Improved Arsenic Accumulation in Pteris Vittata and Its Unique Ability to Acquire Phosphorus

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Improved Arsenic Accumulation in Pteris Vittata and Its Unique Ability to Acquire Phosphorus
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Lessl, Jason T
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Doctorate ( Ph.D.)
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University of Florida
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Soil and Water Science
Committee Chair:
Ma, Lena Q
Committee Co-Chair:
Teplitski, Max
Committee Members:
Turner, Benjamin
Rathinasabapathi, Bala
Guy, Charles L

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arsenic -- lessl -- phytase -- phytate -- phytoremediation -- pteris -- vittata
Soil and Water Science -- Dissertations, Academic -- UF
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Soil and Water Science thesis, Ph.D.
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Abstract:
The fern, Pteris vittata L. (Chinese brake), is capable of accumulating arsenic (As) and storing it in the aboveground biomass. The physiological aspects pertaining to the connection between As and phosphorus (P) acquisition were studied along with plant mediated effects on soil As mobilization.   The role of phytase enzymes in As tolerance and P acquisition by P. vittata were studied. Enzyme-mediated hydrolysis of phytate in P. vittata extracts was not inhibited by As at 5 mM or by heating at100°C for 10 min.  Phytase in root exudates of P. vittata allowed its growth on media amended with phytate as the sole source of P. Phosphorus in P. vittata tissue grown on phytate was equivalent to control plants with an increase in As uptake.  In three soils, P. vittata phytase retained significantly more activity compared to phytase from wheat and a As-sensitive fern, Pteris ensiformis.    Since P and As compete for uptake by P. vittata roots, we hypothesized that the physiological responses of plant roots in a P-limiting environment would increase As uptake.  We evaluated this by growing P. vittata in three As-contaminated soils amended with phosphate rock (PR) over two years.  Phosphate rock, which provides a long-term supply of sparingly-soluble P, did not limit plant growth compared to the fertilized control.  To our knowledge,frond weights in this study are the largest reported and the first to increase over subsequent harvests.  Frond As concentrations grown increased in PR amended soil, allowing for significantly more As removal than the control.     The affect of P. vittata on As sorption during growth was examined by sequentially fractionating soil As by decreasing availability: soluble, exchangeable,amorphous, crystalline, and residual. Soluble As declined slightly with no change in the exchangeable fraction.  The amorphously bound fraction in all three soils accounted for the majority of As loss.  Since plant As uptake arises from the soluble and exchangeable fractions, we surmised that the amorphous fraction replenishes available As during phytoremediation. Thus, a model based on the ratio of As in the amorphous to available fractions was developed to predict As uptake.
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by Jason T Lessl.
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Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Ma, Lena Q.
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Co-adviser: Teplitski, Max.
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1 IMPROVED ARSENIC ACCUMULATION IN PTERIS VITTATA AND ITS UNIQUE ABILITY TO ACQUIRE PHOSPHORUS By JASON THOMAS LESSL 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 2012

