Yeast strain Debaryomyces hansenii for amelioration of arsenic stress in rice

Ecotoxicology and Environmental Safety 195 (2020) 110480

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Yeast strain Debaryomyces hansenii for amelioration of arsenic stress in rice a,1

a,b,1

a,b

a,b

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Jasvinder Kaur , Vandana Anand , Sonal Srivastava , Vidisha Bist , Pratibha Tripathi , Mariya Naseemc, Sampurna Nandc, Anshua, Puja Khared, Pankaj Kumar Srivastavae, Saraswati Bishtf, Suchi Srivastavab,∗ a

Division of Microbial Technology, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226 001, India Academy of Scientific and Innovative Research, AcSIR, Ghaziabad, 201002, India c Environmental Technology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226 001, India d Chemistry Division, CSIR-CIMAP, Lucknow, India e CSIR- Recruitment and Assessment Board, New Delhi, India f Department of Botany, Kumaun University, Nainital, 263002, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Yeast Arsenic mitigation Arsenate reductase arsR gene Rice Plant growth promotion

Arsenic (As) is a serious threat for environment and human health. Rice, the main staple crop is more prone to As uptake. Bioremediation strategies with heavy metal tolerant rhizobacteria are well known. The main objective of the study was to characterize arsenic-resistant yeast strains, capable of mitigating arsenic stress in rice. Three yeast strains identified as Debaryomyces hansenii (NBRI-Sh2.11), Candida tropicalis (NBRI-B3.4) and Candida dubliniensis (NBRI-3.5) were found to have As reductase activity. D. hansenii with higher As tolerance has As expulsion ability as compared to other two strains. Inoculation of D. hansenii showed improved detoxification through scavenging of reactive oxygen species (ROS) by the modulation of SOD and APX activity under As stress condition in rice. Modulation of defense responsive gene (NADPH, GST, GR) along with arsR and metal cation transporter are the probable mechanism of As detoxification as evident with improved membrane (electrolyte leakage) stability. Reduced grain As (~40% reduction) due to interaction with D. hansenii (NBRI-Sh2.11) further validated it's As mitigation property in rice. To the best of our knowledge D. hansenii has been reported for the first time for arsenic stress mitigation in rice with improved growth and nutrient status of the plant.

1. Introduction Arsenic (As), a group I carcinogen is toxic to all forms of life. It has been considered as one of the major global environmental pollutant due to its predominant occurrence in the form of arsenite (As III) and arsenate (As V). The metalloid enters the farming system through natural geochemical processes, use of As-based pesticides, combustion of fossil fuels and irrigation with As-contaminated groundwater (Smedley and Kinniburgh, 2002; Meharg and Hartley‐Whitaker, 2002; Wang and Mulligan, 2006). Rice is the main staple food for about half of the world's population. However, millions of people are at risk of As poisoning due to consumption of rice and rice based products (Meharg and Rahman, 2003; Awasthi et al., 2017). Rice accumulates arsenic more efficiently than other crops due to its requirement for excess water, which facilitates the conversion of stable and bound forms of arsenic into more mobile arsenate (As V) and arsenite (As III) forms (Williams et al., 2009; Su et al.,

2010; Xu et al., 2008; Stroud et al., 2011). Different strategies involving development of As tolerant varieties, transgenic, bioremediation and nutrient supplementation are being used for reduced arsenic uptake in rice (Zhao et al., 2009; Wu et al., 2011; Matsumoto et al., 2015; Shaibur et al., 2013). Roots, which act as interface for plant and soil, are the primary organ for acquisition of both metals and mineral nutrients. Different mineral nutrients (Fe, S, P, Si and Zn) are known to play an important role in decreasing As accumulation in edible plant parts either due to competition with their analogues or complexation with other metal ion (Dahlawi et al., 2018). Similarly, microbes present at rhizosphere are known to improve plant growth and development in heavy metal contaminated soils in various crops (Tripathi et al., 2013; Srivastava et al., 2011; Lampis et al., 2015; Ahmad et al., 2012; Dixit et al., 2015). Several microorganisms belonging to different genera viz. Aeromonas, Exiguobacterium, Acinetobacter, Bacillus, Pseudomonas, Acidithiobacillus, Deinococcus and Desulfitobacterium are capable of

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Corresponding author. Division of Microbial Technology CSIR-National Botanical Research Institute Rana Pratap Marg, Lucknow, 226 001, India. E-mail address: [email protected] (S. Srivastava). 1 Sharing equal authorship. https://doi.org/10.1016/j.ecoenv.2020.110480 Received 7 November 2019; Received in revised form 11 March 2020; Accepted 13 March 2020 Available online 20 March 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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arsenite (NaAsO2) and sodium arsenate (Na3AsO4) through KMnO4 assay as described by Salmassi et al. (2002). In brief, arsenic resistant yeast strains were cultured for 72 h (0.6 O.D.) in chemically defined media (CDM) containing 100 μg ml−1 Na3AsO4. One ml of 72 h grown culture was mixed with 20 μl of 0.01 mol L−1 KMnO4 followed by rigorous vortexing. Change in colour from yellow to pink or vice versa depending on the substrate was observed. The yellow colour indicates the reduction of As(V) to As(III) i.e. presence of arsenate reductase activity. Further, screening of arsenite oxidase activity using sodium arsenite (NaAsO2, AsIII) as substrate indicate oxidation of As(III) to As (V) i.e. presence of pink colour. Methyl transferase ability of the yeast strains were performed by silver nitrate (AgNO3) assay (Singh et al., 2015). Yeast strains were cultured on YPD plates supplemented with 100 μg ml−1As (III) and covered with 0.8% AgNO3 impregnated whatman filter paper followed by incubation at 28 °C. After 72 h, plates were observed for brown colour development due to the formation of silver arsenite or arsenate as compared to control, which remains yellow. Further absorbed, adsorbed or volatile arsenic concentration in selected yeast strain was determined through Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) (Agilent, 7500ce). Yeast cultures were grown in YPD medium spiked with 100 μg ml−1 of As (V) and As (III). After inoculation silver nitrate (0.8% solution) impregnated filter papers were placed beneath the cotton plug of each flask to trap volatile arsenic content in order to measure biovolatilization. After 7 days of incubation, cells were harvested and washed, unwashed pellets and filter paper was collected for the estimation of absorbed, adsorbed and volatile arsenic through ICP-MS analysis. Pellets were dried to a constant weight at 45 °C followed by sample (pellet, broth and filter paper) preparation through microwave digestion procedure using HNO3 (69%, ACS quality Germany) (BERGHOF Speedwave-MWS-3+) (Singh et al., 2015). The total As in digested samples was determined using ICP-MS analysis. Quality control and quality assurance of total As was performed as described earlier by Srivastava et al. (2011).

