Seed priming with Se alleviate As induced phytotoxicity during germination and seedling growth by restricting As translocation in rice (Oryza sativa L c.v. IET-4094)

Ecotoxicology and Environmental Safety 145 (2017) 449–456

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Seed priming with Se alleviate As induced phytotoxicity during germination and seedling growth by restricting As translocation in rice (Oryza sativa L c.v. IET-4094) Debojyoti Moulicka, a b

⁎,1

MARK

, S.C. Santraa, Dibakar Ghoshb,1

Department of Environmental Science, University of Kalyani, Nadia, West Bengal, India ICAR-Directorate of Weed Research, Jabalpur, Madhya Pradesh, India

A R T I C L E I N F O

A B S T R A C T

Keywords: As Interaction Rice Seed priming Se Translocation

Interactive aspect of among selenium (Se) and As (As) to mitigate As induced phytotoxicity in rice during germination and seedling growth has been based on mostly to petriplates and hydroponic mode of experiments. In this investigation we explore the consequences of sowing Se primed rice seeds in As spiked soil. Unprimed, hydroprimed and Se primed rice (IET-4094) seeds sown in As spiked soil, with five replications, arranged in complete randomized design for evaluating the impacts of seed priming on germination and seedling growth as well as As uptake and translocation pattern. Se promotes germination, seedling growth by modulating proline content, lipid peroxidation in root and shoot beside enhancing total chlorophyll content significantly in both As free and As spiked soil as compared to their respective unprimed and hydroprimed counterparts grown alike. Findings also indicates that seed priming with Se was able to execute dual roles i.e. a promotive and antagonistic aspect against As by restricting maximum soil As load to the root (with greater bioconcentration factor) and reducing translocation of As from root to shoot in a more practical and farmer friendly way to mitigate As induced toxicity and enhance germination and growth in rice seedlings.

1. Introduction During cultivation of cereal crops like rice, a preset amount of seeds are sown in to seed bed prepared often adjacent to actual fields with the aim to accomplish a set number of seedlings, that upon transplanting and cultivation can achieve satisfactory or higher yield per unit area. Now if germination and subsequent emergence of seedlings fail to achieve in adequate seedling rate that will further decrease yield upon cultivation (Bleasdale, 1967). In Arsenic (As) contaminated agroecosystems like in Ganga-Meghna-Brhamhaputra basin area as well as in other countries of the world, phytoxicity of As in rice plant is a serious concern (Moulick et al., 2016b; Santra et al., 2013). Rice plant (Oryza sativa. L) has been considered as an efficient As accumulator due to it's ability to absorb As from paddy field soil. The As concentration in rice grain depends upon the ability of rice plants to accumulate As in to their root zone from irrigated field it is generally expressed as bioconcentration factor (BCF) and further it's capability to translocate into the aerial part of the plant, expressed as translocation factor (TF), which is influenced by several soil physicochemical properties such as pH, conductivity, texture (Chen et al., 2016; Dai et al., 2016; Sahoo and

⁎

1

Corresponding author. E-mail address: [email protected] (D. Moulick). Authors sharing Equal Credit.

http://dx.doi.org/10.1016/j.ecoenv.2017.07.060 Received 16 May 2017; Received in revised form 16 July 2017; Accepted 21 July 2017 0147-6513/ © 2017 Published by Elsevier Inc.

Kim, 2013). In case of rice, the TF for As is closed to unity (Kumar et al., 2015; Bhattacharya et al., 2010). Speaking of rice cultivation, generally rice seedlings were uprooted from seed bed and then manually transplanted in to puddled soil under anaerobic condition with continuous flooding i.e. three to four centimeter of standing water, moreover this anaerobic conditions favors conversion of arsenate (AsV) to arsenite (AsIII) (Ghosh et al., 2016; Takahashi et al., 2004). Upon exposure to As, reactive oxygen species (ROS) forms within the plant cell often leads to enhance lipid peroxidation thus increase membrane permeability which may even lead to premature cell and then subsequently death of whole plant (Farooq et al., 2016a, 2016b). As toxicity in rice plant specially by the inorganic As (iAs) species like AsV and AsIII resulted in consequences ranges from inhibition of germination, seedling growth (Moulick et al., 2016b; Shri et al., 2009) to reduction in chlorophyll content, tiller number, test weight (Rahman et al., 2007, 2014; Rauf et al., 2011; Bhattacharya et al., 2013). Till date, numerous mitigation option have tried in order to decrease phytotoxic influence of As by reducing uptake and/translocation in rice seedling. Among the various investigated methods, role of iron plaque (Lee et al., 2013); salicylic acid (Singh et al., 2015); calcium as

