Accepted Manuscript Title: Chlorella vulgaris and Pseudomonas putida interaction modulates phosphate trafficking for reduced arsenic uptake in rice (Oryza sativa L.) Authors: Suchi Srivastava, Sonal Srivastava, Vidisha Bist, Surabhi Awasthi, Reshu Chauhan, Vasvi Chaudhry, Poonam C. Singh, Sanjay Dwivedi, Abhishek Niranjan, Lalit Agrawal, Puneet Singh Chauhan, Rudra Deo Tripathi, Chandra Shekhar Nautiyal PII: DOI: Reference:
S0304-3894(18)30127-4 https://doi.org/10.1016/j.jhazmat.2018.02.039 HAZMAT 19203
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
18-10-2017 2-2-2018 22-2-2018
Please cite this article as: Srivastava S, Srivastava S, Bist V, Awasthi S, Chauhan R, Chaudhry V, Singh PC, Dwivedi S, Niranjan A, Agrawal L, Chauhan PS, Tripathi RD, Nautiyal CS, Chlorella vulgaris and Pseudomonas putida interaction modulates phosphate trafficking for reduced arsenic uptake in rice (Oryza sativa L.), Journal of Hazardous Materials (2010), https://doi.org/10.1016/j.jhazmat.2018.02.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chlorella vulgaris and Pseudomonas putida interaction modulates phosphate trafficking for reduced arsenic uptake in rice (Oryza sativa L.) Suchi Srivastava#1, Sonal Srivastava#1, Vidisha Bist1, Surabhi Awasthi2, Reshu Chauhan2, Vasvi Chaudhry1,3, Poonam C. Singh1, Sanjay Dwivedi1, Abhishek Niranjan1, Lalit Agrawal1, Puneet Singh Chauhan1, Rudra Deo Tripathi3*, Chandra Shekhar Nautiyal1* 1
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Division of Plant Microbe Interaction, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India 2
Plant Ecology and Environment Science Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India Bacterial Genomics and Evolution Lab, CSIR-IMTECH, Chandigarh-160036, India.
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# sharing the first authorship. Corresponding author：
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Tel: +91-522-2297925, e-mail-
[email protected];
+91-522-2205839
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Fax:
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Tel: +91-522-2297825, e-mail-
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Highlights P. putida (RAR) acts as microalgae (C. vulgaris, CHL) growth promoting strain
CHL and RAR improve growth and imparts As and high P tolerance in rice
Synchronized interaction of CHL and RAR impedes arsenic uptake in rice
CHL and RAR modulates P trafficking in rice especially through OsPT11
CHL and RAR modulates mineral nutrient uptake under arsenic stress
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Abstract
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Rice grown in arsenic (As) contaminated areas contributes to high dietary exposure of As
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inducing multiple adverse effects on human health. The As contamination and application of
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phosphate fertilizers during seedling stage creates a high P and As stress condition. The flooded paddy fields are also conducive for algal growth and microbial activity. The present
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study proposes potential role of microalgae, Chlorella vulgaris (CHL) and bacteria, Pseudomonas putida (RAR) on rice plant grown under excess As and phosphate (P)
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conditions. The results show synchronized interaction of CHL+RAR which, reduces As uptake through enhanced P:As and reduced As:biomass ratio by modulating P trafficking.
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Gene expression analysis of different phosphate transporters exhibited correlation with reduced As uptake and other essential metals. The balancing of reactive oxygen species
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(ROS), proline accumulation, hormone modulation, and As sequestration in microbial biomass were elucidated as possible mechanisms of As detoxification. The study concludes that RAR and CHL combination mitigates the As stress during P-enriched conditions in rice by: (i) reducing As availability, (ii) modulating the As uptake, and (iii) improving
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detoxification mechanism of the plant. The study will be important in assessing the role and applicability of P solubilizing biofertilizers in these conditions.
Keywords
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Arsenic, arsenate-phosphate interaction, bioremediation, Chlorella vulgaris, phosphate
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transporters.
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1. Introduction Arsenic (As), having origin of geological, mining and industrial processes has resulted in contamination of the soil and groundwater [1]. Several Asian countries, including Bangladesh, China, and India are reported to have highest As pollution ranging from 50 -500 micrograms, at least 5 times higher to the WHO standard of 10 µg/L [2-5]. The population in the affected areas get exposed to As through consumption of tainted grains, vegetables,
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dairy products and drinking water [6-8]. Food grains produced in the As polluted regions, limited with mineral nutrients pose serious threat to health and economy of these countries
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[9-11].
Many strategies are being practiced to mitigate As contamination through breeding, transgenics, bioremediation and improved fertilization with their own limitations of time, ethics and applicability [12-14]. The excess phosphorous (P) fertilization in pursuit to
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improve productivity is widely reported in India [15, 16], which, is also known to aggravate
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the adverse effect of As by increasing its mobility and bioavailability [17-19]. Phyto-
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availability of As depends on the extent of P-induced As mobilization in soils and P-induced competition for As uptake by roots [20]. It is also known to magnify the As entry due to
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highly expressed transport pathways of their chemical analogues [21]. Application of microbes harbouring As tolerance is an upcoming important strategy in As mitigation [14,
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Rice is more prone to As uptake due to repeated irrigation with As contaminated
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ground water [22]. Flooded rice fields provide ideal conditions for microbial and algal growth, thus regulating As bioavailability through precipitation, complexation, redox
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reactions and nutrient availability [14, 23-25]. Microbes found in the soil are known to increase the P availability to the plants through solubilisation and mineralization processes [26]. Since P is a chemical analogue of As, the microbes affecting P bioavailability will effect As uptake as well. Chlorella vulgaris and Pseudomonas putida are prevalent micro-
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organisms with As mitigation potential [26-30]. Microalgae transform As through biosorption, exclusion, binding to cysteine rich peptides/proteins, methylation, and volatilization [27, 30]. Therefore, bioremediation strategies with appropriate heavy metaladapted rhizobacteria and microalgae have received more attention in wastewater treatment [31-33]. However, application of these commonly used microbes remains unexplored with respect to their synergy and role in As mitigation in rice. The present study investigates the 4
role of microalgae Chlorella vulgaris (CHL) with a P-solubilising, plant growth promoting bacteria, Pseudomonas putida MTCC 5279 (RAR) [34], in arsenic mitigation under high phosphate and As exposed conditions in rice.
