Phycocyanobilin

Functional and mechanistic insights into the differential effect of the toxicant ‘Se(IV)’ in the cyanobacterium Anabaena PCC 7120

Manisha Banerjee a,c,d,*, Prakash Kalwani a,c,d, Dhiman Chakravarty a,c,d, Beena Singh b,c,d, Anand Ballal a,c,d,*

Abstract

Selenium, an essential trace element for animals, poses a threat to all forms of life above a threshold concentration. The ubiquitously present cyanobacteria, a major photosynthetic biotic component of aquatic and other ecosystems, are excellent systems to study the effects of environmental toxicants. The molecular changes that led to beneficial or detrimental effects in response to different doses of selenium oxyanion Se(IV) were analyzed in the filamentous cyanobacterium Anabaena PCC 7120. This organism showed no inhibition in growth up to 15 mg/L sodium selenite, but above this dose i.e. 20–100 mg/L of Se(IV), both growth and photosynthesis were substantially inhibited. Along with the increased accumulation of non-protein thiols, a consistent reduction in levels of ROS was observed at 10 mg/mL dose of Se(IV). High dose of Se(IV) (above 20 mg/L) enhanced endogenous reactive oxygen species (ROS)/lipid peroxidation, and decreased photosynthetic capability. Treatment with 100 mg/L Se(IV) downregulated transcription of several photosynthesis pathways-related genes such as those encoding photosystem I and II proteins, phycobilisome rod-core linker protein, phycocyanobilin, phycoerythrocyanin-associated proteins etc. Interestingly, at a dose range of 10-15 mg/L Se(IV), Anabaena showed an increase in PSII photosynthetic yield and electron transport rate (at PSII), suggesting improved photosynthesis. Se was incorporated into the Anabaena cells, and Se-enriched thylakoid membranes showed higher redox conductivity than the thylakoid membranes from untreated cells. Overall, the data supports that modulation of photosynthetic machinery is one of the crucial mechanisms responsible for the dose-dependent contrasting effect of Se(IV) observed in Anabaena.

Keywords:
Cyanobacteria
Selenium toxicity
Photosynthesis ROS
Anti-oxidants
Differentially regulated genes

Introduction

Selenium (Se), a naturally occurring metalloid, is mostly present in the form of inorganic oxyanions, selenate (SeO4− 2, +6 oxidation state) or selenite (SeO3− 2, +4 oxidation state) in the Earth’s shell. But, in the absence of oxygen i.e. under anoxic conditions, selenide (Se− 2) and elemental selenium (Se0) forms of Se predominate (Mangiapane et al., 2014). Se is an essential trace element required for the normal growth, metabolism, and development of animals, including humans. Se is incorporated as selenocysteine (the 21st amino acid) into over 25 different selenoproteins, such as glutathione peroxidases (GPx), thioredoxin reductase and iodothyronine deiodinase (DIO), which play important roles in regulating normal cellular functions (Labunskyy et al., 2014). Se is also known to exert antioxidative effects, protecting against the deleterious effects of reactive oxygen species (ROS). Although nutritionally important (in a relatively narrow concentration range), higher concentrations of Se are toxic to humans (MacFarquhar et al., 2010). Se enters into the food chain from the Earth’s surface i. e. soil) via plants that absorb and metabolize inorganic Se into a variety of organo-selenium species (seleno-methionine, seleno-cysteine, Me-Se-Cys etc) (El Mehdawi, et al., 2012). Even green algae are known to bioabsorb Se on to their cell surface and internalize it. Se metabolism or bioaccumulation has been studied in Scenedesmus (Umysova et al. 2009´ ), Chlorella (Sun et al. 2014) and Chlamydomonas (Geoffroy et al. 2007).
It is also important to note that, selenium enters into freshwater reservoirs as a runoff from agricultural or mining waste. In these waters, Se is mainly present as selenite or selenate oxyanions, whose ready bioavailability makes them particularly toxic beyond a particular threshold to the aquatic species residing in that environment. Consequently, any aquatic species, including cyanobacteria that naturally reside in these waters, are directly affected by Se pollution (Chavan and Bhattacharjee, 2016). In typical fresh water bodies, the total concentration of all form of selenium usually ranges from 0.01 to 0.1 mg/L, but in certain selenium-contaminated locations, it may even reach as high as 5–50 mg/L (Janz et al., 2011). The primary reason for Se toxicity seems to be the accumulation of malformed, non-specific Se-containing proteins and generation of oxidative stress, where selenium acting as pro-oxidant, reacts with cellular thiols, enhancing production of ROS (Hoewyk, 2013). Toxicity of Se came into attention after a major disaster that occurred at Kesterson reservoir in California, wherein concentration of selenium rose to greater than 1.4 mg/L (Winkel et al., 2012). Presently, Se toxicity is still a major concern in South Dakota (USA), Venezuela, China and India (Bajaj et al., 2011).
Comparative genomic and phylogenetic analyses reveal that Se- utilizing traits are absent in cyanobacteria. Hence, with the exception of Prochlorococcus sp, members of this phyla do not contain a selenoproteome (Santesmasses et al., 2017). But in spite of this, cyanobacteria such as Spirulina (marketed as superfood), are known to take up selenium and incorporate it non-specifically into proteins (e.g. phycocyanin) (Li et al., 2003). In cyanobacteria, earlier studies on Se have only focused on the uptake of this element into cells or monitored the absence of growth at higher levels of Se by measuring the content of photosynthetic pigments (Gouget et al., 2005). However, there is a gaping lacuna in this field because detailed mechanistic study in response to different doses of Se(IV) has not been performed in any cyanobacteria.
Nitrogen-fixing strains of cyanobacteria such as Anabaena, commonly used in paddy fields as biofertilizers, are continuously threatened by the various toxic agents present in the environment. With the advent of increasing Se content being observed in groundwater at agricultural sites (Bajaj et al., 2011), it is vital to understand how Anabaena responds and adapts to this stress. Anabaena PCC 7120 (also known as Nostoc PCC 7120) is a filamentous, nitrogen-fixing cyanobacterium that withstands high doses of gamma radiation or desiccation stress (Singh and Apte, 2018) and possess an arsenal of oxidative stress detoxifying enzymes (Banerjee et al., 2013; Cha et al., 2007; Chakravarty et al., 2019a; Ballal et al., 2020; Tailor and Ballal, 2017). Anabaena PCC 7120 has also been used as a platform for production of proteins (Chakravarty et al., 2019b). Although, effects of toxic metals like U, Cd and Ni (Acharya et al., 2017) have been studied in Anabaena PCC 7120, a detailed study characterizing the effect of the metalloid selenium has not been performed as yet. Selenate (VI) and selenite (IV) are the common water-soluble oxides of selenium found in aquatic bodies. Among the two, selenite is more toxic to the aquatic species. Our initial studies showed that treatment with 0.25 mM (~43 mg/L) sodium selenite severely diminished growth of Anabaena, whereas equimolar dose of sodium selenate (50 mg/L) had virtually no effect on the growth of this organism at the time points tested (Supp. Fig 1). As selenite was observed to be more toxic to Anabaena PCC 7120 than selenate, a thorough analysis of the effect of selenite treatment on Anabaena was carried out. This study provides a detailed mechanistic insight into the differential modulation of photosynthetic parameters or oxidative stress machinery in response to low or high dose of Se(IV) leading to beneficial or toxic effects respectively, in the cyanobacterium, Anabaena PCC 7120.