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2 2012 Jason Thomas Lessl

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3 To Sandi

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4 ACKNOWLEDGMENTS I would like 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 f or the guidance she provided both in my research and professional development. I would also like to thank my committee members Dr. Bala Rathinasabapathi, Dr. Charles Guy Dr. Ben Turner and Dr. Max Teplitski for t heir valuable suggestions and the time they spent on ensuring the smooth progress of my research.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ 4 LIST OF TABLES ................................ ................................ ................................ ........... 8 LIST OF FIGURES ................................ ................................ ................................ ........ 9 ABSTRACT ................................ ................................ ................................ .................. 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ... 12 2 REVIEW OF LITERATURE ................................ ................................ ................... 15 Arsenic in the Environment ................................ ................................ .................... 15 Arsenic Toxicity ................................ ................................ ................................ ..... 16 Remediation of Arsenic Contaminated Soil ................................ ............................ 17 Pteris vittata L. ................................ ................................ ................................ ....... 18 Description and Distributio n ................................ ................................ ............ 18 Arsenic Hyperaccumulator ................................ ................................ .............. 2 0 Rhizosphere Modifications ................................ ................................ .............. 20 Root Induce d Arsenic Mobility ................................ ................................ ......... 22 Phytoremediation of Arsenic Contaminated Soils by P. vittata ............................... 23 3 A NOVEL PHYTASE FROM PTERIS VITTATA RESISTANT TO ARSENATE, HIGH TEMPERATURE, AND SOIL DEACTIVATION ................................ ............ 27 Plant Phytases ................................ ................................ ................................ ...... 27 Materials and Methods ................................ ................................ .......................... 29 Hydroponic Plant Culture ................................ ................................ ................ 29 Seedling and Gametophyte Culture ................................ ................................ 29 Enzyme Collection ................................ ................................ .......................... 30 Phytase and Phosphatase Assays ................................ ................................ .. 30 Arsenic and Phosphorus Analysis ................................ ................................ ... 31 Phytase Arsenic Resistance and Thermostability ................................ ............ 31 Phytase Stability After Mixing With Soils ................................ ......................... 32 Statistical Analysis ................................ ................................ .......................... 32 Results ................................ ................................ ................................ .................. 32 Pteris vittata Phytase Showed Arsenic Resistance and Thermostability .......... 32 Phytase and Phosphatase in P. vi ttata and P. ensiformis Tissues ................... 33 Pteris vittat a Growth on Media Amended with Arsenic and Phytate ................ 34 Phosphorus and As Uptake by P. v ittata Gametophyte ................................ ... 35 Phytase Activity in Pteris vittata Gametophyte and Root Exudate ................... 35 Pteris vittata Phytase Activity was not Deact ivated by Soils ............................ 36

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6 Discussion ................................ ................................ ................................ ............. 37 Arsenic Tolerance in Pteris vittata ................................ ................................ ... 37 P teris vittata Phytase showed As Resistance and Thermostability .................. 38 Phytase and Phosphatase Activity in P. vittata and P. ensiformis Tissues ...... 38 Pteris vittata Growth on Phytate ................................ ................................ ...... 39 Phytase Activity in P. vittata Gametophyte and Root Exudates ....................... 41 Pteris vittata P hytases We re Not Deactivated by Soils ................................ .... 42 4 IMPROVED HUSBANDRY AND PHOSPHATE ROCK AMENDMENTS SIGNIFICANTLY IMPROVE SOIL ARSENIC PHYTOREMEDIATION BY PTERIS VITTATA : A TWO YEAR STUDY ................................ ............................. 51 Phytoremediation Using Pteris vittata ................................ ................................ .... 51 Materials and Methods ................................ ................................ .......................... 54 Soil Collection ................................ ................................ ................................ 54 Experimental Setup ................................ ................................ ......................... 55 Plant Harvest ................................ ................................ ................................ .. 56 Soil and Root Sampling ................................ ................................ ................... 56 Statistical Analysis ................................ ................................ .......................... 57 Results and Discussion ................................ ................................ ......................... 57 Soil Characteristics ................................ ................................ ......................... 57 Harvest Scheme Improved Re growth of P. vittata ................................ .......... 59 Phosphate Rock Amendments Increased P. vittata Biomass .......................... 60 Phosphate Rock Improved Arsenic Uptake in P. vittata ................................ ... 61 Pteris vittata P Acquisition ................................ ................................ ............... 62 Soil Arsenic Removal ................................ ................................ ...................... 64 Conclusion ................................ ................................ ................................ ............. 65 5 ARSENIC DISTRIBUTION IN THE SOIL AND FROND OF PTERIS VITTATA L. DURING PHYTOEXTRACTION HARVESTED OVER TWO YEARS ..................... 78 Arsenic Soil Distribution ................................ ................................ ......................... 78 Materials and Methods ................................ ................................ .......................... 80 Soil Collection ................................ ................................ ................................ 80 Experimental Setup ................................ ................................ ......................... 81 Soil Sampling and Plant Harvest ................................ ................................ ..... 82 Statistical Analy sis ................................ ................................ .......................... 82 Results and Discussion ................................ ................................ ......................... 82 Soil Characteristics ................................ ................................ ......................... 82 Biomass and Arse nic Ac c umulation in Pteris vittata ................................ ........ 83 Soil As Fractionation ................................ ................................ ....................... 85 Predicting Arsenic Uptake in P. vittata Using Sequential Extraction Data ........ 87 Conclusions ................................ ................................ ................................ ........... 89 6 ROLE OF ARSENIC HYPERACCUMULATION IN PTERIS VITTATA ................... 97 Role of Metal Accumulation in Plants ................................ ................................ ..... 97