2. Materials and methods Microalga Chlorella vulgaris (CHL) and growth promoting rhizobacterium P. putida MTCC
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5279 (RAR) were used for co-inoculation experiments. Rice plants were grown in presence of
these microbes both in hydroponic and soil conditions. Under hydroponic conditions different
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physiological parameters, As uptake and expression of selected genes were performed after
21 days of treatment (dpt). In live soil experiment, biochemical estimations were performed after 1 month of arsenic treatment. Physical parameters and arsenic estimation were performed at the time of harvesting. All the experiments were performed in four replicates.
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Detailed material and method is given in supplementary section.
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2.1. In vitro study of C. vulgaris and P. putida interaction
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C. vulgaris (CHL) and P. putida RAR were grown and maintained in mineral salts medium
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(BG-11) and nutrient broth (NB, Hi-media) or minimal growth medium (M9), respectively. Arsenic tolerance of both was determined by monitoring their growth under different arsenic
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concentrations (0, 25, 50, 100 µM) followed by their viable cell count determination (CFU/ml) at different time intervals upto 21 days. These cultures were incubated in
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Erlenmeyer flasks at 25 ± 2 °C with a light intensity of 60 mmol m-2s-1.
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2.2. Growth of rice plant in the presence of Chlorella vulgaris and Pseudomonas putida Oryza sativa cv. Triguna, was grown hydroponically as described earlier [35]. The treatments were control [CONT] (a), RAR (b), CHL (c) and CHL +RAR (d) under 0, 25 and 50µm arsenic concentrations of earlier reports [36, 37], along with 0, 350 and 1750µM KH2PO4 as P
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supplementation. Plants were grown in a growth chamber with 14h light, 28oC/20oC day/night temperature, and 70% relative humidity. Extended hydroponic experiments were performed with selected concentrations of As (50µM) and P (1750µM) and further, pot experiment with live soil was performed in presence of RAR, CHL and CHL+RAR. Arsenic (Na2HAsO4 @ 60 mg Kg-1) stress was given after 1 month of transplantation [8]. 2.3. Physiological assays 5
Different physiological parameters of growth viz. Chlorophyll [38] and P content [39]; stress indicator, proline [40]; lipid peroxidation [41]; and level of hydrogen peroxide [42] have been performed with their standard protocols. Superoxide dismutase (SOD), Glutathione Stransferase (GST) and Glutathione reductase (GR) activity was determined by method of Beauchamp and Fridovich [43], Habig and Jacoby [44] and Smith [45] respectively as the defense response. Hormones (gibberellic acid, indole acetic acid, and abscisic acid) were
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extracted by the modified method of Chen [46] and quantified according to Srivastava [38]. 2.4. Quantification of As and other mineral elements
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Arsenic (As) estimation in inoculums pellet (CHL, RAR, and their combination),
hydroponically (21dpt) and soil grown (at harvesting) plant tissues and soil samples were performed in the samples digested as per the protocol of Dwivedi [47]. As and other mineral
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elements in digested samples were quantified by Inductively Coupled Plasma Mass
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Spectrometer (ICP-MS, Agilent 7500 cx) as described in [47].
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2.5. Q- Real Time (Q-RT) PCR
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In order to study the expression pattern of randomly selected genes, Q-RT PCR of phosphate transporters [48] and stress responsive genes [38, 49] was performed at 21 dpt under As (50
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µM) and As+P (50 µM As+1750 µM P) conditions in rice root and shoot tissues in presence of inoculums (CHL, RAR, CHL+RAR) using gene specific primers (Table 1). Total RNA
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from root and shoot tissues was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using maxima H minus first strand cDNA synthesis
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kit (Fermentas, Thermo Scientific). Real-time PCR analysis was carried out in a 10µL reaction mixture with Quanti-Tect TM SYBR® Green PCR kit (Qiagen) on Stratagene Mx3000P systems, using actin as an internal reference. The reactions were performed using the cycle conditions of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94
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°C for 30s, 60 °C for 30s, and 72 °C for 30s. After obtaining ct value for each reaction, the fold change was calculated by delta-delta ct method. Heat map of the differentially expressed genes was generated using TIGR Multiple Experiment Viewer (TMeV) software and transcript levels were depicted by color scale indicating log2 values.
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3. Results and Discussion Pseudomonas putida (RAR) was found to promote the growth of microalgae Chlorella vulgaris (CHL). The increased growth of CHL in presence of RAR, further enhanced on P enrichment (Fig. 1A), may be attributed to microalgae growth promoting ability of RAR due to production of various plant growth promoting substances as reported earlier [34, 50]. Tolerance towards 100µM arsenic concentration up to 21 days was observed for both CHL
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and RAR strains, attaining 6.1 and 9.22 Log10CFU/ml respectively (Fig. 1B and C). The present study explored the combined effect of arsenic tolerant CHL and RAR in improving the
growth of rice plant during As stressed conditions. The study mainly focussed on the possible
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role of the microbial inoculation in modulating the P transport system of rice and various
biochemical and physiological processes to impart the enhanced As tolerance under P enriched hydroponic conditions. The effect of microbial inoculation was further validated
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under greenhouse conditions for amelioration of As stress.