2. Materials and methods

2.1. Culture and growth conditions

The Anabaena PCC 7120 was cultivated in BG-11 liquid medium (pH 7.0) with combined nitrogen (17 mM NaNO3, BG-11N+) under continuous illumination (30 µE m− 2 s− 1), with shaking (100 rpm) or as still cultures at a temperature of 28 ◦C ± 2 ◦C. Survival of the organism was assessed by spotting the respective cultures on BG-11N+ agar plate.

2.2. Culturing of cell with Se (IV)

Sodium selenite (Sigma Life Science, Cat: 214485, Lot# MKBZ0222V) was used to make a 100 mg/mL stock in double distilled water. The solution was filter sterilized and stored at -20 ◦C. A predetermined dose of sodium selenite [ranging from 0–100 mg/L, (0–0.58 mmol/L) final concentration)] was administered to equal volumes of Anabaena culture (density 2–3 µg chlorophyll a/mL) by diluting the stock solution to the desired concentration. This ensured equal amount of starting chlorophyll a density (per mL) for control (i.e. no sodium selenite added) as well as the treatment groups. At the given time points, aliquots were removed and analyzed for different parameters.

2.3. Measurement of growth, chlorophyll a and absorption spectrum

Growth of Anabaena PCC 7120 was measured by monitoring OD (750 nm) at the depicted time points. To determine the content of chlorophyll a, cells from one mL culture were harvested by centrifugation. After discarding the supernatant , 1 mL of 90% methanol was added to the cell pellet and the mixture was thoroughly vortexed to extract the chlorophyll a. The absorbance of this solution was monitored at 664.5 nm with a spectrophotometer. Absorption spectrum (350–700 nm) of Anabaena cultures was monitored with a spectrophotometer after 72 h of treatment with various doses of sodium selenite (0–100 mg/L). All the experiments were repeated at least thrice.

2.4. Protein electrophoresis, Western blotting and immunodetection

Total cellular proteins from Anabaena cultures [control or Se(IV)- treated cells] were extracted in 20 mM Tris buffer pH 7.5, using a bead beater (MP Biomedicals, lysing matrix Cat No 6540–428). One third volume of the lysing matrix was added to the culture suspension prior to bead beating. Protein samples, prepared by adding Laemmli’s gel loading buffer, were electrophoretically separated on denaturing 12% polyacrylamide gels and electroblotted on to a nitrocellulose membrane as described earlier (Kalwani et al., 2020). Immunodetection was carried out with the respective antisera (i.e. anti-2-Cys-Prx or anti-MnSOD). Western blot analysis was repeated thrice with consistent results and representative blots are shown. Integrated optical density (IOD) of each band was calculated using “Image J” software and IOD was normalized with that of the loading control.

2.5. In vivo detection of ROS

The content of reactive oxygen species (ROS) was measured with the fluorescent probe dichlorodihydrofluorescein diacetate (DCHFDA) as described earlier (Banerjee et al., 2015). Anabaena PCC 7120 cultures (3 μg chlorophyll a mL− 1) were treated with different concentrations of sodium selenite (0–100 mg/L) for 1 hour and subsequently 10 μM DCHFDA was added to cells, which were further incubated for 20 min with shaking in dark at 25 ◦C. Subsequently, fluorescence emission (λex = 490 nm, λem = 520 nm) of the control or treated cells was measured. Experiments were repeated thrice and consistent results were obtained.

