is worthy to mention that there is no such study which demonstrates the functioning of MT and GABA in interaction to deal with toxicity amelioration, especially in cyanobacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMelatonin and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eregulate light-harvesting components, photosynthetic oxygen yield, PS II photochemistry, and respiration under Cd stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth performance of photosynthetic organisms is regulated by their light-harvesting component, photosynthesis, and respiration, and they are considered as stress indicators. HMs have been shown to cause negative impact on light-harvesting components such as Chl \u003cem\u003ea\u003c/em\u003e, Cars, and phycobiliproteins (PC, PE, and APC, a main light-harvesting complex of PS II) in cyanobacteria. Cd at 5 \u0026mu;M caused significant negative impact on Chl \u003cem\u003ea\u003c/em\u003e, Cars, and phycobiliprotein in both the test cyanobacteria, and the effect was greater in \u003cem\u003eAnabaena\u003c/em\u003e sp. (Table 1)\u003cstrong\u003e.\u003c/strong\u003e These results are in consonance with the earlier findings of Singh et al. (2018) in different \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. and also in the previous study of Ahad and Syiem (2019) in \u003cem\u003eNostoc muscorum\u003c/em\u003e Meg 1 under Cd stress. The damaging effect of Cd on Chl \u003cem\u003ea\u003c/em\u003e may be correlated with the (i) negative impact on Chl \u003cem\u003ea\u0026nbsp;\u003c/em\u003ebiosynthesis, including \u0026delta;-aminolevulinic acid dehydrogenase and protochlorophyllide reductase (Parmar et al. 2013), (ii) activation of Chl \u003cem\u003ea\u003c/em\u003e degrading enzyme, i.e., chlorophyllase, and (iii) substitution of Mg and Fe, crucial elements for the synthesis of chlorophyll (Aziz et al. 2015). Further, the impact of Cd may destabilize the thylakoid membrane in which the light-harvesting pigments are integrated (Verma and Prasad 2021b). The light-harvesting pigment, i.e, carotenoids, are primarily responsible for the protection of the photosynthetic machinery however, due to higher doses of Cd, their content declines as a result of downregulation in biosynthesis and upregulation in the damaging process (Chen et al. 2022). The greater impact on phycobiliprotein (PE, PC, and APC) could be explained on the basis of their localization on the outer surface of the thalakoid membrane as well as their strong affinity with Cd due to their proteinaceous nature. Similar results were also reported by Goswami et al. (2015) in \u003cem\u003eAnabaena doliolum\u003c/em\u003e Ind1 where Cd was shown to induce the damaging effect on Chl \u003cem\u003ea\u003c/em\u003e, Cars, PC, PE, and APC content. The addition of MT and GABA individually recovered the photosynthetic pigments, which could be correlated with the possible positive effect of these signaling molecules on the biosynthesis of pigment and stabilization of the thylakoid membrane. Furthermore, both the signalling molecules caused substantial reduction in Cd accumulation (Fig. 1b), thereby significantly lowering of ROS (Table 2), which contributed to the recovery in photosynthetic pigments (Table 1). In the presence of a biosynthetic inhibitor of endogenous MT (CPA), GABA appears to be less efficient in the restoration of photosynthetic pigment under stress. This might have occurred due to further rise in ROS content following more intracellular Cd accumulation.\u003c/p\u003e\n\u003cp\u003eThe growth of photoautotrophs is mainly depends on the leading anabolic process, such as photosynthesis. Hence, in order to understand the impact of test metal on growth was explored by analyzing photosynthetic oxygen evolution and PS II photochemistry. Cadmium caused substantial inhibition to the photosynthetic oxygen evolution rate in both the test cyanobacteria, and the damage was more pronounced in \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. (Fig. 1c). In consonance to our study, Verma and Prasad (2021b) also reported significant decline in oxygen evolution rate in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. and explained that Cd impaired the functioning of the oxygen-evolving complex by replacing the calcium ion. Further, it was also correlated with the Cd-induced damage to the photosynthetic electron transport system, light-harvesting complex, and downregulation in the gene expression responsible for D1 and D2 proteins synthesis (Song et al. 2025). Exogenous application of MT and GABA majorly recovered photosynthetic oxygen evolution rate under Cd stress, and this effect was greater with GABA. The similar normalization in photosynthetic activity was also observed after MT exposure under Cd and Al stress, as reported in the study of Sami et al. (2020) in \u003cem\u003eBrassica napus\u003c/em\u003e L., and with GABA exposure in cyanobacteria under UV-B stress (Pandey et al. 2022). Wherein, it was suggested that this recovery occurred as a result of improvement in the functioning of the oxygen-evolving complex, PSII assembly, and photosynthetic