ARTICLE HIGHLIGHTS
Environmental stress induces phytohormones synthesis in soil microorganisms. The present work is an experimental evidence of the temperature-induced synthesis of phytohormones Indole-3-acetic acid (IAA) in filamentous and single-celled cyanobacteria. Effect of ambient (25°C) and non-ambient temperatures (15°C, 35°C, and 45°C) on IAA production was studied. The results indicate that at non-ambient temperatures, the amount of stress generated are directly proportional to amount of IAA produced.
1. INTRODUCTION
The cyanobacteria are the microorganisms that are best suited to a variety of harsh environments. The earliest known photosynthetic species are cyanobacteria, which initially appeared 3.5 billion years ago. In addition to terrestrial, freshwater, and marine ecosystems, ice shelves, bare rocks, hot springs, and arctic and antarctic lakes, cyanobacteria can be found in a variety of situations [1]. Temperature stress is one evident stress brought on by seasonal and daily variations in climate [2]. Climate change and global warming increase the importance of temperature as one of the major stress factors that could be examined to understand the survival strategy of various extremophiles [3,4]. Based on their tolerance to temperature, cyanobacteria are classified into thermophilic, mesophilic, and psychrophilic. Hence, cyanobacteria are utilized as model organisms for determining defense strategies against stresses [5]. Defense strategies depend on how the species react to and adapt to heat stress. Cyanobacteria encounter both short- and long-term stress episodes, and they can adapt to the stress by changing their morphology, metabolism, and genetic makeup [6,7].
Many agricultural soils are home to cyanobacteria, which help with biological nitrogen fixation, phosphate solubilization, and mineral release to increase soil fertility and crop yield [8-10]. However, many cyanobacteria are also known to release a variety of biologically active substances, such as proteins, vitamins, carbohydrates, amino acids, polysaccharides, and phytohormones, which act as elicitor molecules to promote plant growth and aid them in the defense against biotic and abiotic stress [11,12]. This is in addition to naturally fertilizing and balancing the mineral nutrition in the soil. Auxins, gibberellins, cytokinins, and ethylene, which are involved in plants’ growth and development, are known to be significantly accumulated and released by various genera of cyanobacteria and algae [13-15]. Indole-3-acetic acid (IAA), a well-known auxin promotes plant development by controlling root elongation, tropic response, cell division, elongation, and differentiation [16]. IAA production has been demonstrated in a variety of cyanobacteria [17-19]. IAA-containing cyanobacterial extracts cause rooting and shooting in explants [20]. It has been noted that cyanobacteria can produce IAA by both tryptophan-dependent and tryptophan-independent pathways [21]. The following three IAA production pathways used by microbes are: Indole-3-acetamide (IAM) pathway, indole-3-acetonitrile (IAN) pathway, and indole-3-pyruvic acid (IPA) pathway. Bacteria use the IAM and IPA pathways more frequently than the IAN pathway, and they are all tryptophan-dependent [22]. L-tryptophan, a precursor to IAA, is needed by a number of cyanobacterial strains to produce the phytohormone. Cyanobacteria are a significant part of the flora in agricultural fields, thus it is crucial to understand how they contribute to the generation of IAA under temperature stress. In addition, stress alters physiological, biochemical, and morphological responses and negatively impacts cell growth and development [23].
We therefore set out to screen IAA-producing cyanobacterial strains and examine how temperature affects growth (as measured by cell density and chlorophyll A content), biochemical parameters (as measured by carbohydrate, protein, lipids, and extracellular polysaccharide), and stress markers such as malondialdehyde (MDA), Catalase (CAT), and ROS. As a result, their prospective use as a biostimulant would be established by the measurement of IAA produced by these cyanobacterial species under temperature stress.
2. MATERIALS AND METHODS
2.1. Sample Collection, Isolation, and Purification
Cyanobacterial samples were isolated from soil crust of coastal regions of Balasore district, Odisha. Crusts were inoculated on petri plates with BG-11 agar media and incubated under fluorescent light of 7.5 W/m2 intensity. After 7 days of incubation, some cyanobacterial colonies were seen growing in the media plate. These cyanobacterial colonies were purified by repeated culturing and transfer to a new media plate. A total of seven cyanobacterial strains were isolated following this method. Subsequently, the purified strains were transferred to Erlenmeyer flasks containing 25 mL of the same medium under the same conditions for maintenance. Axenic cyanobacterial cultures were obtained by treatment with an antibiotic mixture of 200 μg/mL ampicillin and 100 μg/mL streptomycin.