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calcium chloride (Rahman et al., 2015); inorganic phosphate (Choudhury et al., 2011); co application of selenite and phosphate (Kumar et al., 2013); silicon fertilizer in soil (Lee et al., 2014) have been reported so far. Beside these, by utilizing Se's ability to mitigate heavy metal induced toxicity including As also investigated in prokaryotes, due to having analogous role in various metabolic functions (Stolz et al., 2006). Among the various plant species including rice plant, the role of selenium (Se) to execute antagonism against wide ranges of heavy metals such including AsV by inhibit their uptake and/translocation in plant system in in vitro condition were also investigated. Based on the outcomes of hydroponic experiment (mostly), Se is found to execute anatagonism to restore heavy metal and mettaloid induced phytotoxicities in field crops (Feng et al., 2013, 2009a, 2009b; Kumar et al., 2015; Malik et al., 2012; Srivastava et al., 2009) and in petripate (Shri et al., 2009; Moulick et al., 2016b) so far. Seed priming technology is popular pre-sowing practice of seed enhancement where seeds, after hydrating partially in order permit metabolic events to take place with out germination followed by redried close to actual weight (Bradford, 1986). Seed nutripriming is a particular seed treatment where various micro-nutrients were used in priming solution as priming agents (Singh, 2007). When compared with the unprimed seeds, the primed seeds exhibited faster germination in more synchronized fashion (Farooq et al., 2009) due to have relatively less imbibition time (Brocklehurst and Dearman, 2008) and greater accumulation of germination promoting metabolites (Farooq et al., 2006). Earlier, there are reports published about the positive consensuses of seed priming technology with a wide range of priming agents to alleviate various abiotic stresses in wide range of agriculturally important crops (Jisha et al., 2013). According to the opinion of Whalley and Finch-Savage (2006, 2010) and Hadas (2004) that in seedbed, seeds and later upon germination seedlings, used to experience multiple stresses as multiple soil environmental factors (including distict physicochemical and textural properties) are usually active due to complex nature in seed bed environment. Among the factors associated with seedbed environment studied so far, there is hardly any reports exits that describe the influence of As present in the seed bed soil on germination, seedling growth and As uptake pattern along with impact seed priming with Se as a potential mitigation option on above is also rare. We set this experiment in order to asses the consequences of sowing unprimed and primed rice seeds with water and selenium prior to sow in As free as well as in As spiked soil on (1) germination and seedling growth, (2) soluble protein, proline content and extent of lipid peroxidation in both root and shoot beside total chlorophyll content in leaves, finally (3) As accumulation and translocation pattern in order to further enrich our understanding of interactive aspect exist between Se and As when Se is supplemented in seeds though seed priming technology.

Table 1 Physicochemical properties, textural profile and As and Se content of garden soil, tap water (used for irrigation), intact seeds prior to sowing. As recovery from standard reference materials (Rice Flour SRM-1568a and San Joaquin Soil SRM-2709) and Se content in mean ± SD format (n = 5) in root and shoot of tested rice variety on 14 DAS (days after sowing) grown in As free soil. Specifications Garden soil (Before As treatment) Organic carbon pH (soil:water of 1:2) EC (soil:water of 1:2) Total nitrogen Available phosphorous Available As Available Se Total As Total Se Soil texture Sand Slit Clay Texture Tap water pH As Se Rice flour SRM-1568a Certified value 0.29 ± 0.03 mg As Kg−1 0.38 ± 0.04 mg Se Kg−1 San Joaquin soil SRM-2709 10.5 ± 0.3(µg As g−1) 1.57 ± 0.08(µg Se g−1) Rice seeds (Intact) As Se Se content after seed priming Root Unprimed – Primed with 0.247 ± 0.013 mg Kg−1 0.5 mg Se L−1 0.489 ± 0.005 mg Kg−1 Primed with 0.75 mg Se L−1 0.614 ± 0.023 mg Kg−1 Primed with 1.0 mg Se L−1

Remarks (values)

0.984 ± 0.09(%) 6.2 ± 0.2 0.059 ± 0.04 μmho/cm 0.015 ± 0.05(%) 10.664 ± 0.011 mg/kg < 0.003 mg/L(BDL) < 0.001 mg/L (BDL) < 0.003 mg/Kg(BDL) < 0.001 mg/Kg(BDL) 6.55 ± 0.02(%) 21.110.704(%) 72.34 ± 1.338(%) Clay Loam 6.8 ± 0.2 < 0.003 mg L−1 (BDL) < 0.001 mg L−1 (BDL) Obtained Value 0.279 ± 0.05 mg As Kg−1 0.366 ± 0.11 mg Se Kg−1 10.144 ± 0.52 µg As g−1 1.52 ± 0.012 µg Se g−1 < 0.003 mg L−1 (BDL) < 0.001 mg L−1 (BDL) Shoot – 0.031 ± 0.018 mg Kg−1

TF – 0.1255

0.097 ± 0.009 mg Kg−1

0.1983

0.123 ± 0.016 mg Kg−1

0.2003

#TF, translocation factor; TF were calculated using the mean value only; BDL – below detection limit.

attention was given to ensure that no chaffs were left among the unprimed as well as in primed seed lots. 2.2. Experimental lay out Before starting the experiment soil organic carbon content was determined according to the methodology of (Walkley and Black, 1934), available phosphorus (by colorimetric method), and total nitrogen (alkaline permanganate method), pH and conductivity were determined according to the protocol of Trivedy and Goel (1986) and Nelson and Bremner (1972). Soil texture was determined by using pipette method described by Kettler et al. (2001). For determining metal content in garden soil, soil were acid digested according to the method of Moulick et al. (2016a) using aqua regia in block digestion method. For determining available (phytoavailable) metal content in garden soil, 1.0 g of soil dissolved in 10.0 mL of 0.1 N HCl (MERCK, ACS Grade, Germany) for 24 h and then filter through Whatman no. 42 filter paper and stored the filtrate in 4 °C in a polyethylene bottle until quantification of metals using flow injection hydride generation atomic absorption spectrometer (FI-HG-AAS, Perkin Elmer A Analyst 400) according to the external calibration given by Koreňovská (2006) (Table 1). For determining As and Se content tap water was first filter through Whatman no. 42 filter paper and then metal content was