growing in heavy metal contaminated soil and water (Rehman et al., 2010; Anderson and Cook, 2004; Srivastava et al., 2018; Suresh et al., 2004). They tolerate higher concentration of arsenic by developing a number of detoxifying mechanisms, which include efflux, adsorption, absorption or complexation. These microbes have evolved necessary genetic components for their survival under such environmental conditions. High level of arsenic resistance in microbes is conferred by the arsenic resistance (ars) operon consisting of arsR & arsD as regulatory, arsA & arsB as transmembrane efflux and arsC, encoded as aresenate reductase, responsible for the conversion of As (V) to As (III) (Mukhopadhyay et al., 2002; Oremland and Stolz, 2005). Under anaerobic condition, involvement of periplasmic As (III) oxidase to transform As (III) to As (V) is also known (Silver and Phung, 2005). Recently, As (III) S-adenosyl-L-methionine (SAM) methyltransferase gene from different bacterial strains has also been identified for the volatilization purposes (Qin et al., 2006). Microorganisms oxidize or reduce the two major forms of arsenic as a part of their respiratory or phototrophic processes and are known to be responsible for arsenic contamination of soil and water (Dhar et al., 2011; Stuckey et al., 2015; Fendorf et al., 2010). On the other hand, these rhizospheric microbes are also known to enhance mineral availability and production of phytohormones, thereby increasing the plant biomass (Čapek et al., 2018; Ahemad and Kibret, 2014; Vurukonda et al., 2016). Thus, application of As tolerant microbes offers an upcoming technology for reduced As uptake as compared to other strategies (Fendorf et al., 2010; Lee, 2013; Zhang et al., 2017). Yeast strain Saccharomyces cerevisiae has been well studied model organism for its arsenic detoxification ability (Shah et al., 2010; Todorova et al., 2010). Series of detoxification steps are known to reduce arsenic toxicity in S. Cerevisiae through extrusion and vacuole sequestration (Tsai et al., 2009; Wysocki et al., 2001). Other yeasts, such as Schizosaccharomyces pombe, Trichosporon spp., Rhodotorula spp., and Cryptococcus humicolus have been studied in terms of the accumulation, biosorption, and oxidation-reduction processes (Button et al., 1973; Salgado et al., 2012; Ilyas et al., 2014). Involvement of mainly three gene clusters viz. Transcription factor (ARR1), arsenate reductase (ARR2), and arsenic extrusion transporter (ARR3) has been reported in yeast strains for As tolerance (Tsai et al., 2009; Ghosh et al., 1999). Yeast strains with different plant growth promotary attributes (PGP) have been extensively used for plant growth promotion (Fu et al., 2016). However, their implication for mitigation of As stress has not been well studied. The present study involves the characterization of yeast strains for arsenic tolerance and presence of different PGP traits. The potential yeast strain Debaryomyces hansenii (NBRI-Sh2.11) has been further used for As mitigation studies in rice.

2.3. Growth of yeast isolates under different concentrations of arsenic Arsenic tolerance of selected yeast strains was determined by monitoring their growth under different concentrations of sodium arsenate [As(V); 0, 250, 500, 1000 μg ml−1] and sodium arsenite [As(III); 0, 50, 100, 250 μg ml−1] in a 150 ml Erlenmeyer flasks containing 50 ml YPD with the initial titer of about 104-105 Log10 CFU ml−1. The flasks were incubated at 28 °C in a refrigerated incubator shaker (New 110 Brunswick Scientific, Edison, NJ, USA) at 180 rpm. Viable cells (CFU ml−1) were counted at different time intervals up to 10 consecutive days by serial dilution plating method on YPD plates in triplicate (Nautiyal et al., 2013).

2. Materials and methods 2.1. Isolation, screening and characterization of yeast isolates The yeast strains were isolated from rhizospheric soil samples collected from cultivated fields of different parts of India. Yeast strains were isolated by serial dilution plating method on yeast extract peptone dextrose agar plate followed by their incubation for 48 h at 28 °C (Algabr et al., 2014). Further to screen arsenic tolerance, isolates were streaked on different concentration of sodium arsenate [Na3AsO4; As (V): 0, 50–1000 μg ml−1] and sodium arsenite [NaAsO2; As (III)] [0, 5–500 μg ml−1]. Arsenic tolerant yeast strains were selected for further study and effect of arsenic toxicity on the morphology of yeast strains were also observed through calcofluor staining as described by Rasconi et al. (2009).

2.4. Characterization of yeast strains for plant growth promoting traits Phosphate solubilization ability of yeast strains was determined as reported earlier (Nautiyal et al., 2003). Yeast strains were grown in NBRI-P media for 48 h at 28 °C in a rotary shaker at 180 rpm. The solubilised phosphate was quantified by molybdenum blue method after 72 h of incubation (Fiske and Subbarow, 1925). Auxin production by yeast strains was determined using salkowski's reagent in the 72 h old cultures grown in tryptophan supplemented medium (Limtong et al., 2014). In brief, 1.5 ml of culture supernatant was mixed with 10 mM orthophosphoric acid and 1.0 ml of salkowski's reagent. The intensity of the pink colour developed in the mixture was quantified by taking absorbance at 530 nm. Zinc solubilization and siderophore production in yeast strains were performed as reported earlier (Fasim et al., 2002; Schwyn and Neilands, 1987).

2.2. In vitro study of arsenic detoxification ability Arsenic tolerant yeast strains were also characterized for their oxidation and reduction properties of the respective substrates, sodium 2

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2.5. Molecular identification of yeast strains

2.9. Biochemical assays

For molecular identification, genomic DNA was isolated using Qiagen DNA mini kit followed by PCR amplification of the internal transcript spacer (ITS) region using ITS-1 and ITS-4 primers in a PCR reaction mixture (20 μL) containing 10 × PCR buffer (2.0 μL), dNTPs (2 μL) (2.5 mM each), 10 μM primer pair (1 μL each), 5 U μL−1 Taq DNA polymerase (0.3 μL) (Genei, india), DNA template (10–25 ng) (2 μL), and PCR grade water (12.8 μL) at PCR conditions of, initial denaturation at 95 °C for 10 min; 40 cycles of 95 °C for 30s, 56 °C for 45s, 72 °C for 1 min and 72 °C for 10 min for final extension. PCR product was purified using Qiagen PCR purification kit according to manufacturer's instructions. Sequences of purified PCR product were determined through Sanger's sequencing method at the Central Instrumentation Facility of CSIR-NBRI. Nucleotide sequence similarities were determined using the basic local alignment search tool (BLAST; National Centre for Biotechnology Information database; http://www. ncbi.nlm.nih.gov/BLAST).

