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Arsenic Hyperaccumulation by Pteris Vittata L. Arsenic Transformation, Uptake and Environmental Impact

Permanent Link: http://ufdc.ufl.edu/UFE0042493/00001

Material Information

Title: Arsenic Hyperaccumulation by Pteris Vittata L. Arsenic Transformation, Uptake and Environmental Impact
Physical Description: 1 online resource (117 p.)
Language: english
Creator: MATHEWS,SHINY
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: AQUAPORIN -- ARSENIC -- HYPERACCUMULATOR -- INSECT -- OXIDASE -- PTERIS -- REDOX -- SAISSETIA -- SAP -- STERILE -- TRANSPORTER -- UPTAKE
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ARSENIC HYPERACCUMULATION BY PTERIS VITTATA L.- ARSENIC TRANSFORMATION, UPTAKE AND ENVIRONMENTAL IMPACT By Shiny Mathews May 2011 Chair: Lena Q. Ma Major: Soil and Water Science The arsenic (As) hyperaccumulating fern, Pteris vittata (Chinese brake fern), is capable of taking up arsenate (AsV) and arsenite (AsIII). The physiological aspects pertaining to the transformation of As in the media of the fern, its uptake mechanisms, speciation in the biomass and the impact of the hyperaccumulated arsenic on insects were studied. The role of the fern and microbes in AsIII oxidation in the growth media and the location of AsIII oxidation and AsV reduction in P. vittata biomass were studied. Arsenic speciation was performed in the growth media, roots, rhizomes, rachis, pinnae, fronds, and sap of P. vittata. Arsenite was rapidly oxidized in the growth media by microbes and was then further oxidized in the roots of P. vittata. Arsenate reduction mostly occurred in the rhizomes and pinnae of P. vittata. Arsenite translocation from the roots to the fronds was more rapid than arsenate. The mechanism of AsIII uptake was hypothesized to be via aquaporin transporters and was studied using competitors of AsIII uptake, glycerol and antimonite (SbIII), and also an inhibitor of aquaporin transporter, silver nitrate (AgNO3). The presence of glycerol or SbIII had no impact on AsIII or AsV uptake by P. vittata. However, the presence of 0.01 mM AgNO3 reduced the AsIII concentrations in the fronds and roots respectively, indicating that AsIII uptake might be via an aquaporin transporter different from the glycerol and SbIII transporters. Arsenic hyperaccumulation by P. vittata may serve as a defense mechanism against herbivore attack. A study was conducted to examine the effects of arsenic concentrations on scale insect (Saissetia neglecta) infestation of P. vittata. Scale insects were counted as percentage fallen from the plant to the total number of insects after 1 week of As-treatment. The higher arsenic concentrations in the fronds resulted in higher percentage of dead and fallen scale insects indicating that arsenic may help P. vittata defend against herbivore attack.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SHINY MATHEWS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Ma, Lena Q.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042493:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042493/00001

Material Information

Title: Arsenic Hyperaccumulation by Pteris Vittata L. Arsenic Transformation, Uptake and Environmental Impact
Physical Description: 1 online resource (117 p.)
Language: english
Creator: MATHEWS,SHINY
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: AQUAPORIN -- ARSENIC -- HYPERACCUMULATOR -- INSECT -- OXIDASE -- PTERIS -- REDOX -- SAISSETIA -- SAP -- STERILE -- TRANSPORTER -- UPTAKE
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ARSENIC HYPERACCUMULATION BY PTERIS VITTATA L.- ARSENIC TRANSFORMATION, UPTAKE AND ENVIRONMENTAL IMPACT By Shiny Mathews May 2011 Chair: Lena Q. Ma Major: Soil and Water Science The arsenic (As) hyperaccumulating fern, Pteris vittata (Chinese brake fern), is capable of taking up arsenate (AsV) and arsenite (AsIII). The physiological aspects pertaining to the transformation of As in the media of the fern, its uptake mechanisms, speciation in the biomass and the impact of the hyperaccumulated arsenic on insects were studied. The role of the fern and microbes in AsIII oxidation in the growth media and the location of AsIII oxidation and AsV reduction in P. vittata biomass were studied. Arsenic speciation was performed in the growth media, roots, rhizomes, rachis, pinnae, fronds, and sap of P. vittata. Arsenite was rapidly oxidized in the growth media by microbes and was then further oxidized in the roots of P. vittata. Arsenate reduction mostly occurred in the rhizomes and pinnae of P. vittata. Arsenite translocation from the roots to the fronds was more rapid than arsenate. The mechanism of AsIII uptake was hypothesized to be via aquaporin transporters and was studied using competitors of AsIII uptake, glycerol and antimonite (SbIII), and also an inhibitor of aquaporin transporter, silver nitrate (AgNO3). The presence of glycerol or SbIII had no impact on AsIII or AsV uptake by P. vittata. However, the presence of 0.01 mM AgNO3 reduced the AsIII concentrations in the fronds and roots respectively, indicating that AsIII uptake might be via an aquaporin transporter different from the glycerol and SbIII transporters. Arsenic hyperaccumulation by P. vittata may serve as a defense mechanism against herbivore attack. A study was conducted to examine the effects of arsenic concentrations on scale insect (Saissetia neglecta) infestation of P. vittata. Scale insects were counted as percentage fallen from the plant to the total number of insects after 1 week of As-treatment. The higher arsenic concentrations in the fronds resulted in higher percentage of dead and fallen scale insects indicating that arsenic may help P. vittata defend against herbivore attack.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SHINY MATHEWS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Ma, Lena Q.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042493:00001


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1 ARSENIC HYPERACCUMULATION BY PTERIS VITTATA L. ARSENIC TRANSFORMATION, UPTAKE AND ENVIRONMENTAL IMPACT By SHINY MATHEWS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Shiny Mathews

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3 To my daughter Lisa Annie Johnson

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4 ACKNOWLEDGMENTS I would like to take this opportunity to thank my advisor Dr. Lena Q. Ma for letting me be a part of her group and for her support and encouragement. I am grateful to her for the guidance provided both for my research and professional development. I would also like to thank my committee members Dr. Bala Rathinasabapathi, Dr. Robert Stamps, Dr. Charles Guy and Dr. Max Teplitski for their valuable suggestions and the time they spent on ensuring the smooth progress of my research. I am grateful to Dr. Tim Martin and Dr. Carlos Gonzales for the training provided on the pressure chamber. I am obliged to Dr. Rao Mylavarapu, Dr. John Thomas and Dr. Andy Ogram for their help in the research related aspects. My research and lab training was very much influenced by the dedicated efforts of our laboratory manager Dr. Uttam Saha. I thank him and Dr. Mri ttunjai Srivastava and their families for all the help from the day I arrived in the U S till now. The Biogeochemistry of Trace Metals group was like a huge family to me. At this moment I thank my friends Anhui, Shuhe, Xiao w e n, Shawn, Dr. Ding, Dr. Ullah Edmund, Xin, Hao, Tan, Jay, Piyasa, Ling and Ying for ongoing support in research and in personal matters. Special thanks also to Moshe, Akua and Abel for their help in research. My roommates and friends in Gainesville played a major role in creating a home away from home atmosphere. I would like to convey my gratitude to Pragyan, Bini, Biji, Georgy, Vivek, Sudhamshu, Ramesh, Nithin, Bharath, Manu, Anand, Rajesh, Phalgun, Deepak and all my friends. I thank my previous supervisors Dr. P. Venugopalan, Dr. L Suseela Devi Dr. Suguna Yesodharan and Dr. Sivanandan Achary for their encouragement and help during the application process to the University of Florida.

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5 I would never have been able to achieve anything in my life without the love and support of my par ents, inlaws and husband. With their constant encouragement and prayers I have been able to achieve my goal. I would like to take this opportunity to express my love to them and dedicate this research to my family especially to my daughter Lisa who lear ned to understand my responsibilities and my absence throughout these 4 years. I thank God for keeping me safe and strong and for helping me achieve my objectives.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 REVIEW OF LITERATURE .................................................................................... 16 Arsenic: O rigin and Occurrence .............................................................................. 16 Arsenate and Arsenite in the Environment .............................................................. 17 Arsenate Reduction .......................................................................................... 18 Arsenite Oxidation ............................................................................................ 20 Applications of Arsenic ............................................................................................ 22 Toxicity and Health Effects ..................................................................................... 23 Inor ganic Arsenic .............................................................................................. 23 Organic Arsenic ................................................................................................ 24 Arsenic Remediation ............................................................................................... 24 Pteris vittata in Phytoremediation ........................................................................... 27 Life Cycle .......................................................................................................... 28 Physiology ........................................................................................................ 28 Arsenic Uptake ................................................................................................. 29 Microflora Associations ..................................................................................... 30 Insect Deterrence ............................................................................................. 31 3 ARSENIC TRAN SFORMATION IN THE GROWTH MEDIA OF P VITTATA THE ROLE OF MICROBES OR ROOT ENZYMES ................................................ 32 Arsenic Transformations ......................................................................................... 32 Materials and Methods ............................................................................................ 33 Experimental Setup .......................................................................................... 33 Arsenic Speciation under Natural Conditions ................................................... 34 Arsenic Speciation under Sterile Conditions ..................................................... 35 Arsenic Speciation in Sonicate Extract ............................................................. 36 Arsenic Analysis ............................................................................................... 37 Data Analysis ................................................................................................... 38 Results and Discussion ........................................................................................... 38 Arsenic Sp eciation under Natural Conditions ................................................... 38 Media arsenic speciation............................................................................ 38 Biomass arsenic speciation ........................................................................ 39 Arseni c Speciation under Sterile Conditions ..................................................... 45

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7 Arsenite Oxidation in Sonicate d Extract ........................................................... 47 Future Research ..................................................................................................... 49 4 ARSENIC TRANSF ORMATION AND SPECIATION IN PTERIS VITTATA BIOMASS AND XYLEM SAP .................................................................................. 51 Arsenic Speciation .................................................................................................. 51 Materials and Methods ............................................................................................ 53 Results an d Discussion ........................................................................................... 53 Xylem Sap Arsenic Speciation ......................................................................... 54 Biomass Arsenic Speciation ............................................................................. 54 Research Findings .................................................................................................. 56 5 UPTAKE AND TRANSLOCATION OF ARSENITE AND ARSENATE BY PTERIS VITTATA L.: EFFECTS OF GLYCEROL, ANTIMONITE AND SILVER .... 59 Arsenic Uptake and Translocation .......................................................................... 59 Materials and Methods ............................................................................................ 62 Experimental Setup .......................................................................................... 62 Time Dependent Uptake Study ........................................................................ 63 Competition and Inhibition Study ...................................................................... 63 Data Analysis ................................................................................................... 64 Results and Discussion ........................................................................................... 64 Effects of Glycerol and SbIII on AsIII Uptake .................................................... 65 Effects of Silver Nitrate on AsIII Uptake ........................................................... 68 AsIII Oxidation in the Media and P. vittata ........................................................ 76 Research Findings .................................................................................................. 80 6 ARSENIC REDUCED SCALEINSECT INFESTATION ON ARSENIC HYPERACCUMULATOR PTERIS VITTATA L. ...................................................... 81 Insect Detterence .................................................................................................... 81 Materials and Methods ............................................................................................ 85 Experiment Setup ............................................................................................. 85 Arsenic Treatment and Scale Counting ............................................................ 85 Chemical and Data Analysis ............................................................................. 86 Result s and Discussion ........................................................................................... 86 Arsenic Toxicity in Scales ................................................................................. 86 Arsenic Concentration and Scale Death ........................................................... 87 Findings and Future Directions ............................................................................... 91 7 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................... 93 APPENDIX A EFFECT OF ARSENIC LOADING ON ARSENIC HYPERACCUMULATION BY PTERIS VITTATA ................................................................................................... 97

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8 B COMPARISON OF ANTIMONY ACCUMULATION IN PTERIS VITTATA AND PTERIS ENSIFORMIS ............................................................................................ 99 LIST OF REFERENCES ............................................................................................. 101 BIOGRAPHY ............................................................................................................... 117

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9 LIST OF FIGURES Figure page 1 1 pE/pH diagram of As in water system at 25C .................................................... 18 1 2 Pathways for the reductive dissolution of sorbed arsenic. .................................. 19 1 3 Hypothetical model of an arsenite oxidase enzyme ............................................ 21 3 1 One month old P. vittata sporophytes ................................................................. 34 3 2 Propagation of P. vittata under sterile conditions ................................................ 36 3 3 As speciation when treated wit h 0.10 mM AsIII for 24 h. .................................... 41 3 4 Arsenic speciation when treated with 0.27 mM AsIII for 8 d. .............................. 43 3 5 Arsenic speciation when treated with0.27 mM AsV for 8 d ................................. 44 3 6 As speciation when treated with 0.10 mM AsIII for 1 and 14 d under sterile conditions.. ......................................................................................................... 48 3 7 As speciation in the media with root sonicate of P. vittata .................................. 50 4 1 Schematic of different parts of P. vittata ............................................................ 57 4 2 Scholander pressure chamber ............................................................................ 57 4 3 Arsenic speciation in the sap and tissue of P. vittata treated with 0.1 mM As V for 8 d ................................................................................................................. 58 5 1 Arsenic speciation in the media and root of P. vittata .exposed to 0.1 mM AsIII or AsV for 1, 2, 4, 6 and 24 h. ............................................................................. 66 5 2 Effect of glycerol on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h ......................................................................................................... 69 5 3 Effect of glycerol on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h .......................................................................................................... 70 5 4 Effect of SbIII on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h ......................................................................................................... 71 5 5 Effect of SbIII on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h .......................................................................................................... 72 5 6 Concentration of Sb in P. vittata when treated with 0.1 mM AsIII and different concentrations of SbIII for 1 h ............................................................................. 73

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10 5 7 Effect of AgNO3 on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h. ........................................................................................................ 77 5 8 Effect of AgNO3 on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h. ......................................................................................................... 78 5 9 Effect of AgNO3 on arsenic speciation in the fronds and roots of P. vittata after exposing to 0.1 mM arsenic for 2 h ............................................................ 79 6 1 Scale Insect: Saissetia neglecta ......................................................................... 84 6 2 The total number of fallenscale and intact scale on the fronds of P. vittata after 1 week of arsenic exposure ...................................................................... 88 6 3 Arsenic concentrations in the fronds of P. vittata fallen scales and intact scales. ................................................................................................................ 92 A 1 Effect of arsenic loading when treated with As for 15 d on frond arsenic concentration in P. vittata ................................................................................... 97 A 2 Effect of arsenic loading when treated with As for 15 d on root arsenic concentration in P. vittata .................................................................................. 98 B 1 Concentration of Sb in P. vittata when treated with different concentrations of Sb ....................................................................................................................... 99 B 2 Concentration of Sb in P. ensifomis when treated with different concentrations of Sb ......................................................................................... 100

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy A RSENIC HYPERACCUMULATION BY PTERIS VITTATA L. ARSENIC TRANSFORMATION, UPTAKE AND ENVIRONMENTAL IMPACT By Shiny Mathews May 2011 Chair: Lena Q. Ma Major: Soil and Water Science The arsenic (As) hyperaccumulating fern, Pteris vittata (Chinese brake fern) is capable of taking up arsenate ( AsV) and arsenite ( AsIII) The physiological aspects pertaining to the transformation of As in the media of the fern, its uptake mechanisms, speciation in the biomass and the impact of the hyperaccumulated arsenic on ins ects were studied. The role of the fern and microbes in AsIII oxidation in the growth media and the location of AsIII oxidation and AsV reduction in P. vittata biomass were studied. Arsenic speciation was performed i n the growth media, roots, rhizomes, rachis, pinnae, fronds, and sap of P. vittata Arsenite was rapidly oxidized in the growth media by microbes and was then further oxidized in the roots of P. vittata Arsenate reduction mostly occurred in the rhizomes and pinnae of P. vittata Arsenite trans location from the roots to the fronds was more rapid than arsenate. The mechanism of AsIII uptake was hypothesized to be via aquaporin transporters and was studied using competitors of AsIII uptake, glycerol and antimonite (SbIII) and also an inhibitor of aquaporin transporter, silver nitrate (AgNO3). The presence of glycerol or SbIII had no impact on AsIII or AsV uptake by P. vittata However, the

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12 presence of 0.01 mM AgNO3 reduced the AsIII concentrations in the fronds and roots respectively, indicating t hat AsIII uptake might be via an aquaporin transporter different from the glycerol and SbIII transporters. A rsenic hyperaccumulation by P vittata may serve as a defense mechanism against herbivore attack. A study was conducted to examine the effects of arsenic concentrations on scale insect ( Saissetia neglecta) infestation of P. vittata Scale insects were counted as percentage fallen from the plant to the total number of insects after 1 week of As treatment. The higher arsenic concentrations in the fronds resulted in higher percentage of dead and fallen scale insects indicating that arsenic may help P. vittata defend against herbivore attack.

