Effect of water management and silicon on germination, growth, phosphorus and arsenic uptake in rice
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Ecotoxicology and Environmental Safety
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E ?ect of water management and silicon on germination,growth,phosphorus and arsenic uptake in rice
Zahida Zia a ,Ha ?z Faiq Bakhat a ,Zul ?qar Ahmad Saqib b ,Ghulam Mustafa Shah a ,Shah Fahad c ,?
,Muhammad Rizwan Ashraf d ,Ha ?z Mohkum Hammad a ,Wajid Naseem a ,Muhammad Shahid a
a
Department of Environmental Sciences,COMSATS Institute of Information Technology Vehari,Pakistan
b
Saline Agriculture Research Center (SARC),Institute of Soil &Environmental Sciences,University of Agriculture,Faisalabad,Pakistan c
College of Plant Science and Technology,Huazhong Agricultural University,Wuhan,Hubei,China d
Department of Entomology,University of Agriculture,Faisalabad,Sub-campus Burewala/Vehari,Pakistan
A R T I C L E I N F O
Keywords:Germination Aerobic
Anaerobic cultivation As uptake
A B S T R A C T
Silicon (Si)is the 2nd most abundant element in soil which is known to enhance stress tolerance in wide variety of crops.Arsenic (As),a toxic metalloid enters into the human food chain through contaminated water and food or feed.To alleviate the deleterious e ?ect of As on human health,it is a need of time to ?nd out an e ?ective strategy to reduce the As accumulation in the food chain.The experiments were conducted during September-December 2014,and 2016to optimize Si concentration for rice (Oryza sativa L .)exposed to As stress.Further experiment were carried out to evaluate the e ?ect of optimum Si on rice seed germination,seedling growth,phosphorus and As uptake in rice plant.During laboratory experiment,rice seeds were exposed to 150and 300μM As with and without 3mM Si supplementation.Results revealed that As application,decreased the germination up to 40–50%as compared to control treatment.Arsenic stress also signi ?cantly (P <0.05)reduced the seedling length but Si supplementation enhanced the seedlings length.Maximum seedling length (4.94cm)was recorded for 3mM Si treatment while,minimum seedling length (0.60cm)was observed at day7by the application of 300μM As.Silicon application resulted in 10%higher seedling length than the control treatment.In soil culture experiment,plants were exposed to same concentrations of As and Si under aerobic and anaerobic conditions.Irrigation water management,signi ?cantly (P ?0.05)a ?ected the plant growth,Si and As con-centrations in the plant.Arsenic uptake was relatively less under aerobic conditions.The maximum As con-centration (9.34and 27.70mg kg DW ?1in shoot and root,respectively)was found in plant treated with 300μM As in absence of Si under anaerobic condition.Similarly,anaerobic condition resulted in higher As uptake in the plants.The study demonstrated that aerobic cultivation is suitable to decrease the As uptake and in rice exo-genous Si supply is bene ?cial to decrease As uptake under both anaerobic and aerobic conditions.
1.Introduction
Rice is one of the major food crops in Southern Asia where groundwater contaminated with As is used for the irrigation of paddy ?elds (Dixit et al.,2016).It has been estimated that 1000t of As per year are added to the agricultural soils as a result of applying As loaded ground water for irrigation purposes (Duxbury and Panaullah,2007).Bioavailability of As to plant is governed by biological,chemical and physical processes and their interactions altering metal speciation and behavior in soil-plant systems (Bakhat et al.,2017).Concentration of As in plants depends on plants root ability to uptake and transport it from soil to roots/shoots.The most widely adopted conditions to cultivate the rice in ?eld are water submerged conditions.Anaerobic conditions of the paddy ?elds facilitate the reductive dissolution and release of the adsorbed arsenate (As V )in the soil stoma water (Bakhat et al.,2017).Furthermore,anaerobic conditions in paddy soils usually lead to the reduction of As V into more mobile arsenite (As III )(Takahashi et al.,2004;Punshon et al.,2016).
Rice is an e ?cient crop in As uptake in comparison to other cereal crops (Bhattacharya et al.,2009;Su et al.,2009).Studies showed that As concentrations in rice plant depends on As presence in soil and/or irrigated groundwater in addition to other factors governing the As mobility and uptake in plant-rhizosphere.In rice,As is taken up by plant roots using macro-nutrient transporters;As V via the phosphate while As III through Si transporters (Ma et al.,2008).Arsenate is a chemical analog of phosphate and shares the uptake pathway in rice
https://www.sodocs.net/doc/8a12541265.html,/10.1016/j.ecoenv.2017.06.004
Received 24February 2017;Received in revised form 31May 2017;Accepted 2June 2017?
Corresponding author.
E-mail addresses:faiqsiddique@https://www.sodocs.net/doc/8a12541265.html,.pk (H.F.Bakhat),shah_fahad80@https://www.sodocs.net/doc/8a12541265.html, ,shah.fahad@https://www.sodocs.net/doc/8a12541265.html, (S.Fahad).
Ecotoxicology and Environmental Safety 144 (2017) 11–18
Available online 06 June 2017
0147-6513/ ? 2017 Elsevier Inc. All rights reserved.
with the same transporters(Chen et al.,2017).Therefore,antagonistic e?ects of phosphate on As V uptake and vice versa are probable in rice plant.Silicon can in?uence As III uptake competitively as As III shares the same uptake pathway as Si(Sanglard et al.,2016).The rice Si trans-porter LSi1is permeable to As III and it acts as As III in?ux transporter. Silicon transporter LSi2is an e?ux transporter of Si which transports Si/As III from exodermis to endodermis and towards the stele of the root cells.Redox status(governed by soil moisture contents)of paddy?elds is another important factor controlling the As bioavailability to rice plant.Changing soil redox status through water management in paddy ?elds has been supposed to be one of sustainable solution to reduce As accumulation in rice.A remarkable e?ect of aerobic conditions turns
the speciation pattern towards As V in comparison to As III which has prominently higher solubility,plant availability,and toxicity (Takahashi et al.,2004).