2. Materials and method 2.1. Description of tested rice variety and seed priming method For this experiment rice seeds of IET-4094 variety, popularly known as Khitis was chosen and obtained from Regional Rice Research Station, Chinsurah, Hoogly, West Bengal, India. The initial seed moisture content was found to be 10.09% (dry weight basis); from morphological point of view, Khitis is a long and slender type (L/B of 2.3 ± 0.01) variety. Surface sterilization and seed priming was done by following the methodology described by Moulick et al. (2016b); using sodium selenite (Anhydrous Na2SeO3 salt, purchased from HIMEDIA; molecular weight (MW) 172.94; 99% purity) in 1: 5 seed to priming solution of desired strength (0.5, 0.75 and 1.0 mg Se L−1) for 24 h. Here hydropriming was carried out using the double distilled water only by adopting the procedure described above. Moreover during the course of surface sterilization and later (during the priming treatments) special 450

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2.6. Bioconcentration factor and translocation factors

quantified alike the method discussed above. The collected soil (As and Se free) for the experimentation had “clay loam” texture was hammered in order to broken large soil chunks. Then the broken soil was passed through 2.0 mm stainless steel sieve to remove any unwanted foreign material. After that, 350.0 g soil was poured into the plastic cups (16 cm diameter; 20 cm height) and maintained the soil depth of 15 cm uniformly. To fulfill the proper moisture requirement for germination of rice seed 70 mL water (tap water for As free treatment; As solution with desired concentration i.e. 5, 10 and 15 mg As Kg−1) was poured in each cup. The As and Se content in tap water for seed germination were to be below detection limit (< 0.0003 and < 0.001 mg/L for As and Se, respectively) (Table 1). On next day 20 rice seeds were sown in cup soil for each treatment (20 treatment combinations) The desired As solution (5, 10 and 15 mg As L−1) were made from stock solution of arsenate (sodium arsenate; MW-312.01, obtained from MERCK; Germany). Arsenate was chosen as a source of As because under anaerobic condition with low pH (acidic) arsenate can be easily converted into arsenite (Bhattacharya et al., 2013). The experiment was arranged in factorial complete randomized design (fCRD) with five replicas (in five different cups contain 20 seeds each). The total hundred cups were kept in the net house floor under natural condition similar to that of nearby paddy field.

Bioconcentration factor (BCF) for As content was calculated as the ratio of tAs content in root and shoot to soil As content; whereas translocation factor (TF) values were computed as the ratio of shoot As content to root As content in 14 days after sowing (DAS) old rice seedlings (Rauf et al., 2011). 2.7. Statistics SAS (for windows version 9.4) software used for all statistical analysis. Difference among the various treatment combinations were separated by using mean value (n = 5) as well as applying two way ANOVA following the general linear model procedure. Further treatment means were separated with the use of Tukey's Honest Significant Difference (HSD) test at a 5% level of significance. 3. Results and discussion 3.1. Impacts of seed priming and As stress on germination and seedling growth on 14DAS A significant (at p < 0.001 level) inhibition on rice seed germination by 21.25% (5.0 mg As Kg−1), 37.22% (10.0 mg As Kg−1) and 54.38% (15.0 mg As Kg−1) was noticed in unprimed seeds germinated in As spiked soil in a dose dependent manner. An analogous trend can also be seen in reduction of intact seedling's bio mass as well as root and shoot length at 14 DAS, among the seedlings of unprimed seeds when compared with the control. A considerable, 48%, 40% and 21% decrease in intact biomass was noticed among the seedlings of unprimed seeds grown in 15.0, 10.0, 5.0 mg As Kg−1 stress respectively,

2.3. Observations After 14 days all the treatments were evaluated for the following parameters in five replication. Final germination percentage (FGP) was computed according to the formula of Ellis and Roberts (1981), root and shoot length of rice seedlings were measured by the methodology of Moulick et al. (2016b), intact seedling dry weight was computed according to the formula of Kharb et al. (1994).

Table 2 Effects of various seed priming treatments on germination and seedling growth in As free and As stressed condition at 14 DAS.

2.4. Biochemical attributes In this experiment biochemical attributes were analyzed in both root and shoots of all the treatments in five replication, soluble protein content was estimated by adopting the methodology described by Lowry et al. (1951), free proline content estimated according to the method of Bates et al. (1973), where as assessment of lipid peroxidation carried away by measuring of malondialdehyde content (MDA) using the protocol given by Heath and Packer (1968). Fully expanded leaf were considered for total chlorophyll estimation using the methodology described by Arnon Daniel (1949).