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3.1 Effect of combined inoculation of P. putida and C. vulgaris on O. sativa
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Combined effect of CHL and RAR (CHL+RAR) on the growth of rice plant, exposed to As
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and P stress was examined under hydroponic conditions. It was found that CHL+RAR enhanced the growth and resulted in 44.78% and 72.06% increase in dry weight at 25 and
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50µM of As treatment respectively, which was further improved by 33 and 14 % on P enrichment under As stressed (50 µM As+350 µM P and 50 µM As+ 1750 µM P) conditions (Tables S1A, B), henceforth, 50 µM As and 1750 µM P concentrations was chosen to
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monitor the effect of microbial inoculations on plant growth. It was observed that 22% reduced dry weight due to As stress was improved by 33% on CHL+RAR inoculation at 21
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dpt (Table 2; Fig. 2A). Better growth of rice plant due to combined inoculation of CHL+RAR, may be attributed to their auxin producing [34, 51], microalgae growth promoting [50], arsenic tolerance (Fig. 1B, 1C) and heavy metal detoxification abilities [52, 53].
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An exponential growth of the microbes (bacteria and microalgae) observed on
rhizosphere as compared to extended lag phase of CHL under in vitro flask experiments was probably due to constitutive nutrient supply as root exudates, leading to plant growth promotion (Figs. S1A, B; Fig. 1B) [28, 54]. Effect of combined inoculation was further evident from arsenic stressed (CHL+RAR+As) plants with higher shoot length (5.51%), root length (28.64%), and dry weight (18.93%) under green house conditions as compared to control (Table 3; Fig. 2B). Chlorophyll, carotenoid and proline, serve as physiological 7
markers for plant health and development. Decline in level of chlorophyll and carotenoid due to arsenic stress under hydroponic conditions was rectified on microbial inoculation and P amendment (Table 2), which was also witnessed by two-way ANOVA based correlation analysis (Tables S3A, B). 3.2. C. vulgaris and P. putida modulate ROS mediated signalling Arsenic toxicity is known for excessive accumulation of reactive oxygen species (ROS)
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resulting in lipid peroxidation (LPX), enzyme inactivation and DNA damage [14, 55]. ROSmediated damage to lipids [malonadialdehyde (MDA) content] was reduced by the presence
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of CHL+RAR under As and As+P conditions in root, indicating the protective role of P and
the microbes against As stress. However, increased H2O2 content in both CHL+RAR+As and CHL+RAR+As+P conditions, appeared to maintain redox state for stress mitigation as reported earlier (Fig. S3A; Fig. S3B) [56]. Earlier studies of As stress in rice have reported
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redox modulations in early stages of stress [38, 57], whereas the present study shows the
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persistence of the ROS activity over a period of 21 days of As stress in presence of microbes.
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metabolism for acclimation purposes [56].
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Maintenance of redox state for longer period of time may enable the plant to adjust their
Different enzymatic processes are known to reduce arsenic stress by the involvement of
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glutathione and superoxide dismutase [58]. Higher GST, GR and SOD activities were found in rice roots under As+P conditions. CHL alone during As stressed condition kept the level of GST and GR elevated. However, CHL+RAR treatment resulted in higher level of GR only, as
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compared to control (Fig. 3A, B). SOD activity was significantly greater in all treatments under As+P conditions, while, presence of As did not have any effect on SOD level
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irrespective of presence of microbial inoculations even after 21 days of As stress (Fig. 3C). Higher glutathione S-transferase (GST) activity during P-enriched conditions has been shown to be crucial in regulating As-induced oxidative stress. Lower accumulation of GST in CHL+RAR+As+P treatment emphasizes the importance of microbe-mediated management
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[12, 59]. Elevated levels of H2O2 in CHL+RAR+As+P treatment irrespective of improved biomass, indicates alternative mechanism for ROS balancing such as proline accumulation as reported earlier [38, 60]. The higher proline content (3702µM), prominently observed in P enriched conditions (Table 2), is probably due to involvement of oxidative pentose phosphate (OPP) pathway in proline synthesis [60]. 3. 3. C. vulgaris and P. putida modulate hormone mediated signalling 8
Rice roots in presence of different treatments showed differential accumulation patterns of indole acetic acid (IAA), gibbrellic acid (GA) and abscisic acid (ABA) (Fig. 4). Shoot IAA content was negatively affected by the presence of As, whereas, root IAA content increased under As and As+P conditions. CHL+RAR inoculation maintained the shoot IAA level under all the three conditions (CONT, As, As+P), however, root IAA level was negatively affected during As stress (Fig. 4B). Improved plant biomass observed due to CHL+RAR coinoculation in hydroponically grown rice was evident with higher auxin content in RAR
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treatment [38]. However, it was observed that plant growth promotary, As tolerant RAR was unable to improve the auxin level in root and shoot under As stress condition. Albeit,
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presence of CHL (CHL+RAR+As/As+P) restored the auxin levels similar to control supports the auxin producing ability of Chlorella [51]. GA content was found to be higher in As alone conditions, which was reduced by the treatment of CHL+RAR and P, however, As+P treatments led to slightly higher accumulation of GA in root tissues (Fig. 4A). Shoot GA
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content was higher due to CHL+RAR inoculation under both As and As+P conditions. It can
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be deduced from previous report [61], that heavy metal detoxification associated with higher
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GA content, supported the plant growth under As stress. Arsenic stress elevated the shoot ABA content whereas, CHL+ RAR inoculation reduced its accumulation in both As and
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As+P conditions (Fig. 4C). Higher ABA content in As treated plant indicates its role as a stress marker in accordance to earlier report [62] and further reduction by microbial
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inoculation emphasised their role as a stress ameliorator [12, 52].