Therefore to eliminate the As associated risks,integrated ap-proaches are needed to cultivate the rice especially in those areas that are facing higher contamination of As in ground water or in soil.Among these strategies aerobic rice production with judicious fertilization of nutrient can be a solution to tackle this speci?c problem(Bakhat et al., 2017).Aerobic rice cultivation is a revolutionary way of rice production and requires only50%of the water required for irrigated rice produc-tion for achieving yield levels of4–6Mg t ha?1(Anil et al.,2014).In addition to water saving,uptake of As can be decreased by plant through aerobic rice production.Understanding of the combined e?ects of rice cultivation under aerobic condition with exogenous Si is critical strategy to eliminate the elevated uptake of the toxic metalloid. Therefore,present study was conducted;1)to investigate the e?ect of two rice cultivation method on As uptake in rice and,2)to determine the e?ect of optimum Si on rice germination,phosphorus and As uptake in rice.
2.Materials and methods
Highly pure analytical-grade reagents and chemicals were pur-chased and used for the solutions preparation.Standard stock solution of As was prepared by dissolving sodium arsenite(Na-AsO2,M.W. 129.91,BDH,England)and sodium arsenate(Na2HAsO4·7H2O;MW: 312.01,BDH,England)in ultra-pure water while solutions of required concentrations were prepared by further dilution of this stock solution. Silicon solution was prepared using sodium trisilicate solution(Sigma Aldrich).
2.1.Optimization experiment
Optimization experiments were performed to determine the growth response of rice variety KSK-133against As stress in presence of various levels of Si.Sand prewashed with acid(growing media)was?lled in the pots and seedlings were transferred.Nutrient solution was applied as described by Zhu et al.(2009)to ful?ll the nutritional requirements. After one week of transplantation,to make most e?ective use of the situation or resources,four sets of experiments were arranged in a completely randomized design with di?erent concentrations of Si and As.These treatments were as:Set1)0mM Si(control),0.25mM Si, 1mM Si,2mM Si,3mM Si;Set2)0μM As(Control),50μM As,100μM As,150μM As,300μM As;Set3)0mM Si+0μM As(Control),0.5mM Si+150μM As,1mM Si+150μM As,3mM Si+150μM As;Set4) 0mM Si+0μM As(Control),0.5mM Si+300μM As,1mM Si+ 300μM As,3mM Si+300μM As.The nutrient solution was applied fortnightly to compensate the nutrient depletion in the growth medium. After two months of treatments application,the plants were harvested and washed with deionized water.Afterward,these were separated into root and shoot and fresh weights of the fractions were recorded using analytical balance(AS220R Radwag,Europe)https://www.sodocs.net/doc/8a12541265.html,boratory experiment to determine e?ect of Si supplementation on seed germination and seedling growth
In this experiment,seeds in the petri plates lined with?lter paper were soaked with the solution of0mM Si+0μM As,0mM Si+150μM As,0mM Si+300μM As,3mM Si+0μM As,3mM Si+150μM As, 3mM Si+300μM As.Total six treatments with three replicates were performed during the experiment.In this experiment,germination percentage,root and shoot length were recorded.
2.3.Pot trail and experimental set up
Rice variety KSK-133nursery was grown in pots.After the estab-lishment,the seedlings were transplanted to soil pots.Soil was taken from the research farm of the COMSATS Institute of Information Technology,Vehari campus.The soil was thoroughly mixed and sieved using a4mm mesh to remove plant parts and other debris.Soil was processed for physico-chemical properties(Table1).The soil was al-kaline in nature with low in available phosphorus and organic matter contents.Afterwards,7kg of soil were?lled in pots and basal doses of fertilizers nitrogen,phosphorus,and potassium@170kg,90kg and 60kg per hectare,respectively were added.Nitrogen was added in three split doses(At1st day of nursery transplantation(DAP),30DAP and45DAP)while full dose of potassium and phosphorus was added at the pot?lling stage.The treatment were arranged as completely ran-domized design with four repeats.
2.3.1.Seedlings transplantation
In each pot,?ve seedlings were transplanted and thinned in to two after establishment of the seedlings.After thinning,the pots were di-vided into two groups.Twenty four pots were kept under?ooded conditions to maintain anaerobic environment with treatments;T1-Si0As0,T2-Si0As150,T3-Si0As300,T4-Si3As0,T5-Si3As150and T6-Si3As300.Other twenty four pots were kept moist with lesser amount of water to maintain aerobic conditions with the Si and As treatments;T7-Si0As0,T8-Si0As150,T9-Si0As300,T10-Si3As0,T11-Si3As150and T12-Si3As300.
2.3.2.Plants harvesting and growth attributes measurements
Three months after the treatments application,plant height and number of tillers per plant were recorded.Plants were harvested,wa-shed with deionized water and separated into roots and shoots.The fresh weights of roots and shoots were measured by using weighing balance.The samples were kept in oven at78°C till constant weight and then the dry weights were recorded.