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13 CHAPT ER 1 INTRODUCTION The ubiquitous presence of arsenic (As) in the environment through natural or anthropogenic sources has lead to a wide range of research areas in its occurrence, toxicity and remediation. Arsenic is found in geological formations and is r eleased to the atmosphere, water and soil either naturally through volcanoes ( Signorelli, 1999) or by As mining for commercial purposes (Smedley and Kinniburgh, 2002). These pathways result in the transformation of stable nonbioavailable arsenic to more bioavailable and toxic forms. Arsenic in water is of major environmental concern in terms of human, animal and plant health due to its solubility and bioavailabi lity. Once As enters a cell, it acts by binding onto thiol groups or replaces phosphates in the biochemical pathway and behaves as a carcinogen by creating chromosomal abberations Due to the health effects caused by As, strict regulations have been imposed by the WHO setting the standards for As in drinking water as 10 g L1. The introduction of strict regulations requires efficient and cost effective remediation methods for As removal from water. The existing remediation methods include oxidation, coagulation, lime softening, ion exchange, nano filtration, reverse osmosis, electro dialysis and phytoremediation (Mohan and Pitmann, 2007) Plants sensitive to As are either killed or have stunted growth when exposed to high concentrations. However, a certain group of plants called hyperaccumulators are able to tolerate and accumulate high concentrati ons of arsenic in its tissue. The use of these plants to accumulate high concentrations of As from the soil or water in the biomass, which can be harvested, is termed phytoremediation.

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14 The first hyperaccumulator of arsenic, Pteris vittata L ( Chinese brake fern ) was discovered by Ma et al ( 2001) This fern produces a large plant biomass compared Thlaspi sp and Brassica sp., to and is efficient in arsenic uptake. The fern can accumulate as much as 2.3% arsenic in its biomass and tolerate arsenic concentrat ions as high as 1, 500 mg kg1 in soil. P. vittata is unique in its As hyperaccumulating capacity and hence may have a different mechanism of uptake and tolerance to As compared to other plants. There are several hypotheses set forth as to why certain plant s are able to hyperaccumulate As (Boyd, 2004). These include metal tolerance, competition with neighboring plants, drought tolerance and defense against herbivores. Pteris vittata can be used in phytoremediation of arsenic contaminated sites and also as a model plant in the study of several physiological mechanisms in arsenic uptake and metabolism that can be related to other living organisms. One of the major pathways of As movement in an ecosystem is plant uptake. Arsenic may undergo changes in the rhizosphere in the presence of bacterial communities and root exudates before being taken up by the fern. This transformation can be of commercial value if the arsenic is converted to less toxic forms. The uptake and translocation of As in the fern depends on t he species of As in the media. Since P. vittata is unique in its ability to accumulate high concentrations of arsenic in its biomass it may have developed a unique uptake mechanism specific for As uptake. This may be different from other plants where As is mainly taken up by phosphate or water channels. Once accumulated the As is transformed to AsIII and sequestered in the vacuoles in the frond of P. vittata The accumulation of As in the biomass of P. vittata may have a negative impact on the herbivores th at feed on the fern. This is based on the hypothesis of defense mechanism

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15 of plant hyperaccumulation where the metal in the tissue itself or an organic chemical produced as a result of hyperaccumulation or a combined effect might result in repulsion or death of insects feeding on it. Hence, it is important to understand the threshold level of hyperaccumulated arsenic beyond which herbivores are impacted. This can also imply that plant hyperaccumulation can be a natural pest control measure if the concentrat ions of arsenic accumulated in the insects are not high enough to affect the food chain. It is important to understand the fate of arsenic in the growth media and rhizosphere of P vittata its uptake, biomass speciation and insect defense mechanism and hence the major objectives in this research w ere to understand the 1) effect s of the rhizosphere of P. vittata on As speciation in the media, 2) localization of oxidation or reduction of arsenic in the tissue, 3) uptake and translocation of arsenic and 4) i mpact of arsenic hyperaccumulation on insect infestation in P. vittata.

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16 CHAPTER 2 REVIEW OF LITERATURE Arsenic: Origin and Occurrence Arsenic is a carcinogenic metalloid of major environmental concern. It ranks the 20th in elemental abundance in the earths crust with an average concentration of 23 mg kg1 (Tanaka, 1988), 14th in seawater and 12th in the human body (Mandal and Suzuki, 2002). Arsenic is extracted for commercial purposes from lead and copper ores which have 23 % arsenic and from gold ores (11% As). China is the worlds largest producer of As at 30,000 tons per year and the United States is a major consumer (U S GS, 2008). The background concentration of As in the U S does not exceed 15 mg kg1 but concentrations from 0.2 to 40 mg kg1 hav e been reported by Walsh et al. ( 1977) The known natural oxidation states of As include 3, 0, +3 and +5, of which the most abundant and of environmental concern are +3 [ arsenite ( AsIII ) ] and +5 [ arsenate ( A sV ) ] (Cullen et al 1989; Johnson and Hiltbold, 1969; Chatterjee et al 1999). Arsenic is present in over 245 sulfide (chalcophilic) minerals associated with ultramafic early forming rocks (Abzalov et al., 1997) These minerals include arsenopyrite (FeAsS), arsenolite (As2O3), realgar (AsS), olivinite (Cu2OHAsO4), cobaltite (CoAsS), proustite (Ag3AsS3) and orpiment (As2S3) and are found in high temperature hydrothermal veins and in pegmatites or coarse igneous or sedimentary rocks (Allard, 1995; Reimann and deCaritat, 1998). The presence of these minerals in soils is determined by the geological history of a particular soil (KabataPendias and Adriano, 1995). Volcanoes are a major source of arsenic on the earths surface through magma or fumaroles (Signorelli, 1997).

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17 Arsenate and Arsenite in the Environment Arsenate with a negative charge is strongly adsorbed to the sur face of minerals, such as ferrihydrite and alumina, which reduces arsenate mobility. Its adsorption decreases with increasing pH, due to the increasing negative surface potential, leading to repulsion (Mahimairaja et al., 2005). Arsenite, on the other hand, is neutral at pH of 4 9 and hence has a weaker pH dependence on adsorption. It adsorbs less strongly and to fewer minerals, which makes it more mobile (Smedley and Kinniburgh, 2002). Both AsV and AsIII form similar surface complexes with goethite by bidendate binuclear complexes with 2 adjacent iron octahedral corner sites (Manning, 1998). The activity of AsV and AsIII is controlled by surface complexation reactions on metal hydrooxide and clay minerals of Al, Mn and Fe (Goldberg, 1986). The species of arsenic in water depends on the pH and redox potential existing in that system as indicated in the pE/pH diagram (Figure 1 1). In aerobic conditions As exists predominantly as arsenate (H3AsO4, H2AsO4 -, HAsO4 2and AsO4 3-) and under submerged and reduced environments as arsenite (H3AsO3, H2AsO3 and HAsO3 2-) (Onken and Hossner, 1996; Yan et al., 2000; Abedin et al., 2002) The species of As that predominates in a system depends on the capacity of the environment to reduce or oxidize arsenic in the presence of electron donors or acceptors based on abiotic or biotic factors. The abiotic factors include the presence of chemical oxidants or reductants and biotic processes requires living organisms particularly microbes.

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18 Figure 11. pE/pH diagram of As in water system at 25C (Smedley and Kinniburgh, 2002) Arsenate Reduction Arsenic adsorbs on both iron and aluminum oxides or hydroxides but iron oxide is the most important sink of both AsIII and AsV in aquatic and terrestrial environments. When arsenate is present in submerged soils, it may get directly reduced to AsIII abiotically in the presence of sulfide as an electron donor at pH < 5 (Rochette et al., 2000). Once AsIII is formed, the total soluble As decreases as a result of the formation of AsIII sulfide complexes. There are 2 biotic pathways by which AsV adsorbed on metal oxide minerals are converted to AsIII. In the first pathway FeIII oxides undergo a reductive dissolution allowing the release of AsV into the aqueous phase as indicated in Figure 12 (Inskeep et al 2002). Here microbes utilize FeIII as a terminal electron acceptor (Jones et al.,

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19 2000). For example, the FeIII reducing Shewanella alga can release AsV from scorodite at 35 M h1 with 10 mM lactate as carbon source (Cummings et al., 1999). These bacteria however, cannot reduce AsV to AsIII. The released AsV gets reduced to AsIII by biotic or abiotic pathways. Arsenate reduction occurs primarily by the action of dissimilatory arsenic respiring prokaryotes (DARPs) such as Sulphospirillum that respire AsV and release AsIII (Oremland and S tolz, 2005) A specific anaerobic bacteria Sulphospirillum barnessi is capable of both reductive dissolution of FeIII and reduction of AsV by using FeIII and AsV as terminal electron acceptors (Zobrist et al., 2000). This reductive dissolution of FeIII and subsequent release of AsV depends on crystallinity and surface area of the substrate. The dissolution is faster for amorphous ferrihydrate than crystalline goethite (Jones et al., 2000). Figure 12. Pathways for the reductive dissolution of sorbed arsenic through reduction to arsenite (left) or degradation of substrate (right) (Inskeep, 2002).

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20 In the second pathway of AsV reduction from iron oxides, the AsV first gets reduced to AsIII and then is released upon reductive dissolution of FeIII oxides (I nskeep et al 2002) (Figure 12). Certain microbes can reduce AsV to AsIII under oxic and anoxic conditions. These microbes do not use AsV as an electron source. Instead they follow an arsenic resistance mechanism where AsV enters into the cell, undergoes reduction to AsIII and is finally effluxed from the cell. These arsenic resistant bacteria are known to have an ars operon, which contains genes that encode for arsenate reductase (ArsC) that reduces AsV to AsIII. This ars operon detoxifies arsenic by the efflux of produced AsIII (Macur et al., 2001; Kaur et al 2009). Arsenite Oxidation The rate of oxidation of AsIII to AsV with O2 is very slow at neutral pH but is faster at highly alkaline or acidic solutions (Kolthoff, 1921). It is stable in water at 25C (Tallman and Shaik, 1980) with a life of 1 year at pH levels less than 9 (Eary and Schramke, 1990). The predominant abiotic oxidation mechanism of AsIII is by manganese minerals (MnIII and MnIV) (Oscarson et al 1983; Brannon and Patrick, 1987). The MnIII oxide has been shown to oxidize AsIII and adsorb both AsIII and AsV species (Chiu and Hering, 2000) whereas Mn IV oxides adsorb AsV (Tani et al., 2004). There are two types of AsIII oxidizers, the chemoautotrophic arsenic oxidizers (CAO) and the heterotrophic arsenic oxidizers (HAO). The chemoautotrophs obtain energy by the oxidation of electron donating groups, using CO2 as C source whereas heterotrophs require organic carbon as C source (Kulp et al., 2004). Several bacterial strains are known to oxidize AsIII to AsV using respiratory and nonrespiratory enzymatic systems (Oremland and Stolz, 2003). An AsIII oxidase enzyme on the outer

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21 surface of the cytoplasmic membrane in these bacteria is responsible for this oxidation (Ilyaletdinov and Abdrashitova, 1981). For example, the AsIII oxidase enzyme of the bacteria Alcaligenes faecalis is a Femolybdenum complex with azurin and cytochrome c, which acts as an electron acceptor from AsI II for AsIII detoxification (Figure 13). The molybdenum centre contains an oxygen atom which results in the oxidation of AsIII to AsV. The electrons released then pass through an electron transport chain to the FeS complex in the enzyme and finally to t he electron acceptor azurin or cytochrome c (Ellis et al., 2001). Microbially mediated AsIII oxidation substantially reduces the half life of AsIII to 1.8 hr. (Philips and Taylor, 1976). Figure 13. Hypothetical model of an arsenite oxidase enzyme (Ander son et al., 1992; Ellis et al., 2001) Considering a real scenario, As that is present in the reduced form in minerals as AsS, As2S3 and FeAsS are attacked by CAOs resulting in the oxidation of AsIII and iron and sulfide. The construction of wells accelerates this process in the presence of oxygen or fertilizers like nitrates. The AsV is then adsorbed on oxidized mineral

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22 surfaces like ferrihydrite or alumina. DARPs respire the adsorbed AsV, resulting in the release of AsIII into aquifers (Oremland and St olz, 2003) This leads to high arsenic concentrations in drinking water in areas like West Bengal Ganges river delta regions where millions of people are exposed to ground water As contamination (Madhavan and Subramanian, 2006). Research during the last 19 years has shown that nearly 569,749 square km in Bangladesh and eastern India with a population of about 500 million are at risk of arsenic poisoning (Chakraborti et al 2001). Other countries affected include China, Mongolia, Nepal, Afghanistan, Pakistan, Argentina, Chile and several parts of the United States and Europe. Applications of Arsenic The wide uses of arsenic are mainly based on its toxicity. Calcium and lead arsenates are used as herbicides and insecticides (Abernathy, 1983). Application of P fertilizers to soils previously contaminated with lead arsenate has resulted in the release of arsenic to shallow groundwater (Peryea, 1991). Arsenic is also used as a feed additive for poultry as roxarsone for increased growth rates due to its action against intestinal parasites. It is also of medicinal value. Salvarsan or salvation by arsenic was used for the treatment of syphilis phenylarsenic acid for trypanosomal infections, and As2O3 for treatmen t of leukemia (Zhu et al., 1999; Jones, 2007). Other applications include its use in circuits and semiconductors, transistors, bullets, fireworks, paper, pesticides, pigments and metal adhesives (Ishiguro, 1992). A major portion of arsenic currently used in the United States (~ 96%) is imported f rom China as As2O3, and the remaining as As metal. Arsenic was used in the United States mainly for agriculture and further for pressure treating lumber with chromate copper arsenate. Regulations by the EPA on the use of arsenic in agriculture and in

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23 residential areas have however significantly reduced the use of arsenic for land application or wood treatment (Jones, 2007). Toxicity and Health Effects Inorganic Arsenic The natural abundance of this metalloid and anthropogenic sources has made it ubiquitous and hence a threat to the biosphere. I n a 1984 health assessment, the U S EPA classified arsenic as a class A human carcinogen, and Smith et al (1992) showed arsenic as a prominent source of cancer mortality in the world. Due to the devastating impact of ars enic contamination in the environment, the U S EPA and the WHO in 2006, adopted a new permissible limit for arsenic in drinking water at 10 g L1, replacing the old standard of 50 g L1. Arsenite is more mobile and more toxic to biota and plants than ar senate (Korte and Fernando, 1991) and the more toxic arsine gas is produced under highly reduced conditions ( Buchet and Lauwerys, 1981). The major route of arsenic poisoning is by ingestion via drinking water and food or inhalation. Inhalation exposure can be from the smelters or mining activities Arsenic forms stable bonds with S and C in organic compounds and can react with sulfhydryl groups of cysteine in proteins and result in enzyme inactivation. Arsenite retained in the body has the capacity to inact ivate sulfhydryl groups (thiols), consequently increasing the reactive oxygen species (ROS). Glutathione reductase is an enzyme that protects the cells from these oxidants that can affect DNA replication and repair The presence of ROS results in cell damage due to the inhibition of glutathione reductase and can result in cancer (Cuzick et al., 1992). Most of the symptoms are related to the skin due to its high keratin content which contains several sulfhydryl groups to which AsIII binds