2.9.1. Proline, sugar and chlorophyll content Biochemical assays viz. proline, sugar and total chlorophyll were estimated in the hydroponic as well as soil grown shoot tissues of rice as described earlier (Srivastava et al., 2018). In brief the content of proline in leaf was estimated after 15th, 30th and 45th days of arsenic stress using ninhydrin method and expressed as μmol g−1 FW (Bates et al., 1973). The total chlorophyll content in leaf was determined after 15th, 30th and 45th days of arsenic stress following the method of Lichtenthaler (1987) by taking the absorbance of the 80% acetone suspension of the leaf was recorded at 645 and 663 nm on the UV-VIS spectrophotometer (PerkinElmer, USA). Sugar accumulation in rice tissue was estimated as per protocol of Dubois et al. (1956). In brief, 0.1 g shoot samples were crushed in 80% methanol and the obtained supernatant after centrifugation was mixed with equal volume of 5% phenol and 2.5 volume of conc. sulphuric acid followed by its absorbance at 640 nm.

2.6. Amplification of arsenate reductase gene (arsC)

2.9.2. Malondialdehyde, hydrogen peroxide content and electrolyte leakage Different biochemical assays as indicator of oxidative stress were measured in terms of, hydrogen peroxide (H2O2), malondialdehyde (MDA) and electrolyte leakage (EL) by their standard protocols (Heath and Packer, 1968; Singh et al., 2006). In brief, for determination of H2O2 content 100 mg leaf tissue was homogenized in 1 ml of 0.1% TCA followed by centrifugation. Leaf extract (0.5 ml) was mixed with 0.5 ml of 10 mM phosphate buffer (pH 7.0) and 1 ml of 1 M KI solution. Finally, absorbance of mixture was taken at 390 nm. MDA content was determined by the use of thiobarbituric acid (TBA) test which determines malondialdehyde (MDA) as an end product of lipid peroxidation. Plant tissue (100 mg) crushed in 2 ml of extraction buffer [TCA (10%) containing 0.25% TBA] was heated at 95 °C for 30 min followed by snap chilling on ice. The absorbance of the supernatant was taken at 532 and 600 nm. For determination of EL; fresh leaves were washed with deionised water in order to remove surface – adhered electrolytes and used for determination of EL (Das and Sarkar, 2018). Cleaned leaves were kept in closed vials containing 10 ml of deionised water and incubated at 25 °C on a rotatory shaker for 24 h and electrical conductivity of the solution (L1) was estimated subsequently. The final EC i.e., L2 of the sample was determined after autoclaving at 15 lbs pressure for 20 min and after equilibrium at 25 °C. The EL was determined as follows: EL (%) = (L1/L2) × 100.

On the basis of qualitative screening, selected yeast strain with arsenate reductase activity was used for the amplification of arsenate reductase gene (arsC). PCR amplifications of arsC gene were performed with the primers amlt-42-f and amlt-376-r in a reaction mixture as described above. The thermal cycling parameters for arsC, was: 95 °C for 5 min followed by 35 cycles at 95 °C for 30s, 60 °C for 1 min, 72 °C for 1 min; followed by a final extension at 72 °C for 10 min (Zhang et al., 2015). The amplified PCR products were confirmed through agarose gel (1.2%) electrophoresis for presence of arsC genes. 2.7. Effect of D. hansenii inoculation on growth of rice plant under arsenic stress Arsenic stress ameliorative property of the NBRI-Sh2.11 was checked through in vitro and in vivo studies using rice as a host plant. Initially, pre-germinated rice seedlings were grown on 0.8% plain agar plates spiked with different concentration of As (III) (0, 5, 10, 20, and 50 μg ml−1) in absence and presence of NBRI-Sh2.11. After 7 days of seedling transfer plates were observed for the effect of NBRI-Sh2.11 inoculation on the growth of rice under arsenic stress conditions. Hydroponic experiments were performed as described earlier (Srivastava et al., 2018). In brief, 7 days old seedlings were transferred to Hewett media. After three days of seedling transfer, NBRI-Sh2.11 treatments were given @1% using log phase grown culture (pelleted and suspended in Hewett media, 0.8 O.D.). After 48 h of NBRI-Sh2.11 treatments, 20 μg ml−1 of arsenic stress was given. Plants were allowed to grow in controlled conditions, 28 °C/20 °C day/night temperature, and 70% relative humidity for 7 days. For greenhouse experiments, plants were grown in live soil in earthen pots. The experiment was performed in 6 replicates of each treatment. NBRI-Sh2.11 was inoculated to the rice plants at the time of transplantation. Sodium arsenite (AsIII, 20 mg kg−1) was added after 30 days of transplantation of rice seedlings. Different treatments were (i) Cont. (ii) As(III) (iii) NBRISh2.11 and (iv) NBRI-Sh2.11 + As. Sampling of the rice tissues was done after 15th, 30th and 45th days of arsenic stress.

2.10. Defense enzyme assays Defense enzyme activities in the rice leaves of flag leaf stage were assayed in the extract prepared by crushing the plant tissue(s) in 0.1 M potassium phosphate buffer containing, 0.1 mM EDTA, 1% polyvinyl pyrrolidone (PVP), PMSF and dithiothreitol. Supernatants after centrifugation of the extracted tissues were used for different enzymatic assays. Superoxide dismutase (SOD) activity was determined by measuring the inhibition in the photochemical reduction of nitroblue tetrazolium as described earlier (Srivastava et al., 2018). The activity of ascorbate peroxidase (APX) was determined using the method of Nakano and Asada (1981). Catalase activity was measured according to Aebi (1984) by recording the decrease in absorbance of H2O2 at 240 nm in a 3 ml reaction mixture containing 50 mM phosphate buffer (pH, 7); 20 mM, H2O2 and enzyme extract. Total guaiacol peroxidase (GPX) activity was measured in reaction mixture containing 50 mM phosphate buffer (pH 6.6), guaiacol and enzyme extract. The increase in absorbance was measured at 470 nm for 1 min (Hemeda and Klein, 1990).

2.8. Determination of yeast root colonization ability Root colonization ability of the yeast strain NBRI-Sh2.11 was determined by trypan blue staining method as per the protocol of Pathare et al. (2016) of the hydroponically grown rice plants. Further, for quantitative determination of effect of arsenic stress on colonization of yeast strain, rice plant roots were crushed in 0.85% saline and rhizospheric colonization of yeast strains were determined by serial dilution method as described earlier (Srivastava et al., 2018).