2.2. Detection of IAA
Salkowski colorimetric method was used to screen IAA-producing cyanobacteria [24]. 100mg of pure cultures of cyanobacterial isolates were inoculated into BG-11 medium and incubated at RT for 14 days. The cultures were kept in an aseptic environment and supplemented with various concentrations of filter sterilized tryptophan (0.1, 0.25, 0.5, 1, 2.5, and 5mg/ml) followed by incubation under 12:12 h light: Dark cycle conditions. After 14 days of incubation, the cultures were centrifuged at 10,000 RPM for 10 min and the supernatant was collected for IAA assay. The supernatant was then exposed to Salkowski’s colorimetric assay and the absorbance was measured at 535 nm.
2.3. Ultraviolet-Visible (UV-Vis) Spectrum of IAA Produced by Cyanobacteria
The spectral analysis of cell-free supernatant was done at variable wavelength using a UV-Vis spectrophotometer. The spectrum was run from 190 nm to 600 nm using appropriately diluted sample and compared with the standard IAA (11 μg/ml and 33 μg/ml).
2.4. Polyphasic Identification of Cyanobacterial Strains
Three cyanobacterial isolates that showed maximum IAA production were identified based on morphology following Komárek [25] and further confirmed by 16S rRNA gene sequencing. For phylogenetic analysis, total DNA was isolated using phenol-chloroform method. A part of the 16S rRNA gene was amplified using cyanobacterial universal primer 106F (5’CGGACGGGTGAGTAACGCGTGA3’) and 1387R (5’TAACGACTTCGGGCGTGACC3’). 20 μL reaction mixture was prepared which comprises 10 μL of SRL Taq Mix polymerase chain reaction (PCR) 2X Master Mix, 2.5 μL of the template DNA, 1 μL each of 10 mM of forward and reverse primers, and 5.5 μL of sterile water. The PCR thermal cycle comprised an initial denaturation step at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min 30 s, 72°C for 1 min 30 s, and the final extension at 72°C for 15 min. The 1281 bp amplified product was purified using Qiagen Gel purification kit. The gel-purified PCR products were submitted to Eurofins Genomics India Pvt. Ltd, Bengaluru, India, for sequencing. Sequences of 16S rRNA gene were compared with NCBI sequence database [GenBank] through BLAST (www.ncbi.nlm.nih.gov/BLAST).
2.5. Exposure of Cyanobacterial Strains to Temperature Stress
A seed culture was set up with 5 ml BG11 broth to which three axenic cyanobacterial cultures were inoculated and incubated for 3 days under fluorescent light of 7.5 W/m2 intensity at 25 ± 2°C under 12:12 h light: Dark cycle conditions. Once the cells are in log phase, they are sub cultured into four 50 ml BG 11 broth and again incubated for 5 days same conditions till the cells are in log phase. After 5 days, the four flasks were shifted to 15°C, 25°C, 35°C, and 45°C, respectively, and incubated under fluorescent light of 7.5 W/m2 for 12:12 h light: Dark cycle conditions. After 15 days, samples were harvested for biochemical analysis and estimation of growth.
2.6. Estimation of Growth
To estimate the total biomass, 1 ml culture was taken and homogenized. The optical density of the homogenized cultures was measured at 720 nm following which the samples were dried and the dry weight was estimated. In the case of cyanobacteria, total chlorophyll content is an indirect measurement of growth. For this, 1 ml culture is centrifuged at 10,000 rpm. To the pellet, 4 ml methanol is added and vortexed thoroughly. The mixture is incubated at 60°C water bath for 1 h. After incubation, the tubes are cooled and centrifuged at 10,000 rpm and the clear supernatant is taken for measuring OD at 665 nm.
2.7. Estimation of Macromolecular Content
The total carbohydrate content was estimated using Anthrone method [26]. Total protein content was estimated using Lowry method [27]. The total lipid was estimated using Vanillin-Phosphoric acid reagent [28]. Exopolysaccharide (EPS) estimation was done by using Anthrone reagent [29]. MDA content was estimated by protocol proposed by Heath and Packer, 1968 [30], CAT assay was done as per the protocol in the cited literature [31], and ROS estimation was done by protocol proposed by Able et al. [32].
3. RESULTS AND DISCUSSION
3.1. Screening of IAA Producing Strains
Seven cyanobacterial isolates were purified and named as FMU-PC1 to FMU-PC7. These isolates were tested for their ability to produce IAA using varying amounts (0.1, 0.25, 0.5, 1, 2.5, and 5 mg/ml) of tryptophan as a precursor. Production of IAA was concentration-dependent and increased with increasing concentration of L-tryptophan in the medium [Figure 1a]. This suggests that the tryptophan-dependent route for IAA synthesis is present in all strains. Spectral analysis using UV-visible spectrometry was done as a qualitative check for the sample purity and identity. The maximum absorption wavelength (λmax) of IAA obtained from the cell-free supernatant of cyanobacterial samples was compared with the λmax of standard IAA. The λmax of purified IAA was obtained in the range of 290–294 nm whereas that of standard IAA was obtained at 290 nm which confirmed the purity and quality of IAA produced by selected cyanobacterial species [Figure 1b].