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24 (Styblo et al., 1996). Arsenate does not react with sulfhydryl groups but since it is an analog to phosphate it substitutes phosphates in ATP synthesis and cell function. Other health hazards related to arsenic include the organs th at directly function with arsenic excretion such as the gastrointestinal tract, the circulatory system, liver, kidney and skin (Hughes, 2002). Symptoms include hyperpigmentation, hyperkeratosis, loss of hair and skin cells, neuropathy and skin cancer. Org anic Arsenic Methylation acts as a detoxifying mechanism where the inorganic arsenic is converted to mono methylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) as the methylated forms are less reactive with tissues and are excreted from the body (Braman and Foreback, 1973). Here a methyl group from S adenosylmethionine is added to trivalent As for methylation (Stolz et al., 2006). Certain f ungi, yeast and bacteria can methylate arsenic to arsine gas which is a deadly form of arsenic that affects the nervous system. Marine organisms have higher arsenic content in their tissue compared to terrestrial organisms. Shrimps and lobsters are observed to have methyl arsine concentrations as high as 200 mg kg1 (Chapman, 1926). The methylated form of As in mammal s (Aposhian, 1997), fungi, and algae (Edmonds and Francesconi, 1993; Cullen and Reimer, 1989), undergoes further transformation by its incorporation into organic molecules and form arsenocholine, arsenobetaine or arsenosugars (Edmonds and Francesconi, 1993) Arsenic Remediation There are several methods to remediate arsenic from contaminated soils and water which include physical, chemical and biological techniques. Soil physical remediation methods include excavation, capping, soil solidification, soil fl ushing and

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25 vitrification whereas chemical methods include electrokinetics. Compared to soils, As is more problematic in surface and ground waters as it is easily ingested via drinking water. Arsenic remediation in water employs granular metal oxide adsorbent method, ion exchange or coagulationmicrofiltration methods. The granular metal oxide adsorbent method uses activated alumina, ironoxide coated sand and granular ferric hydroxide and works on the principle of adsorption. However, all the above methods are only effective if AsIII is first converted to AsV resulting in a negative charge for better adsorption (Clifford and Ghurye, 2002). This can be achieved by using oxidants such as chlorine, ozone or permanganate (Frank and Clifford, 1986). Ion exchange technology is the best available technology recommended by U S EPA (Ghurye et al., 1999). Here strong base anion exchange resins like polystyrene divinylbenzene polymers are used. In the remediation process of coagulation and microfiltration, As is adsorbed on hydrolyzing metal salts such as ferric chloride or alum (Brandhuber and Amy, 1998). Biological treatments in soil and water include microbial remediation and phytoremedi a tion. The microbial remediation of As involves the reduction and oxidation of arsenic and hence is not an effective detoxification mechanism. Here certain bacteria such as Pseudomonas arsenitoxidans can gain energy in the presence of AsIII by oxidizing AsIII and Sulfurospirillum arsenophilum and Sulfurospirillum barnessii can reduce AsV (Inskeep et al., 2002). Phytoremediation is the use of hyperaccumulator plants that can tolerate high concentration of contaminants in their tissue to remediate soil ad water Co nventional methods of arsenic remediation would cost billions of dollars in the U S (Salt et al.,

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26 1995) and hence less expensive methods of remediation are required for a long term remediation strategy. Phytoextraction of one acre of sandy loam soil to a depth of 50 cm will cost $60,000100,000 compared to the cost of excavation and storage at $400,000 (Salt et al., 1995) Another advantage is that the contaminants can be remediated in situ. There are different kinds of phytoremediation techniques based on the media that is to be remediated and on the required end result. Phytoextraction extracts metals from the soil into the harvestable parts of the plants. This depends on the rate of uptake and the biomass of the plant. Rhizofiltration requires plants that have rapidly growing roots to absorb and precipitate heavy metals from solution. Here surface sorption occurs by chelation, ion exchange and adsorption (Salt et al., 1995). A soil contaminated with metals results in erosion and further spread of the cont amination. To minimize this effect plants can be used to stabilize the soil and hence prevent further movement of the contaminants through a process called phytostabilization. The plants may also be able to convert the contaminants from a soluble oxidation state to an insoluble oxidation state. Heavy metal contaminants in soil exist in several forms such as free soluble metal ions, ions occupying exchange complexes, organically bound metals, precipitated or insoluble complexes and in silicate minerals. Phy toremediation can be efficiently used only when the metals are present in available form (Salt et al 1995). Chelating agents, pH, root exudates and microbes can influence the bioavailability of metals in soils (Harter, 1983; Uren, 1981; Blaylock and James, 1994). Hyperaccumulators can take up metals by using metal chelating molecules like siderophores with mugeneic and avenic acid (Kinnersley, 1993). Plant roots may have

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27 metal reductases that increase the metal availability or may have the capacity to ac idify the soil environment (Salt et al. 1995). The metals once taken up by the roots are either stored in the roots or translocated symplastically into the xylem vessels. Hence the symplastic transport is the rate limiting step in translocation of metals. Once the metals enter the plant system detoxification can occur either by chelation, compartmentalization or precipitation (Mathys, 1977; Krotz et al 1989; van Steveninck et al 1994). With the rising remediation expenses using traditional methods, phy toremediation is a promising method of remediating heavy metal contaminated soil and water. Improvements can be made in phytoremediation techniques by introducing genes that can enhance hyperaccumulation. Efforts should also be taken in following appropriate agronomic practices with addition of amendments which can improve phytoextraction. A total of 450 angiosperms have been identified so far as heavy metal hyperaccumulators of As, Cd, Co, Cu, Ni, Pb, Sb, Se, and Zn (Rascio and Navari Izzo, 2010).The hyperaccumulators should be capable of accumulating >10 mg g1 of Zn, >1 mg g1 As, Co, Cr, Cu,Ni, Pb, Sb, or Se and >0.1 mg g1 Cd in the aerial parts without phytotoxic damage (Verbruggen et al., 2009). Twenty five % of the hyperaccumulators belong to the family Brassicaceae and genera Thlaspi and Alyssum (Macnair et al., 1998). The major accumulators of arsenic are Pteris vittata, P. cretica, P. multifida, P. oshimensis, P. bh aspericaulis and P. fauriei (Wang et al., 2007) and Pityrogramma calomelanosis (Francesconi et al., 2002). Pteris vittata in Phytoremediation The discovery of the first arsenic hyperaccumulator fern Pteris vittata L. (Chinese brake fern) by Ma et al (2001) has made it possible for arsenic remediation. In a study

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28 by Natarajan et al. (2008), the fern was capable of reducing the arsenic concentration levels in water from 130 to 10 g L1 in 8 h. Life Cycle P vittata belongs to the phylum Pteridophyta. They are tetraploid vascular plants with haploid chromosome number n = 58 (Beri and Bir, 1993; Srivastava et al. 2007). Similar to other ferns it follows an alternation of generation between a tetraploid sporophyte generation and a diploid gametophyte generation (Xie et al., 2009). The sporophyte stage of the fern is well adapted to moist and shady environments. Fern leaves are called fronds and if the stems are underground they are referred to a s rhizomes. Emerging new fronds are called croziers or fiddleheads. When fertile fronds mature, repro ductive structures called sori are formed on the undersurface of the pinnae(divisions of the compound frond) (Bondada et al., 2006). A sorus contains clusters of sporangia, which contain tetraploid spore mother cells that divide by meiosis to produce diploid cells. These diploid cells are released from the pinnae of the sporophyte and germinate to form a prothallus. The prothallus houses both the antheridia, the male reproductive structures and the archaegonia, the female reproductive structures (Gumaelius et al., 2004). Upon fer tilization, the zygote divides rapidly to form an embryo and further division results in a sporophyte. Both the sporophyte and the gametophyte are capable of As hyperaccumulation (Ma et al., 2001 and Gumaelius et al., 2004). Physiology In a normal plant heavy metal phytotoxicity may result in changes in the physiological processes at cellular or molecular levels as a result of inactivation of enzymes, blocking functional groups and disrupting membrane integrity (Rascio and

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29 Navari Izzo, 2010). A hyperaccu mulating plant may be able to overcome these effects and accumulate large concentrations of metals in its tissue. The arsenic hyperaccumulating fern P. vittata is capable of accumulating up to 2.3% As in the fronds (Ma et al., 2001). Studies indicate that once As is taken up by P. vittata As is translocated to the shoots (Cao et al., 2004), sequestered in the pinnae (96% of total As) and are stored in the vacuoles of the upper and lower epidermis, as revealed by EDXA analyses (Lombi, et al. 2002). Studies also show that As may be sent to tiny hairs that guard the reproductive cells near the edge of the fronds (Pickering et al., 2006). The hyperaccumulator P vittata can take up 4.8 to 5.6 times higher arsenic than the non hyperaccumulator Pteris ensiformis Once taken up, P. vittata is highly efficient in translocating As from the roots, rhizomes and the fronds compared to P. ensiformis which was ineffective in reducing AsV to AsIII (Singh and Ma, 2006). In a study where excised P. vittata tissue was expos ed to As, AsIII predominated in the fronds and AsV in the roots (Tu et al., 2004b). These ferns were also found to accumulate As in the tissue when sprayed with AsV and AsIII on the pinnae surface (Bondada et al., 2004). This indicates that the fern can readily take up As through specified transporters which may be different for AsV and AsIII. Compared to the fronds lower concentrations of As are stored in the roots. Root exudates released by P. vittata may act as chelators which enhance metal uptake, tr anslocation and resistance. The dissolved organic carbon released P. vittata roots may change the rhizosphere pH which might result in an increase in As uptake (Gonzaga et al., 2006) Arsenic Uptake Arsenate is an analogue of inorganic P and hence its transport into cells is assumed to be via a P transporter. Bacteria with a defective P uptake system exhibited

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30 increased tolerance to AsV (Willisky and Malamy, 1980). AsV uptake in carcinoma cells inhibited P uptake, indicating a common transport system (Huang and Lee, 1996). AsIII on the other hand, is neutral and hence its uptake is mediated by aquaporins which allow the passage of neutral solutes. The main function of aquaporins is in osmoregulation by transport of water molecules and neutral solutes. The pores of the channels are believed to be narrow so that water molecules move through a single file (Maurel and Chrispeels, 2001). A glycerol transporter or aquaglyceroporin, GlpF, mediates SbIII uptake in E. coli as a mutation in the gene resulted in increased tolerance (Sanders et al 1997). Saccharomyces cerevisae Fps1 gene mediates the uptake and efflux of glycerol from yeast cells (Sutherland et al., 1997) and it is homologous to the E. coli GlpF gene. Disruption of this Fps1 gene confers AsIII and SbIII tolerance. Aquaporins in E. coli (AQP Z) are water selective whereas aquaglyceroporins in E. coli (Glpf) can transport neutral solutes such as glycerol or urea (Gomes et al., 2009). Mercury a nd silver block the water channels by binding to a cysteine residue in the pore (Kuwahara et al.,1997). Microflora Associations P. vittata has unique characteristics of arsenic hyperaccumulation and it may also house a number of microorganisms on its phyll osphere and rhizosphere, that may aid in the transformation or hyperaccumulation of arsenic. Microbes have a highly resistant system against AsIII and AsV and both AsV reducing and AsIII oxidizing system exists in microbes for the detoxification of As. An arsenic resistant proteobacterium was isolated from the fronds of P. vittata grown in an As contaminated site (Rathinasabapathi et al., 2006). The bacterium was resistant to AsIII AsV and antimony. The plant roots and microbes result in a combined release of carbon in the

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31 form of sugars, organic acids and amino acids. Several AsV resistant bacteria were identified from the rhizosphere of P. vittata by Huang et al. (2010). These include Naxibacter sp., Mesorhizobium sp., Methylobacterium sp., Enterobacter s p and etc. Insect Deterrence There may be several hypotheses why a plant would hyperaccumulate high concentrations of arsenic. The phenomenon of hyperaccumulation could have developed over a period of time during evolution in order to evade certain herbi vores prevalent in the environment. There are studies which do and do not indicate a positive correlation between metal accumulation and herbivore deterrence. Laboratory trials indicat ing insect deterrence may not be seen in a field situation (Noret et al. 2007). Deterrence also depends on the mode of feeding in herbivores (Jhee et al., 2005). It is hence important to understand the herbivore defense mechanism in P vittata using different test herbivores and pathogens in lab and field conditions and also monitor the synthesis of organic constituents in the presence of As.

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32 CHAPTER 3 ARSENIC TRANSFORMATION IN THE GROWTH MEDIA OF P VITTATA THE ROLE OF MICROBES OR ROOT ENZYMES Arsenic Transformations Of the two predominant inorganic forms of arsenic, AsV and AsIII, AsIII is more toxic (Smith et al., 1992). This makes AsIII a greater environmental concern than AsV in terms of its environmental occurrence and transformation pathways. Both AsV and AsIII can complex with iron oxides like goethite by forming bidendate, binuclear complex by complexing with two adjacent iron octahedral corner sites (Manning et al., 1998; Parfitt et al.,1975). However, AsV has a strong affinity for other metal hydroxides like aluminum and clay minerals (Goldberg, 1986). Since A sIII exists as a neutral species at pH < 9.2, it is less strongly adsorbed onto minerals in aquifers and soils making it more mobile, bioavailable and hence more toxic than AsV (Hering, 1996; Bhattacharya et al., 2004). This difference in their environment al behavior and toxicity makes it important to understand arsenic speciation and transformation in the environment. The presence of plants in a soil system with different forms of As may have an effect on its speciation. P vittata has an extensive fibrou s root system. The roots release exudates which can form a carbon source and house a number of microbial communities (Al Agely et al., 2005). In nature, several AsIII oxidizing and AsV reducing bacteria have been identified from different sources, which c ontrol arsenic transformation in the rhizosphere (Leblanc et al., 1995; Blum et al., 1998; Stolz et al., 1999). Arsenate can be remediated by adsorption, ion exchange or coagulation based on the fact that it is charged. However, AsIII is a neutral species at normal waste water pH levels (H3AsO3) and is more difficult to remediate than AsV because of its low affinity for

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33 adsorbents. The AsIII has to be first converted to AsV by pretreatment before conventional remediation methods such as precipitation, ion exchange, lime softening or coagulation are carried out. This oxidation of AsIII requires oxidants such as ozone, H2O2, manganese oxides (Hug and Leupin, 2003; Driehaus et al., 1995) or TiO2 (Bissen et al., 2001). Due to the presence of a pretreatment proc ess in remediation, these techniques are often costly which requires further processing. This intermediate step of AsIII oxidation by chemical means can be avoided if existing phytoremediation methods can be used to oxidize AsIII in the media. The rhizosphere of P. vittata exude organic acids (Tu et al 2004a) and enzymes nourishing a number of microbial communities which may alter AsIII stability and hence oxidize AsIII. Arsenate is taken up via the P transporters in P. vittata (Wang et al., 2002). The transporters involved in AsIII uptake are unknown in P. vittata but research in a number of plants and microbes shows that AsIII is taken up via aquaglyceroporins (Meng et al., 2004; Isayenkov and Maathuis, 2008). Knowledge of arsenic speciation in the media is hence important as it controls the arsenic uptake mechanism by P. vittata (Mathews et al., 2010). The objective of this study was to understand the dynamics of arsenic transformation in the growth media and P. vittata tissues with specific objectives (1) to determine arsenic transformation in the growth media in the presence or absence of P. vittata and (2) to examine the role of microbes and plants in AsIII oxidation in the growth media. Materials and Methods Experimental Setup Mature P. vittata wit h spores on the underside of the fronds were collected and placed in plastic containers for a week. The spores that settled at the bottom of the

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34 containers were then spread on germination soil mix (Jungle growth mix) or sand: vermiculite mix (1:1) and sprayed with 0.2x Hoaglands solution (HS) daily to maintain the moisture of the germination mix After 4 7 d ays green algae like growth was observed followed by the gametophytic stage. In about 23 weeks the sporophytes develop by the fertilization of the ma le and the female gametes in the gametophytic stage (Figure 31). The sporophytes were then transplanted to potting mix in separate pots and watered with 0.2x HS for 34 months until ready for use. Figure 31. One month old P. vittata sporophytes Arsenic Speciation under Natural Conditions Ferns 4 months of age with 1518 cm frond length were acclimatized in HS at 0.2strength with pH adjusted to 5.7 with 1 mM KOH MES buffer for 2 weeks. They were maintained under constant aeration with a 12 h photoperiod and a photon flux of 350 mol m2 s1 using cool and warm white fluorescent lamps. The temperature was maintained at 23 28C and relative humidity at 70%. After two weeks of acclimation, the ferns were transferred to opaque containers containing 1 liter of test solution spiked with AsV (Na2HAsO4 .7H2O) or AsIII (Na2AsO3) (Sigma, St. Louis, MO). The arsenic was