Seed priming treatment

Arsenic stress (mg As Kg−1)

Final germination (%)

Biomass (g)

Root length (cm)

Shoot length (cm)

Unprimed

0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0

(1)

92.56a-c 72.89d-g 58.11g-i 42.22i 93.45ab 76.05c-f 60.11f-h 48.67hi 99.56a

0.125cd 0.098f-h 0.075ij 0.065j 0.135C 0.1f-h 0.078ij 0.069j 0.176b

9.03de 6.57g-j 5.47i-k 4.97k 9.87dc 6.62hi 5.73i-k 5.2jk 10.7bc

5.2de 3.23h-k 2.5kl 2.1l 5.67d 3.27h-k 3.07h-l 2.33kl 6.8c

5.0 10.0 15.0 0.0

86.37a-d 73.22d-g 69.07e-g 100.0a

0.109d-f 0.083h-j 0.071j 0.217a

7.77e-g 6.17h-k 5.93h-k 12.1ab

4.0f-h 3.1h-k 2.8j-l 8.67b

5.0 10.0 15.0 0.0

87.78a-d 78.49b-e 73.33d-g 98.44a

0.12c-e 0.089g-i 0.08ij 0.19b

8.3ef 7.27f-h 6.37g-k 12.3a

4.53e-g 4.93d-f 3.63h-j 10.5a

5.0 77.78b-e 10.0 69.0e-g 15.0 71.33d-g ANOVA F-VALUES

0.106e-g 0.079ij 0.075ij

6.67g-i 6.03h-k 5.73i-k

4.03f-h 3.87g-i 3.0i-l

69.96*** 822.42*** 22.86***

32.64*** 368.74*** 5.31***

120.08*** 631.43*** 24.14***

Hydroprimed

0.5 mg Se L−1

2.5. Acid digestion of soil and rice seedlings and quality assurance Prior to start acid digestion (block digestion) process, all the glass and stainless steel apparatuses were dipped in chromic acid solution for 24 h and rinsed with double distilled water and oven dried. Different plant parts i.e. root and shoot were digested using 5.0 mL tri-acid mixture having 70% perchloric acid, nitric acid and sulpuric acid (all ACS grade) by following the methodology of Moulick et al. (2016b); where as soil samples were digested using aqua regia along with SRMs (standard reference material, Item no. 1568a rice flour, and Item no. 2709 – San Joaquin Soil both purchased from NIST, USA), beside reagent blanks all in five replication (Rahman et al., 2007). Total As (tAs) and Se content in soil, tap water and seedlings (root, shoot) along with in SRMs were analyzed by adopting the external calibration using FIHG-AAS, Perkin Elmer AAnalyst 400 (flow injection hydride generation atomic absorption spectrometer) with 0.5% NaBH4 (obtained from MERCK; Germany) dissolved 0.05% NaOH and 10% (v/v) solution of HCl (Koreňovská, 2006), the recovery of As and Se from two SRMs after acid digestion were found to be more than 95% (Table S1).

0.75 mg Se L−1

1.0 mg Se L−1

Sources of variation Se As Se × As

25.94*** 126.92*** 2.85*

(1) Indicates mean values (n = 5). The values with same letter cases are not significantly different at p < 0.05 level. *, ** and *** denotes values are significant at p < 0.05, 0.01 and 0.001 levels respectively. DAS – days after sowing.

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indicating a significant (at p < 0.001 level) phytotoxic effect of As stress on seedling growth. At 14 DAS, root and shoot length seedlings of unprimed seeds grown in As spiked soil gets repressed considerably, the inhibition of root length in As stress was found to be greater than the inhibition of shoot length. As inhibited root elongation by 46%, where as 35% inhibition noticed in shoot elongation in seedlings of unprimed seeds, when compared with the control (Table 2; Fig. 1(a–b)). Findings from the current investigation regarding As induced inhibition of germination, reduction of root and shoot length along with biomass in a linear fashion (5.0–15.0 mg As Kg−1) analogous with the findings of Jin et al. (2010); Tripathi et al. (2007). Whereas, the root growth inhibited greatly than the shoot growth under As stress can be ascribed to the fact that roots experience more As content than shoot (Shree et al., 2009) (Table 2; Fig. 1(a–b)). On the contrary, a 6.66–27.67% restoration in germination% (FGP), 3.86–17% enhancement in intact seedlings biomass, 3.23–22.53% longer root length, beside greater shoot length exhibited by seedlings of primed seeds (with Se), when compared with their unprimed and hydroprimed counter parts germinated (and grown) in similar As stress regimes. These trend of greater germination %, longer root and shoot length of rice seedlings (Se primed seeds), suggesting towards a low to highly significant antagonistic aspect of Se against As induced toxicity (Se × As) may ascribed behind these trend. This trend of enhanced germination, higher seedling growth may be due to stimulation of carbohydrate metabolism (Malik et al., 2010; Moulick et al., 2016b) and to the selenium's antioxidative role along with ability to interact with As by selenium primed seeds in a highly significant way (Table 2, Fig. 1). Another interesting trend can be observed when the results were examined and compared among the selenium primed seeds germinated and grown alike control in As free environment. Further, statistical analysis points out towards the positive and highly significant (at p < 0.001 level) involvement of seed priming with Se in promoting germination and seedling growth in As free environment in promoting germination and seedling growth (Table 2; Fig. 1(a–c)). More over the trend of having greater germination and enhanced seedling growth in As free as well as in As contaminated environment is in good agreement with the findings of Moulick et al. (2016b), Khaliq et al. (2015) and Shree et al. (2009) (Table 2; Fig. 1(a–c)). 3.2. Impacts of seed priming with Se and As stress on selected biochemical attributes in rice seedlings at 14 DAS

Fig. 1. Effects of various seed priming treatments and As stress on (a) final germination percentage, (b) seedling biomass accumulation and (c) root and shoot length in rice seedlings (14 DAS); column with the same letter (effects of seed priming- with lower case; As stress- with upper case) were not significantly different at p < 0.05.