electron transport system (Jahan et al. 2021; Li et al. 2021; Jahan et al. 2023). Further, after masking the role of endogenous MT (due to the addition of CPA, an endogenous inhibitor), GABA induced reversal in the inhibitory effect of Cd in photosynthetic activity (oxygen yield) was declined. This effect might have occurred due to less positive effect on the oxygen-evolving complex, D1 and D2 protein, light-harvesting complex, and the photosynthetic electron transport system in both the cyanobacteria under Cd stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eIn order to pinpoint the negative impact on PSII-mediated oxygen evolution rate under Cd stress was further elaborated by observing PS II photochemistry (Chl \u003cem\u003ea\u0026nbsp;\u003c/em\u003efluorescence kinetics) in both the cyanobacteria with the presence and absence of MT/GABA (Fig. 2a,b). Cd treatment damaged PS II photochemistry as indicated by lowering in maximum quantum yield \u0026nbsp;(Fv/Fm or Phi Po), the size and number of active reaction centers (Fv/F\u003csub\u003e0\u003c/sub\u003e),\u0026nbsp;yield of electron transport per trapped exciton (Psi_\u003csub\u003e0\u003c/sub\u003e), quantum yield of electron transport (Phi_Eo), and overall performance index (PI\u003csub\u003eABS\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003ein both the cyanobacteria. The damaging effect was noticed because of direct impact on the oxygen-evolving complex, PS II assembly, and thylakoid membrane, as reported in earlier findings (Verma and Prasad 2021b). The Cd-induced damage to the reaction centre and thylakoid membrane was also revealed by increased energy fluxes, i.e.,\u0026nbsp;absorption flux per reaction center (ABS/RC), dissipation energy per reaction center (DI\u003csub\u003e0\u003c/sub\u003e/RC), electron transport flux per reaction center (ET\u003csub\u003e0\u003c/sub\u003e/RC), and trapped energy flux per reaction center (TR\u003csub\u003e0\u003c/sub\u003e/RC). The significant improvement in PS II photochemistry following MT/GABA treatments, which occurred as a consequence of reversal in inhibitory/damaging effect on PS II reaction centre, antenna pigment, and thylakoid membrane. GABA appears to be more efficient in normalizing the PS II efficiency under Cd stress. These results are also in consonance with the earlier findings, where PS II photochemistry was found to be improved by GABA in cyanobacteria under UV-B stress (Pandey et al. 2022) \u0026nbsp;while MT strengthened PS II in wheat seedlings exposed to salt stress (Ke et al. 2018). The study based on the endogenous MT inhibitor (CPA) revealed that the efficiency of GABA \u0026nbsp;in recovering the PS II photochemistry under Cd stress was more profound with the conjugation of MT.\u003c/p\u003e\n\u003cp\u003eUnder stress conditions, respiratory rate is found to increase to maintain the energy loss due to decline in photosynthetic activity in cyanobacteria as some components of electron transport are shared in photosynthesis and respiration (Singh and Prasad 2024). In the current study, it was also noticed that the respiratory rate in both tested cyanobacteria exposed to Cd stress significantly increased (Fig. 1d). The findings of Verma and Prasad (2021b) in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. under Cd stress are also in consonance with the current study. Carfagna et al. (2013) repoted that in \u003cem\u003eChlorella sorokiniana\u003c/em\u003e treated with Pb and Cd substantial increase in respiratory oxygen consumption was the result of an adaptation approach to make up for the energy loss due to decreased photosynthetic activity. Hence, they have suggested that \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esorokiniana\u0026nbsp;\u003c/em\u003ecells use respiration to produce extra energy to support cell metabolism. Nonetheless, in the current study, after\u0026nbsp;exogenous supplementation of MT, oxygen consumption showed a declining trend due to improved photosynthetic rate (Fig.\u0026nbsp;1c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In exogenously supplied GABA, the declining trend further continued, but it was still higher than that of the controls. Similar result was also reported in cyanobacteria with exogenous GABA by Pandey et al. (2022) under UV-B stress. The interactive role of MT and GABA towards the normalization of respiratory rate even under Cd stress was clearly demonstrated by masking the effect of endogenous MT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMelatonin and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egamma-aminobutyric acid\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;downregulate oxidative stress by stimulating antioxidant enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder aerobic conditions, all living organisms, including cyanobacteria and plants, generate reactive oxygen species [superoxide radical (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e), hydroxyl radical (OH\u003csup\u003e.