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35 provided in deionized (DI) water in all experiments to minimize P competition for AsV unless otherwise indicated. All experiments were performed in triplicates. In the first experiment, AsIII oxidation in the growth media was determined in solution containing 0.10 mM AsIII with and without P. vittata Both plant and solution samples were tak en after 24 h. In the second experiment, AsIII oxidation and AsV reduction were determined in the growth media containing 0.27 mM AsIII or AsV with or without P. vittata Aqueous solution samples were taken at intervals of 1 h, and 1, 2, 4 and 8 d and plant samples were taken after 8 d. Arsenic Speciation under Sterile Conditions Efforts were made to sterilize 4 month old P. vittata from microbes. These ferns were previously raised in potting mix (Jungle growth mix, ) The intact P. vittata fronds and root s were washed thoroughly in tap water and then rinsed in autoclaved water three times. These were then dipped in 10% bleach for 7 minutes and then immersed in autoclaved cooled water. The ferns were then placed in Murashige and Skoog media (Macro+ Micron utrients) with 2% (w/v) sucrose and 0.8% agar. In another trial the ferns were washed in tap water and rinsed in autoclaved water five times. Here, the ferns were dipped in 10% bleach for 10 minutes. The ferns survived in both intact plant sterilization tr ials. The first set with 7 minutes of 10% bleach treatment indicated immediate microbial contamination in the media. In the second trial with 10% bleach treatment for 10 minutes, white cloudy liquid was observed in the agar medium after 2 d indicating micr obial contamination. As a result of the failed sterilization of the intact ferns the ferns were raised under sterile conditions from the spore stage. Surface sterilization of spores was done using 10% bleach and 0.5% Tween 20 for 4 min and followed by four washes with sterile

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36 water. The spores were then germinated in autoclaved magenta boxes with sterile MS media and 2% (w/v) sucrose and 0.8% (w/v) agar at pH 5.7 in sterile G7 boxes. The gametophytes were formed in 2 weeks and they were subsequently subcultured into fresh media every 2 weeks. After two months, the ferns of 5 7 cm in size were placed in autoclaved containers with the roots inside 5 mL of 0.10 mM sterile AsIII solution. They were then placed in sealed autoclaved G7 boxes for 1 d and 14 d under aseptic conditions. A separate set of containers containing 5 mL of 0.10 mM AsIII without P. vittata was placed in the G7 box as a control to understand the speciation of AsIII after 14 d in the absence of the fern. The growth media and P. vittata ti ssue were sampled for arsenic speciation. A B Figure 32. Propagation of P. vittata under sterile conditions ( A ) germinated spores a nd ( B ) P. vittata sporophyte Arsenic Speciation in Sonicate Extract Arsenic transformation in the media can be due the presence of specific enzymes in the root s or due to the effect of microbial activity or both. For this, a study was done where the root extracts were tested with As to understand the speciation of AsIII Here

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37 0.1 g of root samples of P. vittata wer e cut to 1 cm and sonicated in 10 mL DI water for 2 h. The sonicate was either used as such or boiled at 100C for 10 minutes to inhibit m filter to remove microbes. Following this 5 mL of the sonicate was used to mak e a final concentration of 1.33 M AsIII and 1.33 M AsV mixture of 100 mL. The media was analyzed after 1h, 1d, 4d and 8d for arsenic speciation. Arsenic Analysis The growth media samples were analyzed immediately for arsenic speciation. The media was diluted and speciated for AsV and AsIII directly using an arsenic speciation cartridge (Waters SPE cartridge), which retains arsenate (Meng et al., 2001). For fern arsenic analysis, the ferns were harvested and separated into fronds and roots. P. vittata ro ots were placed under running distilled water, rinsed with ice cold phosphate buffer (1 mM Na2HPO4, 10 mM MES and 0.5 mM Ca (NO3)2, pH 5.7) and washed once again with distilled water to remove arsenic adsorbed on the root surface. The ferns were then flashfrozen in liquid nitrogen and stored at 80C. For arsenic speciation, the samples were ground using liquid nitrogen. 0.1 g of this fern sample was extracted with methanol:water (1:1 v/v) under sonification for 2 h (Zhang et al., 2002). By this method bot h total As and AsIII will be obtained and the difference between them gives AsV. Since AsV and AsIII are predominant in the growth media (100%) and in P. vittata biomass (>95%), other arsenic species were not considered in this study. For total arsenic analyses a part of the sample was ovendried (65C for 2 d). The air dried plant tissue was ground (20mesh), digested with concentrated HNO3 (1:1, v/v), and followed by 30% H2O2 for arsenic determination (U S EPA, 1983; method 3050). Arsenic in the growth media and digested plant tissues were determined by a

PAGE 38

38 graphite furnace atomic absorption spectrophotometer (GFAAS; Varian 240Z, Walnut Creek, CA). In addition, standard reference materials from the National Institute of Science and Technology (Gaithersburg, MD) and appropriate reagent blanks, internal standards and spikes were used to ensure method accuracy and precision, which was within 100 20% of the expected quality control checks. Data Analysis The treatment effects were determined by analysis of variance according to the linear model procedure of the Statistical Analysis System (Freund et al., 1986). Treatment means were separated by Duncans multiple range tests using a level of significance of p < 0.05. Results and Discussion Arsenic Speciation under Natural Conditions Media arsenic speciation The impact of fern roots and associated microbes on As transformation in the growth media were examined. The tissue As concentrations were also analyzed to understand the difference in uptake in the presence of AsV or AsIII in the media. Arsenite was stable in the solution at 1 d (Figure 33 A) in the absence of P. vittata It has been shown that abiotic AsIII oxidation in water in the presence of atmospheric oxygen is slow at 25C (Frank and Dennis, 1986; Scott and Morgan, 1995), and this is supported by its long half life of 1 year in water (Eary and Schramke, 1990). However, in the presence of P. vittata roots in the solution, >67% AsIII in the media was oxidized to As V after 1 d (Figure 33 A). Arsenic tra nsformation in the growth media, with and without P. vittata was monitored for 8 d where P. vittata grew in solutions containing 0.27 mM AsIII or AsV.

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39 After 1 d of the experiment 18% of AsIII was oxidized to AsV (Figure 34 A). Compared to the experiment using 0.10 mM AsIII (Figure 33 A) which had 67% oxidized, the oxidation rate in the experiment using 0.27 mM AsIII was slower i e. 18% (Figure 34 A). Hence, it is indicative that there is a capacity of AsIII to be oxidized in the growth media, and this oxidation is concentration dependent. The data also suggests that for most hydroponic experiments where AsIII is supplied to P. vittata AsIII oxidation occurs within 1 d. In the case of AsV, no reduction to AsIII was observed with or without P. vittata even after 8 d (Figure 35 A). This indicates that AsV is stable under natural oxidized conditions and also due to the constant aeration used in the experiment. From this experiment it is clear that P. vittata is critical for AsIII oxidation in the growth medi a since no oxidation occurred in its absence. Biomass arsenic speciation The speciation of As in the media may or may not have an effect on As speciation in the biomass. It is known that AsV dominates in the roots of P. vittata whereas AsIII dominates in t he fronds (Wang et al., 2002; Singh and Ma, 2006). When P. vittata was treated with 0.1 mM AsIII, after 1 d, 60% of As in the roots were AsV whereas up to 90% in the frond was AsIII (Figure 3 3 B) Here 70% of AsIII was oxidized to AsV in the media (Figure 3 3 A). To better understand the dynamics of arsenic transformation in the biomass of P. vittata root and frond samples were collected for arsenic speciation over an 8d period where P. vittata grew in 0.27 mM AsIII or AsV solution. Arsenic concentrations in the roots and fronds increased with increasing exposure time from 1 h to 8 d (Figures 3 4 and 35). Since no P was supplied in the growth media, one can compare the uptake rate of AsIII and AsV by P. vittata without the competitive effect of P on AsV. However,

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40 there were no significant differences in the uptake rates between AsIII and AsV by P. vittata from 1h to 8d (p < 0.05). Since AsIII and AsV were stable during 1 h of exposure in the growth media (Figures 3 4A and 35 A ), those data are more compell ing. After 1 h of exposure, the arsenic concentrations in the roots and fronds were 7 and 12 mg kg1 for the AsIII treatment (Figures 3 4B and 34 C) compared to 5 and 17 mg kg1 for the AsV treatment (Figures 3 5B and 35 C). Similar data were obtained after 8 d of exposure, where the arsenic concentrations in the roots and fronds were 23 and 73 mg kg1 respectively for AsIII treatment (Figures 3 4B and 34 C) compared to 37 and 58 mg kg1 for AsV treatment (Figure s 3 5B and 35 C). Though arsenic species in the growth media did not impact plant uptake rate by P. vittata they directly impacted arsenic species in the roots. For example, in the AsV treatment, all arsenic was present as AsV in the growth media (Figure 35 A), and also in the roots (Figure 35 B ). On the other hand, in the AsIII treatment, after 1 and 2 d exposure, 100% and 82% of arsenic was present as AsIII in the growth media (Figure 34 A), which resulted in 50% and 21% AsIII in the roots (Figure 34 B). After 4 d and 8 d of exposure, there was no AsIII detected in the growth media or in the roots (Figures 3 4 A and 3 4 B). Unlike the roots, AsIII dominated in the fronds (Figures 3 4C and 3 5 C). Though there was no detectable AsIII in the roots from either AsIII or AsV treatment after 1 h exposure (Figures 3 4B and 35 B), 75% and 47% AsIII were detected in the fronds (Figures 3 4C and 35 C). This is consistent with the literature that most arsenic is present as AsV in the roots and AsIII in the fronds in P. vittata (Singh and Ma, 2006). Based on the data, the following inferences were made (1) both AsIII

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41 Figure 33. As speciation when treated with 0.10 mM AsIII for 24 h A) the growth media and B) P. vittata tissue. 0 2 4 6 8 10 Control media (without fern) Media (with fern) Media As mg/L Total As As III 0 20 40 60 80 100 Fern roots Fern frondsTissue As mg/kg A B

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42 and AsV were translocated from the roots to the fronds as both AsIII and AsV were detected in the fronds; (2) AsIII was translocated more rapidly than AsV as relatively more AsIII was detected in the fronds treated with AsIII than AsV; and (3) the AsIII in the fronds came from two sources: translocation of AsIII from the roots and reduction of AsV in the fronds. The amounts of AsIII in the fronds were 60 89% for the AsIII treatment compared to 47 98% for the AsV treatment. Hence, the data may argue against the hypothesis that AsV is reduced to AsIII in the roots (Su et al., 2008). First of all, though no AsIII was detected in the roots in all AsV treatments (Figure 35 B), 47 98% of AsIII was det ected in the fronds (Figure 35 C). If AsV were reduced in the r oots, then at least some AsIII should be detected. Secondly, after 1 d and 2 d of exposure, 50% and 21% AsIII was detected in the roots of t he AsIII treatments (Figure 34 B) compared to no AsIII in the AsV treatments (Figure 35 B). Yet the increased AsIII concentrations in the roots in the AsIII treatment didnt translate to greater arsenic concentrations in the fronds (Figure 34 C). After 1 d and 2 d of exposure, arsenic concentrations in the fronds were 26 and 54 mg kg1 for the AsIII treatment compared to 25 and 33 mg kg1 for the AsV treatments (Figures 3 4C and 35 C). The data clearly showed that (1) P. vittata was able to take up both AsIII and AsV and translocated them from the roots to the fronds, and (2) regardless of arsenic species supplied, AsV dominated in the roots while AsIII dominated in the fronds. This would indicate the presence of an arsenite oxidizing enzyme in the roots and arsenate reducing enzyme in the fronds of the fern. The activity of microbes in the oxidation of As III is another possibility.

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43 Time Figure 34. Arsenic speciation when treated with 0.27 mM AsIII for 8 d. A) growth media, B) roots and C) fronds of P. vittata 0 20 40 60 80 100 No plant 1h 1 d 2 d 4d 8 d Media As mg/L As Total As III 0 20 40 60 80 100 1 h 1 d 2 d 4 d 8 d Root As mg/kg FW 0 20 40 60 80 100 1 h 1 d 2 d 4 d 8 d Frond As mg/kg FW A B C

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44 Time Figure 35 Arsenic speciation when treated with0.27 mM AsV for 8 d. A) growth media, B) roots and C) fronds of P. vittata 0 20 40 60 80 100 No plant 1 h 1 d 2 d 4 d 8 d Media As mg/L Total As 0 20 40 60 80 100 1 h 1 d 2 d 4 d 8 d Root As mg/kg FW 0 20 40 60 80 100 1 h 1 d 2 d 4 d 8 d Frond As mg/kg FW As Total As III B C A

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45 Though the data did not support the hypothesis that AsV reduction occurs primarily in the roots, they supported the hypothesis of faster translocation of AsIII than AsV from the roots to fronds. Except for 1 h data, arsenic translocation was more rapid with AsIII than AsV treatment. For example, the translocation factor, which is defined as the ratio of arsenic concentrations in the fronds to the roots, for AsIII treatment was 2.5 3.9 compared to 1.4 1.6 for the AsV treatment (Figures 3 4B C and 35 B C). To help determine the location of arsenic reduction in P. vittata arsenic speciation was conducted in the roots and fronds of P. vittata after growing in 0.10 mM AsIII for 1 d (Figure 3 1 B). Though only ~33% AsIII was present in the growth media (Figure 33 A), ~42% and 86% of AsIII were detected in t he roots and fronds (Figure 33 B), respectively. Since no difference was observed in the uptake rate of AsIII and AsV by P. vittata in the absence of P the data were consistent with the hypothesis that AsIII reduction occurred in the roots. This was possible since rhizomes were not separated from the roots in this experiment. Arsenic Speciation under Sterile Conditions It is unclear whether P. vittata or microbes are responsible for AsIII oxidation. Arsenite oxidizing bacteria may require specific carbon sources to oxidize AsIII. For example, a strain of AsIII oxidizing bacteria N 26 can grow heterotrophically in the presence of carbon sources like acetate, succinate, fumarate, pyruvate, malate, mannitol, sucrose, glucose, arabinose, fructose, trehalose, raffinose, maltose, xylose, galactose, lactate, salicin, glycerol, lactose and inositol but not on citrate, sorbitol or rhamnose (Ehlrich, 2002). Hence, P. vittata may have played three roles: (1) it provides the microbes with the C sources via root exudates; (2) it exudes enzymes to oxidize AsIII; and (3) its roots provide the source of microbes for AsIII oxidation. Though

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46 microbial oxidation of AsIII in both oxic and anoxic environment has been reported, no report is available on AsIII oxidation by plant roots (Oremland and Stolz, 2003; Kulp et al., 2008). To separate the effect of plant from microbes, arsenic speciation in the growth media was determined under sterile conditions. It was observed that no AsIII oxidation occurred with or without P. vittata after 14 d (Figure 36 A). It should be noted that the ferns were grown under aseptic conditions from the spore stage indicating no impact of microbes on its rhizosphere. Hence, microbes and not P. vittata were directly involved in AsIII oxidation in the growth media. However, under natural conditions, P. vittata may have facilitated microbially mediated AsIII oxidation by providing the microbes associated with the roots (Al Agely et al., 2005) and the required carbon sources via root exudates (Tu et al., 2004a). Here, the original source of microbes would be f rom the potting mixture or the soil where P. vittata grew before transferring to hydroponic system. Since AsIII was unstable in water and possibly in the soil solution in the presence of P. vittata efforts should be made to monitor arsenic species in the growth media while comparing AsIII and AsV uptake by P. vittata However, the data from the sterile experiment indicates that AsIII was stable and it clearly supports the hypothesis that AsIII was predominantly oxidized i n the roots including rhizomes. After growing P. vittata in the sterile media containing 0.10 mM AsIII for 1 d, no AsV was present in the media (Figure 36 A). However, 35% AsV was p resent in the roots (Figure 36 B). The result s suggested that once taken inside the roots, AsIII was oxidized to AsV in the roots. Similarly, 48% AsV was present in the roots after 14 d exposure though AsV was

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47 absent in the growth media (Figure 36 B). There was a higher concentration of AsIII in the roots in the sterile experiments as the rhizomes were too small to be separated from the roots. But the presence of 48% AsV clearly supports the hypothesis that AsIII was predominantly oxidized in the roots including rhizomes. Arsenic reduction predominantly occurs in the pinnae based on studies using excised P. vittata (Tu et al., 2004b). After exposing excised P. vittata to 0.67 mM AsV for 2 d, 86% and 24% AsIII was detected in the excised pinnae and roots including rhizomes. Hence, AsV reduction occurs in both the roots and pinnae and the pinnae have much more reducing power than the roots since the arsenic concentration in the excised pinnae is 15fold greater than that in the roots. The fact that 92% and 61% AsIII were detected in the pinnae and roots after exposing excised P. vitt ata to 0.67 mM AsIII for 2 d was consistent with the hypothesis that AsIII oxidation occur red in the roots (Tu et al., 2004b). This indicates more oxidation in the roots and more reduction in the pinnae regardless of whether AsIII or AsV wa s supplied to P. vittata Arsenite Oxidation in Sonicated Extract To further study the effect of microbial or root enzymatic activity on arsenic transformation, the sonicated extract of the roots were tested in a mixture of AsV and AsIII solution. The P. vittata root sonicated extract maintained at room temperature (RS) 1 h, 43% in 1d and 100% after 4d indicating that either the root extract or microbes have resulted in AsII I oxidation (Figure 37). Boiling the sonicate (RSB) to 100C for 10 minutes may have inhibited enzymatic activity in the AsIII and AsV mixture, which showed a decrease in oxidation by 16% 29% compared to the sonicated extract at room temperature for 1 h.