2.6. Lipid peroxidation

For malondialdehyde (MDA) estimation, cell free extracts (300 µl, 500 µg total protein) in 10 mM potassium phosphate buffer (pH 7.0) were prepared using bead beater and reacted with 900 µL of TBA reagent (0.375% 2-thiobarbituric acid, 0.25 M HCl, and 6 mM Na2EDTA). 15% trichloroacetic acid was added (to precipitate the proteins) and the reaction mixture was incubated at 95 ◦C for 20 min, cooled to room temperature, and centrifuged (10,000 rpm, 10 min). MDA equivalents in the supernatant were estimated by measuring fluorescence (λex =530 nm; λem = 590 nm). The lipid peroxidation values are calculated as nmoles of MDA equivalents per mg protein using 1, 1, 3, 3-tetra methoxy propane as standard. The content of MDA equivalents for control was considered as 100% and the other values were calculated accordingly (Chakravarty et al., 2016). Samples were harvested after 16 or 72 h of exposure to different doses of sodium selenite. Experiments were repeated at least twice.

2.7. Determination of non-protein thiols

The content of non-protein thiols (wherein GSH is the major component) of cells was measured using DTNB. After 3 h of Se(IV) treatment, the cells were suspended in 0.2 mL of 10 mM Tris/HCl, pH 7.4 and disrupted by bead beating. The lysates (containing equal amount of total protein) were treated with 10% TCA to precipitate cellular proteins. The supernatant obtained after centrifugation (14,000 g, 20 min) that contained all the small-molecule thiols was mixed with 10 μM DTNB and incubated for 10 min. Absorbance was monitored at 412 nm against appropriate control samples (i.e. without DTNB). Pure GSH (MP Biomedicals) was used to prepare the standard curve. Experiments were repeated at least twice and consistent results were obtained.