 | Figure 1: (a) Tryptophan concentration dependent indole-3-acetic acid production by seven cyanobacterial pure cultures obtained from coastal regions of Balasore district of Odisha, India and (b) ultraviolet-visible spectral analysis of indole-3-acetic acid of cyanobacterial samples. [Click here to view] |
Out of seven strains, FMU-PC5 produced the highest concentration of IAA and the lowest level was observed in FMU-PC7. Since under stress conditions, different strains behave differently by altering their growth, metabolism, and physiology, we therefore selected three strains one unicellular (FMU-PC2) and two filamentous (FMU-PC1 and FMU-PC5) cyanobacteria for further study.
3.2. Molecular Analysis of the IAA-Producing Strains
Molecular characterization by 16S rRNA confirmed that FMU-PC1 pure cultures showed 100% similarity to Westiellopsis sp. TPR-29 (GenBank: MT350511.1), FMU-PC5 showed 100% similarity to Hapalosiphon sp. Ryu2-7DN_D3 (GenBank: LC325255.1), and FMU-PC2 showed 100% similarity to Chlorogloeopsis fritschii PCC 6912 (GenBank: MK953013.1).
3.3. Effect of Temperature on Growth
According to estimates, global warming may cause an increase of 0.2°C every 10 years [33]. Climate change-related abiotic stress induces cyanobacteria, which are known for their remarkable adaptability to many environmental situations, to change their metabolic function [5]. Temperature variation influences a wide range of biological processes, including the physiology and metabolism of cyanobacteria. The maximum biomass formation was observed at 25°C since this is the optimal temperature for cyanobacterial growth. As an indirect indicator of growth, chlorophyll A concentration is likewise reported to be maximum at 25°C. Both biomass (dry cell weight) [Figure 2a] and chlorophyll A concentration [Figure 2b] gradually decreased when temperature rose to 35°C and 45°C. The presence of chlorophyll A shows that the cells were still alive even though the dry cell weight had decreased. It is interesting that exposure to low temperatures, that is, 15°C, had little impact on growth. Cyanobacteria are widely known for their ability to live at extremely low temperatures, even down to 20°C [34]. This may be because desaturation of fatty acids in the membrane happens at low temperatures and activates certain enzymes that increase the efficiency of transcription and translation, enabling growth and metabolism at below-ambient temperatures [5]. This trend was obtained for all three strains Westiellopsis sp. TPR-29, Hapalosiphon sp. Ryu2-7DN_D3 and C. fritschii PCC 6912. Hapalosiphon sp. Ryu2-7DN_D3 and Westiellopsis sp. TPR-29 were more resistant to higher temperature, whereas C. fritschii PCC 6912 showed better growth at 15°C.
 | Figure 2: (a) Dry weight of cyanobacterial samples and (b) chlorophyll A content of cyanobacterial samples under different temperature. [Click here to view] |
3.4. Effect of Temperature on Carbohydrate Content
It was observed that 25°C was the ideal temperature for promoting growth and carbohydrate buildup because, at this temperature, cells’ capacity to use carbon and nitrogen rises dramatically. Higher temperature influences cellular physiology and results in denaturation of vital metabolites (enzymes/proteins), which lowers CO2 fixation, while lower temperatures during growth result in reduced electron transport [35]. As a result, the carbohydrate content was lower at 15°C and 35°C than it should have been. While at 45°C, there was a drastic fall in carbohydrate content, indicating that higher temperatures impair an organism’s ability to photosynthesize [Figure 3a]. All strains showed this tendency, with the exception of C. fritschii PCC 6912, whose carbohydrate content was nearly the same at 15°C and 25°C, showing that this species has a wide range of tolerance for low temperatures and can live and photosynthesize even at lower temperatures.
 | Figure 3: (a) Total carbohydrate content; (b) total protein content; (c) total lipid content; and (d) EPS content of cyanobacterial samples under different temperature. [Click here to view] |
3.5. Effect of Temperature on Protein Content
Under high-temperature stress, proteins frequently aggregate and desaturate, disrupting their transport and activities [5]. This is one of the main causes of the low protein content at 35°C and 45°C. All of the three strains showed this pattern [Figure 3b]. However, compared to the ambient temperature of 25°C, the protein concentration was considerably lower at 15°C. Low protein content may be a result of the cellular metabolism slowing down at non-ambient temperatures like 15°C, which also affects growth rate and total cell biomass, as seen in our study.