\u003c/sup\u003e), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eetc.] during the respiratory, photosynthetic process, and membrane electron transport even under normal conditions. Hence, causes oxidation to protein, lipid, and nucleic acid (Sabahi et al. 2018; Smirnoff and Arnaud 2019). These organisms are equipped with enzymatic (superoxide dismutase, peroxidase, catalase, glutathione \u003cem\u003eS\u003c/em\u003e transferases, etc.) and non-enzymatic (ascorbate, glutathione, flavonoid, proline, cysteine, etc.) antioxidants, thereby scavenging ROS efficiently to maintain homeostasis under normal conditions (Verma and Prasad 2021a; Zhu et al. 2023). However, under stress, ROS production was excessively increased, which could not be controlled by the antioxidant system, thereby cell homeostasis was disturbed. Under Cd stress, oxidative biomarkers [superoxide radical (SOR; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and malondialdehyde equivalents content (MDA)] were increased despite of accelerated activity of SOD, POD, CAT, and GST in both the test cyanobacteria (Table 3). As a consequence of this, the rate of lipid peroxidation was also enhanced as evidenced by higher levels of MDA equivalent content (Table 2). Similar results were also reported by Verma and Prasad (2021a) under Cd stress in cyanobacteria and by Zulfiqar et al. (2024) in \u003cem\u003eMatthiola incana\u003c/em\u003e L. where increased oxidative stress caused cellular damage, thereby declined vital metabolic pathways and finally reducing the growth. Under similar stress conditions, when cyanobacterial cells were treated with exogenous MT, the activity of enzymatic antioxidants i.e. SOD, POD, CAT, and GST, were further augmented (Table 3), and concurrently, the ROS level tended to bring down significantly (Table 2). As a result of this, damage to cyanobacteria was lessened. Hence, MT fostered as an alleviating role in reducing the Cd toxicity on photosynthesis and growth in both cyanobacteria. It has also been reported that MT directly scavenges ROS \u0026nbsp;and also by improving the antioxidant system in soybean under osmotic stress (Jahan et al. 2023). Another signaling molecule, GABA, induced more profound effect on alleviating Cd toxicity on lipid peroxidation by keeping the ROS level under limits. This could occur as a result of further enhancement in the activity of the enzymatic antioxidant system (Table 3). It has also been demonstrated that exogenous GABA application enhanced the gene expression related with \u003cem\u003esod\u003c/em\u003e and \u003cem\u003ecat\u003c/em\u003e in green alga \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e exposed to salinity and high-light stress conditions (Li et al. 2021). Further, rise in ROS production/accumulation in the presence of endogenous MT inhibitor (CPA) suggests the role of MT for the significant functioning of GABA in Cd toxicity alleviation in test cyanobacteria. The study reveals that GABA, being the prime player, acts downstream to MT in upregulating growth, and PS II photochemistry by attenuating oxidative stress in \u003cem\u003eN. muscorum\u003c/em\u003e and \u003cem\u003eAnabaena\u003c/em\u003e sp. under Cd stress. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.4\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the growth of both cyanobacteria was significantly hampered due to excessive intracellular accumulated Cd induced toxicity on light-harvesting pigments and PS II photochemistry. Under this condition, the accelerated activity of enzymatic antioxidants could not keep oxidative stress biomarkers under control. The signaling molecules MT as well as GABA tended to restrict the entry of Cd significantly inside the cell, thereby strengthening effects on pigments and PS II photochemistry supported the growth of cyanobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The finding further reveals that GABA is a primary player and acts downstream to MT in relieving the cyanobacteria from Cd-induced toxicity. The study pinpoints towards the engineering of cyanobacteria by involving molecular approaches so that MT and GABA-enriched cyanobacteria can support the paddy crops in Cd stress, in particular, and stresses in general.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABS/RC : Absorption flux per reaction center\u003c/p\u003e\n\u003cp\u003eANOVA : Analysis of variance\u003c/p\u003e\n\u003cp\u003eAPC : Allophycocyanin \u003c/p\u003e\n\u003cp\u003eCars : Carotenoids \u003c/p\u003e\n\u003cp\u003eCAT : Catalase\u003c/p\u003e\n\u003cp\u003eCd : Cadmium \u003c/p\u003e\n\u003cp\u003eCDNB : 1-chloro-2, 4-dinitrobenzene\u003c/p\u003e\n\u003cp\u003eChl \u003cem\u003ea \u003c/em\u003e: Chlorophyll \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCPA : 4-Chloro DL-phenylalanine\u003c/p\u003e\n\u003cp\u003eDAB : 3, 3\u0026apos; diaminobenzidine \u003c/p\u003e\n\u003cp\u003eDDW : Double-distilled water\u003c/p\u003e\n\u003cp\u003eDIo/RC : Dissipation energy per reaction center\u003c/p\u003e\n\u003cp\u003eDW : Dry weight \u003c/p\u003e\n\u003cp\u003eETo/RC : Electron transport per reaction center\u003c/p\u003e\n\u003cp\u003eFv/Fo : Size and number of active reaction centers)\u003c/p\u003e\n\u003cp\u003eGABA : Gamma-aminobutyric