2.8. RNA sequence analysis

RNA sequencing ( next generation sequencing, NGS) was carried out at a commercial facility (AgriGenome Pvt. Ltd., Inda). The control or the Se(IV)-treated Anabaena cells were stored in Trizol (Invitrogen) at -80 ◦C prior to analysis. After extraction, total RNA was cleaved into short fragments, which were employed for cDNA synthesis. Subsequently, library was constructed and sequenced using Illumina HiSeqTM. The raw reads generated (stored in FASTQ format) were processed, assembled and aligned to Anabaena PCC 7120 genome (GCA_000009705.1) using Galaxy tool (online pipeline available at https://usegalaxy.org/) as described by Afgan et al., 2018. Two biological replicates along with two technical replicates were processed for RNA-seq analysis. Using cuff-merge option in Galaxy tool, cuff-links assemblies were generated. Final data files (gene FPKM matrices and differential expression of genes) generated by Galaxy were used to produce visualization of scatter plots, heatmap, volcano plots and MA plots. Volcano plots and MA (M, log ratio; A, mean average) plots were generated using FPKM matrix in Biojupies, an automated RNAseq data visualization server with P-value <0.05 with fold change: log2FC>2 to log2FC<-2. Scatter plots were generated using FPKM values in Origin. Heatmap was obtained by plotting log10FPKM values for all three conditions (Control, 0 mg/L; 10Se, 10 mg/L; 100Se, 100 mg/L). Table 1, Supplementary Table 2 and 3 were constructed using false discovery rate (FDR) adjusted p-value<0.05 (the default FDR for Cuffdiff is 5%) and fold change, log2FC>2 to log2FC<-2 as filters. Plot for 13 differentially expressed genes was created by log10 (number of transcripts). Level of significance was verified by Student t-test (Graphpad prism 7.05). For alternate representation and better clarity, different volcano plots and MA plots were generated using Biojupies. 2.9. Quantitative RT PCR Total RNA, from the control or the 100 mg/L Se(IV)-treated Anabaena cells, was extracted using the TRIzol® Reagent (Invitrogen, extraction steps were performed according to the manufacturer’s instructions) and employed for cDNA synthesis. RNA was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific) and its quality and integrity was verified by resolving it on the formaldehyde agarose gel as described (Banerjee et al., 2012b). The RNA samples (~1 µg) were treated with 1 U of RNase-free DNase (Sigma Aldrich) according to manufacturer’s instructions. For cDNA synthesis, 500 ng RNA along with the Readyscript™ cDNA synthesis mix (Sigma Aldrich) was incubated in a final volume of 20 µl at 42 ◦C for 1 h. Real time qPCR reaction was carried out using KAPA SYBR® FAST qPCR Master Mix (2X) Kit (KAPABIOSYSTEM) on Master cycler ep realplex Real-time PCR instrument (Eppendorf, Germany). Following thermal cycling conditions were used: 94 ◦C (2 min),45 cycles of 94 ◦C (10 s), 60 ◦C (15 s), 68 ◦C (20 s). Each reaction was performed in triplicate using 100 ng of total RNA and the corresponding primer sequences (with amplicon size <250 bp). The sequences of the six selected genes were obtained from KEGG and primers were synthesised using the primer 3 software to amplify a portion of gene (primer sequence in Supp Table 1). Specificity of the PCR amplification was assessed by melting curve analysis or by visualizing the products on 2.0 % agarose gels. For each gene (-RT) control was used under the same conditions to ascertain the baseline and threshold value for the subsequent analysis. The absolute quantification was obtained from the software in terms of Ct values. RNaseP RNA gene (rnpB), which is a housekeeping gene in Anabaena PCC 7120, was employed as an internal control (Srivastava et al., 2017). Semiquantitative RT PCR and qPCR analysis showed no variation in expression of the rnpB transcript in the control, 10Se and 100Se RNA samples (Supp. Fig 2). The relative fold change quantification of a target gene in 100Se compared to the control cells is expressed in terms of 2− ΔΔCt values after normalization with the internal control (rnpB gene). Experiments were repeated twice. 2.10. Light microscopy, SEM and TEM Control Anabaena PCC 7120 or the Se(IV)-treated cells were visualized at 40X or 100X magnification on a Carl Zeiss Axioscop 40 microscope. Images were captured with a charge-coupled device (CCD) Axiocam MRc (Zeiss) camera. Filaments lengths were counted manually. To observe the cell surface morphology, scanning electron microscopy (using camscan MV2300CT/100; operating voltage, 20 KeV) was performed. The control or Se(IV)-treated cells were harvested, washed thrice with water, dried, layered on carbon sheet and sputerred with gold (SEG, Korea). The surface was photographed by SEM while the elemental analysis was carried out using EDS (Oxford Xmas 80). Ultrastructural features of the selenite-treated as well as the control cells were viewed with the Libra 120 plus Transmission Electron Microscope (TEM, Carl Zeiss). Samples were processed for TEM as described earlier (Kulkarni et al., 2013). 2.11. PAM fluorimetry Chlorophyll fluorescence was measured using a Daul-PAM-100 fluorometer (Heinz Walz GmbH, Effletrich, Germany). After 48 h of treatment with various doses of sodium selenite (0–50 mg/L), an aliquot of cells (2.0 mL, 3-5 μg chlorophyll a/mL) was removed, washed with and re-suspended in fresh BG11 medium. Measurements were performed using the automated program (data acquisition software v1.19) provided with the Dual-PAM software and PSII activities were quantified by chlorophyll fluorescence changes. After the samples were dark adapted for 5 min, FV/FM values were obtained and electron transport rates (ETRs) in PSII [ETR(II)] were recorded during the measurement of the light response curve with increasing illumination. In the light response curve, sequence of illumination steps was carried out with increasing saturation pulse of photosynthetically active radiation (PAR). The ETR(II) is a relative measure of the rate of electron transport (rate of charge separation at PS II reaction centers). Experiments were repeated at least twice. 