2.11. Estimation of As and other mineral elements Arsenic and other mineral element estimation in the oven dried tissues of rice plant (root, shoot and grain) (0.25 g) grown under arsenic 3

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3.2. Effect of arsenic stress on growth kinetics of yeast strains

stress conditions in presence of NBRI-Sh2.11, and soil samples were initially digested through microwave digestion procedure as described earlier (Singh et al., 2015). The total As in digested samples was determined using ICP-MS analysis as per the standardized protocol of Srivastava et al. (2011).

Growth kinetics of arsenic tolerant yeast isolates (NBRI-B3.4, NBRIB3.5 and NBRI-Sh2.11) was studied in the presence of different concentration of AsV (0, 250 and 500 μg ml−1) and AsIII (0, 250 and 500 μg ml−1). All the three-yeast strain grown in presence of both arsenate and arsenite showed extended lag phase as compared to control (Supp. Fig. 2). NBRI-Sh2.11 was the only yeast strain showing exponential growth in presence of 250 and 500 μg ml−1 of sodium arsenite as compared to control condition.

2.12. Quantitative real time (Q-RT) PCR Real time (Q-RT) PCR analysis of different abiotic stress responsive genes was performed in flag leaf stage rice shoot tissues of different treatments viz. Control, As(III), NBRI-Sh2.11 and NBRI-Sh2.11 + As using specific primers viz. NADPH oxidase (OsNox, Os08g35210), GST (Os10g38590), GR (XM015771323), metal cation transporter (ZIP2, Os03g29850) and arsenate reductase (KC687096) (Supp. Table 3). Total RNA was extracted from different treatments by using RNAeasy Plant Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized using H minus first strand cDNA synthesis Kit (Fermentas, Thermo Scientific). The real-time PCR analysis was carried with SYBR green Premix Ex Taq (Agilent) on Stratagene Mx3000 P systems, using actin as an internal reference. The reactions were performed using the cycle conditions of an initial denaturation at 94 °C for 5min, followed by 35 cycles of 94 °C for 30s, 60 °C for 30s, and 72 °C for 30s. After obtaining value for each reaction, the fold change was calculated by delta-delta ct method.

3.3. Arsenic toxicity on the morpho-physiological features of yeast cells Fluorescence microscopy performed using calcofluor silver white staining clearly showed morphological changes such as flocculation of all the three selected yeast strains when grown in presence of As (V) and As (III) (100 μg ml−1) (Supp. Fig. 3). Yeast strains NBRI-B3.4 and NBRIB3.5 showed higher flocculation as compared to NBRI-Sh2.11. However, yeast strains grown under normal growth condition showed smooth dispersed cell. 3.4. Arsenic detoxification study in yeast strains In order to observe the arsenic detoxification mechanism, total arsenic content adsorbed on the surface, absorbed inside the cell and in the culture, media was analysed through ICPMS analysis. Higher adsorption of arsenic content, estimated in terms of unwashed pellet, was observed in both NBRI-B3.4 and NBRI-B3.5 grown in the presence of As (III) as compared to As (V), (Fig. 1A). Among all the three yeast strains, NBRI-B3.5 showed higher absorbed arsenic content in presence of both As (V) and As (III), estimated in terms of washed pellets, however, no accumulation of arsenic was found in NBRI-B3.4 and NBRI-Sh2.11. NBRI-Sh2.11 adsorb, absorb and expel both forms of arsenic (100 μg ml−1 AsIII and AsV), after 7 days of incubation, as evident from the higher concentration in the media supernatant. It depicts different mechanism of arsenic tolerance in NBRI-Sh2.11 as compared to the other two strains, NBRI-B3.4 and NBRI-B3.5 (Fig. 1A). All the three strains were able to survive in arsenic supplemented media up to 7 and 10 days of exposure showing their resistance towards arsenic (Fig. 1B, Supp. Fig. 2).

2.13. Soil chemical analysis Different soil elements viz. available nitrogen, phosphorus, sulphur, sodium, potassium, and calcium and microbial biomass carbon (MBC) of rhizospheric soil of greenhouse grown plants was determined as per the standard protocol. Available nitrogen content in soil was determined as per the protocol of Subbiah and Asija (1956) using kjeldahl method. Phosphorus and sulphur content in soil was estimated as per the protocol of Olsen et al. (1954) and Rehm and Caldwell (1968). Sodium, potassium and calcium were estimated in soil according to Toth and Prince (1949). Ammonium acetate (1 N) extracted K and Ca was analysed through flame photometer. Microbial Biomass Carbon (MBC) of the soil was estimated by subtracting the values of non-fumigated from fumigated soil as per the protocol of Vance et al. (1987).

3.5. Plant growth promoting attribute of yeast isolates

2.14. Statistical analysis

To assess the As amelioration ability in plant system, selected yeast strains were also characterized for different plant growth promoting traits (Supp. Table 2). Yeast strains NBRI-B3.4 and NBRI-B3.5 showed almost similar auxin production (45.38 and 42.79 μg ml−1) as compared to NBRI-Sh2.11 (23.76 μg ml−1). P-solubilization was found to be higher in NBRI-B3.5 (23.75 μg ml−1) followed by NBRI-B3.4 (17.42 μg ml−1) and NBRI-Sh2.11 (13.42 μg ml−1). NBRI-B3.5 was found to have siderophore production activity and yeast strains NBRIB3.5 and NBRI-Sh2.11 both were screened as zinc solubilizers.

One-way analysis of variance (ANOVA) was performed to identify the significantly different treatments using SPSS 16.0. 3. Results 3.1. Identification and characterization of yeast isolates The arsenic tolerant yeasts strains were isolated from 14 district of India, by determining their tolerance using different concentrations of As (III) and As (V). Yeast strains exhibiting maximum resistance up to 500 μg ml−1As (III) and 1000 μg ml−1of As (V) (Supp. Table 1) were selected for further characterization. Microscopic analysis showed that out of 50 yeast isolates, only three yeast species i.e. NBRI-B3.4, NBRIB3.5 and NBRI-Sh2.11 were having Saccharo type morphology. Change in colour of the KMnO4 from pink to yellow of the yeast culture grown in CDM media containing sodium arsenate @100 μg ml−1 predict the presence of arsenate reductase activity in the selected yeast strains. Further characterization of yeast strains for the presence of arsenic resistant gene i.e. arsenate reductase, arsenite oxidase and arsenite methyltransferase genes showed that all the three strains showed only arsenate reductase activity as evident by the amplification of 350bp arsR gene (Supp. Fig. 1).