acid\u003c/p\u003e\n\u003cp\u003eGST : Glutathione-\u003cem\u003eS\u003c/em\u003e-transferase\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e : Hydrogen peroxide \u003c/p\u003e\n\u003cp\u003eHM : Heavy metal\u003c/p\u003e\n\u003cp\u003eICP-MS : Inductively coupled plasma mass spectrometry \u003c/p\u003e\n\u003cp\u003eMDA : Malondialdehyde \u003c/p\u003e\n\u003cp\u003eMT : Melatonin\u003c/p\u003e\n\u003cp\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e : Superoxide radical\u003c/p\u003e\n\u003cp\u003eOH\u003csup\u003e\u0026minus; \u003c/sup\u003e : Hydroxyl radicals \u003c/p\u003e\n\u003cp\u003ePC : Phycocyanin\u003c/p\u003e\n\u003cp\u003ePE : Phycoerythrin\u003c/p\u003e\n\u003cp\u003ePhi_Eo : Quantum yield of electron transport\u003c/p\u003e\n\u003cp\u003ePhi_Po : Quantum yield of primary photochemistry\u003c/p\u003e\n\u003cp\u003ePI\u003csub\u003eABS\u003c/sub\u003e : Overall performance index \u003c/p\u003e\n\u003cp\u003ePOD : Peroxidases \u003c/p\u003e\n\u003cp\u003ePsi_o : Yield of electron transport per trapped exciton\u003c/p\u003e\n\u003cp\u003eROS : Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSOD : Superoxide dismutase \u003c/p\u003e\n\u003cp\u003eTRo/RC : Trapping flux per reaction center\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors read and approvd this manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAuthors have no competing intrests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eSheo Mohan Prasad is thankful to CSIR, New Delhi, India, for providing financial support through the Emeritus Scientist Scheme, File No. 21/1166/24/EMR-II.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSMP designed the experiments. SP,VKS and SRV performed the experiments. SP analyzed the data and wrote the manuscript. SP, SRV and SMP finalized the draft.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful to Head, Department of Botany, University of Allahabad, Prayagraj, India for providing the necessary facilities. SP and VKS are thankful as AU research scholars to the University Grant Commission, New Delhi. SMP is thankful to CSIR, New Delhi, India, for providing financial support through the Emeritus Scientist Scheme, File No. 21/1166/24/EMR-II.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eData will be provide on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAebi II (1984) Catalase in vitro. 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Sci Total Environ 896: 165200. \u003c/li\u003e\n\u003cli\u003eZulfiqar U, Jiang W, Xiukang W, Hussain S, Ahmad M, Maqsood MF, Ali N, Ishfaq M, Kaleem M, Haider FU, Farooq N (2022) Cadmium phytotoxicity, tolerance, and advanced remediation approaches in agricultural soils; a comprehensive review. Front Plant Sci 13:773815. \u003c/li\u003e\n\u003cli\u003eZulfiqar F, Moosa A, Ali HM, Hancock JT, Yong JWH (2024) Synergistic interplay between melatonin and hydrogen sulfide enhances cadmium-induced oxidative stress resistance in stock (\u003cem\u003eMatthiola incana\u003c/em\u003e L.). Plant Signal Behav 19(1):2331357.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eon the photosynthetic pigments: chlorophyll \u003cem\u003ea\u0026nbsp;\u003c/em\u003e(Chl \u003cem\u003ea\u003c/em\u003e) and carotenoids (Car), and phycobiliproteins: phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) in \u003cem\u003eNostoc muscorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Anabaena\u0026nbsp;\u003c/em\u003esp. after cadmium treatment. Data signifies the mean\u0026plusmn;standard error of three replicates, each with three independent experiments (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 3). Values within same column followed by different superscripts have significant difference at \u003cem\u003eP\u0026lt;0.05\u0026nbsp;\u003c/em\u003elevel according to Duncan\u0026rsquo;s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"1110\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"11\" valign=\"top\" style=\"width: 1110px;\"\u003e\n \u003cp\u003ePhotosynthetic pigments [\u0026micro;g (mg dry weight) \u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cem\u003eNostoc muscorum\u003c/em\u003e\u0026nbsp; ATCC 27893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 496px;\"\u003e\n \u003cp\u003e\u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. PCC 7120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;Chl \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCars\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eAPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;Chl \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCars\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eAPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e15.74\u0026plusmn;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e7.23\u0026plusmn;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e54.05\u0026plusmn;0.