3.6. Effect of Temperature on Lipid Content
The generation of fatty acids by cyanobacteria can be drastically impacted by temperature. The lipid content of the cell often changes in response to stresses including temperature, salt, desiccation, and photoinhibition [36]. In the present study, it was observed that elevated temperature led to increase in lipid content [Figure 3c]. Maximal lipid concentration was found at 35°C, while maximal biomass formation is seen at 25°C. All of the strains’ total lipid contents increased along with the temperature as it went from 15°C to 35°C. Increased lipid production in response to temperature is likely caused by the requirement to stabilize membranes to sustain vital physiological functions. However, it was fascinating to note that very little lipid accumulation occurred at 45°C.
3.7. Effect of Temperature on Exopolysaccharide Content
EPSs provide a milieu that is structurally stable and well-hydrated for colonization of cyanobacteria. In addition, it offers chemical and physical defense against biotic and abiotic stressors. It is believed that EPS excretion serves as a physiological reaction to changes in the environment, enabling cyanobacteria to keep up their fitness while simultaneously supporting the expansion of other cohabiting organisms [37]. In this study, it was observed that the generation of EPS increased when the temperature rose from 15°C to 35°C. At 35°C, the highest EPS production was observed [Figure 3d]. According to earlier research, filamentous cyanobacteria produce their most EPS between 27°C and 34°C. Higher levels of EPS are produced and deposited at higher temperatures as a response to anticipated desiccation because desiccation is typically associated with greater temperatures [29].
3.8. Effect of Temperature on Stress Markers
Cyanobacterial physiology and metabolic behavior are predominantly impacted by abiotic stressors. As a result, a number of stimuli and other defense mechanisms are known to be induced in response to stress. One of them is reactive oxygen species (ROS) which are produced in response to various metabolic stress conditions. ROS can be reduced by a variety of enzymes, including superoxide dismutases (SOD), CAT, and guaiacol peroxidase, as well as non-enzymatic compounds such as carotenoids and glutathione reductase [38]. However, excessive free radical production can cause oxidative stress, which can harm cellular nucleic acids and structural integrity [39]. However, with an effective defense and repair system, cyanobacteria may recover from oxidative damages. This study looked at how different temperatures affected the levels of ROS [Figure 4a], CAT [Figure 4b] and MDA [Figure 4c]. In all of the non-ambient temperature conditions, as was to be predicted, the levels of all three stress markers were high, with the greatest levels being seen at 45°C [Figure 4a-c]. Antioxidant marker production suggests that non-optimal temperature causes a general stress response by generating ROS.
 | Figure 4: (a) Reactive oxygen species content; (b) catalase activity; and (c) malondialdehyde content of cyanobacterial samples under different temperature. [Click here to view] |
3.9. Effect of Temperature on IAA Production
IAA is a natural auxin that is synthesized in many species of non-seedling plants, bacteria, fungi, and algae [40]. A number of cyanobacteria have been reported to produce IAA. Several works have reported the effect of pH, salinity, incubation time, and concentration of Tryptophan on IAA production [41,42], but there are almost no records of effect of temperature on IAA synthesis by cyanobacteria. Effect of temperature on IAA production by various bacteria [43] and yeast [44] has been well established; hence, this study explored the role of temperature as an inducer of IAA production by cyanobacteria. It was observed that in all three strains IAA production was comparatively higher in non-ambient temperatures like (15°C, 35°C, and 45°C) than 25°C which is the optimum temperature for cyanobacterial growth. It was expected that at higher temperatures IAA production might be high, but in the case of Hapalosiphon sp. Ryu2-7DN_D3 and Westiellopsis sp. TPR-29, elevated amounts of IAA production were observed even at 15°C. Only C. fritschii PCC 6912 showed higher IAA titers at higher temperature. At 15°C, the IAA production by C. fritschii PCC 6912 was much less than the amount produced at optimum temperature. Overall highest titers of IAA were observed in Hapalosiphon sp. Ryu2-7DN_D3 followed by Westiellopsis sp. TPR-29 and C. fritschii PCC 6912 under various temperatures [Figure 5].
 | Figure 5: Indole-3-acetic acid produced by cyanobacterial samples under different temperature. [Click here to view] |
A two-way ANOVA was performed to analyze the effect of temperature on IAA production across different cyanobacterial strain. The amount of IAA produced by different strains at different temperature was statistically significant with a P-value of 2.51851e-14 (P < 0.05). Difference in IAA production by three cyanobacterial strains was also statistically significant with a P-value of 7.01295e-11 (P < 0.05). Similarly, statistically significant variation was observed in IAA production at various temperatures (P-value of 4.77249743587743e-17). The detailed two-way ANOVA analysis is presented in Table 1. This experiment indicates that any fluctuation in environmental temperature induces the cyanobacterial species to synthesize phytohormones like IAA as a defense mechanism to protect itself from metabolic stress.