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7 Materials and Methods ................................ ................................ ........................ 102 Fern and Gametophyte Setup ................................ ................................ ....... 102 Herbivory Setup ................................ ................................ ............................ 103 Allelopathy Experiment ................................ ................................ .................. 104 Statistical Analysis ................................ ................................ ........................ 104 Results and Discussion ................................ ................................ ....................... 104 The Effect of Arsenic on P. vittata Growth ................................ ..................... 104 Arsenic as a Defense against Herbi vory ................................ ........................ 106 Allelopathy ................................ ................................ ................................ .... 106 LIST OF REFERENCES ................................ ................................ ............................ 112 BIOGRAPHICAL SKETCH ................................ ................................ ......................... 125

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8 LIST OF TABLES Table page 3 1 Enzyme activities in tissues of P vittata and P. ensiformis ................................ 44 3 2 Plant growth on modified MS media ................................ ................................ .. 45 3 3 Co ncentration of P and As in P. vittata g ametophyte ................................ ......... 46 4 1 Selected physiochemical pro perties of soils used in this study .......................... 67 4 2 Physicochemical properties of soils amended with phosphate rock or control treatments following two years of growth with P. vittata ................................ ..... 68 4 3 Arsenic concentration and uptake in P. vittata frond biomass ............................ 69 4 4 Bioconcentration factor ................................ ................................ ...................... 70 4 5 Frond P concentration ................................ ................................ ....................... 71 4 6 Multi harvest phytoremediation studies with P. vittata grown in moderately contaminated soils ................................ ................................ ............................. 72 5 1 S elect characteristics of soils used in this study ................................ ................ 90 5 2 Arsenic distribution in harvested P. vittata fronds ................................ .............. 91 5 3 Elementa l analysis of harvested P. vittata fronds ................................ .............. 92 5 4 The ratio of amorphous:available arsenic in soil over two years ....................... 93 6 1 Pteris vittat a s pore g rowth ................................ ................................ ............... 108

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9 LIST OF FIGURES Figure page 3 1 Pteris vittata p hytase was resistant to arsenate ................................ ................. 47 3 2 Pteris vittata phyt ase was resistant to heat shock ................................ ............. 48 3 3 Presence of phytate increased phytase activity in P. vittata ............................... 49 3 4 Pteris vittata phytase activity was resi stant to soil inactivation ........................... 50 4 1 Harvested frond biomass from P. vittata increased at each six month harvest .. 73 4 2 Two year old P. vittata roots growing in phosphate rock amended CCA soils contained abundant ad ventitious roots and root hairs ................................ ........ 74 4 3 Total P uptake in P. vittata frond biomass ................................ ......................... 75 4 4 Soil As concentrations declined at a linear rate in soils over two years ............. 76 4 5 Soil As removed from the top 0 1 5 and bottom 15 30 cm fractions of CCA, DVA and DVB soils with phosphate rock and control amendments after two years of P. vittata growth ................................ ................................ ................... 77 5 1 Relative distribution of arsenic in the soluble, e xchangeable, amorphous, crystalline and residual fractions at time of planting. ................................ .......... 94 5 2 Arsenic in the soluble, exchangeable, amorphous, crystalline and residual fractions of CCA, DVA, and DVB soi ls during two years of growth with P. vittata ................................ ................................ ................................ ............... 95 5 3 Predictive model based on the natural logarithm of measured bioconcentration (BC) ratios between amorphous and available soil As fractions to predict As uptake in P. vittata ................................ ......................... 96 6 1 Pteris vittata growing in soil spiked with 0, 25, 50 and 100 mg kg 1 arsenic. .... 109 6 2 Spodopt era latifascia caterpillar and adult moth ................................ .............. 110 6 3 The natural logarithm of soil arsenic concentrations at the surface soil and at a 20 cm depth taken at the base of P. vittata and 50 cm away ........................ 111