2.12. Subcellular fractionation Control (i.e. untreated) or 48 h Se(IV)-treated Anabaena 7120 cells were harvested, washed with buffer (0.8 M sucrose, 5 mM CaCl2, 5 mM MgCl2, 10 mM EDTA, 10 mM MES-NaOH, pH 6.35) and centrifuged at 4000 g at 4 ◦C for 15 min. Cells were incubated in cell disruption buffer (0.8 M sucrose, 5 mM CaCl2, 5 mM MgCl2, 10 mM MES-NaOH, and 1 mM phenyl sulphonyl fluoride pH 6.35) for 1 h and disrupted by a freeze thaw cycle followed by sonication. Cell extracts were centrifuged at 12,000 g at 4 ◦C for 20 min. The pellet contained unbroken cells, cell wall and other debris. Supernatant containing thylakoid membrane was subjected to ultracentrifugation at 1,00,000 g at 4 ◦C for 45 min to separate the blue coloured supernatant containing phycobilisomes and soluble proteins (soluble fraction) from the thylakoid membranes that formed a green pellet. This pellet was resuspended in 200 μl of wash buffer and stored at -80 ◦C. Chlorophyll a content of the thylakoid preparation was measured. 2.13. Cyclic voltammetry studies Cyclic voltammograms were obtained using a PGSTAT 302 N system. 0.1 M phosphate buffer, pH 7, was used as the supporting electrolyte for all systems. Prior to the voltametric scan, the solutions were de-aerated using argon gas. The electrode was polished over a micro-cloth with 0.05 μm alumina paste after each run. Three electrodes consisting of calomel electrode (reference electrode) and platinum as working as well as the counter electrode were used in the chemical cell. Thylakoid membranes (from control or Se-enriched Anabaena) at a concentration of 2.5 μg/mL chlorophyll a was resuspended in 0.1 M phosphate buffer and scanned. Experiments were repeated twice and representative plots were presented. 2.14. Estimation of Selenium using AAS Estimation of selenium was carried out according to the method described by Gota et al., 2016, with appropriate modifications. Different fractions of control or selenium-treated Anabaena cells were subjected to closed vessel microwave nitric acid digestion. During this process, all form of Se (organic or inorganic) present in the sample was converted to Se(IV). Using optimized GF program of AAS, Se content in the sample was determined and signal was measured in the peak area mode. Calibration graph was prepared for the standards in the range from 10 to 200 ng mL− 1. Samples were suitably diluted with nanopure water to obtain Se concentrations within the linear range of calibration. Instrumental outputs of ng/mL were suitably corrected for the dilution and normalized with respect to the chlorophyll a content. For the estimation of the selenium by AAS in the spent medium of differentially treated Anabaena cell and corresponding control similar method of nitric acid digestion was carried out. Reagent blanks were prepared along with the samples for blank correction. Experiments were repeated twice. 2.15. Statistical analysis Standard deviation (SD) is shown in all the graphs as error bar. To determine significant differences, “one way ANOVA” test with Tukey post hoc test (GraphPad Prism 7.04 software) was used. Student’s ’t’ test (GraphPad Prism 7.04) was employed to compare control with the 10 mg/L Se(IV) treatment group for cyclic voltammetry experiments. For the comparison of the distribution of the filament sizes in response to different doses of sodium selenite, χ2 test (https://www.graphpad. com/quickcalcs/chisquared2/) was performed. As depicted in the figures, * denotes P<0.05;** denotes P<0.01; and *** denotes P<0.001. Lack of ‘*’ symbol indicates no statistical difference between control and treatment groups. 3. Results 3.1. High doses of Se(IV) are detrimental to the growth of Anabaena PCC 7120 To glean insights into the effect of Se(IV) treatment on its growth, Anabaena PCC 7120 filaments were incubated with different doses of sodium selenite (0–100 mg/L) and their growth was assessed as a measure of chlorophyll a content and optical density (OD750) (Fig. 1A and 1B). Up to 15 mg/L dose of Se(IV), there was no inhibition in growth when compared with the control (i.e. untreated) cells. Above this concentration, significant decrease in growth was found. A minor increase in the growth was observed in the culture treated with 10 mg/L sodium selenite at the 72 h time point. In cultures treated with 20 mg/L Se(IV) or more, bleaching of pigments were observed, which was in agreement with the reduction in chlorophyll a content or OD750 shown in Fig. 1A and B. After 72 h, Se(IV)-treated or control cells were spotted on BG- 11N+ agar plate to assess their viability (Fig. 1C). At selenite concentration above 20 mg/L, a reduction in viability was observed. After 72 h, equal volumes of above-mentioned cultures were pipetted out into a 24- well plate and photographed. Clearly, as seen in Fig. 1D, at higher concentrations of Se(IV) i.e. above 20 mg/L, a profound bleaching of photosynthetic pigments was observed (Fig. 1D). 3.2. High doses of Se(IV) lead to major changes in ultrastructure and morphology As the dose of 10 mg/L had no detrimental effect on Anabaena, this concentration of Se(IV) was designated as low dose (in this and subsequent sections), whereas dose in the range of 30–100 mg/L of Se(IV) was labelled as high dose. The morphological changes in Anabaena on treatment with increasing doses of sodium selenite were initially assessed by light microscopy (Fig. 2A). After 72 h, when treated with 15 mg/L or lower dose of Se(IV), Anabaena filaments appeared similar to control filaments. However, above this dose, a gradual Se(IV) concentration-dependent decrease in the filament length was observed. At concentrations of 30 mg/L or more, a distinct reduction in the filament length was observed (Supp. Fig 3). Electron microscopic analysis was performed with the control and the representative doses in the range where beneficial (10 mg/L) or detrimental effects (100 mg/L) of Se(IV) respectively, were observed. Under the transmission electron microscope, 10 mg/L selenite-treated cells did not show any visible ultrastructural changes and resembled the control cells. But, discrete alterations were observed in the 100 mg/L treated Anabaena cells, wherein, instead of regular thylakoid membranes, bead like structures were visualized. On the other hand, there was no variation in number of thylakoid membranes, which were 3–4 membranes per stack, in both control or 10 mg/L Se(IV)-treated cells. (Fig. 2B). Scanning Electron Microscopy along with Energy Dispersive Spectroscopy (SEM-EDS) analysis revealed no deposition of Se even on the cell surface of 100 mg/ L Se(IV)–treated Anabaena cells (Fig. 2C, Supp. Fig 4). 