Table 1: Two-way ANOVA analysis on effect of temperature on IAA production in various cyanobacterial species.
| Source of variation | SS | df | MS | F | P-value | F crit |
|---|---|---|---|---|---|---|
| Cyanobacterial strains | 3244089 | 2 | 1622045 | 47.97648 | 7.01E-11 | 3.259446 |
| Temperature | 9472744 | 3 | 3157581 | 93.39425 | 4.77E-17 | 2.866266 |
| Interaction | 7922317 | 6 | 1320386 | 39.0541 | 2.52E-14 | 2.363751 |
| Within | 1217130 | 36 | 33809.16 | |||
| Total | 21856281 | 47 |
ANOVA: Analysis of variance, IAA: Indole-3-acetic acid
Previously, it has been reported that among many compounds that cyanobacteria produce, IAA promotes growth and prevents organisms from environmental stress such as desiccation, osmotic, cold shock, and heat shock. Further, IAA activates the antioxidant activity in the cell by inducing the expression of antioxidant enzymes such as CAT, SOD, and peroxidase [45]. A similar results have also been obtained in the present study which corroborates with the cited literature.
4. CONCLUSION
The present study showed that temperature stress acts as an elicitor for IAA production by cyanobacterial strains. On the basis of habitat and tolerance of cyanobacteria toward temperature, they are classified as thermophilic, mesophilic, and psychrophilic; hence, these temperature ranges (15°C, 25°C, 35°C, and 45°C) were selected for the study [46]. As expected at optimum temperature of 25°C, the biomass and chlorophyll content as well as macromolecular content was highest for all selected species. Whereas at non-ambient temperatures, there was a concomitant decline in all growth and biochemical parameters, indicating the effect of temperature stress on physiology of the strains. As the strains were exposed to higher temperature, it resulted in reduction of growth and macromolecular contents whereas ROS and MDA content increased significantly at 45°C with a concomitant increase in antioxidant enzymes like CAT. Increase of ROS, MDA, and CAT levels at non-ambient conditions clearly emphasizes that fluctuations in incubation temperature elicit stress response in cyanobacteria. While these observations were similar across the selected temperature ranges for all three strains, but in terms of tolerance, Hapalosiphon sp. Ryu2-7DN_D3 was most tolerant to variations in temperature. The higher tolerance of Hapalosiphon sp. Ryu2-7DN_D3 is evident from the fact that it had higher growth as well as biomolecular content as compared to other strains during temperature stress. Whereas the unicellular cyanobacteria, C. fritschii PCC 6912 was most sensitive to temperature change. Most importantly, the IAA content was observed to be higher in non-ambient conditions (15°C, 35°C, 45°C). Hapalosiphon sp. Ryu2-7DN_D3 and Westiellopsis sp. TPR-29 produced almost similar levels of IAA at 15°C, 35°C, and 45°C. However, C. fritschii PCC 6912 produced IAA only at 25°C, 35°C, 45°C and the levels decreased at 15°C. Production of IAA at non-ambient temperature indicates that abiotic stress induces phytohormone production in cyanobacterial strains as a defense strategy to protect itself against changing environmental conditions.
Apart from protecting itself, cyanobacteria coexist with plants and mutually benefit each other. They not only fix nitrogen but also synthesize phytohormones like IAA that helps them in root colonization and parallelly improves plant vigor [47]. IAA isolated from yeast has been known to inhibit the growth of weeds and therefore can be used as a herbicide and replace the chemical herbicides [48]. Similarly, it has been observed that usage of IAA or cyanobacterial biofertilizer ameliorates the atrazine toxicity in paddy crops [45]. Role of cyanobacteria as biofertilizers and soil stabilizers in agriculture has been well proved where it promotes cell division and plant elongation [49]. Even they are known to protect plants from pathogenic infections [50]. Cyanobacteria has been known to increase saline tolerance in rice, where strains isolated from saline soils have led to increase in root length and promoted seedling growth and yield [51]. Gibberellic acid produced by cyanobacteria has also led to increase in shoot dry weight and carotenoid content in case of saline-stressed rice [52]. A significant contribution of cyanobacteria is the restoration of drylands in case of severe drought situations. Priming the soil with cyanobacterial strains increases water retention capacity of soils and thereby increases germination and seedling growth [53]. These characteristics of cyanobacteria are an advantage for increasing agricultural productivity. Like salinity and drought, temperature fluctuation is also an abiotic stress that inhibits plant growth, resulting in low agricultural yields. Hence, we propose that when soil temperature becomes unsuitable for plant growth, using cyanobacterial extracts as bio-stimulants may induce plant growth promotion by synthesizing various phytohormones.