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3.4. Effect of C. vulgaris and P. putida on arsenic uptake The present study has focussed specifically on role of microbes in As translocation. Consistent increase in As uptake in hydroponically grown rice after 7, 14 and 21 dpt was
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lowered in both root and shoot in the presence of CHL+RAR, exposed to 50 µM As, followed by reduction on P amendments (350, 1750 µM) (Table S2, Fig. S2). These observations led to the hypothesis that the presence of RAR and CHL in the growth medium reduced the As
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uptake by roots. Extended hydroponic experiments with selected concentrations (50 µM As + 1750 µM P) also showed lesser arsenic uptake in CHL+RAR treatment (Fig. 5A). Contrary to that As uptake was higher in the microbial biomass (CHL+RAR) both in As and As+P conditions (Fig. 5B). Presence of P and CHL+RAR decreased the As uptake by 48.15 % and 77.7% respectively in rice roots compared with As control (Fig. 5C). P supplementation is known to increase the As translocation [20]. Present study also observed enhanced As
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translocation during As+P condition, which was decreased due to CHL+RAR inoculation by 38.99% (Fig. 5D). However, the As uptake reduced due to its enhanced sequestration in the microbial biomass, reducing its availability as reported earlier [63], using C. pyrenodoisa. The reactions taking place at the interface of the root surface may convert the available P into unavailable complexes or precipitate them by chelation causing P deficit like conditions even in high P doses as evident from earlier report of fixation of available P
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sources [64]. However, the microbial activity are probably able to maintain an equilibrium of the available and unavailable forms of P so that the plant is able to uptake P, as evident by improved P content in As stressed plants as compared to respective controls (CONT, As)
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(Table 2; Fig. 5D). A competitive involvement of P, magnified by RAR, is probably operative
in rice plant, which resulted in enhanced growth and reduced level of arsenic. Phosphate and As ratio (P:As) depicting the competitiveness of the two ions was found to be enhanced by
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the availability of P and microbial inoculations (Table 2). The observation is supported by the arbuscular mycorrhizal fungus (AMF) mediated maintenance of higher P:As ratio for
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amelioration of As toxicity [8, 12]. Relationship of P:As and As:biomass ratio with reduced
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arsenic uptake [8], is well correlated with lower As:biomass ratio observed in present study.
(As:biomass) (Tables 2 and 3).
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Probably, CHL+RAR inoculation mediated enhanced biomass dilute the arsenic concentration
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Differential pattern of As and P accumulation observed in green house grown rice plants is well correlated with hydroponic experiments. Shoot arsenic content was lowest in
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CHL+RAR treatment along with high P content. Translocation of P was enhanced in presence of RAR and its combination with CHL (CHL+RAR) and conversely for As. Higher P:As and reduced As:biomass ratio of both root and shoot tissues observed in the presence of
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combination (CHL+RAR) is in accordance to the report of Li[8] (Fig. 5E-G; Table 3). Soil and grain arsenic content lowered by the presence of RAR, CHL and CHL+RAR as compared
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to As alone supports the As stress ameliorative property of the inoculums.
3.5. Effect of C. vulgaris and P. putida on other mineral uptake Arsenic mediated limitation of mineral nutrients in arsenic contaminated rice is known (10, 11, 47). Considering this, present study has also performed the correlation analysis to find out the effect of CHL+RAR inoculation on mitigation of arsenic induced reduction of different 10
microelements. Analysis of correlation coefficient (r2 values) of mineral nutrients in response to As and As+P conditions showed that, a general negative correlation observed in the presence of As, was decreased by P supplementation in roots for uptake of Mn, Fe, Co, Ni and Mo, which was unaffected for Cu and Zn. However, shoot showed similar pattern of uptake in all the minerals except for Fe and Zn. Thus, P supplementation overcame the inhibitory effect of As in mineral nutrients acquisition. The translocation was profoundly affected for Zn by RAR, Fe by CHL and Cu by CHL+RAR in roots. Positive correlation for Fe
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and Mo in CHL, and negative for Co and Ni in CHL+RAR was observed in shoot (Table 4; Figs. S4A-D). Translocation of mineral elements in the presence of CHL/RAR/As/P showed
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involvement of competition and/or modulations of the transporters. A general decrease observed in the uptake of Fe in the presence of As may be associated with Fe plaque formation responsible for sequestering minerals, metalloids and heavy metals magnified in
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the presence of CHL+RAR as reported earlier [65-67].