3.3. Low doses of Se(IV) reduce ROS formation and stimulate production of non-protein thiols in Anabaena When compared with the untreated control cells, ROS generation (after 1 h) was found to be significantly enhanced at higher concentrations of Se(IV) i.e. 20–50 mg/L. Interestingly, cells treated with 10 mg/L sodium selenite showed consistently reduced levels of ROS than the corresponding control cells (Fig. 3A). Lipid peroxidation, another indicator of oxidative stress, was also monitored in Se(IV)-treated Anabaena cultures. In comparison to the control or cells exposed to low doses of Se (IV), significant enhanced levels of malondialdehyde (MDA; an end product of lipid peroxidation) were observed with 50 mg/L sodium selenite after 72 h of exposure (Fig. 3B). Expression of different ROS- responsive proteins such as 2-Cys-Peroxiredoxin (2-Cys-Prx) or superoxide dismutase (MnSOD) was assessed with specific antiserum (Fig. 3C). Interestingly, expression of 2-Cys-Prx was reduced at the concentration of 10–15 mg/L Se(IV). MnSOD, the membrane bound superoxide dismutase from Anabaena, was induced at higher concentration of sodium selenite (30–50 mg/L). Surprisingly, cells treated with 10–15 mg/L sodium selenite showed considerably increased formation of non-protein thiols, which was in good agreement with the reduced levels of ROS observed at lower concentrations of Se(IV) (Fig. 3D). 3.4. Low doses of Se(IV) enhance photosynthesis in Anabaena Photosynthetic activity of the selenite-treated Anabaena cells was monitored by measuring the PSII-mediated electron transport rates (ETRs) (Fig. 4A). Light curve of the cells treated with increasing concentration of sodium selenite (0–100 mg/L) did not follow a linear decrease in ETR. In agreement with the previous data (Fig. 1A), treatment with lower dose of sodium selenite (5–10 mg/L) resulted in a statistically significant enhancement of ETR with increasing light intensity (Fig. 4A and 4B). On the other hand, Se(IV) doses above 10 mg/L caused a gradual decrease in ETR and at 100 mg/L selenite dose, and virtually zero ETR was observed after 48 h of exposure (Fig. 4A). When compared with the untreated control, an increase in Fv/FM ratio was observed in cells exposed to low doses of sodium selenite (5–20 mg/L), whereas a decline in this ratio was observed at high doses of Se(IV) (i.e. above 30 mg/L) (Fig. 4C). Sodium selenite doses above 30 mg/L led to destruction of pigments as observed by the reduction in absorbance corresponding to chlorophyll a and phycocyanin in the spectra obtained from Anabaena cells (Fig. 4D and Fig. 4E). 3.5. Selenium is incorporated into Anabaena and Se-enriched thylakoids show higher redox conductivity A three-day old Anabaena culture was treated with 10 mg/L sodium selenite (for 48 h) and subsequently fractionated into different subcellular components. On analysis, about 15.05±3.2 ng Se/µg of chlorophyll a was observed in the whole cells of Anabaena. Further fractionation showed that 66.6% of the total incorporated Se presented itself in the insoluble fraction (cellular debris, membranes etc.), while rest was incorporated into the soluble fraction. Around 6% of the total incorporated selenium was present in the thylakoid membrane fraction (Fig. 5A). Furthermore, reduction in the concentration of selenium in the supernatant of the Anabaena cultures also indicated the incorporation of Se into the Anabaena (Supp. Fig. 5). In a control experiment, 100 mg/L sodium selenite was added to BG11 medium (without Anabaena cells) and incubated for 3 days. In this case, no precipitation was observed and no change in the Se(IV) concentration was seen (Supp. Fig. 5), indicating no interaction/interference between the components of the BG11 medium and Se(IV). The redox behavior of the thylakoid membrane was investigated by cyclic voltametry. The cyclic voltammetric current-potential curves of 0.1 M aqueous phosphate buffer solution exhibited anodic peak at 0.09 V vs calomel electrode at a scan rate of 25 mV/s, which arises due to the formation of platinum oxide, usually observed in electro-oxidation of aqueous solution at the platinum electrode. In the presence of the control thylakoid membranes (obtained from cells not exposed to 10 mg/L sodium selenite), this peak was suppressed and formation of additional peak was not observed. However, in presence of selenium-enriched thylakoid membranes, the suppression of the anodic peak due to platinum oxide (compared to the corresponding control) was considerably reduced, indicating the presence of an electrochemically conductive membrane (Fig. 5B). This was further confirmed by following the current at the anodic peak at different scan rates (25 mV/s -150 mV/s). As seen in Fig. 5C and 5D, the increase in the scan rate was accompanied by increase in the peak current. The plot of peak current as a function of square root of the scan rate was linear confirming the electrochemical reaction to be controlled by diffusion. Additionally, the slope of the linear plot derived from selenium-enriched thylakoid (5.6 × 10− 5) was considerably higher than the wild type (3.7 × 10− 5), further confirming that the Se-enriched membrane possessed higher electrochemical activity (Supp. Fig. 6). 