5. ACKNOWLEDGMENTS
The authors acknowledge P.G. Department of Biosciences and Biotechnology, Fakir Mohan University, Balasore, for providing the research facilities. The authors sincerely appreciate the kind cooperation of P.G. Department of Environmental Science, Fakir Mohan University, Balasore, for their assistance. The Centre of Excellence for Bioresource Management and Energy Conservation Material Development, Fakir Mohan University is acknowledged for providing fellowship to Dhanesh Kumar.
6. AUTHOR’S CONTRIBUTIONS
Conceptualization: Shubhashree Mahalik and Dhanesh Kumar; Data acquisition/analysis: Priyanka Behera and Dhanesh Kumar; Data analysis/interpretation/drafting manuscript: Shubhashree Mahalik; Critical Revision of the manuscript: Shubhashree Mahalik and Dhanesh Kumar; and Supervision and final approval: Shubhashree Mahalik.
7. FUNDING
The work is funded by Centre of Excellence for Bioresource Management and Energy Conservation Material Development (Under Odisha Higher Education Program for Excellence and Equity (OHEPEE) Assisted by World Bank, Fakir Mohan University, Balasore-756089, Odisha, India.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
All the data is available with the authors and shall be provided upon request.
10. DATA AVAILABILITY
All datasets were generated and analyzed in the present study.
11. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
REFERENCES
1. Sukenik A, Zohary T, Padisák J. Cyanoprokaryota and other prokaryotic algae. In:Encyclopedia of Inland Waters. Vol. 1. Netherlands:Elsevier Inc.;2009. 138-48. [https://doi.org/10.1016/B978-012370626-3.00133-2]
2. Rezayian M, Niknam V, Ebrahimzadeh H. Stress response in Cyanobacteria. Iran J Plant Physiol 2019;9:2773-87.
3. Panda AK, Mishra R, Miglani R, Dewali S, Kumar A, Bora S, et al. Extremophilic diversity and climate change. In:Biodiversity. United States:CRC Press;2023. 41-53. [https://doi.org/10.1201/9781003220398-4]
4. Babu P, Chandel AK, Singh OV. Survival mechanisms of extremophiles. In:Extremophiles and their Applications in Medical Processes. 1st ed. Cham:Springer;2015. 9-23. [https://doi.org/10.1007/978-3-319-12808-5_2]
5. Yadav P, Singh RP, Rana S, Joshi D, Kumar D, Bhardwaj N, et al. Mechanisms of stress tolerance in Cyanobacteria under extreme conditions. Stresses 2022;2:531-49. [https://doi.org/10.3390/stresses2040036]
6. Dash SK, Pandey JK, Jena M, Biswal B. Effect of heat stress and the recovery potential of heterocystous Cyanobacterium, Anabaena iyengarii Bharadwaja 1935. J Pure Appl Microbiol 2020;14:2467-76. [https://doi.org/10.22207/JPAM.14.4.24]
7. Rachedi R, Foglino M, Latifi A. Stress signaling in Cyanobacteria:A mechanistic overview. Life (Basel) 2020;10:312. [https://doi.org/10.3390/life10120312]
8. Pathak J, Rajneesh MPK, Singh SP, Haeder DP, Sinha RP. Cyanobacterial farming for environment friendly sustainable agriculture practices:Innovations and perspectives. Front Environ Sci 2018;6:7. [https://doi.org/10.3389/fenvs.2018.00007]
9. Chittora D, Meena M, Barupal T, Swapnil P, Sharma K. Cyanobacteria as a source of biofertilizers for sustainable agriculture. Biochem Biophys Rep 2020;22:100737. [https://doi.org/10.1016/j.bbrep.2020.100737]
10. Ramakrishnan B, Raju MN, Venkateswarlu K, Megharaj M. Potential of microalgae and Cyanobacteria to improve soil health and agricultural productivity:A critical view. Environ Sci Adv 2023;2:586-611. [https://doi.org/10.1039/D2VA00158F]
11. Singh S. A review on possible elicitor molecules of Cyanobacteria:Their role in improving plant growth and providing tolerance against biotic or abiotic stress. J Appl Microbiol 2014;117:1221-44. [https://doi.org/10.1111/jam.12612]
12. Gonçalves AL. The use of microalgae and Cyanobacteria in the improvement of agricultural practices:A review on their biofertilising, biostimulating and biopesticide roles. Appl Sci 2021;11:871. [https://doi.org/10.3390/app11020871]
13. Pichler G, Stöggl W, Carniel FC, Muggia L, Ametrano CG, Holzinger A, et al. Abundance and extracellular release of phytohormones in aero-terrestrial microalgae (Trebouxiophyceae, Chlorophyta) as a potential chemical signaling source1. J Phycol 2020;56:1295-307. [https://doi.org/10.1111/jpy.13032]
14. Lu Y, Xu J. Phytohormones in microalgae:A new opportunity for microalgal biotechnology?Trends Plant Sci 2015;20:273-82. [https://doi.org/10.1016/j.tplants.2015.01.006]
15. Liaimer A, Bergman B. Phytohormones in Cyanobacteria:Occurrence and perspectives. In:Biology of Plant-microbe Interactions. St Paul:International Society for Molecular Plant Microbe Interactions;2004. 394-7.