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3.6. Effect of C. vulgaris and P. putida on targeted gene expression
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The present study has focussed specifically on role of P trafficking in As translocation. The differences in P and As contents in root and shoot represented that microbial inoculums in
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presence of arsenic affected P transporters (Fig. 5C, D, F,G). Expression pattern of the representative genes of phosphate transport (6), stress response (5) and arsenic detoxification
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(4), showed differential response for root and shoot tissues (Table 1). Among the selected genes of P transport, most of the genes were upregulated in RAR, CHL and CHL+RAR under As+P conditions (3 to 70 fold; Fig. 6; Fig. S5a). The OsPT2, a
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low affinity P transporter responsible for direct P uptake and phosphate transporter 3 (OsPT3), a mitochondrial P transporter, showed higher expression in root and mixed expression in shoot under As and As+P conditions. Treatments with CHL and CHL+RAR
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resulted in upregulation of OsPT2 and OsPT3 in shoot under As+P conditions. The OsPT2 is a root stele specific gene in rice involved in Pi signalling, acquisition and loading of P and As into the xylem [68, 69]. The gene expression increased in the rice roots in presence of RAR in all the combinations with CHL, As and P, except in RAR+As treatment indicating creation of Pi deficiency as reported earlier [70]. The deficiency created in presence of As (control As) was overcome by RAR, a P solubilizer resulting in unaltered OsPT2 expression in RAR+As. The bacterium Pseudomonas has been reported to possess two types of P binding proteins 11
(PBP), one is high affinity P binder which acts under low P conditions and the other is low affinity which acts under high P conditions and are also able to discriminate between As and P, and selectively take up P in bacterial cells [71]. The ability of RAR to efficiently colonize rice roots and influence plant growth, and plausible presence of the two PBP components in RAR could explain the low expression of OsPT2 in RAR+As treatment and over expression in P+As/CHL+P+As treatment. The modulated RAR expression of PBP components probably maintains the high P or P deficit like conditions by selective absorption of As/P in different
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treatments. The gene OsPT3, is known to have a role in redox homeostasis, alternate
respiration pathway and leaf and flower development [72]. Its higher expression in root as
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compared to shoot in response to both biotic (RAR, CHL and CHL+RAR) and abiotic interactions (As and As+P) emphasizes high P mediated multifold modulations of mitochondrial functions in rice root system [48].
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The gene OsPT5, a Pi transporter playing central role in P homeostasis, is upregulated during As stress, also responsible for root As uptake as reported earlier [73, 74]. Contrary to
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earlier report higher OsPT5 expression was observed with significant reduction in As
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accumulation by As, RAR, CHL and RAR+CHL both in root and shoot. It is interesting to note
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that under normal P conditions the microbial inoculation is reducing the P uptake in presence of As, whereas, under high P condition the inhibitory effect of As seems to be compensated
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such that both P and As uptake increased significantly as compared to As treatment. This indicates that, it is probably the availability and form of the As/P and not the microbes directly, which are involved in the regulation of OsPT5 expression. The gene OsPT11 is a Pi
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transporter exclusively reported to be expressed in presence of AMF fungi irrespective of P supplementation [68, 75]. However, the present study showed that As could modulate
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expression of OsPT11 in presence of P (As+P). The changes in expression levels of OsPT11 observed in presence of CHL and CHL+As emphasize that the presence of CHL increased the OsPT11 expression in roots under control conditions, which was maintained in presence of As, As+P and RAR+As+P for decreasing the As uptake/accumulation in roots. To the best of
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our knowledge, the present study reports higher expression of OsPT11 for the first time in presence of microalgae which improved P:As ratio (Tables 2 and 3) as reported by Li [8]. OsPHR2, the negative regulator of phosphate transport and OsPHO2, the gene responsible for P accumulation in root were downregulated (0.1-0.44 fold) under As coditions. However, these genes were upregulated in root under CHL+RAR+As condition.
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Over-expression of these genes in all As+P treatments as compared to As indicated the competitive role of Pi over As resulting in increased uptake of both P and As in shoots as evident by earlier report that over-expression of OsPHR2 under Pi sufficient condition results in P accumulation [76]. However, due to the complex nature of the OsPHO2 expression and function for P uptake and translocation, the results in the present study are insufficient for a conclusive inference. The upregulation of PHR2 and OsPHO2 a Pi transport regulator [77], may be due to induced expression of high and low affinity phosphate transporters [21, 69,
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78].
Among defence responsive genes, glutathione reductase (OsGR, Os02g56850), the
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key player of metalloid tolerance, got exceptionally downregulated in root and overexpressed in shoot. Glutathione S transferase (GST) and other oxidative stress responsive genes NADPH dependent FMN reductase (FMN, Os05g42190), NADPH Oxidase (NOX,
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Os08g35210) and peroxidase (PER) were having similar expression pattern in root, however, NADPH dependent FMN reductase have differential expression pattern in shoot among the
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four treatments (Fig. 6, Fig. S5b). Higher expression of GST, GR, NOX, PER, FMN in
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CHL+RAR (As+P) treatment emphasizes the importance of microbe-mediated management
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of oxidative stress. Higher expression of above mentioned genes co-regulated with their enzyme activities (GST, GR and SOD) during As+P condition seems to provide higher
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antioxidant potential with reduced As uptake as reported with As+Se+P conditions [12, 59, 77]. Proline accumulation, an alternate ROS balancing mechanism, probably due to the involvement of oxidative pentose phosphate (OPP) pathway of proline synthesis [38, 60],
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prominently higher in P-enriched conditions was further enhanced due to microbial inoculations. Upregulation of FMN and NOX under As+P conditions ensure the availability
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of NADPH for the OPP pathway of ROS balancing (Fig. 6 and S5a) [79]. Expression of arsenate reductase (ARSR, KC687096) and MYB transcription factor
(Os10g41260) was higher (3 to 7 fold) in CHL and CHL+RAR treated root during As
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exposure (Fig. 6 and Fig. S5c). However, P enrichment (As+P) upregulated its expression predominantly in shoot tissues (Fig. 6 and Fig. S5c). Arsenate reductase (Ars R, KC687096), responsible for reducing AsV to AsIII, showed higher expression in CHL+RAR treated roots as one of the amelioration mechanisms followed by lower As transportation in shoot is in accordance to the report of Meadows [80]. MYB transcription factors (XM015757381) are known to regulate various metabolic pathways by upregulating the phenylpropanoid pathway
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during abiotic and biotic stresses [81]. Their upregulation in CHL and CHL+RAR treated rice roots emphasizes its multi-regulatory role at interaction site (Fig. 6 and S5a). Expression of gibberellin 20 oxidase (G20O, Os01g66100) was significantly higher during As+P condition (8 and 3 fold) and inoculums treatment (5 and 10 fold) in root and shoot (Fig. S5c). Enhanced plant growth correlated with higher GA content by microbes, even under high As concentration, may be attributed to expression of gibberellic acid (G20O,
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Os01g66100) [61]. The expression of OsZIP2 (Os03g29850), a metal cation transporter, did not alter during As stress; however, P supplementation upregulated its expression (4 fold) in roots (Fig. S5c). Treatments with CHL and CHL+RAR further resulted in higher expression
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of OsZIP2 in both root and shoot under As+P conditions. Its higher expression in root during As+P conditions, depicts symporter activity of phosphate in metal transportation, irrespective
of the microbial colonization, as reported for E. coli [82]. Similarly, enhanced co-
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translocation of Zn with As in CHL+RAR treatment correlates well with higher transcription
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of OsPT11 and metal cation transporter (Os3G29850) [83].