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10 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 IMPROVED ARSENIC ACCUMULATION IN PT ERIS VITTATA AND ITS UNIQUE ABILITY TO ACQUIRE PHOSPHORUS By Jason Thomas Lessl August 2012 Chair: Lena Q. Ma Cochair: Max Teplitski Major: Soil and Water Science The fern, Pteris vittata L. (Chinese brake), is capable of accumulating arsenic (As) and storing it in the aboveground biomass. The physiological aspects pertaining to the connection between As and phosphorus (P) acquisition were studied along with plant mediated effects on soil As mobilization. T he role of phytase enzymes in As tolerance an d P acquisition by P. vittata were studied. Enzyme mediated hydrolysis of phytate in P. vittata extracts was not inhibited by As at 5 mM or by heating at 100C for 10 min. Phytase in r oot exudates of P. vittata allow ed its growth on media amended with ph ytate as the sole source of P Phosphorus in P. vittata tissue grown on phytate was equivalent to control plants with an increase in As uptake. In three soils P. vittata phytase retained significantly more activity compared to phytase from wheat and an As sensitive fern, Pteris ensiformis Since P and As compete for uptake by P. vittata roots, we hypothesized that the physiological responses of plant roots in a P limiting environment would increase As uptake. We evaluated this by growing P. vittata i n three As contaminated soils amended with phosphate rock (PR) over two years. Phosphate rock, which provides a

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11 long term supply of sparingly soluble P, did not limit plant growth compared to the fertilized control. To our knowledge, frond weights in thi s study are the largest reported and the first to increase over subsequent harvests. Frond As concentrations grown increased in PR amended soil allowing for significantly more As remova l than the control Soil arsenic sorption during P. vittata growth was examined by sequentially fractionat ing soil As by decreasing availability: soluble, exchangeable, amorphous, crystalline, and residual. Soluble As declined slightly with no change in the exchangeable fraction. The amorphously bound fraction in all t hree soils accounted for the majority of As loss. Since plant As uptake arises from the soluble and exchangeable fractions, a model based on the ratio between available and amorphous fraction s were used. This model takes into account the redistribution o f As to more available fractions during phytoremediation and allowed for accurate prediction of As uptake by P. vittata over two years.

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12 CHAPTER 1 INTRODUCTION During the last century, vast portions of the earth's natural resources have been appropriated to sustain its growing population. A chief consequence of this has been the deterioration of soil ecosystems from exposure to toxic chemicals. Yet even as the productivity of our soil declines, more is expected of it due to our growing population. In t he United States al one, there are 1,210 registered superfund sites contaminated with arsenic (U.S. Environmental Protection Agency, 2004) Arsenic is a deadly toxin and is linked to cancer of the bladder, kidney, liver, lung, and prostate (ATSDR, 2009) Tens of millions of people are p otentially exposed to excessive levels of arsenic (Ng et al., 2003) Due to its deadly toxicity and carcinogenicity, arsenic is ranked by the Agency for Toxic Substances & Disease Registry as the #1 most dangerous chemical in the environment (ATSDR, 2007a) Development of novel remediation techniques are required to safely meet the demands of a burgeoning society. Phyto remediation using Pteris vittata (Chinese brake fern) offers a simple, non invasive, and cost effective method to remediate arsenic contaminated soil compared to traditional clean up techniques. This application is made possible by the unique capacity of P. vittata to hyperaccumulate arsenic in its aboveground biomass (Ma et al., 2001) This feature allows the plant to tolerate an otherwise lethal environment. The capacity to remove high concentrations of arsenic from soil and water offers an easy, non invasive, and cost effective remediation method compared to traditional clean up techniques (U.S. Environmental Protection Agency, 2002)