2.3.3.Chemical analysis
The plant samples(root and shoot)were acid digested with nitric and perchloric acid as reported by Miller(1998).Brie?y,one gram plant sample was taken in conical?ask,kept overnight after adding 5mL concentrated HNO3and5mL HClO4.Next day again5mL of concentrated HNO3were added and plant material was digested on hot plate by increasing the temperature slowly to120°C for2h and Table1
Physicochemical properties of soil used for the experiment.Values are the means of three replicates±Standard Error.
Characteristics Values
Soil Texture Silt loam
Electrical conductivity(dS m?1) 1.88±0.06
pH-H2O extract7.78±0.04
Organic matter(%)0.60±0.05
Available phosphorous(mg kg?1soil) 6.44±0.10
Available potassium(mg kg?1soil)123.34±9.79 Saturation percentage37.27±1.47
afterwards the temperature was increased to180°C for an hour.After digestion,plant material was cooled and made the volume50mL with distilled water(Miller,1998).Digest was?ltered with Whatman?lter paper No.42and proceeded for As determination through hydride generation atomic absorption spectroscopy(Talukder et al.,2012).
Speci?c As uptake(SAU)was calculated using the formulae given in Eq.(1)(Zhiyan et al.,2008).
=
?×+?×
R
SAU oot As Concentration RootBiomass Shoot
As Concentration SootBiomass
RootBiomass(1)
Vanadate molybdate method using UV–visible spectrophotometer was used to determine phosphorus concentration in plant samples (Chapman and Pratt,1961).To prepare ammonium vanadate-mo-lybdate reagent,ammonium heptamolybdate[(HH4)6MoO24·4H2O] (22.5g)was dissolved in400mL distilled water and1.25g of ammo-nium metavanadate(NH4VO3)in300mL hot distilled water.These solutions were mixed in1L volumetric?ask and allowed the mixture to cool at room temperature.Concentrated HNO3(250mL)was added to this mixture,cooled at room temperature and made volume1L with distilled water.Five mL of each of the?ltered digested sample and ammonium vanadomolybdate were taken into50mL volumetric?ask, diluted to50mL volume with distilled water and kept for half an hour for the stabilization of the developed blue color.Phosphorus was measured with spectrophotometer Model UV Winlab Lambda25 (Perkin-Elmer Instruments,USA).Instrument was calibrated with a series of P standard solutions(0,0.5, 1.0, 2.0, 4.0, 6.0,8.0and 10mg L?1)and standard curve was drawn.The absorbance of light by the test samples was recorded at410nm wavelength and an actual concentration was obtained by comparing with standard calibration curve.
For the quanti?cation of Si,plant material was digested in3mL of 65%HNO3and2mL30%H2O2.The Te?on tubes were put on hot plate.After?rst step10mL of20%NaOH were added and material was heated for1h.The digested material was?ltered and sample diluted to 100mL.The?ltrate was used to determine Si by color method.One mL of sample was taken in50mL?ask added with0.26N HCl(1.60mL), (NH4)6Mo7O24(10%)(1mL),10%tartaric acid(1.60mL),and redu-cing agent(0.80mL).Color development was completed within1h and absorbance was measured at600nm at spectrophotometer.The redu-cing agent was prepared by dissolving250mg Na2SO3,125mg1-amino-2-naphthol-4-sulfonic acid,and7.5g NaHSO3in50mL of dis-tilled water(Elliott and Snyder,1991).
2.4.Statistical analysis
Completely randomized design was used to analyze the obtained experimental data.The experimental data were subjected to analysis of variance using SAS(2004)(SAS/STAT9.1).Multiple comparisons were done using least signi?cant di?erence test.For all analyses,a P-value of less than5%(P<0.05)was interpreted as statistically signi?cant. 3.Results
3.1.Optimization of silicon for rice growth under arsenic stress
Silicon improved the plant growth as measured in terms of shoot growth.Shoot growth increased gradually by increasing Si concentra-tion up-to1mM,while a decreasing trend in growth was observed beyond1mM Si concentration(up-to3mM).Maximum shoot weight (18.68g plant?1)was observed at1mM Si supply in comparison to 10.81g plant?1observed in control treatment(Fig.1).A gradual de-crease in shoot growth was recorded with increasing concentration of As(0μM to300μM).Minimum shoot growth was measured at300μM As(2.73g plant?1)nearly75%lower as compared to control(10.81g plant?1).A distinct e?ect of Si to alleviate the As toxicity was observed for both low and high As treatments(As150μM and300μM).In-creasing Si supplementation under150μM and300μM As exposure resulted a gradual increase in shoot growth;however,the e?ects were more pronounced with higher Si supply(3mM).For As150μM,there was about159%increase in shoot biomass with3mM Si as compared to its lower concentration0.5mM.Similarly,an increase of140%(5.75 Vs13.84)was observed for higher Si as compared to lower Si treatment under higher As(300μM)exposure condition.From the results of the optimization experiment,it can be concluded that1mM Si supply is optimum under no-As-stress while3mM Si supply yielded maximum plant biomass under As stress conditions.Therefore,for further ex-perimentation,3mM Si was used as the optimum concentration for alleviation of As toxicity in rice plants(Fig.1).