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e8.72\u0026plusmn;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e8.90\u0026plusmn;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e15.01\u0026plusmn;0.26\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.74\u0026plusmn;0.11\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e49.72\u0026plusmn;0.86\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e7.90\u0026plusmn;0.13\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e8.10\u0026plusmn;0.14\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCd\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e12.38\u0026plusmn;0.21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5.98\u0026plusmn;0.09\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e40.52\u0026plusmn;0.70\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.68\u0026plusmn;0.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e6.55\u0026plusmn;0.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e11.35\u0026plusmn;0.19\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5.40\u0026plusmn;0.09\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e36.10\u0026plusmn;0.62\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5.81\u0026plusmn;0.10\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5.72\u0026plusmn;0.09\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCd+ MT\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e13.81\u0026plusmn;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.58\u0026plusmn;0.11\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e45.83\u0026plusmn;0.77\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e7.55\u0026plusmn;0.13\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7.38\u0026plusmn;0.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e12.44\u0026plusmn;0.21\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5.79\u0026plusmn;0.10\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e39.76\u0026plusmn;0.68\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.44\u0026plusmn;0.11\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6.30\u0026plusmn;0.10\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCd + GABA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e14.61\u0026plusmn;0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.85\u0026plusmn;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e49.70\u0026plusmn;0.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e8.16\u0026plusmn;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7.99\u0026plusmn;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e13.23\u0026plusmn;0.23\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.04\u0026plusmn;0.10\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e43.70\u0026plusmn;0.75\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e7.09\u0026plusmn;0.12\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6.79\u0026plusmn;0.11\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eCd+GABA+ CPA \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e14.15\u0026plusmn;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.72\u0026plusmn;0.11\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e46.85\u0026plusmn;0.81\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e7.83\u0026plusmn;0.13\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7.62\u0026plusmn;0.13\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e12.88\u0026plusmn;0.22\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5.91\u0026plusmn;0.10\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e41.52\u0026plusmn;0.72\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e6.71\u0026plusmn;0.11\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6.56\u0026plusmn;0.11\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid on the contents of SOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand MDA equivalents contents in \u003cem\u003eNostoc muscorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Anabaena\u0026nbsp;\u003c/em\u003esp. after cadmium treatment. Data signifies the mean\u0026plusmn;standard error of three replicates, each with three independent experiments (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 3). Values within same column followed by different superscripts have significant difference at \u003cem\u003eP\u0026lt;0.05\u0026nbsp;\u003c/em\u003elevel according to Duncan\u0026rsquo;s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"1058\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 1058px;\"\u003e\n \u003cp\u003eSOR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u0026nbsp;\u003c/sub\u003eand MDA equivalents contents\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Treatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 383px;\"\u003e\n \u003cp\u003e\u003cem\u003eNostoc muscorum\u003c/em\u003e\u0026nbsp; ATCC 27893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 495px;\"\u003e\n \u003cp\u003e\u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. PCC 7120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eSOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003eSOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 383px;\"\u003e\n \u003cp\u003e[nmol (mg dry weight) \u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 495px;\"\u003e\n \u003cp\u003e[nmol (mg dry