3.6. Identification of yeast isolates The three yeast strains were identified based on ITS sequencing. The BLAST analysis of showed 99% homology NBRI-B3.4 to Candida dubliniensis (MN629347) and NBRI-B3.5 to Candida tropicalis (MN629348). Strain NBRI-Sh2.11 was identified as Debaryomyces hansenii (MN629349) having close relatedness with Debaryomyces fabri (Supp. Fig. 4). 3.7. Effect of D. hansenii inoculation on growth of rice under arsenic stress condition The effect of yeast inoculation on the growth of rice plants exposed 4

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Table 1 Effect of D. hansenii inoculation on physiological and biochemical status of rice plant grown under arsenic stress conditions.

Hydroponic

Physical parameters

Biochemical estimations

Greenhouse experiment

Physical parameters

Biochemical estimations

Treatments

Cont.

As

Sh2.11

Sh2.11 + As

Root length (cm) Shoot length (cm) Dry weight(g) Shoot/Root Biomass Total Chlorophyll (mgg-1) Sugar(μggq) Proline (μM) Root length (cm) Shoot length (cm) No. of Tillers No. of Spikes Dry weight(g) Total Chlorophyll 15 30 45 Sugar 15 30 45 Proline 15 30 45

3.5 ± 0.19 13.08 ± 0.16 0.048 ± 0.02 0.938 3.166 ± 0.00 35.15 ± 4.55 1.21 ± 0.11 16.5 ± 2.52 67 ± 0.96 1.33 ± 0.84 1.16 ± 0.74 20.72 ± 0.73 2.9 ± 0.26 2.23 ± 0.04 1.60 ± 0.00 57.8 ± 2.9 181.9 ± 1.8 195.2 ± 0.8 42.07 ± 2.25 34.04 ± 0.71 25.57 ± 0.38

3.766 ± 0.37 11.70 ± 0.50 0.0348 ± 0.01 1.058 1.938 ± 0.03 88.95 ± 5.15 1.65 ± 0.11 14.33 ± 1.87 60.5 ± 2.85 1.5 ± 1.02 1 ± 0.63 14.67 ± 1.11 2.48 ± 0.04 1.58 ± 0.08 1.75 ± 0.22 56.8 ± 4.2 212.2 ± 1.8 250.6 ± 0.6 95.59 ± 1.32 61.49 ± 3.13 64.68 ± 0.33

4.58 ± 0.37 15.08 ± 0.71 0.059 ± 0.02 0.613 3.226 ± 0.11 45.55 ± 3.15 1.26 ± 0.05 19.66 ± 1.38 78.16 ± 2.7 3.16 ± 1.01 2.83 ± 0.90 34.15 ± 3.31 3.72 ± 0.04 2.46 ± 0.18 2.23 ± 0.10 123.9 ± 0 160.8 ± 4.1 193.9 ± 5.9 55.165 ± 2.25 56.92 ± 2.91 11.27 ± 0.71

4.50 ± 0.16 15.7 ± 0.65 0.078 ± 0.03 0.555 2.727 ± 0.02 39.1 ± 2.6 1.21 ± 0.00 20.83 ± 0.60 74.83 ± 1.08 1.83 ± 1.16 1.83 ± 1.16 30.57 ± 3.08 3.21 ± 0.07 2.04 ± 0.08 1.79 ± 0.20 105.9 ± 8.9 259.6 ± 0.1 297.9 ± 8.4 87.54 ± 1.65 49.55 ± 1.21 26.89 ± 3.24

Day Day Day Day Day Day Day Day Day

(Fig. 1C). Root colonization of the yeast strain using trypan blue staining showed adherence of NBRI-Sh2.11 around the root surface (Supp. Fig. 5) Further on, hydroponic experiment performed under 20 μg ml−1 As (III), stress conditions showed better root length in NBRISh2.11 and NBRI-Sh2.11 + As condition as compared to control, however, reduction in root length was not observed under As stressed condition (Fig. 1D; Table 1). Approximately, 10.55% reduction in shoot

to As stress was analysed under in vitro and in vivo conditions in presence of D. hansenii (NBRI-Sh2.11), as other two strains NBRI-B3.4 and NBRI-B3.5, identified as C. tropicalis and C. dubliniensis reported to be opportunistic pathogen. For initial screening plants grown under arsenic stress condition in petriplates showed ~50% growth inhibition at 50 μg ml−1of arsenic stress, however, plants inoculated with yeast strain (NBRI-Sh2.11) showed improved growth at same stress of arsenic

Fig. 1. A: Total arsenic content in pellet, growth medium and silver nitrate impregnated filter paper (volatile arsenic) of the three selected yeast strains and their growth in the YPD media supplemented with 100 μg ml−1 of Na3AsO4 (AsV) and NaAsO2 (AsIII) after 7th day of inoculation B: Growth pattern of yeast strains (B3.4, B3.5 and Sh2.11) (B) under in vitro condition. Vertical bars indicate mean ± S.D. of three replicate C: Growth of rice under in vitro conditions in presence of D. hansenii (NBRI-Sh2.11) under arsenic (As III; 0, 5, 10, 20, 50 μg ml−1) stress condition after 7th day of seedling transfer; D: Growth of rice plant under hydroponic (7 dpt) E: Greenhouse conditions under control, arsenic (As III, 20 μg ml−1), D. hansenii (NBRI-Sh2.11) and D. hansenii + arsenic [As III (20 μg ml−1)+NBRI-Sh2.11] conditions after 30th day of arsenic treatment. 5

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Fig. 2. Superoxide dismutase (SOD) (A); Guaicol peroxidase (GPX) (B); Ascorbate peroxidase (APX) (C); Hydrogen peroxide (H2O2) (D); malondialdehyde (MDA) activities (E) and electrolyte leakage (EL) (F); in shoot of rice grown under greenhouse conditions after 30th day of arsenic treatment. Treatments were control, arsenic (As III, 20 μg ml−1), D. hansenii (NBRI-Sh2.11) and D. hansenii + arsenic [As III (20 μg ml−1) +NBRI-Sh2.11]. Vertical bars indicate mean ± S.D. of three replicate. Fig. 3. Expression analysis of defense responsive (NADPH oxidase, GST and GR), metal cation transporter and arsenate reductase (arsR) genes in shoot of rice grown under greenhouse conditions after 30th day of arsenic treatment. Treatments were control, arsenic (As III, 20 μg ml−1), D. hansenii (NBRI-Sh2.11) and D. hansenii + arsenic [As III (20 μg ml−1) +NBRI-Sh2.11. Vertical bars indicate mean ± S.D. of three replicates.

6

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3.8. Modulation of defense enzyme activities by the inoculation of D. hansenii

Table 2 Correlation (r2 values) of different mineral uptake with As uptake in different parts of rice plants treated with D. hansenii under greenhouse conditions.