3.2.E?ect of silicon supplementation on seed germination and seedling growth of rice under arsenic stress
Germination of seeds was recorded to see the e?ect of Si on rice seeds subjected to As stress(Fig.2A).Increasing concentration of As resulted in a continuous decrease in germination percentage in com-parison to control.Arsenic exposure at both concentrations(As150μM and As300μM),germination of seeds was decreased about40–50%in comparison to control.At higher As level(As300μM),the germination was lowest(almost30%in comparison to control treatment).Although the e?ect of Si application was not signi?cant(P<0.05)but Si sup-plementation raised the germination of seeds almost8–10%as com-pared to control(Fig.2A).Silicon application showed maximum ger-mination at the beginning but later its e?ect was lowered as compared to control.In addition,Si application increased the germination of seeds notably in presence of As in comparison to only seed subjected to As stress.
Seedling length was measured to check the e?ect of Si and As ap-plication on rice seeds(Fig.2B).Si-supplementation improved the seedlings length throughout the experimental period as compared to control.Addition of As inhibited the shoot length.In comparison to control,As stress resulted in a decrease of3and6folds in seedlings length.Root growth decreased with increasing concentration of As.In the absence of Si supplementation(0mM Si),decrease in root growth was almost95%for150μM As as compared to control.Silicon sup-plementation(3mM Si)increased the root growth about32%as com-pared to control.There was no positive e?ect of Si application on root growth under As stress(Table2).
3.3.Plant growth attributes
Results showed that two cultivation systems markedly di?er for the number of tillers plant?1.Maximum numbers of tillers were observed when plants were grown under anaerobic conditions with additional Si supply.While,As stress inhibited the number of tillers in both treat-ments(0mM Si and3mM Si)at both concentrations(150μM As and 300μM As)under anaerobic conditions.Under aerobic conditions,the e?ects of As stress were less pronounced.No positive e?ects of Si were observed on numbers of tillers upon Si-supplementation in aerobic cultivations.
Plant height was signi?cantly a?ected by As application in both cultivation systems.Maximum plant height(57.00cm)was observed when plants were grown with exogenous Si supply(3mM Si)under anaerobic conditions.At lower level,As stress signi?cantly reduced the plant height in anaerobic conditions.While under aerobic conditions, the maximum plant height(39.63cm)was observed in control treat-ment supplied with Si(Silicon3mM+Arsenic0μM)and minimum height(32.13cm)was observed when plants were treated with higher level of As and Si(Silicon3mM+Arsenic300μM).There was no positive e?ect of Si on height of plant under As stress in both cultivation systems(Table3).
Maximum plant fresh biomass was observed when plants were supplied with exogenous Si supply(Silicon3mM+Arsenic300μM). Under anaerobic conditions As application in growth medium resulted in a sharp decline in plant fresh biomass and the two levels of As(150 and300μM)were statistically non-signi?cant to each other.In contrast to anaerobic condition,di?erent levels of As showed a gradual de-creasing e?ect on the fresh biomass in aerobic cultivation.There was a continuous decrease in fresh shoot biomass with increasing con-centration of As in presence or absence of Si.Silicon application under As stress did not show any positive e?ects on plant fresh biomass in both cultivation systems.
The aerobic cultivations system results in markedly less plant dry biomass as compared to anaerobic cultivation system(Table3).Under anaerobic conditions,exogenous Si application resulted in maximum dry weight.Arsenic presence in the root medium sharply declined the plant dry mass at both levels in comparison to control treatment under anaerobic conditions.While,under aerobic conditions maximum plant dry biomass was observed in control treatment.There was a decreasing
Fig.1.Response of rice variety KSK-133to silicon,arsenic and the combination.Four set of treatments were applied are represented as:A)0mM Silicon(control),0.25mM Silicon,1mM Silicon,2mM Silicon and3mM Silicon;B)0μM Arsenic(Control),50μM Arsenic,100μM Arsenic,150μM Arsenic and300μM Arsenic;C)0mM Silicon+0μM Arsenic(Control), 0.5mM Silicon+150μM Arsenic,1mM Silicon+150μM Arsenic,3mM Silicon+150μM Arsenic;and D)0mM Silicon+0μM Arsenic(Control),0.5mM Silicon+300μM Arsenic, 1mM Silicon+300μM Arsenic,3mM Silicon+300μM Arsenic.The completely randomized design was used for analysis of variance and mean values were compared using least signi?cant di?erence at5%level of probability.Bars with in a single plot sharing same letter did not di?er signi?cantly from each other.Values are the means of three replicates±
SE.
Fig.2.E?ect of silicon and arsenic application seed germination percentage(A)and seedling length(B).Seeds of rice variety KSK-133were socked in distilled water for36h and were put on?lter paper moistened with di?erent concentration of Si and As.Data was recorded at3rd day for germination and at6th day of seed socking.The completely randomized design with factorial arrangements was used for analysis of variance and mean values were compared using least signi?cant di?erence at5%level of probability.Germination percentage and seedling lengths were signi?cantly a?ected by time(p=0.0001and0.0001,respectively)and treatment(p=0.0001and0.0001,respectively)while interaction between treatment and time was non-signi?cant for germination percentage and seedling lengths with p values1.0and0.26,respectively.Values are the means3replicates±SE.
trend in dry weight with increasing concentration of As in the presence or absence of Si under aerobic conditions.There was no positive e?ect of Si on dry weight of plants under As stress in both aerobic and anaerobic cultivation conditions.
Fresh weight of rice roots was recorded to evaluate the e?ect of Si and As application on roots weight under aerobic and anaerobic con-ditions.Maximum root fresh weight was observed in plant supplied with Si under control conditions,while between cultivations systems, anaerobic system produced higher biomass as compared to aerobic conditions.A continuous decrease in weight was observed with the increasing concentration of As in the root medium in both cultivation systems.There was no positive e?ect of Si application under As stress in both water systems as compared to controlled treatments(Table3). There was a clear decrease in dry weight under aerobic conditions as compared to anaerobic conditions.In both water systems,low root weights were measured as compared to control,while Si application showed a positive e?ect in root weight only under controlled condi-tions.There was no remediation by Si application under As stress in both water systems.A continuous decrease in weight was measured with increasing concentration of As.
