Microcl 21

In brief, the root and shoot samples (0.1 g) were independently homogenized with 100 mM phosphate (pH 7.0) in a mortar and pestle. The homogenate was centrifuged at 8000 rpm for 10 min (MicroCL 21, Thermo Scientific, United States)., and the transparent portion was collected for analysis. We first analyzed the SOD activity by mixing the plant extract (100 µl) with 0.1 mM of EDTA, 50 mM of NaHCO₃ (pH 9.8), and 0.6 mM of epinephrine^{32 (link)}. After 4 min, the adrenochrome confirmation was read at 475 in a spectrophotometer. Secondly, the plant extract (100 µl) was supplemented with 1 ml of assay solution with 0.1 mM EDTA, 0.1 mM H₂O₂, 0.5 mM ascorbic acid, and 50 mM phosphate^{33 (link)}. The absorbance of the mixture was then observed at 290 nm and thereafter calculated for APX activity by extinction coefficient (2.8 mM⁻¹ cm⁻¹). Besides, 100 µl extract was mixed up with 100 mM phosphate buffer and 6% hydrogen peroxide solution. The absorbance was read twice in 30 s interval and thereafter calculated for the activity of CAT using a coefficient of 0.036 mM⁻¹ cm⁻¹). Lastly, 100 mM phosphate, 1 mM EDTA, 20 mM GSSG and 0.2 mM NADPH added individually to 100 μL of plant extract. The reaction was initiated by GSSG, and absorption was reduced by NADPH-oxidation at 340 nm. The calculation of GR activity was then performed by the extinction coefficient (6.12 mM⁻¹ cm⁻¹)^{34 (link)}.

The Phytoxigene™ CyanoDTec (Diagnostic Technology, Belrose, Australia) multiplex quantitative real-time PCR assay was applied to determine the gene copies of the 16s rRNA gene (total cyanobacteria) and the mcyE, cyrA, sxtA genes (microcystins, cylindrospermopsin, saxitoxins, respectively). The kit was used according to the manufacturer’s instructions and PCR was carried out in a Smartcycler II system (Cepheid). In brief, a volume of water sample (1 to 15 mL, depending on visual cell density) was filtered through a Nucleopore 25 mm, 0.8 µm filter (Whatman, Little Chalfont, UK) using a syringe and filter holder. DNA extraction of filters was carried out using BioGx bead lysis tubes (Diagnostic Technology) in a BeadBug bead beater (Benchmark Scientific). Extracts were centrifuged (MicroCL 21, Thermo Fisher Scientific, Waltham, MA, USA) and proceeded to qPCR. Quantitation of gene copies was based on calibrations with the Phytoxigene™ CyanoNAS standards (100–1,000,000 copies per reaction).

The ultimate consequence of damage of the cell membrane integrity in both root and shoot were measured by using a conductivity meter with some modifications^{26 (link)}. Root and shoot surface components were washed with deionized water. Thereafter, the freshly harvested samples were transferred into a beaker filled with 20 ml deionized water and kept at 25 °C for 2 h. Later, the solution’s electrical conductivity (EC1) was calculated. Afterward, the samples were heated in a water bath for 20 min at 95 °C then soothed at 25 °C before recording the final electrical conductivity (EC2). The electrolyte leakage was then determined as follows: = (EC1/EC2) × 100 (%).
Evans blue was determined to account for the cell death rate with some modifications^{27 (link)}. The entire fresh root and shoot were placed into 2 mL of Evan's blue mixture for 15 min. The samples were then mixed with 80% ethyl alcohol and placed at room temperature for 10 min. Afterward, the solutions were incubated in a water bath for 15 min at 50 °C and were further centrifuged for 10 min at 12,000 rpm (MicroCL 21, Thermo Scientific, United States). The supernatant was then transferred into a new centrifuge tube before measuring the absorbance at 600 nm. Eventually, the % of cell death was calculated.