16. Spaepen S, Vanderleyden J, Remans R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 2007;31:425-48. [https://doi.org/10.1111/j.1574-6976.2007.00072.x]
17. Jaiswal A, Das K, Koli DK, Pabbi S. Characterization of Cyanobacteria for IAA and siderophore production and their effect on rice seed germination. Int J Curr Microbiol Appl Sci 2018;7:212-22.
18. Duong TT, Nguyen TT, Van Dinh TH, Hoang TQ, Vu TN, Doan TO, et al. Auxin production of the filamentous cyanobacterial Planktothricoides strain isolated from a polluted river in Vietnam. Chemosphere 2021;284:131242. [https://doi.org/10.1016/j.chemosphere.2021.131242]
19. Ahmed M, Stal L, Hasnain S. Production of indole-3-acetic acid by the Cyanobacterium Arthrospira platensis strain MMG-9. J Microbiol Biotechnol 2010;20:1259-65. [https://doi.org/10.4014/jmb.1004.04033]
20. Gayathri M, Kumar PS, Prabha AM, Muralitharan G. In vitro regeneration of Arachis hypogaea L. and Moringa oleifera lam. using extracellular phytohormones from Aphanothece sp. MBDU 515. Algal Res 2015;7:100-5. [https://doi.org/10.1016/j.algal.2014.12.009]
21. Tan CY, Dodd IC, Chen JE, Phang SM, Chin CF, Yow YY, et al. Regulation of algal and cyanobacterial auxin production, physiology, and application in agriculture:An overview. J Appl Phycol 2021;33:2995-3023. [https://doi.org/10.1007/s10811-021-02475-3 https://doi.org/10.1007/s10811-021-02561-6]
22. Patten CL, Glick BR. Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 1996;42:207-20. [https://doi.org/10.1139/m96-032]
23. Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 2013;14:9643-84. [https://doi.org/10.3390/ijms14059643]
24. Gang S, Sharma S, Saraf M, Buck M, Schumacher J. Analysis of indole-3-acetic acid (IAA) production in Klebsiella by LC-MS/MS and the Salkowski method. Bio Protoc 2019;9:e3230. [https://doi.org/10.21769/BioProtoc.3230]
25. Komárek J. Süßwasserflora von mitteleuropa, Bd. 19/3:Cyanoprokaryota. In:3. Teil/3rd Part:Heterocytous Genera. Heidelberg:Spektrum Academischer Verlag;2013.
26. Hedge JE, Hofreiter BT. Estimation of carbohydrate. In:Methods in Carbohydrate Chemistry. New York:Academic Press;1962. 17-22.
27. Waterborg JH. The Lowry method for protein quantitation. In:The Protein Protocols Handbook. Totowa, NJ:Humana Press;2009. 7-10. [https://doi.org/10.1007/978-1-59745-198-7_2]
28. Byreddy AR, Gupta A, Barrow CJ, Puri M. A quick colorimetric method for total lipid quantification in microalgae. J Microbiol Methods 2016;125:28-32. [https://doi.org/10.1016/j.mimet.2016.04.002]
29. KvíderováJ, Kumar D, LukavskýJ, Kaštánek P, Adhikary SP. Estimation of growth and exopolysaccharide production by two soil Cyanobacteria, Scytonema tolypothrichoidesand Tolypothrix bouteillei as determined by cultivation in irradiance and temperature crossed gradients. Eng Life Sci 2019;19:184-95. [https://doi.org/10.1002/elsc.201800082]
30. Heath RL, Packer L. Photoperoxidation in isolated chloroplasts:I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 1968;125:189-98. [https://doi.org/10.1016/0003-9861(68)90654-1]
31. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121-6. [https://doi.org/10.1016/S0076-6879(84)05016-3]
32. Able AJ, Guest DI, Sutherland MW. Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of Phytophthora parasitica var nicotianae. Plant Physiol 1998;117:491-9. [https://doi.org/10.1104/pp.117.2.491]
33. Hare B, Meinshausen M. How much warming are we committed to and how much can be avoided?Clim Change 2006;75:111-49. [https://doi.org/10.1007/s10584-005-9027-9]
34. Ma J, Qin B, Paerl HW, Brookes JD, Hall NS, Shi K, et al. The persistence of cyanobacterial (Microcystis spp.) blooms throughout winter in Lake Taihu, China. Limnol Oceanogr 2016;61:711-22. [https://doi.org/10.1002/lno.10246]
35. Juneja A, Ceballos RM, Murthy GS. Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production:A review. Energies 2013;6:4607-38. [https://doi.org/10.3390/en6094607]
36. Singh SC, Sinha RP, Hader DP. Role of lipids and fatty acids in stress tolerance in Cyanobacteria. Acta Protozool 2002;41:297-308.