weight) \u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e4.7\u0026plusmn;0.08\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e7.72\u0026plusmn;0.13\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.89\u0026plusmn;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e5.20\u0026plusmn;0.09\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e8.20\u0026plusmn;0.14\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e0.94\u0026plusmn;0.01\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eCd\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e5.97\u0026plusmn;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e10.06\u0026plusmn;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1.21\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e6.80\u0026plusmn;0.11\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e10.90\u0026plusmn;0.18\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.31\u0026plusmn;0.02\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eCd+ MT\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e5.38\u0026plusmn;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8.98\u0026plusmn;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1.08\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e6.09\u0026plusmn;0.10\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e9.71\u0026plusmn;0.16\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.17\u0026plusmn;0.02\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eCd + GABA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e5.00\u0026plusmn;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8.29\u0026plusmn;0.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.98\u0026plusmn;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e5.68\u0026plusmn;0.09\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e9.11\u0026plusmn;0.15\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.06\u0026plusmn;0.01\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eCd+GABA+ CPA \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e5.19\u0026plusmn;0.09\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8.66\u0026plusmn;0.15\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1.03\u0026plusmn;0.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e5.89\u0026plusmn;0.10\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e9.45\u0026plusmn;0.16\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.11 \u0026plusmn;0.02\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Impact of exogenous supplementation of melatonin and gamma-aminobutyric acid\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e on the activity of enzymatic antioxidants SOD, POD, CAT, and GST in \u003cem\u003eNostoc muscorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Anabaena\u0026nbsp;\u003c/em\u003esp. after cadmium treatment. Data signifies the mean\u0026plusmn;standard error of three replicates, each with three independent experiments (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 3). Values within same column followed by different superscripts have significant difference at \u003cem\u003eP\u0026lt;0.05\u0026nbsp;\u003c/em\u003elevel according to Duncan\u0026rsquo;s multiple range test (DMRT). Control, Cd = cadmium, MT = melatonin, GABA = gamma-aminobutyric acid, CPA = 4- Chloro DL Phenylalanine\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"1080\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Treatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 460px;\"\u003e\n \u003cp\u003e\u003cem\u003eNostoc muscorum\u003c/em\u003e \u0026nbsp;ATCC 27893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 481px;\"\u003e\n \u003cp\u003e\u003cem\u003eAnabaena\u0026nbsp;\u003c/em\u003esp. PCC 7120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003ePOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eGST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003ePOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003eGST\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 460px;\"\u003e\n \u003cp\u003e(U mg\u003csup\u003e-1\u003c/sup\u003e protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 481px;\"\u003e\n \u003cp\u003e(U mg\u003csup\u003e-1\u003c/sup\u003e protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e11.5\u0026plusmn;0.19\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e25.20\u0026plusmn;0.43\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e12.66\u0026plusmn;0.21\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e46.00\u0026plusmn;0.79\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e9.93\u0026plusmn;0.17\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e23.50\u0026plusmn;0.40\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e11.83\u0026plusmn;0.20\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e44.50\u0026plusmn;0.77\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eCd\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e15.81\u0026plusmn;0.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e32.80\u0026plusmn;0.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e16.85\u0026plusmn;0.29\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e58.05\u0026plusmn;1.