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48 Figure 36. As speciation when treated with 0.10 mM AsIII for 1 and 14 d under sterile conditions. A) growth media with and without P. vittata and B) P. vittata roots and fronds. 0 2 4 6 8 10 14 d sterile no plant 1 d sterile with plant 14 d sterile with plant Media As mg/L Total As As III 0 5 10 15 20 25 30 35 40 1 d root 1 d shoot 14 d root 14 d shoot Tissue As concentration ug/g A B

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49 However, filtration of the sonicate d extract with a 0.2 m filter indicated an oxidation similar to the sonicated extract that was boiled. At 8 d, the sonicated extract when boiled or filtered resulted in an oxidation of 533% of the AsIII after 8 d compared to 100% oxidation after 8 d when untreated root sonicated extract was used. Hence a decrease in microbial population by filter sterilization or boiling resulted in a decrease in oxidation of AsIII as enzymatic activity is not affected by filtration. This reduction in AsIII oxidation indicates a predominant microbial role in oxidizing AsIII. Future Research The results shed new insights into the dynamics of arsenic transformation in the growth media and P. vittata tissues. It was clearly demonstrated that AsIII oxidation occurred in the media and the roots of P. vittata This research has made use of the hydroponic conditions rich with microbes, sterile conditions and root extracts. The results of the study indicate the arsenite oxidation to be microbially mediated. This study will open the door to a number of research ideas such as the presence of a novel arsenite oxidizing bacteria in the rhizosphere of P. vittata the characterization of root exudates in P. vittata and identify the carbon source required for the growth of these b acteria and also to investigate the use of this fern in arsenite oxidation and remediation in water treatment plants. While arsenate reductase and cytosolic triosephosphate isomerase from the fronds were previously implicated in arsenate reduction (Ellis et al., 2006; Rathinasabapathi et al., 2006), others have reported arsenate reductase activities in protein extracts from the roots with rhizomes (Duan et al., 2005; Liu et al., 2009).

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50 Figure 37. As speciation in sonicated extract of P. vittata root s incubated for 8 d with 1.33 M AsV and 1.33 M AsIII (untreated (RS), boiled (RSB) and filtered (RSF)) A) 1 h trea tment B) 1 d treatment C) 4 d treatment D) 8 d treatment 0 50 100 150 200 250 No plant tissue RS 1h RSB 1h RSF 1h Media As ug/L As Total As III 0 50 100 150 200 250 RS 1d RSB 1d RSF 1d Media As mg/L 0 50 100 150 200 250 RS 4d RSB 4d RSF 4d Media As mg/L 0 50 100 150 200 250 RS 8d RSB 8d RSF 8d Media As mg/L A B C D

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51 CHAPTER 4 ARSENIC TRANSFORMATION AND SPECIATION IN PTERIS VITTATA BIOMASS AND XYLEM SAP Arsenic Speciation Under natural conditions plants growing in soils uncontaminated with arsenic have a low translocation factor of As less than 0.1 (Cullen and Reimer, 1998). Unlike hyperaccumulators these plants, which are called excluders, have restricted uptake and restricted translocation of As (Zhao et al 2010). It is important to understand the speciation of arsenic in plants to understand its toxicity to herbivores at higher trophic levels (Zhao et al. 2010). Plants growing in As contaminated environments take up As and the species of As depends on plant species. Arsenic is predominantly present as A sIII in many plants (Dhankher et al., 2002). Unlike other plants, hy peraccumulators of As contain AsIII as uncomplexed species due to low phytochelatin content in tissue (Raab et al. 2004). It is believed that P. vittata takes up AsIII and AsV by the root s (Wang et al., 2002; Fayiga et al., 2005), translocates AsIII and AsV from the roots to fronds (Kertulis Tartar et al., 2005; Singh and Ma, 2006), reduces AsV to AsIII in the fronds (Bondada et al., 2004; Tu et al., 2004b), and transports AsIII into vacuoles for storage (Lombi et al., 2002). In the biomass of P. vittata inorganic arsenic species AsV and AsIII account for more than 95% of the total arsenic with minimal organic species (Zhang et al., 2002), therefore, these two species are considered in most arsenic speciation studies. To better understand the different parts in P. vittata and the localization of As in the fern tissue a schematic of the fern has been provided (Figure 41). The below ground biomass of this fern includes rhizomes (horizontal stems) and roots whereas their aboveground biomass consists of the fronds (leaves).Fronds are composed of a stipe

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52 (leaf petiole), rachis ( continuation of central stalk ) and pinnae (leaflet s) that are attached to the rachis Based on the fact that arsenic i s predominantly present as AsIII in excised pinnae exposed to 0.67 mM AsV (86% AsIII ) (Tu et al., 2004), and in pinnae supplied with foliar AsV at 1.3 mM (65 86% AsIII ) (Bondada et al., 2004), it is hypothesized that AsV reduction occurred primarily in the pinnae. Unlike pinnae, both AsIII oxidation and AsV reduction occur in the roots including rhizomes. After 1 d exposure, 61% of the arsenic is present as AsIII in excised roots exposed to 0.67 mM AsIII (71% AsIII in the growth media) and 76% as AsV in exc ised roots exposed to 0.67 mM AsV (97% AsV in the growth media) (Tu et al., 2004). Hence, it is hypothesized that AsIII oxidation occurs in the roots exposed to AsIII (71% AsIII in the growth media compared to 61% AsIII in the roots), and AsV reduction occurs in the roots exposed to AsV (97% AsV in the growth media compared to 76% AsV in the roots) (Kertulis Tartar et al., 2005). On the other hand, regardless of arsenic species supplied (0.5 M AsIII or AsV) AsIII dominates the xylem sap of P vittata Here, Su et al. (2008) hypothesized that AsV reduction mainly occurs in the roots, and the reduced AsIII is then rapidly translocated to the fronds. The data are consistent with Duan et al. (2005) who showed arsenic reductase activity in the roots but not in the fronds. However, in both experiments, they did not separate the roots from the rhizomes. Therefore, it is unclear whether AsV reduction occurs in the roots and/or rhizomes. The overall objective of this study was to understand the dynamic s of arsenic transformation in different parts of the fern and in the sap of P. vittata. The specific objectives were to investigate the location of arsenic oxidation and reduction in the sap and biomass of P. vittata The results from this study should shed light on the mechanisms of arsenic hyperaccumulation by P. vittata

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53 Materials and Methods Six month old P. vittata with 8 12 fronds (50 60 cm in height) were used. Larger ferns were chosen to ensure easier sap collection. After acclimation, the ferns gr ew in 0.2strength HS containing 0.10 mM AsV for 8 d. Sap was collected by 2 different methods. Young succulent fronds (cut 1 cm above the rhizomes) were used to collect xylem sap via a portable Scholander pressure chamber (PMS 1000, MPS Instrument Co., Co rvallis, OR). Also, sap oozing out of the cut where the frond was separated 1 cm above the rhizomes was collected using a pipette and considered sap from the rhizomes. A total of 50 L sap was obtained from each of the cut fronds. Separately, fresh fronds, rhizomes and roots from the same ferns, which were used for sap collection, were used for arsenic speciation. The pinnae, rachis stipe and the roots (without rhizomes) were divided into three sections, i.e., upper, middle and lower 1/3 based on their length (Figure 41) to understand the location of arsenic transformation. The rhizomes as a whole were used for As speciation. Results and Discussion Arsenic was supplied as AsV in the hydroponic media and there was a predominance of AsV in the roots. Based on the fact that AsV dominates the roots and AsIII dominates the fronds of P. vittata it i s hypothesized that AsV reduction occurs primarily in the fronds whereas AsIII oxidation occurs mainly in the roots. Su et al. (2008) conducted sap arsenic analysis and proposed that arsenic reduction occurs in the roots, and the reduced AsIII is then rapidly translocated to the fronds. However, in their experiment, they did not separate the rhizomes from the roots; therefore the sap they collected was actually from t he rhizomes.

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5 4 Xylem Sap Arsenic Speciation To better understand the location of arsenic transformation in P. vittata arsenic speciation was conducted in the sap collected from the fr onds and rhizomes after exposure to 0.10 mM AsV for 8 d (Figure 43 A). Sa p from the fern rhizome and the frond were used to understand the speciation of arsenic in the fern. The efforts to collect sap from the roots of large fern plants were unsuccessful. The arsenic concentration in the rhizomes sap was 8fold great er than that in the growth medium i.e., 61 compared to 7.5 mg L1 (Figure 43 A). This is consistent with the observation of Su et al. (2008) who reported 18 51 times greater arsenic concentration in the sap than the initial concentration of 5 M in the growth media. Though arsenic concentration in the rhizomes sap was much lower than that in the frond sap, i.e., 61 versus 650 mg L1, AsIII dominated both saps (Figure 4 3 A).This is consistent with the fact that when treated with AsIII or AsV most plants have a predom inance of AsIII in its xylem sap (Zhao et al., 2009). But this characteristic varies from plant to plant as rice loads AsIII into xylem more efficiently than wheat or barley (Su et al., 2008). This may indicate a highly developed AsIII reducing system in t he roots. The roots and shoots of rice indicate AsV reduction but since AsIII is present in the xylem sap, the root may be an important location for AsV reduction (Duan et al., 2007). However, in the case of the fern sap analysis performed just above the r hizome indicates a predominant AsIII which puts forth the question whether the rhizomes are key centers of AsV reduction and not the roots. Biomass Arsenic Speciation The roots were separated into lower, middle and upper third after separating from the r hizomes (Figure 41).Total arsenic concentrations decreased from 82, to 70, and to

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55 50 mg kg1 as it traveled from the lower to middle and to upper roots (Figure 43 B). However, regardless of the root location, AsV dominated the roots with 92 93% being AsV. In most experiments reported in the literature, the roots and rhizomes are not separated (Singh and Ma, 2006). This is partially because many people are unaware the presence of rhizomes and partially because, for young ferns, rhizomes are not easily separable from the roots. Rhizomes are actually underground stems from which the roots and fronds arise (Figure 41). They also transport water and nutrients up and down its length (Foster, 1984). However, more AsIII was present in the frond sap (86% AsIII) than the rhizome sap (71% AsIII), again suggesting further AsV reduction in the fronds. Arsenic speciation data in the rhi zome sap (71% AsIII; Figure 43 A) was consistent with that in the rhizom e tissue (68% AsIII; Figure 43 B). The data suggests that when arsenic was translocated from the roots (7% as AsIII) into the rhizomes (71% as AsIII), some of the AsV was reduced to As III in the rhizomes (Figure 43 B). After the mixture of arsenic (71% AsIII) was translocated from the rhizomes into the pinnae, some of the AsV was reduced to AsIII in the pinnae (>90% as AsIII) (Figure 43 B). Since rhizomes are underground stems for ferns, t hey may have similar function as the fronds (Figure 41) The hypothesis that arsenic reduction occurred in the pinnae is also supported by the arsenic speciation data in the rachis ( main leaf stalk with attached leaflets), which was separated into lower, middle and upper third (Figure 41). The lower portion of the rachis included the stipe ( petiole without leaflets), and fewer pinnaes compared to the middle and upper rachis. As arsenic moved from the lower to upper rachis, arsenic

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56 concentrations increased from 47 to 160 and to 244 mg kg1 with proportionally more being AsIII (23%, 71% and 72%). The data are consistent with those of Pickering et al. (2006) who reported 76% AsIII in the rachis based on Xray absorption spectroscopy analysis. The fact that much higher AsIII was observed in the upper rachis (71 72% AsIII where more pinnae are located) than the lower rachis (23% AsIII wit h much fewer pinnae) supports the hypothesis that arsenic reduction occurred in the pinnae. If arsenic were mainly reduced in the rhizomes and then translocated from the rachis to the pinnae, then one would not expect substantial changes in arsenic speciation along the rachis. Though pinnae were separated from the rachis, the arsenic concentrations and speciation in the rachis was probably affected by pinnae, which had much greater arsenic concentration (463 588 mg kg1) and was dominated by AsIII (90 100% ) (Figure 43). The fact that substantially more AsIII was present in pinnae (90 100%) than that in rachis (23 72%) is consistent with the hypothesis that arsenic reduction occurred in the pinnae. AsV reduction predominately happened in the rhizomes and the pinnae of P. vittata though limited AsV reduction also occurred in the roots. The results also open new questions into the tissue distribution of arsenic reducing and oxidizing enzymes in P. vittata Research Findings The results reported here are consi stent with the fact of the presence of arsenate reducing enzymes in the fronds and rhizomes but not in the roots. The presence of an arsenite oxidizing enzyme in the fern roots have to be studied using molecular techniques.