37. Rossi F, De Philippis R. Role of cyanobacterial exopolysaccharides in phototrophic biofilms and in complex microbial mats. Life (Basel) 2015;5:1218-38. [https://doi.org/10.3390/life5021218]
38. Rajput VD, Harish, Singh RK, Verma KK, Sharma L, Quiroz-Figueroa FR, et al. Recent developments in enzymatic antioxidant defence mechanism in plants with special reference to abiotic stress. Biology (Basel) 2021;10:267. [https://doi.org/10.3390/biology10040267]
39. Fasnacht M, Polacek N. Oxidative stress in bacteria and the central dogma of molecular biology. Front Mol Biosci 2021;8:671037. [https://doi.org/10.3389/fmolb.2021.671037]
40. Varalakshmi P, Malliga P. Evidence for production of Indole-3-acetic acid from a fresh water Cyanobacteria (Oscillatoria annae) on the growth of H. annus. Int J Sci Res Publ 2012;3:1-5.
41. Prasanna R, Joshi M, Rana A, Nain L. Modulation of IAA production in Cyanobacteria by tryptophan and light. Pol J Microbiol 2010;59:99-105. [https://doi.org/10.33073/pjm-2010-015]
42. Boopathi T, Balamurugan V, Gopinath S, Sundararaman M. Characterization of IAA production by the mangrove Cyanobacterium Phormidium sp. MI405019 and its influence on tobacco seed germination and organogenesis. J Plant Growth Regul 2013;32:758-66. [https://doi.org/10.1007/s00344-013-9342-8]
43. Bessai SA, Bensidhoum L, Nabti EH. Optimization of IAA production by telluric bacteria isolated from Northern Algeria. Biocatal Agric Biotechnol 2022;41:102319. [https://doi.org/10.1016/j.bcab.2022.102319]
44. Scarcella AS, Junior RB, Bastos RG, Magri MM. Temperature, pH and carbon source affect drastically indole acetic acid production of plant growth promoting yeasts. Braz J Chem Eng 2017;34:429-38. [https://doi.org/10.1590/0104-6632.20170342s20150541]
45. Ahmad N, Yasin D, Bano F, Fatma T. Ameliorative effects of endogenous and exogenous indole-3-acetic acid on atrazine stressed paddy field cyanobacterial biofertilizer Cylindrospermum stagnale. Sci Rep 2022;12:11175. [https://doi.org/10.1038/s41598-022-15415-z]
46. Chen CH, Berns DS. Thermotropic properties of thermophilic, mesophilic, and psychrophilic blue-green algae. Plant Physiol 1980;66:596-9. [https://doi.org/10.1104/pp.66.4.596]
47. Hussain A, Hamayun M, Shah ST. Root colonization and phytostimulation by phytohormones producing entophytic Nostoc sp. AH-12. Curr Microbiol 2013;67:624-30. [https://doi.org/10.1007/s00284-013-0408-4]
48. Bunsangiam S, Thongpae N, Limtong S, Srisuk N. Large scale production of indole-3-acetic acid and evaluation of the inhibitory effect of indole-3-acetic acid on weed growth. Sci Rep 2021;11:13094. [https://doi.org/10.1038/s41598-021-92305-w]
49. Arafa AS, El-All AM. Evaluation of the technological properties of the bio-organic colored cotton. J Agron 2013;12:78-85. [https://doi.org/10.3923/ja.2013.78.85]
50. Mazhar S, Hasnain S. Screening of native plant growth promoting Cyanobacteria and their impact on Triticum aestivum var. Uqab 2000 growth. Afr J Agric Res 2011;6:3988-93.
51. El Sheek MM, Zayed MA, Elmossel FK. Effect of Cyanobacteria isolates on rice seeds germination in saline soil. Baghdad Sci J 2018;15:16-21. [https://doi.org/10.21123/bsj.15.1.16-21 https://doi.org/10.21123/bsj.2018.15.1.0016]
52. Rodríguez AA, Stella AM, Storni MM, Zulpa G, Zaccaro MC. Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L. Saline Syst 2006;2:7. [https://doi.org/10.1186/1746-1448-2-7]
53. Muñoz-Rojas M, Chilton A, Liyanage GS, Erickson TE, Merritt DJ, Neilan BA, et al. Effects of indigenous soil Cyanobacteria on seed germination and seedling growth of arid species used in restoration. Plant Soil 2018;429:91-100. [https://doi.org/10.1007/s11104-018-3607-8]