3.4.Phosphorus,silicon and arsenic concentration in shoots and roots
Data recorded for phosphorus concentration in shoots showed a signi?cant e?ect of Si and As addition to the growth medium.Silicon application markedly increased the concentration of phosphorus in shoot tissues.The maximum concentration(3.25mg g?1DW)was ob-served in3mM Si treated plants,whereas the minimum concentration (1.60mg g?1DW)was observed when plants were treated with Silicon 3mM+Arsenic300μM under aerobic conditions.Arsenic presence in root medium signi?cantly decreased the phosphorus concentration in both cultivation systems.At intermediate stress level(150μM As),Si application showed some positive e?ects to mitigate the negative ef-fects of As on phosphorus concentration.While at higher dose of As (300μM As),Si did not produced any signi?cant e?ect on shoot phosphorus concentrations(Table4).Plant grown under anaerobic conditions showed an increase in phosphorus concentration in roots with the increasing concentration of As.Maximum concentration of phosphorus(2.37mg g?1DW)was recorded when plants were stressed with higher dose of As(300μM As)while the minimum concentration (1.00mg g?1DW)was observed in As(150μM As)treatment under aerobic cultivation.Under anaerobic condition plant showed an in-creasing trend with the increasing concentration of As in the growth medium.There was a signi?cant e?ect of Si application on phosphorus concentration in rice roots under both cultivation systems.Under aerobic conditions,maximum phosphorus concentration(2.44mg g?1 DW)was recorded when plants were supplied with Si under no As-stress conditions.In aerobic conditions,a gradual signi?cant decreasing trend
E?ect of silicon and arsenic addition in growth medium on root length.Seeds of rice variety KSK-133were socked in?ltered water for36h and were put on?lter paper moistened with di?erent concentration of silicon and arsenic.Data was recorded at9th day of seed socking.Values are the means of three replicates±SE.Means sharing same letter did not di?er signi?cantly from each other at5%level of probability.
Silicon and arsenic addition to the germination medium
0mM Si3mM Si
Days0μM As150μM As300μM As0μM As150μM As300μM As
9th270.48±40.19b12.86±8.93c 1.90±0.91c356.19±45.61a 6.19±4.24c8.10±3.34c
10th308.10±45.43b14.76±9.64c 6.67±1.67c399.05±47.40a7.14±4.13c12.86±1.65c
LSD for9th day=77.34.
LSD for10th day=83.87.
Table3
E?ect of silicon and arsenic application on number of tillers plant?1,plant height,root and biomass accumulation cultivated under anaerobic and aerobic conditions.Rice plant were grown for3months in pots?lled with soil treated with di?erent concentrations of As and Si.The completely randomized design was used for analysis of variance and mean values were compared using least signi?cant di?erence at5%level of probability.Bars with in a single plot sharing same letter did not di?er signi?cantly from each other.Values are the means of4 replicates±SE.
Treatment Code Interaction(WM×Si×As)Shoot parameters Root parameters
No.of tillers Plant height(cm)Shoot Fresh weight Shoot Dry weight Root Fresh weight Root Dry weight
(g plant?1)(g plant?1)(g plant?1)(g plant?1)
T1Anaerobic-Si0As017.00±1.19ab56.13±0.66a73.46±4.51b30.37±2.47b32.88±2.64b 5.35±0.63b
T2Si0As15018.88±1.01ab56.63±0.72a51.09±3.89c21.55±3.96c32.31±5.62b 5.16±0.58ab
T3Si0As30015.38±0.80bc52.38±2.17ab47.37±5.55c17.54±5.27c24.17±3.50c 4.24±0.45ab
T4Si3As021.38±0.47a57.00±1.51a88.02±8.04a41.46±4.32a51.17±3.73a8.09±0.69a
T5Si3As15014.63±0.67bc47.63±2.77b49.32±2.87c16.74±1.27c35.37±8.75b 4.97±0.87ab
T6Si3As30013.13±1.44de50.25±2.46b46.86±4.18c19.36±1.50c31.47±7.51b 5.35±1.40b Mean16.73±0.7752.93±1.8759.35±4.8424.50±3.1334.56±5.29 5.52±0.77
T7Aerobic-Si0As09.50±0.64f38.25±2.15c21.90±1.59d8.97±0.80d14.24±0.97d 1.94±0.09c
T8Si0As15010.25±0.72ef37.13±0.94cd18.76±1.84d7.31±0.59d12.61±0.99d 1.87±0.14c
T9Si0As3008.25±1.87f35.25±1.51cd16.67±1.88d 6.40±0.46d8.28±1.71d 1.53±0.39c
T10Si3As09.50±0.45f39.63±2.25c21.16±1.11d8.14±0.36d14.85±1.72d 2.16±0.04c
T11Si3As1508.63±0.48f35.50±1.26cd17.70±1.13d7.16±0.53d10.80±1.33d 1.87±0.10c
T12Si3As3007.13±0.48f32.13±1.55d15.47±1.59d 6.33±1.04d8.49±1.27d 1.21±0.16c Mean8.88±0.9336.31±1.6118.61±1.527.38±0.6311.54±1.33 1.76±0.15 Anaerobic cultivation:(T1-T6)
T1=Silicon0mM+Arsenic0mM,T2=Silicon0mM+Arsenic150μM,T3=Silicon0mM+Arsenic300μM,T4=Silicon3mM+Arsenic0mM,T5=Silicon3mM+Arsenic150μM and T6=Silicon3mM+Arsenic300μM
Aerobic cultivation:(T7-T12)
T7=Silicon0mM+Arsenic0μM,T8=Silicon0mM+Arsenic150μM,T9=Silicon0mM+Arsenic300μM,T10=Silicon3mM+Arsenic0μM,T11=Silicon3mM+Arsenic150μM and T12=Silicon3mM+Arsenic300μM
in phosphorus concentration was observed with the increasing con-centration of As in the growth medium.It was also observed that the concentration of phosphorus was a?ected by increasing the con-centration of As from150μM to300μM in the growth medium in the absence of Si,but there was a clear decrease in phosphorus con-centration with increasing concentration of As when it is supplemented with3mM Si(Table4).