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e13.43\u0026plusmn;0.23\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e29.70\u0026plusmn;0.51\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e15.31\u0026plusmn;0.26\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e54.35\u0026plusmn;0.94\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eCd+ MT\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e17.26\u0026plusmn;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e36.11\u0026plusmn;0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e19.63\u0026plusmn;0.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e73.03\u0026plusmn;1.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e14.53\u0026plusmn;0.25\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e32.98\u0026plusmn;0.57\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e17.88\u0026plusmn;0.30\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e68.59\u0026plusmn;1.18\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eCd + GABA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e18.68\u0026plusmn;0.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e39.61\u0026plusmn;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e22.16\u0026plusmn;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e83.32\u0026plusmn;1.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e15.80\u0026plusmn;0.27\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e35.97\u0026plusmn;0.62\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e20.36\u0026plusmn;0.35\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e79.04\u0026plusmn;1.36\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eCd+GABA+ CPA \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e17.98\u0026plusmn;0.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e37.90\u0026plusmn;0.65\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e20.92\u0026plusmn;0.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e78.36\u0026plusmn;1.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e15.21\u0026plusmn;0.26\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e34.41\u0026plusmn;0.59\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e19.10\u0026plusmn;0.33\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e73.91\u0026plusmn;1.28\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Abiotic stress, cyanobacteria, heavy metal, stress mitigation, signaling molecules","lastPublishedDoi":"10.21203/rs.3.rs-7857714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7857714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCyanobacteria, being nitrogen fixers most often beneficially associated with paddy crops and support the productivity. However, the growth of these symbionts is relatively affected by abiotic stresses. In the present study, the impact of Cd toxicity was explored in cyanobacteria \u003cem\u003eNostoc muscorum\u003c/em\u003e ATCC 27893 and \u003cem\u003eAnabaena\u003c/em\u003e sp. PCC 7120, in the presence of signalling molecules melatonin (MT; 90 nM) and gamma-aminobutyric acid (GABA; 40 nM). Cadmium at 5 \u0026micro;M disturbed light-harvesting pigments and photosynthesis (whole cell oxygen evolution and PS II photochemistry), and finally diminished the growth of cyanobacteria. Excessive intracellular accumulation of Cd raised the level of oxidative biomarkers i.e., superoxide radical, hydrogen peroxide, and malondialdehyde equivalents content despite of accelerated activity of enzymatic antioxidants i.e., superoxide dismutase, peroxidase, catalase, and glutathione-\u003cem\u003eS\u003c/em\u003e-transferase. Furthermore, exogenous application of MT and GABA individually supported the growth by improving light-harvesting pigments and photosynthetic activity, and it was more pronounced with GABA. This could occur due to considerable decline in Cd accumulation as a result, the declining trend in the oxidative stress biomarkers was taken place by further augmentation of antioxidant activity. The endogenous MT inhibitor CPA (90 \u0026micro;M) suggested the interactive role of MT and GABA in Cd toxicity alleviation. Hence, the study concludes that GABA, being the prime player in Cd toxicity alleviation, appears to act downstream of MT. Further, more insight is needed by involving molecular techniques to enrich the cyanobacteria with these signalling molecules to use them as a biofertilizer for sustainable agriculture, particularly paddy, even under prevailing stress conditions.\u003c/p\u003e","manuscriptTitle":"Melatonin mediates gamma-aminobutyric acid induced mitigation of cadmium toxicity on growth, PSII photochemistry, and oxidative stress in Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 11:35:23","doi":"10.21203/rs.3.rs-7857714/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4811726c-89be-4fa8-b0be-7a5ec4a7352f","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-23T05:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 11:35:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7857714","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7857714","identity":"rs-7857714","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}