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57 Figure 41. Schematic of diff erent parts of P. vittata including root, rhizome, stipe, pinna, rachis, and frond. Modified from http://www.bioscripts.net/flora/plantas/FI/Pteris%20vittata.jpg Figure 42. Scholander pressure chamber

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58 Figure 4 3. Arsenic speciation in the sap and tissue of P. vittata treated with 0.1mM As V for 8 days. A) Sap from fronds and rhizomes, and B) in the pinnae (P), rachis (Ra), rhizomes (Rh), and roots (R) U, M and L = upper, middle and lower and the lower rachis includes the stipe with few pinnae (see Figure 41) 0 100 200 300 400 500 600 700 800 900 1000 Frond Rhizome Sap As mg/LA As Total As III 0 100 200 300 400 500 600 700 800 900 1000 PU PM PL RaU RaM RaL Rh RU RM RL Tissue As mg/kgB

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59 CHAPTER 5 UPTAKE AND TRANS LOCATION OF ARSENITE AND ARSENATE BY PTERIS VITTATA L.: EFFECTS OF GLYCEROL, ANTIMONITE AND SILVER Arsenic Uptake and Translocation The speciation of As depends on the environmental conditions a plant is in and this greatly affects the uptake rate and mechanism of As into the plant. The mechanisms of plant arsenic uptake depend on arsenic species as they are structurally and chemically different. Most plants take up AsV via phosphate transporters whereas they take up AsIII via aquaglyceroporin transport ers. Arsenate, with dissociation constants 2.2, 6.97 and 11.5 behave as oxyanions in solution, i.e., HAsO4 2and H2AsO4 at pH 57 (Jeon et al., 2009), which are similar to phosphate, HPO4 2and H2PO4 (Teo et al., 2009). As chemical analogs, they compete for entry through the membrane phosphate transport system. Moreover, AsV can repress genes involved in the phosphate starvation response suggesting that AsV may interfere with the phosphate signaling mechanism (Catarecha et al., 2007) and can be taken up by plants instead of phosphate. This competitive effect has been observed in E. coli (Willsky et al 1980) yeast (Yompakdee et al 1996), and plants including barley (Lee, 1988), wheat (Zhu, 2006), rice (Meharg, 2004), H lanatus (Meharg and Macnair, 1992), and Brassica napus (Quaghebeur and Rengel, 2005). Arabidopsis thaliana mutants defective in P transporters are more tolerant to AsV (Catarecha et al., 2007) Studies also indicate that AsV uptake into P. vittata is by the phosphate transporter (Wang e t al ., 2002). Arsenite (H3AsO3), on the other hand, is present as neutral species at pH <9.2 (ODay et al., 2006) and are transported via aquaglyceroporins in E.coli (Meng et al., 2004), yeast (Robert et al., 2001) and human cells (Liu et al., 2002). In plants a nodulin

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60 26like intrinsic proteins (NIPs) are the structural and functional equivalents of bacterial and mammalian aquaglyceroporin (Wallace et al., 2005). Hence a similar mechanism of AsIII uptake is expected in plants as well. In rice roots, Lsi1 (OsNIP 2;1) is a major route of entry for silicic acid (Ma et al., 2006) and AsIII (Ma et al., 2008) and mutation in this protein resulted in a 60% loss of AsIII upt ake. NIP channels in rice, Lsi2, allow bidirectional transport of AsIII as well as the efflux of As from the exodermis and endodermis cells towards the stele (Ma et al., 2008) and mutation to this gene resulted in a dramatic effect on AsIII loading into the xylem (Ma et al., 2007) This may be a prominent step in P. vittata as well, where efficient AsIII efflux systems pump As into the xylem. Several analogues of AsIII compete with it during uptake into living cells. Antimonite ( SbIII) is one such analogue, which is chemically and structurally similar to AsIII. The pKa values of SbIII and AsIII are 11.8 and 9.2, respectively, and hence both exist as neutral solutes [Sb(OH)3 and As(OH)3] in the environment (Meng et al., 2004) and may compete for plant uptake. Several proteins have been identified that transport both AsIII and SbIII and this includes AtNIP 5;1, AtNIP 6;1, AtNIP 7;1, OsNIP 3;2, OsNIP 2;1 and OsNIP 2;2 (Bienert et al., 2008). A similarity also exists among AsIII, SbIII and a specific conformation of glycerol, C3H5 (OH)3, where the 3 hydroxyl groups occupy nearly the same positions as As(OH)3 and Sb(OH)3 with similar charge distribution and volume. The Sb content in the earths crust is low and is elevated in Sb mining areas (Flynn et al., 2003). Though SbIII is not essential for organisms, it is taken up by living cells through the same channel as glycerol and AsIII. The glycerol facilitator of E coli

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61 (GlpF) takes up SbIII and a lack of this transporter in mutants makes them resistant to both SbIII and AsIII (Sanders et al., 1997). Similarly, the glycerol facilitator in yeast (fsp1) can take up both SbIII and AsIII (Wysocki et al., 2001). Mammalian aquaglyceroporins AQP7 and AQP9 also allow SbIII and AsIII to permeate (Liu et al., 2002). The prot eins that have been studied to transport both AsIII and glycerol in plants include AtNIP 1;1, AtNIP 1;2 and AtNIP 6;1 (Kamiya et al 2009 and Tanaka et al., 2008). The smaller diameter of As(OH)3 and Sb(OH)3 is an additional advantage for transit though t he narrowest region of the glycerol transporter channel (Bhattacharjee, 2008). Hence it can be predicted that a glycerol facilitator that transports glycerol may also be responsible for AsIII and SbIII uptake in P. vittata and the presence of SbIII or glyc erol in the substrate may inhibit AsIII uptake by the cells. The function of these aquaporins can be inhibited by chemicals that interfere with the permeation of water and neutral solutes. Some of the known aquaporin inhibitors include mercury, silver, gold, copper, phloretin, tetraethyl ammonium salts and acetazolamide compounds (Haddoub, 2009). Silver is a powerful inhibitor of aquaporins due to their interaction with sulfhydryl groups of cysteine near the conserved NPA motif, which blocks the constricti on regions of the channel. Silver inhibition is rapid and not reversible by mercaptoenthanol (Niemietz and Tyerman, 2002) In human red blood cells, the inhibition of aquaporins was 200 times more potent than mercury compounds (Shaafi, 1977). Silver nitrate has been tested on peribacteriod membranes of soybean nodules, plant plasma membrane vesicles and human red blood cells Compared to the widely used mercurial compounds for aquaporin inhibition, which is toxic to plants (Patra and Sharma, 2000), silver nitrate is less phytotoxic and more specific.

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62 In the presence of transition metals like silver, the pKa of the thiol moiety is lowered from 8.5 to 6 which helps in its co ordination with transition metal ions (Scozzfava et al., 2001). Another amino acid that can interact at silver concentrations of greater than 200 M is histidine (Wells et al 1995). Using AsIII competito rs and aquaporin inhibitors serves as an important preliminary tool to understand the uptake mechanism of AsIII in P. vittata The objective of this paper was to understand if the aquaglyceroporin channels are responsible for AsIII uptake in P. vittata by investigating 1) the competitive effects of glycerol and SbIII, and 2) the inhibiting effect of aquaporin AgNO3 on AsIII and AsV uptake by P. vittata. Materials and Methods Experimental Setup P. vittata ferns, 4 months of age and 1518 cm in height, pur chased from Milestone Agriculture Inc. (Apopka FL, U S A ) were used for this study. Efforts were taken to ensure uniform plants were used for the study. The ferns were acclimatized in an aerated hydroponic system with 0.2 strength HS and pH adjusted to 5. 7 with 1 mM KOHMES [(2 (N morpholino) ethanesulphonic acid] buffer for 2 weeks. During the experiments photon flux of 350 molm2s1 was used using cool and warm white fluorescent lamps with temperature maintained at 2328C and relative humidity 6570%. After acclimatization in HS, the ferns were acclimatized in a solution of 0.5 mM MES (pH 5.7) and 0.5 mM CaCl2 for 1 day. Following this they were transferred to opaque containers containing 1 L of solutions spiked with 0.1 mM AsV (Na2HAsO4.7H2O) or AsIII (Na2AsO3) (Sigma, St. Louis, MO). The arsenic was provided in deionized water in all experiments to minimize P competition for AsV.

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63 Time Dependent Uptake Study An experiment was conducted to study the time dependent uptake of AsIII and AsV into the fern. P. vittata were grown in 1L solution of 0.1 mM AsIII or AsV for 1, 2, 4, 6 and 24 h. The root samples were analyzed to study the short term influx of AsIII into P. vittata The water samples were analyzed to understand AsIII stability in the media in the presence of the fern. The results of both experiments helped to decide the time required for the uptake competition and inhibitor experiments. Competition and Inhibition Study Glycerol [ C3H5 (OH)3] and SbIII (potassium antimonyl tartarate) at concentrations 0, 0.1, 1, 10, and 100 mM were used with 0.1 mM AsIII or AsV. The inhibition studies used silver nitrate (AgNO3) at concentrations 0, 0.001, 0.01 and 0.1 mM against 0.1 mM AsIII or AsV. For the inhibition study the ferns were first treated with AgNO3 for 1 h before addition of AsIII or AsV to ensure prior inhibition of the aquaporin. The concentrations of As, glycerol, SbIII and Ag used showed no phytotoxic effects on P. vittata All experiments were performed in triplicate and the duration of the study was 1h. This was to minimize conversion of AsIII to AsV by oxidation. Following the completion of the experiment and to further clarify the effect of silver nitrate on aquaporin inhibition and AsIII uptake, this experiment was repeated for 2 h using the most effective concentration of AgNO3. After the 1 h or 2 h treatment, the water samples were collected for arsenic speciation. The fern was washed with distilled w ater followed by rinsing in icecold phosphate buffer (1 mM Na2HPO4, 10 mM MES and 0.5 mM Ca(NO3)2, pH 5.7) to ensure As desorption from the root surface. Following this, the plants were again washed with distilled water. The plant parts were blotted dry, weighed and stored in

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64 80C for As speciation analysis. For arsenic speciation in the fern, the samples were ground using liquid nitrogen and extracted with methanol: water (1:1 v/v) under sonication for 2 h (Zhang et al 2002). AsV and AsIII were separated using an a r senic speciation cartridge ( Waters SPE cartridge), which retains arsenate (Meng et al 2001). For total arsenic, air dried fern tissue was ground (20mesh), digested with concentrated HNO3 (1:1, v/v), and followed by 30% H2O2 (U S EPA method 3050) Total As and Sb in the growth media and fern tissues were determined by a graphite furnace atomic absorption spectrophotometer (GFAAS; Varian 240Z, Walnut Creek, CA). In addition, s tandard reference materials from the N ational Institute of Science and Technology (Gaithersburg, MD) and appropriate reagent blanks, internal standards and spikes were used as quality checks and were within 100+ 20% of the expected values. Data Analysis The treatment effects were examined by analysis of variance based on the linear model procedure of the Statistical Analysis System (SAS Institute Inc., 1986) Results and Discussion A preliminary experiment on arsenic stability in the growth media indicates that In the presence of P. vittata A sIII was unstable in the growth media whereas AsV was stable (Figure 51 A). AsIII was stable for 1 h, after which it was gradually transformed to AsV. In comparison, AsV was stable beyond 24 h since it was maintained under an aerated oxidized system (Figur e 5 1 A). The influx of both AsIII and AsV into P. vittata roots was l inear for up to 8 h (Figure 51 B). To minimize AsIII oxidation in the media, 1 h was chosen for all the uptake experiments. During this period, the difference in AsIII and AsV uptake by P. vittata was minimal (Figure 51 B).

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65 Effects of Glycerol and SbIII on AsIII Uptake AsIII uptake in rice (Meharg and Jardine, 2003) and Arabidopsis (Kamiya et al., 2009) is via aquaporins which can transport water or neutral solutes. Aquaporins can either be water channel aquaporins or glycerol transporters (aquaglyceroporin) and are known to allow the passage of water, glycerol, carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and metalloids such as AsIII, SbIII, boric acid, and silicic acid. The major difference between the two is in the constriction regions of the channel. The narrow constriction in water specific aquaporins have a diameter of water molecule at 2.8 and aquaglyceroporins have a diameter of glycerol molecule at 3.4 (Beitz, 2004). The speciation analysis of the growth media samples in the glycerol and SbIII treatments indicated limited AsIII oxidation (210%) (Figures 5 2 A and 54A), indicating that most of the As taken up by the plant was AsIII. This is if there was no oxidati on of AsIII at the surface of the roots due the impact of microbial activity. Separate control experiments with 0.1 mM AsV treatment in the presence of different concentrations of glycerol (Figures 5 3B and 53 C) and SbIII (Figures 5 5B and 55 C) showed a similar trend as AsIII with no significant difference in plant As uptake. This was expected as AsV is taken up by the ferns through a phosphate transporter. Studies by Nagarajan and Ebbs (2007) where 0.1 mM of SbIII had no impact on accumulation of As by P vittata when treated with 0.1mM AsIII for 8 h indicates that the uptake of AsIII by the fern may be by a different mechanism compared to that of an SbIII transporter. The total arsenic concentrations in the fronds with the glycerol treatment (4.6 to 6.3 mg/kg As) were slightly greater than those in the SbIII treatment (4.4 to 5.7 mg/kg As).

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66 Figure 51 Arsenic speciation in the media and root of P. vittata exposed to 0.1 mM AsIII or AsV for 1, 2, 4, 6 and 24 h. A) Media i) As total and AsIII with AsIII treatment ii) As total with AsV treatment, and B) total As concentrations in the roots of P. vittata 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 0 1 2 4 6 24 Media As mg/LTime (h) As Total with As III treatment As III with As III treatment As Total with As V treatment A 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30Root As mg/kg FWHours As III treatment As V treatment B

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67 The root arsenic concentrations were 3.86.2 mg kg1 and 3.94.2 mg kg1 with the glycerol and SbIII treatments, respectively. The increased As uptake in the presence of glycerol may be due to the positive impact it has on microbes. Plant growth promoting rhizobacteria are known to enhance metal accumulation by enhancing plant growth or sequestering metal ions inside the cell walls (Khan, 2009). Glycerol in the media can act as a carbon source for the microbes promoting As accumulation by P. vittata On the other hand, SbIII is known to inhibit microbial activi ty in soil (An et al., 2009), causing reduced As uptake with SbIII compared to glycerol. The arsenic in the fronds was predominantly AsIII ranging from 78 to 96% in both glycerol and SbIII treatments whereas the roots contained almost all AsV (Figures 5 2 B and 53B). In addition to As, SbIII concentrations in P. vittata were analyzed. There was an increase in SbIII concentrations in the fronds and roots with an increase in SbIII concentration in the media (Figure 56).The fact that Sb concentrations in the roots (45 5,742 m g kg1) were much higher than those in the fronds (3.2 13 m g kg1) was observed by Muller et al., 2009. This indicates that P. vittata was an efficient accumulator of SbIII in the roots, but was ineffective in translocating SbIII from the roots to fronds during the 1 h experiment. For example, the highest Sb translocation factor (TF; ratio of Sb in the fronds to roots) in P. vittata was 0.07 (Figure 56). In comparison, the highest As TF in P. vittata treated with AsIII and SbIII was 2.4 (Figure 54) which indicates that, upon uptake, P. vittata translocated 71% As to the fronds and 7% Sb to the fronds. This shows that though AsIII and SbIII are analogs there were differences in

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68 their uptake and trans location in P. vittata It is possible that they were taken up through different aquaporin channels or transporters. Wang et al. (2010) had similar studies on AsIII uptake with competition experiments using analogs of AsIII, silicic acid and boric acid. The treatments had no impact o n the uptake of AsIII which agrees with this study. However, silicic acid [Si(OH)4] has a molecular diameter of 4.38 which is larger than that of As(OH)3 (4.11 ; Ma et al., 2008) and of the ar/R region of the aquaglyceropor in and hence sili cic acid may not be an effective competitor of AsIII in uptake studies. Also, the permeability for boric and silicic acid is not wide spread in all members of the NIPs (Maurel et al., 2008) whereas AsIII permeability is observed in several different subclasses of NIPs (Zhao et al., 2009) In the rice study a dose dependent reduction in AsIII uptake was observed when similar treatments using glycerol and SbIII were performed on excised roots indicating that AsIII was taken up by glycerol transporters (Meharg and Jardine, 2003). The fact that glycerol and SbIII had no effect on AsIII uptake may indicate a separate uptake pathway of AsIII in P. vittata Effects of Silver Nitrate on AsIII Uptake Silver, an aquaglyceroporin inhibitor, was used to understand the mechanisms of AsIII uptake by P. vittata Mercurial compounds are also inhibitors of aquaporin activity which are commonly used for uptake studies However at higher concentrations Hg may be phytotoxic to plants and a reduction in AsIII uptake may result from its phytoxic effect. Moreover, studies indicate that 0.01 mM Hg had no impact on AsIII uptake by P. vittata when treated with 0.015 mM AsIII for 2 d (Wang et al., 2010).