When compared with the control, a considerable decrease in soluble protein, proline and MDA content can be seen in both root and shoot of seedlings of Se primed seeds grown like the control in As free environment. As much as 18.08% and 15.24% lesser proline accumulation; 12.48% and 54.77% reduction in MDA content can be observed in root and shoot of Se primed seedlings respectively, than the control supports the findings of Moulick et al. (2016b). Moreover, a 41.13% (1.0 mg Se L−1), 31.89% (0.75 mg Se L−1), 18.91% (0.5 mg Se L−1) significantly (p < 0.001) greater chlorophyll content can be seen among the Se primed seedlings grown in As free environment alike the control. Where as a dose dependent as well as step wise enhancement in soluble protein, proline and MDA content can be seen with maximum enhancement can be seen in the unprimed seedlings grown in soil having 15.0 mg As Kg−1. Significantly (p < 0.001) greater accumulation of proline by unprimed seedlings grown in As spiked soil than seedlings of control similar with the observation made by Zhao et al. (2009) suggesting an increasing effort has been extended by the untreated (unprimed seedlings here) plants towards the adjustment of water imbalance by maintaining sodium ionic (sodium) homeostasis (BenHassine et al., 2008) or might be due to increase in proline synthesis (Garg and Singla, 2012) (Table 3; Fig. 2c). Like above, lipid peroxidation also enhanced with increase in As stress (in soil) in both root and shoot of unprimed seedlings, may ascribed to the As induced reactive oxygen species (ROS) evoked peroxidation of unsaturated fatty

acids in the cell membranes which in turn leads to formation of end products like MDA (Moulick et al., 2016b). Beside these a gradual decrease (56.04%) in total chlorophyll content can also be noticed compared with the control; suggests about involvement (phytotoxic) of As in modulating the biochemical attributes, this findings is in good agreement with the observations of Rahman et al. (2007) and Duman et al. (2010), might be either due to increase in chlorophyll degradation (chlorosis) or due to inhibition of chlorophyll synthesis (Jain and Gadre, 2004). On the contrary a significant decrease in protein content (36.05%), proline content (11.60%), MDA (29.93%) in roots exhibited by seedlings of Se primed seeds, when compared with their respective unprimed counterparts grown in similar As stress regimes at 14DAS; beside an average of 11.02% enhancement in total chlorophyll content also displayed by leaves of Se primed seedlings grown alike unprimed counter parts in a series of As spiked soil. These findings indicates a noteworthy interactive aspect (Se × As) of seed priming with Se on the above mentioned biochemical attributes of Se primed seedlings grown in As spiked soil from moderate to highly significant way (Table 3; Fig. 2(a–c)). The trend emerge out from statistical analysis as well as biochemical attributes suggests that selenium supplemented through 452

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Table 3 Selected stress induced biochemical attributes and total chlorophyll content of seedlings of hydroprimed, selenium primed and unprimed seeds grown in arsenic free and arsenic spiked soil at 14 DAS. Seed priming treatments

Unprimed

Hydroprimed

0.5 mg Se L−1

0.75 mg Se L−1

1.0 mg Se L−1

Sources of Variation Se As Se × As

As stress (mg Kg−1)

0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 ANOVA F-VALUES

Soluble protein mg g−1FW

Proline content µmoles g−1 FW

MDA content nmoles g−1 FW

Shoot

Shoot

Shoot

Root h-j

Root e-g

ij

i

Root

0.42 0.81d-f 1.14b 1.44a 0.40ij 0.77d-g 1.06bc 1.41a 0.37j 0.67e-g 0.82d-f 1.05bc 0.24j 0.59g-i 0.83de 0.88cd 0.28j 0.62f-h 0.67e-g 0.95b-d

0.73 1.20de 1.49bc 1.80a 0.71h-j 1.06e-g 1.34cd 1.63ab 0.66j 0.91f-h 0.99e-g 1.06e-g 0.583j 0.75h-j 0.88f-h 0.86g-i 0.74h-j 0.87f-i 1.05e-g 1.08ef

0.061 0.099bc 0.136a 0.15a 0.059fg 0.087b-d 0.134a 0.142a 0.056fg 0.081c-e 0.09b-d 0.103b 0.044g 0.073d-f 0.081c-e 0.093b-d 0.055fg 0.091b-d 0.099bc 0.105b

0.566 0.695c-f 0.737c-e 0.892a 0.526i-k 0.661e-h 0.707c-e 0.909a 0.475j-l 0.604f-i 0.679d-g 0.763b-d 0.405l 0.576hi 0.659e-h 0.775bc 0.458kl 0.601g-i 0.673d-g 0.849ab

1.43 4.77e-g 8.09ab 8.78a 1.03i 4.7e-g 7.99ab 7.98ab 0.763i 3.8gh 6.15c-e 5.47c-e 0.417i 3.1h 5.85c-e 6.77b-d 0.76i 3.99f-h 7.23bc 7.21bc