Root

Shoot

Grain

Mn Fe Se Zn Mn Fe Se Zn Mn Fe Se Zn

Cont

As

Sh2.11

Sh2.11 + As

+0.043 +0.604 +0.719 +0.425 +0.998 +0.976 0.000 +0.669 −0.042 +0.214 0.00 −0.045

−0.330 +0.004 +0.677 +0.881 +0.393 +0.108 −0.775 −0.620 +0.049 −0.889 +0.096 +0.585

−0.130 +0.000 +0.997 −0.196 +0.848 +0.150 +0.142 +0.788 −0.09 −0.154 +0.989 +0.286

+0.937 +0.578 +0.921 −0.043 −0.035 +0.701 −0.255 +0.999 +0.844 +0.463 +0.378 +0.017

Different enzymatic processes are known to reduce arsenic stress by the involvement of superoxide dismutase; ascorbate peroxidase & guaiacol peroxidase activities. Higher APX, GPX and SOD activities were also found under As stressed conditions in the present study under greenhouse conditions. The SOD activity was reduced in the presence of NBRI-Sh2.11 under As conditions (Fig. 2A). Presence of NBRI-Sh2.11 reduced the level of APX, GPX and SOD by 50.32%, 80%, 56.29%, under arsenic stress condition respectively, as compared to control (Fig. 2A–C).

3.9. D. hansenii mediated modulation of reactive oxygen species and electrolyte leakage

length under As condition was observed and NBRI-Sh2.11 inoculation was found to enhance the shoot length by 20.03% and dry weight by 62.5% under NBRI-Sh2.11 + As conditions as compared to control (Fig. 1E, Table 1). Among physiological parameters reduction in chlorophyll (38.66%) and enhanced accumulation of sugar and proline by 153.05% and 36.36% was observed under As stressed condition, respectively (Table 1). Inoculation of NBRI-Sh2.11 under As condition was found to modulate the accumulation of proline and sugar approximately similar to control. Total chlorophyll was also found to be better synthesized (2.7 ± 0.02 mg g−1) as compared to As stress (1.9 ± 0.03) (Table 1). Root colonization determined to observe the rhizosphere competence of the yeast strain under arsenic showed approximately similar colonization of NBRI-Sh2.11 alone (5.46 Log10 CFU ml−1) and arsenic stressed (NBRI-Sh2.11 + As) condition (5.38 Log10 CFU ml−1). NBRI-Sh2.11 was found to enhance the growth of rice plant under greenhouse conditions under both control and arsenic stressed (20 mg kg−1 sodium arsenite) conditions (Fig. 1E). Plant growth promoting attribute of NBRI-Sh2.11 was well correlated with increased root length (19.15%), shoot length (16.65%) and dry weight (64.81%) as compared to un-inoculated control (Fig. 1E; Table 1). Besides, the presence of NBRI-Sh2.11 also improved the growth of rice plant in terms of root length (26.24%), shoot length (11.68%) and dry weight (47.53%) under arsenic stress condition as compared to control (Table 1). Accumulated proline measured after 15, 30 and 45th dpt showed a general decline pattern in all the treatments. Arsenic stress was found to enhance the proline accumulation at all time interval. Inoculation with NBRI-Sh2.11 alone didn't raise the proline accumulation under control conditions, however, under As stressed conditions, the level of proline accumulation was always higher as compared to control. Estimation of total sugar showed an increasing trend of accumulation which was more in the presence of As alone followed by NBRISh2.11 + As. Chlorophyll content showing reduced accumulation up to 30th day of arsenic treatment and this decline was delayed by the presence of NBRI-Sh2.11 up to 45th day.

The present study showed an increased level of hydrogen peroxide (H2O2) and malondialdehyde (MDA) under As stress condition. The protective role of NBRI-Sh2.11 was observed due to lowering of H2O2 and MDA under NBRI-Sh2.11 + As condition (Fig. 2D and E). Present study showed the damage of shoot tissue (estimated in terms of EL) by 27.21% under As stress conditions, however, inoculation of NBRISh2.11 (NBRI-Sh2.11 + As) was found to reduce the electrolyte leakage by 20.16% as compared to control (Fig. 2F).

3.10. Modulated expression of arsenic stress responsive genes in presence of D. hansenii Expression of certain genes viz. NADPH oxidase, GST, GR, metal cation transporter and arsenate reductase gene, involved in stress response and arsenic detoxification was monitored in the shoot tissues of rice grown under live soil conditions in presence of arsenic and NBRISh2.11 strain through RT-PCR analysis (Fig. 3). Expression profiling of different genes viz. NADPH oxidase, GST, GR, metal cation transporter and arsenate reductase showed differential expression pattern. All the tested gene were found to be differentially modulated during arsenic stress alone and in presence of NBRI-Sh2.11. Presence of NBRI-Sh2.11 was found to alleviate the expression of both reductases viz. GR and arsR, however, under As alone conditions, expression of these genes were upregulated as compared to control. Expression of GR (0.8-fold) and arsR (0.9-fold) was down-regulated in presence of NBRISh2.11 + As condition. Expression of NADPH oxidase was found to be higher under As stress condition (5 fold), however, treatment with NBRI-Sh2.11 was found to mitigate the As stress and its expression was found to be only, 1.9 fold higher as compared to control. Similarly, NBRI-Sh2.11 alone modulates the expression of GST and metal cation transporter. Presence of NBRI-Sh2.11 mitigate its expression by 0.9 and 0.4-fold higher which was otherwise 1.4 and 1.9-fold higher in presence of As stress as compared to control (Fig. 3).

Table 3 Effect of D. hansenii inoculation on the soil macro and micronutrients under arsenic stress condition. Macronutrient Control As Sh2.11 Sh2.11 ± As Micronutrient Control As Sh2.11 Sh2.11 ± As

Avail. P

Avail. S

8.30 ± 0.10 14.18 ± 1.86 12.78 ± 1.24 11.98 ± 0.88

28.46 23.31 27.99 27.94

± ± ± ±

0.62 3.01 0.13 0.78

As

Mn

2.18 ± 0.0 18.53 ± 0.1 1.94 ± 0.0 17.20 ± 0.0

292.4 257.9 283.8 261.4

K⁺ (μg ml−1)

Ca⁺⁺ (μg ml−1)