Results indicated that plant supplied with exogenous Si had higher concentration of Si in shoot tissues.Maximum shoot Si concentration (35.55mg g?1DW)was observed when plants were grown with Si supplementation under no As-stress.A gradual decreasing trend in shoot Si concentration was observed with the increasing concentration of As in the root medium.While,under aerobic conditions exogenous Si supply had no-signi?cant e?ect on Si concentration with increasing concentration of As.
Data for Si concentration in root showed that plants grown with additional Si supply has higher amount of Si in comparison to no-Si-replete plants.Under anaerobic conditions,Si concentration decreased by the increasing concentration of As.Among treatments,maximum concentration of Si was measured for Si-treated plants(29.04mg g?1 DW)and minimum was measured for Silicon0mM+Arsenic300μM (9.11mg g?1DW).Under aerobic conditions,there is a slight decrease in root Si concentration with the increasing concentration of As. Maximum concentration was measured for Si supplied plant under no stress conditions(22.55mg g?1DW)and minimum for As stressed (11.24mg g?1DW).Silicon supplementation(3Mm Si)increased (36–43%)the concentration of Si in roots as compared to0mM Si.In the absence of Si supplementation,maximum concentration of Si was measured for control(13.98mg g?1DW),and minimum was observed for As300μM treated plants.
Arsenic concentration increased in shoots with increasing con-centration of As in root medium.Increase in concentration was sig-ni?cant in all treatments under both aerobic and anaerobic conditions. Maximum concentration was recorded for T3under anaerobic condi-tions,which is(9.34μg g?1DW)about90%greater than control (0.19μg g?1DW).Silicon supplementation signi?cantly decreased the As concentration under anaerobic conditions,while a signi?cant de-crease was also measured(for150μM As)under aerobic conditions.
In rice roots,an increasing trend of As was recorded with increasing concentration of As in root medium in both cultivated conditions. Maximum concentration was measured for T3(Silicon0mM+Arsenic 300μM;anaerobic conditions),that is about90%higher than the control treatment and minimum concentration was recorded for con-trol.Silicon supplementation(3mM Si)signi?cantly decreased As in roots at both As concentration(150μM,300μM As)as compared to 0mM Si under both cultivated conditions.The comparison of two the cultivation methods showed that As concentration was relatively lower under aerobic conditions than anaerobic conditions.
4.Discussion
4.1.E?ect of silicon supplementation on germination of rice seeds subjected to arsenic stress
During a preliminary experiment,rice seeds were exposed to dif-ferent concentrations of As and Si to determine their e?ects on germi-nation of seeds.Germination of seeds proved to be very sensitive to As contamination(Liu et al.,2005).Decreased germination rate in re-sponse to As exposure in rice has been reported by Shri et al.(2009). These e?ects might be due to of As interaction with the enzymes that are responsible for starch metabolism hence decreasing the germination of rice seeds.In plants,α-amylase,β-amylase and starch phosphorylase are major starch hydrolyzing enzymes(Yang et al.,2001).Energy for germination of seeds and for growth of roots and shoots is provided by sugars metabolism and for this purposeα-amylase converts en-dospermic stored starch into metabolizable sugars(Kaneko et al., 2002).Similarly a signi?cant reduction in seed germination has been reported in wheat due to decreased amylolytic activities of theα-amylase,β-amylase under As exposure(Jha and Dubey,2005;Liu et al., 2005).In addition,Sharma(2012)concluded that poor seed germina-tion due to As was attributed to poor cell wall metabolism and hor-monal signaling.While,an improvement in seedlings length was ob-served when Si was supplemented as compared to As alone at both low and high concentrations.Although the e?ects were minor but these might be attributed due to improvement in defense mechanism and antioxidant systems of seedlings by Si supplementation(Tripathi et al., 2013).
E?ect of silicon and water management on phosphorus,silicon and arsenic concentrations in shoots and roots of the rice plant.Rice Varity KSK-133was grown for3months under aerobic and anaerobic conditions in pots?lled with soil and treated with di?erent concentrations of As and Si.The completely randomized design was used for analysis of variance and mean values were compared using least signi?cant di?erence at5%level of probability.Bars with in a single plot sharing same letter did not di?er signi?cantly from each other.Values are the means of4replicates±SE.