7.43g-i 10.53c 12.73b 17.13a 7.43g-i 9.7c-e 12.4b 16.77a 6.13ij 8.77d-h 9.5c-f 10.13cd 5.2j 7.2hi 8.03f-h 8.97c-g 6.17ij 8.17e-h 10.3cd 12.2b

2.084d 0.988ef 0.953f 0.916f 1.96d 1.07ef 0.948f 0.92f 2.57c 1.196ef 1.05ef 0.923f 3.06b 1.268e 1.093ef 1.07ef 3.54a 1.11ef 1.04ef 0.946f

63.7*** 395.3*** 6.4***

116.8*** 205.6*** 16.6***

66.7*** 249.9*** 8.53***

33.1*** 357.5*** 2.01*

31.6*** 569.1*** 3.21**

174.2*** 431.4*** 19.16***

57.7*** 1104.6*** 33.1***

(1)

h-j

Total leaves chlorophyll mg g−1 FW

(1)

Indicates mean values (n = 5); The values with same letter cases are not significantly different at p < 0.05 level. *, ** and *** denotes values are significant at p < 0.05, 0.01 and 0.001 levels respectively; FW, Fresh weight; DAS – days after sowing.

found to be most effective, similar with he observation of Malik et al. (2012). Later, when Se primed and hydroprimed seedlings were compared with unprimed counter parts grown in similar As stressed conditions, significant reduction in soluble protein, proline and MDA content beside enhancement in chlorophyll content (chl-a,chl-b and chl-

seed priming technology were able to execute promotive and/protective ability even under As stress via execution of antagonism beside being an effective antioxidant towards the As induced stress, from moderate to highly significant manner. Further among the doses of Se (used for seed priming treaments) considered here, 0.75 mg Se L−1 was

Fig. 2. Effects of various seed priming treatments and As stress on (a) soluble protein content in root and in shoot, (b) total chlorophyll content (c) proline content and (d) MDA content in14 days old rice seedlings; column with the same letter (effects of seed priming – with lower case and effects of As stress- with upper case and upper case; were not significantly different at p < 0.05.

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Table 4 Arsenic uptake (expressed as tAs on dry weight basis) patterns in root and shoot of hydroprimed, selenium primed and unprimed seeds grown in arsenic spiked soil at 14 DAS. Seed priming treatments

Unprimed

Hydroprimed

0.5 mg Se L−1

0.75 mg Se L−1

1.0 mg Se L−1

Sources of variation Se As Se × As

As stress (mg Kg−1)

0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 0.0 5.0 10.0 15.0 ANOVA F-VALUES

tAs (mg As Kg−1) Shoot

Root

(1) f

0 0.630e 2.018bc 2.730a 0f 0.598e 1.979bc 2.694a 0f 0.537e 1.707cd 2.269ab 0f 0.397ef 1.347d 2.243ab 0f 0.493ef 1.60cd 2.329ab

0g 2.188f 6.099e 10.664b 0g 2.188f 6.502e 10.712b 0g 2.899f 7.138de 11.340abc 0g 3.050f 8.661c 12.560a 0g 2.848f 7.917cd 11.648b

10.50*** 723.80*** 2.33*

23.07*** 2329.30*** 4.38**

(1) Indicates mean values (n = 5). The values with same letter cases are not significantly different at p < 0.05 level. *, ** and *** denotes values are significant at p < 0.05, 0.01 and 0.001 levels respectively. DAS – days after sowing.

a+b) speaks in favor of utility of Se's antioxidative property towards stressed (As stress) induced oxidative stress, beside the effective execution of Se × As (Table 3; Fig. 2(a–d)). 3.3. Effect of seed priming with Se and hydropriming on As content in root and shoot of rice seedlings With gradual increase in As load in soil from 5.0 to 15.0 mg Kg−1, As content in root and shoot of unprimed seedlings also increased in a linear fashion significantly (at p < 0.001 level), having highest As content in those grown under 15.0 mg Kg−1 As spiked soil. A trend, noticed among all the treatments (irrespective of unprimed, hydroprimed and selenium primed) that As content of seedlings had more As content in root than in shoot (Table 4; Fig. 3a); as plant's roots has been considered as the introductory point, from here As gets transported and/translocated as well as distributed among the various plant parts subsequently, specially in terrestrial land races including rice plant. Upon exposure to As stress by roots and/plant As gets entrapped in the apoplast make significantly influence As build up in plant followed by subsequent release into cytosol (Chen et al., 2005) with greater As build up noticed in roots under anaerobic condition (Bravin et al., 2008), might have explain the higher As content in root than shoot. On the other hand, when root As content were compared among the unprimed and primed seedlings grown in similar As stress regimes, an average 18.19%, 12.54% and 24.31% enhancement in As content can be seen in the roots of 1.0 mg Se L−1, 0.5 mg Se L−1 and 0.75 mg Se L−1 primed seedlings grown in 5.0−15.0 mg As Kg−1 stress; further these trend of higher root As content of unprimed seedlings can also be described as higher bioconcentration or BCF root/soil value. Upon comparison the shoot As content of seedlings of unprimed seeds with the As content of shoots of seedlings of Se primed seeds grown in identical As stress regimes, a significant (p < 0.05) reduction in As content can be observed. Those primed with 0.5 mg Se L−1 reduce As content by 14.76–16.88%; where as 0.75 mg Se L−1 and 1.0 mg Se L−1 and hydroprimed seedlings primed seedlings decrease As content in the shoots by 17.84–36.98%