Avail. N

125.44 ± 4.36 148.88 ± 5.44 97.84 ± 3.88 99.84 ± 4.44

1060.72 1028.72 1000.48 1103.44

0.02 0.36 0.56 0.33

± ± ± ±

7

4.2 10.0 4.1 1.4

± ± ± ±

11.56 2.96 9.92 5.36

± ± ± ±

Fe

Se

13,674.4 ± 112.4 12,069.8 ± 87.9 12633.4 ± 68.2 11897.5 ± 63.2

0.7 0.5 0.5 0.4

MBC (μg gm−1) 0.02 0.02 0.05 0.11

776.16 679.14 873.18 824.67

± ± ± ±

194.04 291.06 97.02 145.53

Zn ± ± ± ±

0.0 0.0 0.0 0.0

38.6 40.4 35.2 41.4

± ± ± ±

0.4 0.3 0.1 0.2

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Fig. 4. Total arsenic content in whole biomass of rice; (A) soil, (B) root, (C) shoot and (D) grain; grown under greenhouse conditions after maturity. Treatments were control, arsenic (As III, 20 μg ml−1), D. hansenii (NBRI-Sh2.11) and D. hansenii + arsenic [As III (20 μg ml−1). Vertical bars indicate mean ± S.D. of three replicates.

been found to have least correlation with As in NBRI-Sh2.11 + As condition as compared to As alone condition. Under arsenic stress conditions Fe and Se found to have negligible to negative correlation in root, shoot and grain. Correlation of Zn uptake under arsenic stress conditions was positively correlated in root and grain, however, shoot shows negative correlation.

3.11. Effect of D. hansenii on arsenic uptake in plants grown under greenhouse conditions Uptake of arsenic and other mineral nutrients in different plant parts viz. root, shoot and grain in the presence and absence of NBRISh2.11 was estimated through ICP-MS analysis (Fig. 4). Arsenic content was found to be higher in roots of rice inoculated with NBRI-Sh2.11 as compared to control As treatment. Contrary to this, the shoot was found to have almost similar arsenic content in both uninoculated and inoculated treatment, 2.01 ± 0.0152 and 1.96 ± 0.001 μg ml−1 in both uninoculated and inoculated treatment respectively. Lower content of arsenic in the rice grain in NBRI-Sh2.11 + As (~40% reduced) treatment as compared to arsenic control treatment was observed.

3.13. Effect of D. hansenii inoculation on nutrient status of soil Soil macro and micro-nutrient analysis under arsenic stress conditions and their modulation due to treatments with NBRI-Sh2.11 has been studied in the present study. Significant enhancement in available P (70.79%) and N (124%) under arsenic stress and 53.93% available P and 1900% of available nitrogen due to NBRI-Sh2.11 inoculation was observed (Table 3). Under combined treatment of arsenic (20 μg ml−1) and NBRI-Sh2.11 increase in available P by 44.30% and available N by 1100% was observed. Available S getting reduced (18%) due to arsenic toxicity was found to be approximately similar in NBRI-Sh2.11 and NBRI-Sh2.11 + As treatments as compared to the control. Level of MBC was found to be higher due to NBRI-Sh2.11 inoculation in alone as well as arsenic stressed conditions by 12.5% and 6.25% respectively. Decrease in As accumulation was found in NBRI-Sh2.11 + As inoculation as compared to As alone treatment. In terms of micronutrients, Mn and Fe were found to be significantly lower in As [Mn (11.79%) and Fe (11.73%)] alone whereas combination of NBRI-Sh2.11 + As [Mn (2.9%) and Fe (0.29%)] showed very less or non-significant decrease as compare to control. Level of Se showed no difference in alone and combined treatment while, Zn was found to be increased in NBRISh2.11 + As (2.47%) treatment.

3.12. Effect of D. hansenii inoculation to other minerals uptake with respect to arsenic Effect of NBRI-Sh2.11 inoculation to other mineral uptake in correlation with arsenic uptake has been studied through coefficient correlation (r2 value) analysis in response to different treatments viz. control, As(III), NBRI-Sh2.11 and NBRI-Sh2.11 + As (Table 2; Supp. Fig. 6). Differential uptake of different microelements with respect to arsenic was observed. Under arsenic stress conditions Iron (Fe) and Selenium (Se) found to have negligible to negative correlation in root, shoot and grain. Correlation of Zinc (Zn) uptake under arsenic stress conditions was positively correlated in root and grain, however, shoot shows negative correlation, due to NBRI-Sh2.11 inoculation. In root all the tested elements showed positive correlation, except for Zn in NBRISh2.11 + As condition as compared to As alone condition. In shoot, manganese (Mn) showed least or no correlation, whereas, Se was found to have less negative correlation in NBRI-Sh2.11 + As condition as compared to As alone condition. Zn was found to have strong positive correlation under NBRI-Sh2.11 + As conditions as compared to negative correlation observed under As alone conditions. In grain, Mn, Fe and Se was found to have positive correlation with arsenic in NBRISh2.11 + As condition as compared to As alone condition. Only Zn has

4. Discussion Considering the health hazard potential of arsenic and need to develop eco-friendly, economically feasible, efficient technologies for reduced arsenic uptake in plants along with transgenic approaches, emphasis on bacteria and filamentous fungi has been given till now 8

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transporter in presence of arsenic and NBRI-Sh2.11 + As corroborated well with positive correlation observed in translocation factor of different metals in relation to arsenic uptake. Present study also showed increased level of hydrogen peroxide and MDA under As stress condition as reported earlier (Nath et al., 2014). Lowering of H2O2 and MDA in rice grown under NBRI-Sh2.11 + As condition indicate the protective role of NBRI-Sh2.11 against As stress. Cell membrane stability of plant is often related to plant towards As tolerance and other abiotic stresses like drought, salinity, pathogen attack, heavy metal etc. Electrolyte leakage (EL) is an indicator of different stressed conditions, like drought, salinity, pathogen attack and heavy metal etc. It induces stresses which leads to generation of reactive oxygen species (ROS), which ultimately cause programmed cell death. The conductivity of EL from leaf cells is considered as an indicator of membrane damage in As toxicity. Present study also showed higher electrolyte leakage in rice under arsenic stress in accordance to earlier report (Das and Sarkar, 2018). Reduction in EL in presence of NBRI-Sh2.11 depicts the role of NBRI-Sh2.11 in minimizing arsenic induced membrane damage in plants. Arsenic stress amelioration in presence of microbial inoculums is reported earlier by Srivastava et al. (2018). In addition, Present study also showed NBRI-Sh2.11 mediated reduced arsenic transportation in rice from root to shoot and its accumulation in grain. Higher As content in root might be due to colonization of NBRI-Sh2.11 and its arsenic extrusion mechanism as discussed above. To ascertain the uptake of different mineral nutrients in different parts of rice, present study also determined the correlation among different minerals with arsenic uptake. Positive correlation observed in grain with respect to arsenic is as per the earlier report of (Williams et al., 2009) that arsenic contaminated rice is limited in mineral nutrients (Williams et al., 2009). Soil macro and micro-nutrient analysis under arsenic stress conditions and in presence of NBRI-Sh2.11 showed reduction in available S (18%) due to arsenic toxicity. Approximately similar quantity of available S in presence of NBRI-Sh2.11 and NBRI-Sh2.11 + As treatments supports the PGPR mediated better nutrient status. Enhanced availability of P, K and N due to arsenic stress was evident in accordance to Jung et al. (2017), who first time reported the direct correlation of As and K. Enhanced availability of N by NBRI-Sh2.11 probably associated with some unidentified attribute of nitrogen mineralization by the strain NBRI-Sh2.11. Better nitrogen availability due to Yeast strain inoculation may be the first report from our study under arsenic stress. Higher MBC associated with NBRI-Sh2.11 inoculation may be attributed with enhanced microbial activity resulting in better growth of rice plant. Higher arsenic content in soil is probably associated with arsenic extrusion property of the strain NBRI-Sh2.11 as evident by better colonization and higher MBC. Lesser content Fe, Mn and Se in arsenic stressed conditions both in presence and absence of NBRI-Sh2.11 supports the need of mineral fertilization for reduced arsenic uptake in order to create competition for their transportation.