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69 Figure 52. Effect of glycerol on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h in the A) media, B) roots and C) fronds 0 2 4 6 8 10 0 0.1 1 10 100 Media As mg/L Total As As (III) 0 2 4 6 8 10 0 0.1 1 10 100 Root As mg/kg FW 0 2 4 6 8 10 0 0.1 1 10 100 Frond As mg/kg FW B C A Glycerol (mM)

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70 Figure 53. Effect of glycerol on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h A) media, B) roots and C) fronds 0 2 4 6 8 10 0 0.1 1 10 100Media As mg/L Total As As (III) 0 2 4 6 8 10 0 0.1 1 10 100 Root As mg/kg FW 0 2 4 6 8 10 0 0.1 1 10 100 Frond As mg/kg FWGlycerol (mM) A B C

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71 Figure 54. Effect of SbIII on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h A) media, B) roots and C) fronds 0 2 4 6 8 10 0 0.1 1 10 100 Media As mg/L As Total As III 0 2 4 6 8 10 0 0.1 1 10 100 Root As mg/kg FW 0 2 4 6 8 10 0 0.1 1 10 100 Frond As mg/kg FWSb III (mM) A B C

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72 Figure 55. Effect of SbIII on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h A) media, B) roots and C) fronds 0 2 4 6 8 10 0 0.1 1 10 100 Media As mg/L As Total 0 2 4 6 8 10 0 0.1 1 10 100 Root As mg/kg FW 0 2 4 6 8 10 0 0.1 1 10 100Frond Asmg/kg FW As Total As III Sb III (mM) B A C

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73 Figure 56. Concentration of Sb in P. vittata when treated with 0.1 mM AsIII and different concentrations of SbIII for 1 h. A) roots and B) fronds 0 1000 2000 3000 4000 5000 6000 7000 0 0.1 1 10 100 Root Sb mg/kg DW 0 2 4 6 8 10 12 14 16 18 20 0 0.1 1 10 100 Frond Sb mg/kg DW Sb III (mM) A B

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74 AgNO3 is a potential inhibitor of aquaporins with reduced phytotoxicity (Niemietz and Tyerman, 2002) and hence was used in this study. The function of the inhibitor can be further substantiated by the fact that the pore diameter of an aquaporin is 2.8 and the ionic radius of Ag+ is 2.5 Hence elements with ionic radii similar to silver may be potent inhibitors of aquaporins. The presence of 0.001 and 0.01 mM Ag had little effect on AsIII oxidation in the media, with 46% AsIII being oxidized after 1 h, which was comparable t o the control at 6% (Figure 57 A). However, at 0.1 mM Ag, the amount of oxidized AsIII increased to 13%. Unlike glycerol or SbIII, the presence of Ag significantly reduced As uptake by P. vittata (Figure 57). As the Ag concentrations increased from 0 to 0.001 and to 0.01 mM, the arsenic concentrations in the roots decreased from 3.8 to 2.5 and to 1.4 m g kg1, (Figure 57 B) and those in the fronds from 5.1 to 5.0 and to 3.0 m g kg1 ( Figure 5 7 C). The impact was most pronounced at 0.01 mM A g, with As reduction being 64% in the roots and 58% in the fronds. The fact that arsenic concentrations in the roots and fronds at 0.1 mM Ag were greater than those at 0.01 mM Ag may be attributable to the increased AsV concentration in the growth media ( 13% compared to 6%). To confirm the impact of Ag at 0.01 mM, the experiment was repeated for 2 h, which showed similar results (Figure 59). At 0.01 mM Ag, arsenic concentrations in the fronds and roots were reduced by 63 and 48% compared to the control (Figure 59 A). Similar to this study, a significant decrease in As accumulation was observed when P. vittata was treated with 10 and 100 M Ag + for 15 d (Nagarajan and Ebbs, 2007). Compared to AsIII uptake by P. vittata the impact of Ag on AsV uptake w as much less. Regardless of the Ag concentrations used, Ag had little impact on arsenic

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75 concentrations in the fronds, ranging from 4.9 to 5.2 m g kg1 (Figure 58 C). However, with Ag concentrations increasing from 0 to 0.001 to 0.01 mM, arsenic concentrations in the roots decreased from 3.8 to 3.5 and 2.7 m g kg1 (Figure 58 B). It seemed that Ag had some impacts on root arsenic concentrations, with the highest reduction at 0.01 mM Ag at 29%. When the time period of the experiment was increased from 1 h to 2 h, 0.01 mM Ag reduced As concentration in the roots by 26% (Figure 59 B). The difference in Ag impact on AsIII and AsV uptake by P. vittata indicates that AsIII uptake was different from AsV and it depended on a pathway, which was inhibited by AgNO3. This study confirms the fact that there was a decrease in AsIII uptake by P. vittata in the presence of AgNO3 but no effect in the presence of glycerol or SbIII. Thi s indicates that AsIII may be taken up by another transporter unique to the fern and the transporter is inhibited by Ag The transporter can be another NIP protein or a protein that has a completely different function compared to that of an aquaporin. Though it is proved that aquaporins or glycerol facilitators are responsible for AsIII uptake in E. coli, yeast or mammals certain other transporters have been recently tested to understand their role in AsIII uptake. A glucose transporter permease and hexose transporter in yeast has been shown to mediate AsIII uptake (Liu et al., 2004; Boles and Hollenberg, 1997). It is seen that in the presence of glucose, AsIII is taken up by the glycerol transporter Fps1p, and in the absence of glucose it is taken up by the glucose transporter. These transporters are analogs to mammalian GLUT permeases which are also know n to take up both AsIII and monomeythylarsenite, MMA (III) (Liu et al., 2006). This indicates that P. vittata may have a separate AsIII uptake pathway.

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76 As III Oxidation in the Media and P. vittata To minimize AsIII oxidation, 1 h was used in this experiment. Based on the preliminary data, little AsIII oxidation was observed in the growth media within 1 h (Figure 51A). However, limited AsIII oxidation occurr ed during the experiment. For example, in the glycerol experiment, 1.78.9% AsIII was oxidized. This means some of the As was taken up as AsV instead of AsIII and it had be to accounted for. In addition to the limited oxidation of AsIII in the growth media (Figure 52 A), AsIII can be oxidized to AsV on the root surface, which are rich in microbial population (Mathews et al., 2010). The oxidized AsV may be taken up by the plant immediately and would not contribute to AsV concentration in the media. This means though no AsV was detected in the media, some of the As was taken up as AsV by P. vittata which may follow the phosphate transporter pathway and therefore would not be inhibited by glycerol or SbIII. This hypothesis was supported by arsenic speciatio n in the roots. Though only 1.78.9% AsV was present in the growth media, almost all As was present as AsV in the roots in the glycerol (Figure 52B) and SbIII ( Figure 5 4 B) treatments. Part of the AsV may be taken up by phosphate transporters in P. vittata In addition, AsIIIoxidized AsV on the root surface has also contributed to AsV concentrations in the roots. In contrast, most of the As in the fronds was present as AsIII, ranging from 69 to 96% in the presence of AsIII and glyc erol (Figure 53 C) Even in the AsV and glycerol treatment, 81 to 85% of the As in the fronds w as present as AsIII (Figure 54 C). Similar data were observed in the AsIII SbIII and AsV SbIII treatment, with 72 to 98 % and 82 to 95% arsenic present as AsIII in the fronds (Figure s 5 4C and 55 C).

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77 Figure 57. Effect of AgNO3 on arsenic speciation in P. vittata when treated with 0.1 mM AsIII for 1 h A) media, B) roots and C) fronds. 0 2 4 6 8 10 0 0.001 0.01 0.1 Media As mg/L As total As III 0 2 4 6 8 10 0 0.001 0.01 0.1 Root As mg/kg FW 0 2 4 6 8 10 0 0.001 0.01 0.1 Frond As mg/kg FW Silver nitrate (mM) A B C

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78 Figure 58. Effect of AgNO3 on arsenic speciation in P. vittata when treated with 0.1 mM AsV for 1 h A) media, B) roots and C) fronds. 0 2 4 6 8 10 0 0.001 0.01 0.1 Media As mg/L As Total 0 2 4 6 8 10 0 0.001 0.01 0.1 Root As mg/kg FW 0 2 4 6 8 10 0 0.001 0.01 0.1 Frond As mg/kg FW As Total As III A C B Silver nitrate (mM)

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79 Figure 59. Effect of AgNO3 on arsenic speciation in the fronds and roots of P. vittata after exposure to 0.1 mM arsenic for 2 h A) AsIII and B) AsV 0 2 4 6 8 10 12 14 16 18 20 Control Frond With Ag Control Root With Ag As concentration mg/kg FW As III Total As 0 2 4 6 8 10 12 14 16 18 20 Control Frond With Ag Control Root With Ag As concentration mg/kg FW A B

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80 Research Findings Aquaglyceroporin competitors such as glycerol and SbIII showed no significant impact on AsIII uptake. However, silver ions had a negative effect on As III uptake which means the transporter of AsIII is different from that of a glycerol or SbIII transporter but is inhibited by silver. This indicates that P. vittata has a different mechanism for AsIII uptake, which needs to be further investigated.

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81 CHAPTER 6 ARSENIC REDUCED SCALEINSECT INFESTATION ON ARSENIC HYPERACCUMULATOR PTERIS VITTATA L. Insect Detterence There are several ways plants protect themselves from natural infestations by herbivores. These include constitutive defenses that w ith physical barriers on the plant surface and the production of allelochemicals to prevent the herbivore from completing its life cycle on the plant (Paiva, 2000). Induced defense mechanisms include both direct and indirect defenses. In direct defenses, plants use secondary metabolites that behave as insect toxins, digestibility reducers or anti nutrients (Baldwin and Preston, 1999), whereas in indirect defenses, plants release volatile compounds after herbivore attack. These volatiles or semio chemicals ( Law and Regnier, 1971) can attract natural enemies of the attacking insect or even induce defense responses in neighboring plants (Pare and Tumlinson, 1999). The above mentioned defense mechanisms demand energy and nutrient resources of a plant, which would otherwise have been used for its vegetative and reproductive development (van Dam and Baldwin, 1998). It might be to overcome this expensive defense mechanism that plant evolution has developed an unusual phenomenon of hyperaccumulation of heavy metals in certain species (Mathews et al., 2009). Hyperaccumulators refer to plants that can accumulate large amounts of elements or compounds such as metals in the aboveground biomass. Heavy metals accumulated in the biomass may influence the plant disease/infestation triangle, which includes pathogens/herbivores, plant susceptibility and a favorable environment (Poschenrieder et al., 2006). Boyd (2007) indicates that hyper accumulators may have

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82 an advantage against pathogens and herbivores in comparison to nonhyperaccumulators. Research has been on going worldwide to understand the ecological benefits and evolutionary basis of hyperaccumulation in relation to biotic stress management. Various hyperaccumulators have been studied to determine if they are better able to defend against infestation by insects and pathogens (Poschenrieder et al., 2006). For example, nickel hyperaccumulator ( Strepthanus polygaloides ) can defend against leaf moths, hoppers, root chewers and mites (Jhee et al., 2005; Jhee et al., 2006) ; zinc hyperaccumulator ( Thlaspi caerulescens ) shows deterrence against hoppers (Behmer et al., 2005), and selenium hyperaccumulator ( Brassica juncea) deters leaf chewer Pieris rapae (Hanson et al., 2003) and phloem feeder Myzus persicae (Hanson et al., 2004). Also, Galeas et al. (2008) found that, selenium hyperaccumulators have lower infestation rates by arthropod load than nonhyperaccumulators under field conditions. However, there are studies that report that certain insects may have developed toleranc e to heavy metals accumulated in plants. For example, Melanotrichus boydi and Plutella xylostella prefers to feed on the Ni hyperaccumulator, Streptanthus polygaloides (Wall et al., 2006) and on the Se hyperaccumulator S. pinnata (Freeman et al., 2006), r espectively. Rathinasabapathi et al., 2007, showed that As hyperaccumulation could deter herbivore damage when the leaf chewing insects, American grasshoppers ( Schistocerca americana), were fed arsenic treated ferns. Arsenic concentrations at 1 mM (sodium arsenate) deterred the grasshoppers from consuming the fronds but at 0.1 mM, arsenic had no deterrence effect. This study was the first to test arsenic -

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83 induced defense mechanism in an arsenic hyperaccumulator. The ability of insects to avoid feeding on the plants indicates that the herbivores are able to taste the difference in plants with or without metals. The experiment was supported by choice studies where lettuce was dipped in water and arsenic solution (1.0mM) and here a similar trend of deterrence was observed. The results were consistent with the hypothesis that arsenic in the tissue was sufficient to deter grasshoppers from feeding on the fern. F erns are very hardy and seldom have pest problems. Though the American grass hopper is polyphagous, it is unlikely that the American grasshopper is a major natural pest of the fern. The major pests of ferns include scales, hemi spherical scales and mealy bugs. Since no natural pest of this fern has been tested for elemental defense hypothesis to date, it is important to test the effect of arsenic hyperaccumulation by P. vittata on a natural infestation where the insects where naturally growing on the fern (Mathews et al., 2009). A study was done focusing on the effects of arsenic on infestation of P. vittata by Carribean black scale ( Saissetia neglecta). The scales are polyphagous, phloem sap sucking insects, 3 5 mm in size (De Lotto, 1969). They commonly inf est Florida citrus (Fasulo and Brooks, 2004), avocado (Pena, 2003), cassava (Pena and Waddill, 1984) and many other crop plants. Unlike the armored scales, these scales do not have a hard protective covering, and hence are called soft scales. The female sc ale insects move on a plant and lay eggs by parthenogenesis underneath the waxy covering. These eggs hatch over a period of 1 3 weeks. The newly hatched scales (crawlers) move around a plant until

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84 they locate succulent new growth where they insert their pi ercing sucking mouthparts into the plant to feed on the sap. Figure 61.Scale Insect: Saissetia neglecta. (Futch et al. 2001) The antennae and legs of adult female scales are reduced and they do not move often. The soft scale secretes a sticky fluid, and hence plants infested by these scales are seen to harbor ants as well. Unlike the armored scale, upon death these scales fall off the plant. Dead scales are normally dried up with no fluid in it (Fasulo and Brooks, 2004). Boyd (2007) elaborated the four steps necessary to study elemental defense of a plant again st herbivores. These include determining if natural enemy performance is reduced by increasing elemental concentrations in the plant, showing that high elemental concentration i n the plant is sufficient to cause the defensive ef fect in artificial media, comparing the fitness between high and low concentration plant attacked by a natural enemy and finally comparing the effectiveness of high and low concentration plant attacked by natural enemies under natural field conditions. The study here starts

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85 with the first approach with the objective to determine the effects of arsenic concentrations in P. vittata on its ability to deter infestation by scale insects. Materials and Methods Experiment Setup P. vittata of similar size were grown in a hydroponic system. They were four month old after transplanting, with 4 5 fronds. The nutrients were supplied as 0.2strength HS with aeration. The plants were grown under a 12h photoperiod with a photon flux of 350 mol m2 s1 using cool and warm white fluorescent lamps with temperature maintained at 25 C and 70% relative humidity. The experiment was performed in triplicates. The ferns were naturally infested with the Caribbean black scale 1 month after transplanting into a hydroponic system. The scales were allowed to multiply on the ferns for 3 months to allow for uniform infestation. A tray covered with a white paper was placed under each plant. The plants were spatially isolated to prevent t he spread of the insects from one plant to the other. Scale counts, visible to the eyes, were taken before the experiment. Arsenic Treatment and Scale Counting Four months after transplanting, the scaleinfested ferns were exposed to 0, 5, 15, and 30 mg kg 1 arsenic as sodium arsenate in the hydroponic solution. The total numbers of scales at the start of the experiment for each treatment were 170 9, 115 21, 252 77, and 195 17, respectively. Following arsenic exposure, the number of fallen scales (presumably dead) was counted on a daily basis for 7 d until no more increase in fallenscales was observed.