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The present study concludes that there exist complex interactions between microbes in rhizospheric regions which affects the availability and competition of nutrients and
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modulate plant uptake channels. The interaction modulated the sensing of the P channel such that the P deficit signalling was induced even in presence of high P conditions to
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accommodate P transportation for reduced As uptake. The study further suggests involvement of multiple mechanisms in the process of reduced As accumulation, including As
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sequestration in microbial biomass, interplay of metal and phosphate transporters, ROS balancing and increased biomass. The study will be useful for further investigations of detailed mechanism of microbial consortia mediated arsenic stress amelioration in
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agricultural crops and development of microalgae and bacteria based bio-inoculum packages.
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Acknowledgement
The study was supported by Network projects, BSC 0204 and INDEPTH- BSC0111 from CSIR, New Delhi, India. So.S and R.C. thanks DST for awarding Research Fellowship.
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Figure captions: Figure 1. Growth of C. vulgaris (CHL) in presence of P. putida (RAR) under phosphate
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Figure 2. Growth of rice in hydroponic conditions (21 dpt) under CONT, As (50 µM) and As+P (50 µM+1750 µM) (A); in green house conditions (30 dpt) treated with As (60mgKg-1)
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Figure 3. Activities of glutathione S- transferase (GST) (A), glutathione reductase (GR) (B)
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CHL+RAR combination under conditions of CONT, As (50 µM) and As+P (50 µM+1750 µM) at 21 days of treatment. Vertical bars indicate mean±S.D. of three replicates. Means
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followed by the same letter were not significantly different at p < 0.05.
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Figure 4. Endogenous level of gibberellic acid (a), indole acetic acid (b) and abscisic acid (c) in root and shoot tissues of rice treated with CHL, RAR and CHL+RAR combination under
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conditions of CONT, As (50 µM) and As+P (50 µM+1750 µM) at 21 dpt. Vertical bars indicate mean±S.D. of three replicates. Means followed by the same letter were not
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significantly different at p < 0.05. Figure 5. Total arsenic content in whole biomass of rice (A), in bioinoculum pellet (B); arsenic and P trafficking in root (C) and shoot (D) tissues of rice treated with CHL, RAR and CHL+RAR under CONT, As (50 µM) and As+P (50 µM+1750 µM) conditions grown hydroponically; (E), arsenic content in soil, husk and grain of rice and As and P trafficking
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in root (A) and shoot (B) tissues of rice on treatment with CHL, RAR and CHL+RAR combination under As stress. Vertical bars indicate mean±S.D. of three replicates. Means followed by the same letter were not significantly different at p < 0.05. Figure 6. Heat map showing expression analysis of phosphate transporters, defence and other
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stress responsive genes in root and shoot tissues of rice treated with CHL, RAR and CHL+RAR combination under conditions of CONT, As (50 µM) and As+P (50 µM+1750
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EP
C
120
CC
ABA content µg g-1 FW
D
a
20
160
cd
b
c
0
c
M
d
A
N
IAA content µg g-1 FW
60
ab
A
cd
U
e
de
SC R
b a ab
0
Fig.4
f
f
80
Root
c
d
d c
80
40
b a
0
a
abcd
a
a
a
a
a f
ab
e
bcd
de
abc
a
abc abcd
a cde
Fig. 5
D
TE
EP
CC
A
SC R
U
N
A
M
IP T
Fig. 6
D
TE
EP
CC
A
SC R
U
N
A
M
IP T
Table Legends: Table 1 List of primers used in the study. Table 2
IP T
Effect of C. vulgaris and P. putida inoculation on biomass, arsenic and P uptake in hydroponically grown rice
SC R
Table 3
Effect of C. vulgaris and P. putida inoculation on biomass, arsenic and P uptake in rice under green house conditions
U
Table 4
N
Correlation coefficient of different metal ion with arsenic uptake during C. vulgaris and P.
A
CC E
PT
ED
M
A
putida inoculation exposed to As and As+P conditions in rice tissues.