Fig. 3. Effects of various seed priming treatments and As stress on (a) total arsenic content (tAs) in root and shoot (b) bioaccumulation factor for As in root and shoot, (c) translocation factor for As from root to shoot in 14 days old rice seedlings; column with the same letter (effects of seed priming – lower case; effects of As stress – with upper case) were not significantly different at p < 0.05.

and 14.64–21.75% and 1.319–5.08% respectively, than the seedlings of unprimed seeds (Table 4, Table 5; Fig. 3(a–c)). Findings also displayed that despite of having higher As content (higher BCFroot/soil value) in Se primed seedlings also had significantly less As content in shoot (less BCFshoot/soil as well as from TF factors) when compared with the As content of unprimed seedling's shoot grown in identical As stress at 14DAS. Though Se × As were found to be of insignificant but the influence of seed priming with selenium is of highly (for BCF root/soil and TF) at (p < 0.001 level) to moderately (p < 0.01 level) for BCFshoot/soil significant type. The results also suggests that 0.75 mg Se L−1 primed seedlings being most efficient and hydroprimed seedlings being lest effective (Table 4, Table 5; Fig. 3(a–c)) in reducing As content in the shoot. The trend of restricting maximum As (with significantly greater As content) to root zone and least being translocated to the shoot by seedlings of selenium primed seedlings partially supports the findings of Malik et al. (2012) and Feng et al. (2009a, 2009b) as well as by Hu et al. (2014) who reported similar observations in mungbean (Phaseolus aureus Roxb.) and Pteris vittata L and Oryza.sativa (rice) respectively. Though a noteworthy difference is also exist in terms of the mode of execution, our current study depicts the impacts of seed priming i.e. 454

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Acknowledgements

Table 5 Bioconcentration factor of arsenic for root (BCFsoil/root), translocation factor (TF) and bioconcentration factor for shoot (BCFsoil/shoot) associated with seedlings of unprimed, hydroprimed, selenium primed seeds grown in arsenic spiked (stress) soil; column with the same letter were not significantly different at p < 0.05. Seed priming treatments

Stress (mg As Kg−1)

BCFsoil/root

TF

BCFsoil/shoot

Unprimed

5.0 10.0 15.0 5.0 10.0 15.0 5.0 10.0 15.0 5.0 10.0 15.0 5.0 10.0 15.0 ANOVA F-Value

0.437g 0.609de 0.710b-d 0.448gf 0.623c-e 0.714b-d 0.579d-f 0.713b-d 0.756a-c 0.610de 0.866a 0.837ab 0.569e-g 0.792ab 0.777ab

0.280a-c 0.351a 0.258a-c 0.270a-c 0.320ab 0.252a-c 0.186bc 0.239a-c 0.200a-c 0.128c 0.155c 0.178c 0.174bc 0.202ac 0.200a-c

0.120c-f 0.208a 0.182a-c 0.119c-f 0.198ab 0.179a-c 0.107d-f 0.170a-d 0.151a-e 0.079f 0.134b-e 0.149a-e 0.098ef 0.160a-e 0.155a-e