3.6. Differential expression of genes in response to selenium Lower doses of Se(IV) (10 mg/L) showed reduced ROS formation whereas higher doses (i.e. over 30 mg/L) were detrimental for Anabaena PCC 7120. To understand the molecular basis of these changes, global alterations occurring in transcriptome of control or the Se-treated Anabaena cells were analyzed using the RNA-seq technique. The differentially expressed genes (DEGs) of Anabaena under lower dose (10 mg/L selenite, denoted as 10Se) or high dose (100 mg/L, denoted as 100Se) of Se(IV) were analysed and compared with control. Scatter plots between FPKM values of different groups showed that number of DEGs was more when Control and 100Se or 10Se and 100Se were compared (Fig. 6A). RNAseq data indicated 138 genes to be differentially expressed [Absolute fold change value: log2 (fold change) >2 for upregulated genes and log2 (fold change) <-2 for down regulated genes], among which 51 were upregulated and 87 were downregulated in 100Se with respect to the control (Table 1, Supp table 2). On the other hand, 39 genes were upregulated and 82 genes were downregulated when 100Se and 10Se were compared (Table 1, Supp Table 3). There was no significant difference in expression of genes between control and 10Se as all gene FPKM values were very near to diagonal center, which indicated less deviation in expression. In contrast, more scattering of FPKM values was observed between 100Se and Control or 100Se and 10Se (Fig. 6A, Supp. Fig. 7A and 7B). All the DEGs (with adjusted P values<0.05 and fold change: log2FC>2 to log2FC<-2) were analyzed and pathways were annotated manually using CYORF or KEGG (Table 1).   Heat map of 138 differentially expressed genes was created using log10FPKM values, which were manually clustered according to pathway annotation (Supp. Fig. 8). Heat map also showed lesser differences between Control and 10Se whereas; a clear disparity was observed when the profile of Control or 10Se was compared with that of 100Se. Multiple genes related to energy metabolism, particularly those encoding components of photosynthesis, oxidative stress responsive genes, transcriptional regulators, heavy metal, iron transport, were differentially expressed in response to high dose (100Se) of sodium selenite (Supp. Table 2). When 100Se was compared with 10Se, similar pathway related genes were found to be differentially expressed (Supp. Table 3). Genes such as pecA, pecC, pecF encoding phycoerythrocyanin- associated proteins; cpcD, cpcE, cpcG1 and cpcG3, encoding phycobilisome rod like proteins; apcC, phycobilisome core linker protein; psbW, psbM, encoding photosystem II related-proteins, all4000, all4002, all4003, encoding photosystem II CP43 protein (PsbC homolog); the Photosystem I associated psaI, psaJ, and psaK genes were all down regulated 2–7 log2 fold in 100Se than in control or 10Se. Nitrogen metabolism related genes like nirA, nrtA, nrtC, narB too showed 3–5 log2 fold decrease in 100Se sample. Expression of receptors/transporters for metals such as Fe (all2674, alr0397, all0388, all0389, all1101) or Cr (all1110) or Cu (alr1627) was reduced by 3–5 log2 fold. Oxidative phosphorylation-related gene (all0948), sulfur metabolism (alr4512) and oxidative stress-responsive genes (encoding catalase, thioredoxin) were upregulated by 3–4 folds after treatment with 100 mg/L sodium selenite. Transcriptional regulators encoded by ORFs alr1629, all3903 were induced under selenium stress. Many other genes encoding unknown protein or hypothetical proteins were also differentially modulated (Supp. Table 2, 3). 4. Discussion Although Se is not an essential element for plants (as selenoproteins are not encoded in the land plant genomes), low doses of Se (0.1 mg kg− 1 soil) are known to promote growth of many plants (Sun et al., 2010). Promotion of growth along with increase in photosynthetic pigments is also reported for the unicellular green algae Chlorella vulgaris with 75 mg/L sodium selenite (Babaei et al., 2017). But, so far, no specific reports on the nutritional requirement of Se or the presence of selenoproteins exist in cyanobacteria. Interestingly, in a manner similar to the eukaryotic plants, the filamentous cyanobacterium Anabaena PCC 7120, showed improved physiological parameters (e.g. increased photosynthesis, reduced ROS content etc.) with low doses of sodium selenite (10 mg/L). On the other hand, concentration of selenite beyond 20 mg/L was toxic to Anabaena, affecting vital parameters such as survival, filament length etc. The considerable damage that occurred to the cellular ultra-structure with high dose of selenite (100 mg/L) also supported this observation. In earlier studies, similar fragmentation of filaments was observed with stresors such as H2O2 and methyl viologen (Banerjee et al., 2012). Other metals such as arsenic, copper, cadmium or stresses such as ionizing radiation are also known to cause fragmentation of filaments and loss in pigments in Anabaena sp. PCC 7120 (Singh et al., 2013; Singh et al., 2014). Thus, when Anabaena is not able to tolerate a stress, it begins to lose its viability, fragmentation of filaments occurs and chlorophyll a content decreases substantially. Interestingly, this study has revealed that 10 mg/L dose of selenite, which is much higher than the concentration found in natural aquatic bodies (0.01–0.1 mg/L) is not only tolerated well by this organism, but also shows a beneficial effect. In the cyanobacterium Synechococcus leopoliensis, exposure to high level of Se (5 g/L) resulted in formation of selenium particles within the thylakoids of this cyanobacterium (Hnain et al., 2013). However, such deposits were not observed in Anabaena, but high dose of selenite, as mentioned earlier, caused ultrastructural damage (Fig. 2B). Cyanobacteria, being the progenitors of chloroplasts, are excellent model systems, especially for plants, to study the various fundamental aspects of environmental stresses. In Anabaena, treatment with high Se (IV) (100 mg/L) caused a collapse in thylakoid structure, and a concomitant loss in the photosynthetic activity was observed (Fig. 2B). Similarly, the first targets of Se cytotoxicity in Chlamydomonas reinhardtii are chloroplasts, wherein Se affects the integrity of stroma, thylakoids and pyrenoids (Morlon et al. 2005). Interestingly, RNAseq analysis showed genes encoding photosynthetic pathways-related proteins to represent a major number of the differentially regulated genes in the 100Se sample, which was in line with the observation that photosynthesis was affected at high doses of Se. Down regulation of genes encoding components of phycobillisome complex, PSI complex or PSII complex (Supp. Table 2) suggests reduced ability of these complexes to be repaired or resynthesized after damage caused by high doses of Se. The other major consequence of selenium toxicity is the increased