Treatment Code Interaction(WM×Si×As)P Conc.(μg g plant DW?1)Si Conc.(mg g plantDW?1)As Conc.(μg g plant DW?1)
Shoot Root Shoot Root Shoot Root
T1Anaerobic-Si0As0 2.53±0.04cd 1.55±0.07cd20.84±0.65d15.46±0.51e0.19±0.14g 1.50±0.28h
T2Si0As150 2.22±0.06ef 1.94±0.10c12.85±0.44f10.79±0.61gh 6.37±0.40c18.60±0.87c T3Si0As300 1.95±0.06fh 2.20±0.07b12.07±0.42f9.11±0.41h9.34±0.55a27.70±1.06a T4Si3As0 3.25±0.17a 1.48±0.08de35.55±0.97a29.04±0.62d0.24±0.11g 1.33±0.28h
T5Si3As150 2.61±0.10c 2.37±0.12ab28.66±0.64b25.97±0.57b 5.11±0.24de13.08±0.88e T6Si3As300 1.92±0.11fh 2.03±0.10b25.04±0.60c22.17±0.81cd 6.87±0.46b22.20±1.05b Mean 2.41±0.09 1.90±0.0922.50±0.6518.76±0.59 4.69±0.3214.07±0.77
T7Aerobic-Si0As0 2.12±0.04eg 1.28±0.07e20.54±0.60d13.98±0.56ef0.34±0.14g 1.08±0.39h
T8Si0As150 1.85±0.05gi 1.00±0.05f17.55±0.67e12.92±1.10fg 3.91±0.39e10.25±0.91f
T9Si0As300 1.69±0.03hi 1.05±0.06f16.04±0.36e11.24±0.77gh 6.06±0.52cd16.00±0.63d T10Si3As0 2.91±0.05b 2.44±0.05d30.74±0.78b22.17±1.54cd0.32±0.16g 1.15±0.14h
T11Si3As150 2.30±0.10de 1.43±0.07de25.64±1.46c22.55±0.48c 2.50±0.26f 5.78±0.46g
T12Si3As300 1.60±0.17i 1.32±0.04e24.23±0.52c19.90±0.85d 4.90±0.44de9.28±0.51f Mean 2.08±0.07 1.42±0.0622.46±0.7317.13±0.88 3.00±0.327.26±0.51 Anaerobic cultivation:(T1-T6)
T1=Silicon0mM+Arsenic0mM,T2=Silicon0mM+Arsenic150μM,T3=Silicon0mM+Arsenic300μM,T4=Silicon3mM+Arsenic0mM,T5=Silicon3mM+Arsenic150μM and T6=Silicon3mM+Arsenic300μM.
Aerobic cultivation:(T7-T12)
T7=Silicon0mM+Arsenic0μM,T8=Silicon0mM+Arsenic150μM,T9=Silicon0mM+Arsenic300μM,T10=Silicon3mM+Arsenic0μM,T11=Silicon3mM+Arsenic150μM and T12=Silicon3mM+Arsenic300μM.
4.2.E?ect of di?erent cultivated conditions(aerobic and anaerobic)on plant physical and chemical attributes
Plant physiological parameters i.e plant height and biomass reduc-tion with increasing concentration of As in both cultivation systems indicates As toxicity in rice plants as reported in many other studies (Islam,1999;Geng et al.,2005).Among the cultivation systems, anaerobic rice cultivation produced higher biomass and the possible reason of this increased growth is the availability of ample amount of water as rice is water loving plant.However,the e?ects of As stress were more prominent under anaerobic conditions than aerobic condi-tions.The possible reason of the e?ects of As toxicity under anaerobic conditions may be due to higher availability of As to rice plants under anaerobic conditions as As III is a dominant As species under reduced conditions(Talukder et al.,2012).Moreover.As III is more mobile in the environment over wide range of pH.Alternatively,the decreased As toxicity under aerobic condition might be due to conditions favoring the binding of As V with mineral oxides and organic materials and less mobility in the soil.Arsenate has a strong a?nity for soil minerals like Fe-hydroxides which leads to decreased As solubility and hence bioa-vailability to plants(Takahashi et al.,2004;Xu et al.,2008).Arsenate becomes less liable then As III,as it is negatively charged over most of the pH ranges so it sorbs on aluminum and iron oxide(WHO, Environmental Health Criteria,2001).Therefore,under aerobic con-ditions As availability has been decreased and ultimately uptake also decreased(Fig.3).
Rice plants responded to the exogenous application of Si as its concentration in roots and shoots increased with higher level of Si in root medium.Bene?cial e?ects of Si application on rice growth(Fig.1) are in line with the?ndings of other studies as reported by Alvarez and Datno?(2001).Increase in plant growth may be attributed to the changes in physiological and morphological characteristics which are facilitated by the presence of Si.Silicon mediated in morphological and biochemical changes has been suggested as a possible reason of in-creased plant growth(Epstein,1999).Kaufman et al.(1979)proposed a “Window-hypothesis”by which Si deposition acts as a window in leaf epidermal cells and enhances the light transmission to mesophyll cells which takes part in photosynthesis.In this way the exogenous Si ap-plication can facilitate the CO2?xation in rice plants(Ma et al.,2002). Silicon functioning as a balancing element for other minerals nutrients may also be the possible reason of its bene?cial e?ects on plants under no-stress conditions(Marschner et al.,1990).