27.72*** 113.20*** 1.84

11.51*** 3.09* 0.61

8.01** 44.90*** 0.57

Hydroprimed

0.5 mg Se L−1

0.75 mg Se L−1

1.0 mg Se L−1

Sources of variations Se As Se × As

The authors are thankful to University of Kalyani for providing Net House and laboratory facility and Ministry of Environment and Forest, Government of West Bengal, India for providing final assistance (SRF (Fellowship)). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.07.060. References Arnon Daniel, I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta vulgaris. Plant Physiol. 24, 1–15. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. BenHassine, A., Ghanem, M.E., Bouzid, S., Lutts, S., 2008. An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress. J. Exp. Bot. 59, 1315–1326. Bhattacharya, P., Samal, A.C., Majumdar, J., Santra, S.C., 2010. Accumulation of As and its distribution in rice plant (Oryza sativa L.) in Gangetic West Bengal, India. Paddy Water Environ. 8, 63–70. Bhattacharya, P., Samal, A.C., Majumdar, J., Banerjee, S., Santra, S.C., 2013. In vitro assessment on the impact of soil As in the eight rice varieties of West Bengal, India. J. Hazard. Mater. 262, 1091–1097. Bleasdale, J.K.A., 1967. The relationship between the weight of a plant part and total weight as affected by plant density. J. Hortic. Sci. 42, 51–58. Bradford, K.J., 1986. Manipulation of seed water relations via osmotic priming to improve germination under stress conditions. Hortic. Sci. 21, 1105–1112. Bravin, M.N., Travassac, F., Le Floch, M., Hinsinger, P., Garnier, J.M., 2008. Oxygen input controls the spatial and temporal dynamics of As at the surface of a flooded paddy soil and in the rhizosphere of lowland rice (Oryza sativa L.): a microcosm study. Plant Soil 312, 207–218. Brocklehurst, P.A., Dearman, J., 2008. Interaction between seed priming treatments and nine seed lots of carrot, celery and onion. II. Seedling emergence and plant growth. Ann. Appl. Biol. 102, 583–593. Chen, H., Yuan, X., Li, T., Hu, S., Ji, J., Wang, C., 2016. Characteristics of heavy metal transfer and their influencing factors in different soil–crop systems of the industrialization region, China. Ecotoxicol. Environ. Saf. 126, 193–201. Chen, T., Yan, X., Liao, X., Xiao, X., Huang, Z., Xie, H., Zhai, L., 2005. Subcellular distribution and compartmentalization of As in Pteris vittata L. Chin. Sci. Bull. 50, 2843–2849. Choudhury, B., Chowdhury, S., Biswas, A.K., 2011. Regulation of growth and metabolism in rice (Oryza sativa L.) by As and its possible reversal by phosphate. J. Plant Interact. 6, 15–24. Dai, Y., Lv, J., Liu, K., Zhao, X., Cao, Y., 2016. Major controlling factors and prediction models for arsenic uptake from soil to wheat plants. Ecotoxicol. Environ. Saf. 130, 256–262. Duman, F., Ozturk, F., Aydin, Z., 2010. Biological responses of duckweed (Lemna minor L.) exposed to the inorganic As species As(III) and As(V): effects of concentration and duration of exposure. Ecotoxicology 19, 983–993. Ellis, R.H., Roberts, E.H., 1981. The quantification of ageing and survival in orthodox seeds. Seed Sci. Technol. 9, 373–409. Farooq, M.A., Gill, R.A., Ali, B., Wang, J., Islam, F., Ali, S., Zhou, W.J., 2016a. Subcellular distribution, modulation of antioxidant and stress-related genes response to As in Brassica napus L. Ecotoxicology 25, 350–366. Farooq, M.A., Gill, R.A., Islam, F., Ali, B., Liu, H., Xu, J., He, S., Zhou, W.J., 2016b. Methyl jasmonate regulates antioxidant defense and suppresses As uptake in Brassica napus L. Front. Plant Sci. 7, 468. http://dx.doi.org/10.3389/fpls.2016.00468. Farooq, M., Basra, S.M.A., Wahid, A., Khaliq, A., Kobayashi, N., 2009. Rice seed invigoration. In: Lichtfouse, E. (Ed.), Sustainable Agriculture Reviews. Springer, Netherlands, pp. 137–175. Farooq, M., Basra, S.M.A., Khalid, A., Tabassum, R., Mehmood, T., 2006. Nutrient homeostasis, reserves metabolism and seedling vigor as affected by seed priming in coarse rice. Can. J. Bot. 84, 1196–1202. Feng, R.W., Wei, C.Y., Tu, S.X., Sun, X., 2009a. Interactive effects of selenium and As on the ir uptake by Pteris vittata L. under hydroponic conditions. Environ. Exp. Bot. 65 (2–3), 363–368. Feng, R.W., Wei, C.Y., Tu, S.X., Wu, F.C., 2009b. Effects of Se on the essential elements uptake in Pteris vittata L. Plant Soil 325 (1–2), 123–132. Feng, R., Wei, C., Tu, S., 2013. The roles of selenium in protecting plants against abiotic stresses. Environ. Exp. Bot. 87, 58–68. Garg, N., Singla, P., 2012. The role of Glomus mosseae on key physiological and biochemical parameters of pea plants grown in As contaminated soil. Sci. Hortic. 143, 92–101. Ghosh, D., Singh, U.P., Brahmachari, K., 2016. 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(1)

Indicates mean of values (n = 5). *, ** and *** denotes values are significant at p < 0.05, 0.01 and 0.001 levels respectively.

supplemented the Se into the intact seeds prior to sow and then exposed to As stress in soil, not by either germinating in growth chamber and exposed separately or even co-exposed in hydroponic culture. Among the different forms of Se such selenite being least efficient in terms of translocation to shoot and restricted to the root zone (Hu et al., 2014; Zhu et al., 2009) analogous to our findings depicted in Table 1. The findings also indicates that only relatively small fraction of Se translocated to the shoot (with small TF value). It may be assumed that upon accumulated to root zone Se in greater amount, traps majority of As (even in greater amount might be as FeAsO4 form) and put an halt to As translocation to the shoot by influencing phosphate transporters or reducing in xylem loading of As (Pickering et al., 2000) or by other means need to be confirmed; this might be a probable mechanistic explanation behind the effectiveness of seed priming with Se (selenite) to mitigate As induced phytotoxicity in rice (Table 4, Table 5; Fig. 3(a–c)).

4. Conclusion The present investigation conclude that in As free environment, seed priming with selenium proved to be growth promotive in terms of higher germination with longer root and shoot length and biomass, compared with the unprimed seeds. For cultivation of field crops like rice it's necessary to have robust seedlings to be transplanted or even in case of direct seeded rice (DSR), in order to ensure higher yield. The findings also suggests that sowing and subsequent events associated with selenium primed rice seeds under As stressed condition provides better protection to the rice cultivation by promoting germination and enhancing seedling growth coupled with reducing As induced oxidative stress (ROS quencher); beside reducing As translocation from soil and root As pool in a significant way. Though the margin between essentiality and toxicity of selenium is very thin, but this article will provoke the august audience to reconsider the concept of essentiality of selenium when applied through seed priming technology in rice and subsequent events upon sowing in As free environment and As stressed condition could be a cheap, farmer friendly option to achieve robust seedling emergence and mitigation for As induced toxicity in rice. 455

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