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86 Chemical and Data Analysis At the end of the experiment, the arsenic con tent in the fallen and intact scales as well as the aboveground biomass of P. vittata was analyzed. The fronds as well as the insects were dried and ground to 20 mesh size and digested with concentrated HNO3 (1:1, v/v), followed by 30% H2O2 for As determination (U S EPA, 1983, method 3050). The As concentration was determined by a graphite furnace atomic absorption spectrophotometer (Varian 240Z, Walnut Creek, CA). Standard reference materials from the National Institute of Science and Technology (Gaithers burg, MD) were used to assess method accuracy and precision (within 100 20%). The arsenic effects were determined by analysis of variance according to the linear model procedure of the Statistical Analysis System (SAS Institute Inc. 1986). Treatment means were separated by Duncans multiple range tests using a level of significance of p < 0.05. Results and Discussion During the 3month infestation by scales, the Chinese brake ferns could withstand the infestation. The underside and base of the leaflets and rachises were most infested by the Caribbean black scales. Ants were also seen in all plants infested with the scales, which is common in the presence of the sweet secretions of scale insects. Arsenic Toxicity in Scales During the 7d arsenic exposure, the number of fallenscales was counted on a daily basis (Figure 61). Arsenic accumulation in the ferns significantly impacted the scale population at high As treatment of 15 and 30 mg L 1. For the control treatment, the number of fallenscales was limited, ranging from 2 to 4 per day. A s imilar trend was observed for the ferns exposed to 5 mg L 1 As, indicating that arsenic in the plant had

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87 little impact on the scales. However, for ferns exposed to 15 30 mg kg 1 As, the impact on the scales was apparent on day 1. Approximately 16 18 fallenscales were observed compared to 5 for the control. There were more fallenscales on day 2 and 3, ranging from 25 to 50 per day. The number of fallenscales was reduced to less than 10 per day after day 5. Pteris vittata i s able to take up arsenic within an hour of exposure and hence would have a significant concentr ation of As by day one (Mathews et al., 2010). After exposing to 10 and 20 mg L 1 As for 1 d in a hydroponic system, the arsenic concentrations in the fronds of P. vittata were 56 and 84 mg kg 1 (Singh and Ma, 2006). In a different hydroponic experiment, the arsenic in the fronds of P. vittata was 165 mg kg 1 after exposing to 7.5 mg L 1 As for 2 d. Hence, the As concentration in the fronds in this experiment would be expected at 56 84 mg L 1 and >165 mg kg 1 after exposing to 15 mg L 1 As for 1 and 2 d. If this is the case, then arsenic concentration >50 mg L 1 in the fronds was toxic to scales and the effect was observed after 1 d of arsenic exposure (Figure 62 ). Arsenic Concentration and Scale Death The total number of fallen and intact scale insects per plant at the end of oneweek experiment is summarized in Figure 62. The control ferns with no As treatment had approximately 29 1 fallenscales and 141 10 int act scales, i.e., 17%. The ferns treated with 5 mg kg 1 As had similar number of fallenand intact scale, indicating limited effect from arsenic. At 15 mg kg 1 As, the number of fallen scale increased significantly to 140 per plant. The number for 30 mg kg 1 As treatment was slightly lower at 120 per plant. Since the ferns were naturally infested with the scales, the number of scales could not be controlled for a given plant. As a result, the total number of scales for each treatment at the beginning of the exper iment was different (Figure 62 A).

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88 To effectively assess the impact of arsenic on scale infestation of P. vittata the percentage of fallenscales to total scales per fern was used (Figure 63). At the end of the experiment after 7 d, the plants, and fallen and intact scales were analyzed for arsenic. Due to the short time used in this experiment, no significant change in plant biomass was observed between treatments. The fresh plant biomass for the four treatments ranged from 5.8 to 7.2 g per plant (data not shown). The total arsenic in P. vittata fronds increased significantly from 5.40 to 812 mg kg 1 as solution arsenic increased from 0 to 30 mg L 1 (Fi gure 63). Figure 62 The total number of fallenscale and intact scale on the fronds of P. vittata after 1 week of arsenic exposure A) Graphical representation B) The number of scale insects fallen from P. vittata fronds exposed to different arsenic concentration for 1 week. 0 50 100 150 200 250 0 5 15 30Number of insectsArsenic concentration in the hydroponic media (ppm) Fallen scales Intact scales

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89 With increase in frond arsenic, the percentage of fallen scales from the plants increased from 17.2% in the control to 55% in the plants treated with 30 mg kg 1 As (Figure 63). There was also a decrease in the ant population associated with the scales and indicates that they might have moved to less contaminated areas. Some ants were also observed dead along with the scale insects (data not shown). It was unclear if this resulted from the arsenic in the fronds or the scale secretions, which the ants were fed on. The majority of the fallenscales were dry with the exception of a few scales of higher instars. The arsenic concentrations in the fallen scales ranged from nondetectable (detection limit 2 g kg 1) in the control to 194 mg kg 1 in the 30 mg kg 1 As treatment (Figure 63). Some scales fell off from the fern while others remained on the plant which may be because female crawlers of the Saissetia sp. are not fastened permanently on the plant until they are ready to lay eggs (Fasulo and Brooks, 2004). Another aspect is that the wax like secretions from scale insects of later instars may harden the insect body and bind it to the plant parts. Approximately 90% of the intact scales were darker brown in color which indicates that they were of later instars compared to the pale greenish color of the earlier instars (data not shown; Futch et al., 2001). Hence the adult scales are fastened on the plant and may remai n on the plant even if they may be poisoned by arsenic. This was indicated by the arsenic concentration of the intact scales on the plant, which ranged from nondetectable in the control to 81 mg kg 1 in 30 mg kg 1 As treatment, which was significantly low er than those in fallen scales. Previous studies by Kertulis et al (2005) indicate that arsenic is transported in P. vittata via the xylem sap. The scale insects tested here were phloem feeders and the presence of

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90 arsenic in the scale biomass is consistent with the fact that arsenic is present in the phloem of the fern as well. The toxic doses of arsenic on aquatic insect species are observed to be 40 mg kg 1for midge (Holcombe et al., 1983) and water bugs (Lanzer DeSouza and Dasilva, 1988). In this study, at the end of 7d experiment, the frond concentration was 90 mg kg 1 when exposed to the lowest As at 5 mg kg 1and no arsenic effect was observed (Figure 63). This corresponded to 47 and 51 mg kg 1 As in the fallen and intact insects, respectively. Rathi nasabapathi et al. (2007) showed that arsenic accumulated at 46 mg kg 1 was sufficient to deter grasshoppers. Other phloem feeders like green peach aphids on a selenium accumulator Brassica indica indicated a 50% reduction in population with a leaf Se conc entration of 1.5 mg kg 1 whereas concentrations above 10 mg kg 1 was lethal (Hanson et al., 2004). Nickel hyperaccumulation by Streptanthus polygaloides had no deterrence effect on phloem feeders but deterred leaf chewing insects (Jhee et al., 2005). The present study indicates the poisoning of phloem feeder of the hyper accumulating fern whereas the previous study by Rathinasabapathi et al. (2007) supports the deterrence against a chewing insect. Chemical accumulation in a plant may result in the synthes is of a variety of organic compounds. These elemental and organic plant compounds may have a joint effect the on defense mechanism (Boyd, 2007). For example, Ni and certain defensive organic compound work together to repel herbivores in Nickle hyperaccumul ators (Jhee et al., 2006). Hence this is an important aspect that is to be considered as it will give an overall insight on elemental defense mechanism.

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91 Considering the research and other works mentioned, it can be inferred that metal toxicity in hyperaccumulators would vary among plant species, metal accumulated and insect species. In addition, the localization of the accumulated metal in the plant is also important. F indings and Fut ure D irections Although arsenic hyperaccumulation in P. vittata w as previously shown to be important in feeding deterrence by grasshoppers (Rathinasabapathi et al., 2007), it was unknown whether arsenic accumulation can deter natural pests of the host plant. In the current study, it was demonstrated that a scale, a natural pest of P. vittata was deterred by the arsenic accumulated in the fronds. This implies that under field conditions arsenic accumulation could have a protective role and hence an evolutionary advantage for P. vittata This study showed, for the first t ime, that scales and ferns can be employed to investigate unanswered questions on potential biological and ecological implications of arsenic hyperaccumulation in P. vittata and related ferns. This study puts forth two important questions regading the threshold level of a heavy metal in the biomass and on impotance of precauitionary measures to be taken while using these plants in phytoremediation. Furture study would involve the use of different pests under lab and field conditions to study the impac t of hyperaccumulated arsenic. The effect of arsenic in the tissue of the insect on organisms of higher tropic levels in the food chain should also be studied in detail.

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92 Figure 63. Arsenic concentrations in the fronds of P. vittata fallen scales and intact scales (left y axis), and % fallenscales (right yaxis) after 1week arsenic exposure. y = 28.702x + 27.195 R = 0.9047 y = 6.1033x 0.2667 R = 0.9399 y = 2.3014x + 20.207 R = 0.7549 y = 1.3295x + 19.881 R = 0.8596 0 10 20 30 40 50 60 70 0 200 400 600 800 1000 1200 0 5 10 15 20 25 30 35 40Percentage of scale insects fallen Frond As (mg/kg)Concentration of Arsenic in the media (mg/kg) Frond As (mg/kg) Fallen and dead scale As (mg/kg) Intact scale arsenic (mg/kg) Fallen and dead scales (%)

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93 CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS The unique mechanism of P vittata as an As hyperaccumulator and its application in phytoremediation has been well established. However, there are many different dimensions to this fern that needs to be understood. This study was useful in determining new aspects of the fern, which can help in proposing further research objectives and also help to harness the additional capabilities of the fern in arsenic remediation and pest control. The impact of P. vittata on AsIII stability in the media was substantial indicating that the presence of the fern in the media induced the AsIII oxidation with complete oxidation of 0.27 mM in 4 d. When growing the fern under sterilized conditions there was no oxidation of AsIII. The sonicated root extract which would contain both extracts from the fern root as well as from the microbes also resulted in 100% oxidation of AsIII in 4 days This oxidation reduced substantially with boiling or filter sterilizing the sonicate. Microbes hence play a substantial role of oxidation in the media. The fern can act as a solid substrate and provide a specific carbon source for microbia l activity or by itself serve as a source of m icrobes. Further research will be to isolate the microbes that are responsi ble for this oxidation and elucidate the specific role that P. vittata has to play in the life cycle of the microbe. For example, there may be a specific carbon source that is required by the microbe in question and hence requires P. vittata as a host. The effect of other As hyperaccumulating plant species on this phenomenon of oxidation is also important. A major obstacle in traditional methods of chemical remediation of AsIII in water is the inability of AsIII to adsorb onto the chemical adsorbents used in remediation. AsV is

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94 negatively charged and can easily be removed by adsorption or precipitation remedial measures. Hence a pretreat ment method is required to first convert the nuetral AsIII to AsV. The use of the fern can effectively avoid an expensive oxidation treatment step and also assist in phytoremediation of water. The study on xylem sap and speciation of As in the biomass of the fern indicates the capacity of the fern to transform As into different species and sequester the As away from other physiological activities in the fern. This capacity should shed light on the As sequestration followed in other organisms including the human body. It is to be noted that the rhizomes play a major role in AsIII and this part of the fern has been given comparatively less importance in arsenic hyperaccumulation studies. The study indicates that different parts of a living organism may have different enzymes for the detoxification of As. The presence of AsIII oxidizing enzymes in the root or endogenous bacteria in the roots of the fern is a possibility that needs to be investigated in detail using molecular techniques. Arsenite is known to be taken up by aquaglyceroporins which can tak e up SbIII as well as glycerol. This indicates that both SbIII and glycerol should compete against the uptake of AsIII in the fern. Also, the aquaporin inhibitor should be able to inhibit AsIII uptake into the fern. Howev er, in this study, there was no impact of glycerol or antimonite on AsIII uptake. This show ed that AsIII may be taken up by a different mechanism unique for P vittata and not via transporters that are responsible for SbIII or glycerol uptake. The inhibition of AsIII uptake by AgNO3 indicates that a certain type of aquaporin may be involved in the uptake of P. vittata and this requires a molecular level analysis of the transporters for further understanding. Once genes responsible for AsIII

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95 uptake are discovered, its presence in other edible plants like rice and seaweeds, which are more susceptible to AsIII uptake, can be elucidated. Once the genes responsible for AsIII uptake are known it may be possible to silence their functioning by mutations in the edible plants and, thereby, prevent AsIII uptake by those plants. Hyperaccumulation of As may have evolved to evade insects that are a common pest of the plants. The study here indicates its impact on a sap sucking insect. Future studies would include its impact on other insect species with different feeding modes and also using laboratory trials as both the fern and the insects would behave differently in field conditions compared to the laboratory conditions. Also, since the fern has the capacity to take up high concentrations of As there are possibilities that As can be taken up by herbivores and then transferred into the food chain affecting many living organisms This requires the adoption of precautionary measures during its use in commercial level phytoremediation where very high concentrations of As are present Under such conditions the use of screens to physically isolate the ferns from the herbivores should be considered. This phenomenon is also of interest because of the positive impacts it m ay have on the environment. The As hyperaccumulator that grow naturally on As contaminated sites or artificially on phytoremediation plots create an above ground biomass that have a high concentration of As. This may prevent the occurrence of pests in that area and can be beneficial for other plants as well. Alternatively, there can be a negative impact of the hyperaccumulator as well. The presence of a hyperaccumulator with high metal concentrations may result in insect attack on the non hyperaccumulators growing in the vicinity indi cating an avoidance mechanism of the fern. The pests of the f ern may turn to other plants as food source.

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96 To conclude, the As hyper accumulator P vittata is a major discovery in the field of phytoremediation. The physiological and molecular mechanisms of As uptake is yet to be studied in great detail to reveal more potentials of this fern as a model plant arsenic hyperaccumulator and detoxifier.

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97 APPENDIX A EFFECT OF ARSENIC LOADING ON ARSENIC HYPERACCUMULATION BY PTERIS VITTATA Figure A 1. Effect of arsenic loading when treated with As for 15 d on frond arsenic concentration in P. vittata The treatments were as follows: T1 0/0/0, T2 5/5/5, T30/10/5, T40/5/10, T55/10/0, T65/0/10, T710/5/0, T810/0/5, T915/0/0, T100 150, T110/0/15, where 0/5/10 can be defined as 0 mg L1 arsenic for the first 5 d, 5 mg L1 As on the next 5 d and 10 mg L1 As in th e last 5 d. The data indicates no significant difference at p<0.05 0 100 200 300 400 500 600 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11Frond As concetration (mg/kg)Treatment

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98 Figure A 2. Effect of arsenic loading when treated with As for 15 d on root arsenic concentration in P. vittata The treatments were as follows: T10/0/0, T2 5/5/5, T30/10/5, T40/ 5/10, T55/10/0, T65/0/10, T710/5/0, T810/0/5, T915/0/0, T100 150, T110/0/15, where 0/5/10 can be defined as 0 mg L1 arsenic for the first 5 d, 5 mg L1 As on the next 5 d and 10 mg L1 As in the last 5 d. The data indicates no significant dif ference at p<0.05 0 100 200 300 400 500 600 700 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 Root As Concentrattion (mg/kg)Treatment

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99 APPENDIX B COMPARISON OF ANTIMONY ACCUMULATION IN PTERIS VITTATA AND PTERIS ENSIFORMIS Figure B 1. Concentration of Sb in P. vittata when treated with different concentrations of Sb A) f ronds B) r oots. The solid bars indicate total Sb and the open bars indicate SbIII 0 0.5 1 1.5 2 2.5 3 3.5 4 0.1 1 10 Frond Sb mg/kgFWSb (mM) 0 100 200 300 400 500 600 700 800 0.1 1 10 Root Sb mg/kgFWSb ( mM) B A

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100 Figure B 2. Concentration of Sb in P. ensifomis when treated with different concentrations of Sb A) f ronds B) r oots. The solid bars indicate total Sb and the open bars indicate SbIII 0 0.5 1 1.5 2 2.5 3 3.5 4 0.1 1 10 Frond mg/kgFWSb (mM) 0 50 100 150 200 250 300 350 400 450 0.1 1 10 Root Sb mg/kgFWSb (mM) A B

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117 BIOGRAPHY Shiny Mathews, the only child of her parents, was born and brought up in Bahrain. After her high school education she went to India to join the Kerala Agricultural Univers ity for her bachelors degree. Following this she obtained an ICAR fellowship for pursuing a master s degree in soil science and agricultural c hemistry at the University of Agricultural S ciences, Bangalore. Her masters thesis was title d Behavior of Alachlor on A lfisols of Banglaore. She was awarded a gold medal for her academic and res earch performance by the university. Further, she went on to qualify for the CSIR fellowship and JN Tata scholarships which are prestigious awards provided by the gov ernment of India. In the year 2006, she joined the University of Florida, Gainesville, Florida to pursue a PhD in soil and w ater science. Here she was a graduate assistant and worked on the physiological studies on the arsenic hyperaccumulator Pteris vitta ta