28
Table 1: List of primers used in the study
6
OsPHR2 OsPT11 (AF536960) OsPHO2
7
10
OsGluta R (XM015771323) Glutathione S- Transferase (Os10g38590) Giberellin 20 Oxidase (Os01g66100) NADPH oxidase (OsNox) (Os08g35210)
11
Peroxidase Precursor (Os01g73200)
12
13 14 15
OsNADP FMN For: TGCACGGCGGCAGCCCGTACGG OsNADP FMN Rev: GCGATGCCAGCGAAGTACTTGC (OsArsR) For: GATCTACGACGCGCACATC (OsArsR)Rev: ACAGTGGAAGACGAGGGTTT Os MYB For: AATTCATACAGATCGCGGCG Os MYB Rev: CGGCGGCCTTTAATCTCTTC OsActin For: GAGTATGATGAGTCGGGTCCAG OsActin Rev: ACACCAACAATCCCAAACAGAG
[69]
This study [38] [38] [38]
OsPER For: CGACCCCACCATGGACAAGTG OsPER Rev: TGGCGGTTTTGCAGGTCGACG OsZIP2 For: CGTTCCTCTCCTGCTTCGGCTA OsZIP2 Rev: CCCATGGAGACGGCGAAGATC
[48]
[69]
[38]
This study [38] This study This study [38]
A
16
Metal Cation Transporter (ZIP2) (Os03g29850) NADPH Dependent FMN Reducatse (Os05g42190) Arsenate Reductase (KC687096) Os MYB (XM015757381) OsActin
[48]
This study
OsGST For: GACGAGCTCATGAAGCAGACGC OsGST Rev: CAGCACGACGTCGACGTAGCCG OsG20O For: TCGAACGGGAGGTATAAGAGCT OsG20ORev: CTGCGGCGTGGCGGCGCTCG OsNox For: GCTGCACAACTATCTCACAAGT OsNox Rev: AGGCCTTGCAAAATGTGTCCTG
PT
9
CC E
8
IP T
OsPT5
[48]
SC R
5
OsPT3
OsPT2 Rev: GTCGACGACGTTGTCCTTG OsPT3 For: CATGCTCATGACGCTGCT OsPT3 Rev: GCGACGTTCTCCTTGGAC OsPT5 For: TCGACGAGCAGGAGAAGG OsPT5 Rev: AATTCCCGGGAGAAGAGC OsPHR2 For: GACCAGAATTGTCTGAAGGTTCTT OsPHR2 Rev: ACGCAATGCCTCAGTGAGAT OsPT11 For: ATCGCCTTCTACAGCCAGAA OsPT11 Rev: GGCCTTGGATATCTGGAACA OsPHO2 For: CGAGAATTTTGTCAAGGAGCA OsPHO2 Rev: TCACGAGCATGTCCAACAA OsGR For: GCTGCTGGTGTTGAAGTTGA OsGR Rev: CGGTTCGTTACATCACCCAC
U
4
OsPT2 For: CACAAACTTCCTCGGTATGCT
N
3
Source
A
2
OsPT2
Primer Sequence
M
1
Primer Details
ED
S/N o.
29
I N U SC R
Table 2: Effect of C. vulgaris and P. putida inoculation on biomass, arsenic and P uptake in hydroponically grown rice
A
Root length (cm)
M
Treatments
Shoot length Dry weight (g) (cm)
Proline (μM)
Shoot/Root Biomass
Translocation factor P
As
P/As ratio Shoot
As/biomass ratio
Root Shoot
Root
0.46±0.02
15.4±2.02
3.10±0.45
0.43
0
0 306.33 2.89
57.95
40.18±0.49
0.59±0.02
27.94±5.44
2.97±0.19
0. 06
0
0 219.04 0.41
44.26
20.42±1.34
37.75±1.00
0.50±0.55
33.93±4.58
3.75±0.14
0.32
0
0 456.21 1.38
44.19
23.75±0.64
0.69±0.05
23.26±3.03
2.58±0.26
0.75
0
0 98.35
42.56
14.70±0.48
34.05±0.62
0.36±0.02
62.48±4.35
5.15±0.16
0.24
0.009 8.80
0.31 266.48 159582.30
15.5±1.01
RAR
22.31±1.17
CHL
34.40±1.24
PT
37.28±0.86
RAR(As)
11.72±0.81
33.45±0.95
0.40±0.02
28.98±0.23
4.20±0.42
0.37
0.008 12.31
0.28 177.19 86799.75
CHL(As)
16.37±0.88
33.95±0.76
0.40±0.02
33.11±5.60
2.96±0.17
0.26
0.011 21.53
0.96 148.34 38555.90
CHL +RAR(As)
17.16±0.88
33.45±1.19
0.61±0.02
36.46±3.50
2.80±0.31
0.23
0.018 19.53
1.55
A
ED
CONT
CONT(As+P)
17.16±0.90
35.65±0.84
0.48±0.05
1819.95±133.01
4.00±0.27
0.47
0.042 8.39
0.76 449.46 42293.16
RAR(As+P)
15.72±0.88
34.45+1.43
0.54±0.05
3506.25±711.10
3.73±0.34
0.42
0.052 11.26
1.40 389.06 27643.37
CHL(As+P)
17.58±1.00
33.77±1.19
0.56±0.02
1191.3±32.67
3.02±0.33
0.35
0.060 10.14
1.74 329.82 16457.00
CHL +RAR(As+P)
14.18±0.76
34.54±1.53
0.58±0.05
3702.6±546.03
3.48±0.22
0.35
0.057 10.67
1.74 299.27 20435.83
CHL +RAR
CC E
CONT(As)
1.04
90.28 13224.12
30
I N U SC R
CONT
RAR
CHL
RAR+CHL As
RAR (As)
CHL (As)