(Verma et al., 2016; Tripathi et al., 2013). Yeast has earlier been used as the model organism to study arsenic detoxification pathway (Rosen, 2002). However, its implication for As stress mitigation in plants is still obscure. Therefore, a number of yeast strains from different rhizospheric soil samples were characterized for arsenic mitigation properties. Based on calcofluor staining, 3 yeast strains, classified as Saccharo types, were further characterized as arsenate reducer based on arsR gene amplification. Arsenic tolerant yeast strains identified as C. tropicalis (NBRI-B3.4) and C. dubliniensis (NBRI-B3.5) in present study have been reported earlier to have resistance for arsenic, lead, mercury and cadmium (Rehman and Anjum, 2011; Muneer et al., 2016). C. tropicalis and C. dubliniensis were not considered for further study because of their opportunistic pathogen record (Ilyas and Rehman, 2015). Another yeast strain identified as Debaryomyces hansenii (NBRI-Sh2.11) also showed arsenate reductase activity, in addition to arsenic tolerance up to 500 μg ml−1 of As (III) and 1000 μg ml−1 of As(V). There are several reports of using yeast strain D. hansenii (NBRI-Sh2.11) for salt tolerance and biocontrol potential (Prista et al., 2005; Sánchez et al., 2018; Medina-Córdova et al., 2016). The present study first time reports D. hansenii as arsenic tolerant strain having arsenate reductase activity. Sustained growth in the media after 10th day of incubation in presence of 500 μg ml−1 of As(III) and As(V), showed higher tolerance level of NBRI-Sh2.11 is contrary to the earlier report of Balsalobre et al. (2003). Probably in order to maintain the growth in media, NBRI-Sh2.11 is applying the mechanism of arsenic extrusion as evident from the higher arsenic content observed in media in the present study. Extrusion, as mechanism of arsenic tolerance has been reported earlier (Čerňanský et al., 2009). Yeast cells are known to possess a remarkable capacity to trigger adhesion in response to different environmental stresses (Verstrepen and Klis, 2006). Presence of flocculant cells in arsenic spiked media demonstrates that arsenic removal ability of the yeast strains is associated with the flocculation property, of the yeast, in accordance to the earlier report that during stress conditions, yeast cells get flocculated and help in removal process from synthetic effluent (Machado et al., 2008). Plant growth-promoting attribute of yeast strains under different stress conditions has already been reported (Ignatova et al., 2015; Fu et al., 2016). Our study also showed better plant growth under normal and arsenic stressed conditions, which may be attributed to auxin producing, arsenic tolerance and heavy metal detoxification abilities of NBRI-Sh2.11 (Srivastava et al., 2018). Sugar and proline, serve as physiological markers for plant health and development (Kaur and Asthir, 2015). Increased sugar level observed under As stressed condition in present study is in congruence with earlier report of enhanced sugar level in plants under different stresses viz. drought, flood, salinity and low temperature (Rosa et al., 2009). Arsenic impaired rate of photosynthesis is widely reported (Abbas et al., 2018). Decrease in level of chlorophyll in present study might be due to low adaptive management of photosystem I and II or reduced availability of precursor for chlorophyll biosynthesis under arsenic stress in plants (Abbas et al., 2018; Maglovski et al., 2019). Earlier studies have reported that arsenic toxicity is known for excessive accumulation of reactive oxygen species (ROS) associated with degradation of biomolecules like lipids, nucleic acid and proteins (Hartley‐Whitaker et al., 2001; Tripathi et al., 2007; Tripathi et al., 2013; Dave et al., 2013). Different enzymatic processes are known to reduce arsenic stress by the involvement of superoxide dismutase, ascorbate peroxidase & guaiacol peroxidase (Meharg and Hartley‐Whitaker, 2002; Nath et al., 2014). NBRI-Sh2.11 mediated reduced defense enzyme activities which were higher under arsenic stress condition demonstrate the potential role of NBRI-Sh2.11 in lowering ROS production and oxidative damage caused by As. Higher enzyme activities of APX, GPX and SOD in correlation with the upregulated expression of defense responsive genes (OsNADPH, GST and GR) and their negative modulation in presence of NBRI-Sh2.11 supports the arsenic stress ameliorating attribute of yeast strain NBRI-Sh2.11 as per the earlier report (Srivastava et al., 2018; Upadhyay et al., 2018). Upregulated expression of metal cation

5. Conclusion Yeast, the model organism which has been reported to have plant growth promotary and biological control potential has not been studied till date for arsenic mitigation in cereal crops. The present study shows the potential of yeast strain D. hansenii (NBRI-Sh2.11) for reduced arsenic transportation in rice. The interaction modulated the physiological and nutrient status of plant along with reduced As accumulation in grain, thereby, improving the plant health. Furthermore, yeast which is highly adaptable to different environmental conditions and have better chance of developing lyophilized formulation (like baker's yeast) offer good potential for As remediation. CRediT authorship contribution statement Jasvinder Kaur: Formal analysis, Methodology, Writing - original 9

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draft. Vandana Anand: Formal analysis, Methodology, Writing - original draft. Sonal Srivastava: Formal analysis, Methodology, Writing original draft. Vidisha Bist: Methodology. Pratibha Tripathi: Methodology. Mariya Naseem: Methodology. Sampurna Nand: Methodology. Anshu: Methodology. Puja Khare: Writing - review & editing. Pankaj Kumar Srivastava: Writing - review & editing. Saraswati Bisht: Writing - review & editing. Suchi Srivastava: Supervision, Conceptualization, Writing - review & editing.

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