production of reactive oxygen species (ROS) i.e. oxidative stress. High doses of Se (50 mg/L) enhanced ROS generation in Anabaena, which correlated well with the induction of SOD (Fig. 3). Another cyanobacterium Spirulina platensis, also shows increased activity of the antioxidative enzymes in response to selenium (Chen et al., 2008). In plants too, increased ROS along with a subsequent increase in anti-oxidant capability was observed with high doses of selenite and in fact, a dose-dependent effect of Se was very evident for production of Se-enriched rice (Dai et al., 2019). RNAseq analysis revealed transcriptional elevation of genes encoding ROS-alleviating proteins such as thioredoxin A and the Mn-catalase, (KatB) in response to high dose of Se in Anabaena. Two transcription regulators, alr1629 & all3903 (Supp. Table 2), which in turn are regulated by the master redox regulator, FurA (Gonzalez et al., 2014´ ), were also induced in the 100Se sample. Thus, high dose of Se, which causes oxidative stress and increased lipid peroxidation, appears to activate genes whose products are components of ROS scavenging machinery. Interestingly, a decrease in production of oxidative stress-inducible 2-Cys-Prx, indicating a reduction in the content of ROS at lower doses of selenite (10 mg/L) was observed (Fig. 3C). The reason for the reduced formation of ROS at lower dose of Se treatment is not clear, but increased accumulation of the non-protein thiols at these doses (Fig. 3D) may play a role in this process. Previous studies have largely described the beneficial or detrimental effect of Se on photosynthetic organisms by measuring growth or monitoring the content of photosynthetic pigments (Hnain et al., 2013). In our present study, we have dissected the molecular basis of this mechanism. Pulse amplitude modulated fluorimetry technique that estimates quantum yield (Fv/FM) of photosystem II (PSII) is known to underestimate the quantum yield in cyanobacteria. The Fv/FM, which is around 0.8 in higher plant leaves, is in the range of 0.3–0.6 in cyanobacteria (Ogawa et al., 2017). These lower values are not due to any inherently inefficient processes, but are largely due to the fluorescence of the phycobilisomes that interferes with the measurements. However, in spite of these issues, PAM method can be gainfully utilized for monitoring alteration in PSII yield in a single species of cyanobacterium under different stressful conditions (as done in this study) when the information is to be used for comparative analysis (Schuurmans et al., 2015). In Anabaena PCC 7120, photosynthetic yield and electron transport rate of PSII increased at low doses of Se (IV), while at higher doses, gradual decrease in these photosynthetic parameters was observed. Cyanobacteria are equipped with well-organized intracellular thylakoid membrane systems, which contain photosynthetic apparatus that performs photosynthesis. Iron-sulfur (Fe-S) clusters are known to play a vital role as electron transfer cofactors in the cytochrome b6f complex or ferredoxin proteins (Jin et al., 2008). Because of their chemical similarities, selenium can substitute sulfur in iron–sulfur proteins located on the thylakoid membrane. Previous studies suggest that both clusters (i.e. [Fe-S] or [Fe-Se]) that are associated with ferredoxin, transfer electrons with almost equal efficiency (Moulis et al., 1982), but excess inclusion of Se in the proteins leads to disruption of the photosynthetic electron transport chain (Geoffroy et al., 2007). In this study too, the dual nature of selenium-related effects were observed i.e. at low doses, incorporation of Se facilitated electron transport rate during photosynthesis (Fig. 4, 5), probably by utilizing both acceptor and donor properties of Se (Lothrop et al., 2014), but excess treatment of Se resulted in diminished photosynthesis, possibly due to the accumulation of the malformed selenoproteins that eventually damage the photosynthetic machinery. Cysteine desulfurases, enzymes that remove sulfur from cysteine for biogenesis of Fe-S cluster, are also known to function as selenolyase. Anabaena PCC 7120 has four cysteine desulfurases (Banerjee et al, 2017), but role of these in metabolism of Se remains unexplored. The cyanobacterial thylakoid membranes serve as a matrix for photosynthetic redox active complexes, catalysing the light-dependent reactions. Interestingly, inclusion of Se into thylakoids resulted in enhancement of the redox conductive properties of the thylakoid membranes (Fig. 5). Agostiano et al. in their electrochemical study of spinach-derived thylakoids have proposed the suppression of platinum oxide peak due to the formation of a low conductive membrane at the electrode–thylakoid interface (Agostiano et al, 1993). On the other hand, appearance of the same peak in the selenium-enriched thylakoid indicates the presence of an electrochemically conductive membrane. Thylakoid essentially functions with the help complexes such as photosystem (PS) I and II and the cytochrome b6/f complex. At this low dose, incorporation of Se into the photosynthetic machinery components may give rise to increase in conductivity. Recently, cyanobacteria have generated a lot of interest for their potential use in generation of eco-friendly power using sunlight and water (Tshortner et. al., 2019). Thus, in principle, cyanobacteria with Se-enriched thylakoids may serve as an improved tool for this energy conversion process. 5. Conclusion Treatment with 10–15 mg/L of Se(IV) resulted in enhanced photosynthesis, whereas higher doses (above 20 mg/L) were detrimental for Anabaena. Detailed analysis showed low doses of Se(IV) to improve the redox status of the cell, and an overall improvement in the photosynthetic efficiency (including redox conductivity of thylakoid membranes) was observed. In contrast to high doses, lower doses of Se(IV) did not significantly alter the general transcriptional status of Anabaena cells. High doses of Se(IV) caused oxidative stress and notably, a sharp reduction in all the photosynthetic parameters was observed, which correlated well with the reduced expression of photosynthetic pathways- related genes. 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