Root length(cm) Shoot length(cm) No. of Tillers No. of Spikes Spike Length Dry weight(g) Shoot/Root Biomass A Chlorophyll B (mg/g FW) Total Sugar (µg/g) Proline (µM) Translocation P factor As Root P:As Shoot Root As: biomass Shoot As conversion factor
11.66±0.33 60.33±1.45 3.66±0.88 3.66±0.88 14.96±0.70 7.92±1.51 0.68821 1.28±0.02 0.52±0.01 1.80±0.03 58.80±0.60 21.61±0.38 0.205 0 0 0 0 0 0
13.00±1.92 62.40±1.20 5.20±0.37 4.20±0.37 15.34±0.62 8.84±1.15 1.018248 0.80±0.03 0.24±0.01 1.04±0.05 45.20±5.20 20.90±0.55 0.128 0 0 0 0 0 0
20.00±0.63 60.00±3.34 3.80±0.66 3.40±0.50 17.46±0.67 8.31±1.10 0.832085 0.61±0.01 0.15±0.01 0.77±0.01 43.00±1.20 15.62±0.00 0.208 0 0 0 0 0 0
14.60±1.12 70.00±3.30 6.00±1.04 8.20±0.66 16.03±0.86 9.95±0.46 1.380722 0.61±0.01 0.17±0.00 0.78±0.01 63.40±1.00 25.41±1.76 0.215 0 0 0 0 0 0
15.33±1.33 60.50±2.21 5.75±1.37 1.75±0.47 14.11±0.44 9.42±1.08 0.876119 0.93±0.03 0.30±0.01 1.23±0.04 49.10±0.10 29.37±1.87 0.217 0.036 0.003 0.337 8505.47 347.38 4.02
14.40±0.97 61.60±1.98 4.50±0.61 3.50±0.76 14.20±0.55 9.97±0.90 0.780785 0.80±0.01 0.26±0.01 1.06±0.00 65.10±9.90 16.88±0.71 0.121 0.119 0.002 0.165 4840.57 738.90 4.137
Arsenic , P modulations
A
PT
ED
M
A
Treatments
CC E
Biochemical estimations
Physical parameters
Table 3: Effect of C. vulgaris and P. putida inoculation on biomass, arsenic and P uptake in rice under green house conditions. .
15.50±0.86 52.25±4.30 3.75±0.85 3.00±0.40 17.62±1.67 6.88±2.81 0.66707 0.29±0.017 0.07±0.00 0.36±0.02 44.50±4.30 16.28±1.65 0.09 0.037 0.00 0.218 15804.56 885.47 2.81
RAR+CHL (As) 15.00±0.57 63.33±2.40 4.33±0.66 3.00±0.00 16.14±0.68 9.42±1.04 0.60477 0.77±0.01 0.27±0.01 1.04±0.02 74.50±3.30 18.86±0.93 0.134 0.027 0.002 0.551 5346.39 238.69 2.441
31
I N U SC R
Table 4: Correlation coefficient of different metal ion with arsenic uptake during C. vulgaris and P. putida inoculation exposed to As and As+P conditions in rice tissues.
ED
PT
Correlation coefficient (r2 values) of mineral nutrients with As during As and As+P conditions ROOT CONT As As+P SHOOT CONT As (-)0.141 (+)0.519 (-)0.875 (-)0.662 Mn (+)0.002 (-)0.498 (-)0.807 (-)0.351 (+)0.670 (-)0.998 (-)0.752 Fe (+)0.070 0 (-)0.478 (+)0.005 (+)0.691 (-)0.945 (-)0.558 Co (-)0.010 (-)0.138 (-)0.721 (-)0.050 (+)0.242 (-)0.925 (-)0.813 Ni (-)0.320 (-)0.142 (-)0.896 (-)0.145 (+)0.787 (-)0.976 (-)0.925 Cu (-)0.22 (-)0.263 (-)0.942 (-)0.074 (+)0.715 (-)0.863 (-)0.810 Zn (-)0.163 (-)0.452 (-)0.964 (-)0.786 (+)0.979 (-)0.982 (-)0.718 Mo (-)0.060 (-)0.228 (-)0.888
CC E
Mn Fe Co Ni Cu Zn Mo
M
A
Correlation coefficient (r2 values) of mineral nutrients with As between different treatments (CHL and CHL+RAR) Root Shoot CONT RAR CHL RAR+CHL CONT RAR CHL RAR+CHL (-)0.198 (-)0.381 (+)0.565 (+)0.545 Mn (+)0.36 (-)0.001 (+)0.751 (-)0.029 (-)0.697 (-)0.998 (+)0.992 (-)0.99 Fe (+)0.192 (+)0.691 (+)0.941 (-)0.596 (+)0.674 (+)0.97 (+)0.963 (+)0.872 Co (+)0.178 (+)0.415 (+)0.145 (-)0.995 (-)0.991 (+)0.85 (+)0.977 (+)0.899 Ni (+)0.328 (-)0.139 (-)0.269 (-)0.998 (-)0.781 (-)0.298 (+)0.389 (+)0.926 Cu 0 (+)0.125 (-)0.136 (-)0.146 (-)0.983 (+)0.037 (+)0.182 (+)0.457 Zn (-)0.268 (-)0.222 (+)0.013 (-)0.115 (-)0.839 (-)0.88 (-)0.963 (-)0.66 Mo (-)0.323 (-)0.215 (+)0.899 (+)0.088
A
Mn Fe Co Ni Cu Zn Mo
As+P (-)0.230 (-)0.489 (-)0.20 (+)0.263 (-)0.563 (-)0.905 (-)0.789
32