Comparison of two cultivated conditions showed that the uptake of As is relatively less under aerobic condition than anaerobic condition in rice roots and shoots.It is evident from many studies that As III,the dominant species under anaerobic conditions,is the major form taken up by the plants as neutral form(As(OH)3)through Si in?ux trans-porters(Xu et al.,2008;Su et al.,2009).Therefore,changing rhizosphere from oxidized(aerobic)to reduced(anaerobic)conditions markedly decrease the As contents in various parts of rice plant (Talukder,2012).On the other hand,As V is a dominant form of As under aerobic conditions,its less availability to plants due to its ne-gative charge and its binding with other minerals explains the low concentration of As in rice roots and shoots(Meharg,2004).Silicon signi?cantly decreased the As concentration in both cultivated condi-tions.Silicon and As III(a dominant form of As under anaerobic condi-tions)compete for the uptake site at root surface.Transporters,Nodulin 26Intrinsic Proteins(NIPs)are responsible for As III uptake in rice roots. As uptake via LSi1and its e?ux via LSi2towards xylem and shoots is a?ected by increase in Si concentration(Ma et al.,2008).In addition,Si nutrition increases the oxidation power of rice roots,hence creation favorable conditions for As immobilization through As III conversation into As V(Fleck et al.,2011).
Arsenic presence in root medium signi?cantly reduced the con-centration of Si in rice roots and shoots under anaerobic conditions and non-signi?cantly under aerobic conditions.The decreased Si con-centration in plant tissues may be due to antagonistic e?ect of As III under anaerobic conditions,while the non-signi?cant decrease in roots As concentration may be due to the presence of As V in rhizosphere which did not have any e?ect on Si transportation.Another possible reason of the decreased Si concentration under aerobic conditions is the less availability of Si due to less amount of water as the Si is taken up in rice roots by aquaporin channels(LSi1)(Ma et al.,2008).In shoots, concentration of Si decreased with increasing concentration of As in both aerobic and anaerobic conditions.The LSi2translocate the Si from roots to shoots,which is also an e?ux transporter for As III.In both cultivated conditions As III a?ected the Si translocation and this decrease may be due to the e?ect of As III on Si translocation from roots to shoots. Majority of the As which is taken up by plant is reduced into As III as Xu et al.(2008)reported that As III accounts for92–99%of the As in roots of rice and tomato.Most of the plants have high capacity to reduce As V into As III through arsenic reductase using glutathione as reductant (Dixit et al.,2016).Arsenite have high a?nity for glutathione and phytochelatins which is an important mechanism for As detoxi?cation. So,a similar antagonistic e?ect may a?ect the Si translocation in shoots under aerobic conditions as well.
A continuous increase in concentration of phosphorus was observed in rice roots under anaerobic condition with increasing concentration of As.The increased phosphorus concentration in rice roots may be due to an increased concentration of As in root medium that may have in-creased the competition between As and phosphorus for sorption sites in soil.So it could be speculated that the increase in As concentration may cause the release of phosphorus and increase its bioavailability to roots.Another possible reason of an increase in phosphorus con-centration in rice roots with increasing concentration of As is its spe-ciation under two di?erent water regimes.Under anaerobic conditions, As III has no antagonistic e?ect on phosphorus concentration. Phosphorus concentration increased with increasing concentration of As demonstrated that its uptake was not restricted due to the presence of As III.While under aerobic conditions,concentration of phosphorus is decreased in rice roots.Arsenate is the analog of phosphorus as both As V and phosphate use same uptake transporter in rice plants.Arsenate use phosphate transport system to pass through plasma membrane (Dixon,1997).So,a decrease in phosphorus concentration may be due to increasing competition between As V and phosphate for transporta-tion in rice roots(Geng et al.,2005).Increase in phosphate nutrition cause the suppression of high a?nity arsenate/phosphate transport system(Dixon,1997).Similarly,phosphate concentration can decrease the increasing concentration of As.Aerobic conditions also promotes the oxidation of substances like iron,manganese and hydrogen sul?de around rhizosphere.Additionally,aerobic condition around rhizo-sphere promotes the iron oxide growth that forms a plaque around the roots(Reddy and DeLaune,2008).This plaque formation under aerobic condition may be a possible cause of phosphorus decrease in roots with
Fig.3.Speci?c arsenic uptake in rice plants variety133-KSK.Rice Variety KSK-133was grown for3months in pots?lled with soil amended with di?erent concentrations of As and Si cultivated under anaerobic and aerobic conditions.The completely randomized design was used for analysis of experimental data and mean values were compared using least signi?cant di?erence at p5%.Bars showing di?erent letter di?er signi?cantly from each other.Values are the means of four replicates±SE.
increasing concentration of As.Phosphorus and As have high a?nities for these Fe-oxides,so and immobilization of these minerals by Fe(III)-oxides may also occurred(Meharg,2004).Silicon supplementation in-creased the concentration of phosphorus in roots and shoots under no As stress.Silicon is taken up by the plants as non-essential elements unlike phosphorus,potassium and nitrogen.However,Si helps to im-prove the plant growth and mitigates environmental stresses(Epstein, 1999;Kim et al.,2011).Increase in Si concentration in root medium may increase the uptake of phosphorus in rice roots.It may be due to desorption of phosphorus from adsorption sites within the soils.
5.Conclusion
It is concluded that the aerobic conditions are more favorable to decrease arsenic uptake in rice.Aerobic conditions decrease arsenic stress on rice growth as compared to anaerobic conditions.Growth attributes were negatively a?ected when there was less availability of water.Silicon supplementation proved bene?cial for growth attributes in the absence of arsenic stress,but under arsenic stress,there was no signi?cant e?ect of silicon on plant growth attributes.However,silicon supplementation signi?cantly decrease the arsenic uptake especially in plants grown under aerobic conditions.Arsenic stress decreased phos-phorus uptake in the rice plant while silicon application partially re-duced the e?ect of arsenic on phosphorus in rice shoot.
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