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13 The success of phytoremediation depends on seve ral factors including the extent capacity to mobilize, absorb, and accumulate metals in shoots. The complexity of this interaction, controlled by climatic condi tions, argues against generic and in favor of site specific phytoremediation approaches. This underlines the importance of understanding the mechanisms and processes that govern metal uptake and accumulation in plants. Due to its high rate of As accumula tion, fast growth and large production of biomass, P. vittata is ideal for phytoremediation. However, factors facilitating the uptake of arsenic into P. vittata are still in question Normally, upon arsenic exposure, plant root growth is inhibited (Meharg and Hartley Whitaker, 2002; Zhao et al., 2008) However, P. vittata roots respond by increasing biomass while mobilizing arsenic in the rhizosphere One possibility is that the increased arsenic availability could be response to low available p hosphorus (P) in the soil. Despite being abundant in the lithosphere, P is one of the most limiting nutrients affecting agricultural production around the world (von Uexkll and Mutert, 1995) A large proportion of soil P is organic (P o ), which is stable, insoluble, and immobile, thus making it unavailable for root uptake (Holford, 1997) The predominant form of soil P o is IPx inositol hexakisphosphate (IP 6 ), which exists in the myo form (phytate; IHP), and is a s table compound highly invulnerable to chemical or enzymatic degradation (Turner et al., 2002) Within the soil fraction, phytate can constitute >50% of P o and >25% of total P (Anderson et al., 1980) Hence, de spite its abundance, P is one of the most unavailable and inaccessible macronutrients in the soil, and frequently limits plant growth (Holford, 1997) During periods of low availability of soil P, plants respond at the

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14 morphological, physiological and molecular level. One such response is u tilization and s ecretion of ph osphomonoesterases into the rhizosphere which can improve phosphate availability (Raghothama, 1999; Vance et al., 2003) High affi nity transporters are generall y accepted as entry points for phosphate which is also the proposed route for arsenic uptake by P. vittata (Meharg and Macnair, 1992) Since phosphate and arsenic are ch emical analogues, P. vittata must make efficient use of P due to arsenates inherent toxicity Thus, root mediated response s of P vittata during phosphate starvation may be unique, making it a poten tial model plant in understanding how r oot s can serve to enhance the availability and use of P. D educing the mechanisms behind a rsenic tolerance and uptake would be potentially valuable for developing crops that tolerate inhospitable environments. Using P. vittata the major objectives in this research were to 1) u nderstand the role of phy tase in phosphorus acquisition and arsenic uptake, 2) d evelop plant management strategies to maximize growth and arseni c uptake over several seasons and 3) evaluate the impact on soil arsenic mobilization during phytoremediation

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15 CHAPTER 2 REVIEW OF LITERA TURE Arsenic in the Environment Arsenic is a ubiquitous element that ranks 20th in abundance in the earth's crust (Mandal and Suzuki, 2002) Arsenic occurs naturally in over 200 different mineral forms, of which approximately 60% are arsenates, 20% sulfides and the remaining 20% includes arsenides, arsenites, oxides, silicates and elemental arsenic (Wedepohl, 1969) Arsenate (AsV) and arsenite (AsIII) are the most common inorganic forms in the environment. Soil As concentrations in the USA are estimated to range between 0.1 55 mg kg with an average of 7.2 mg kg (Allard et al., 1995) However, arsenic concentrations in soil may be much higher, primarily due to anthropogenic contributions from arsenical pesticides, fertilizers, burning of fossil fuels; chromate copper arsenic (CCA) treat ed wood and disposal of industrial and animal wastes (Nriagu and Pacyna, 1988) For nearly five decades (1930 to 1980), arsenic based pesticides were applied on agricultural land throughout the United States, adding > 10 ,00 0 tons of arsenic to the soil each year (Welch et al., 2000) Arsenic does not breakdown and can readily accumulate in soil (Davenport and Peryea, 1991) Despite being a known issue, arsenic accumulation in soils is a continual problem In the U.S., approximately 100 metric tons of arsenic based feed additives (roxarsone ) are fed to chickens every year. Arsenic laced laced poultry litter is later spread on agricultural fields at a rate between 9 20 metric tons ha 1 yr 1 (Cortinas et al., 2006; Hileman, 2007) By 2002, m ore than 90% of all outdoor wooden structures in the U .S. were treated with arsenate pesticide (Gray and Houlihan, 2002) With high concentrations of arsenic (~1,200 mg kg 1 ), treated wood has a long life span (20 50 years) and acts as a source of arsenic

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16 contamination in the vicinity (Stook et al., 2004) Even though CCA wood was banned f or residential use in 2004, ~6.1 10 6 kg of As is used annually for wood treatments in the U.S. (Brooks, 2012) Normally, soil As concentrations exceeding the limit result s in regulatory actions at industrial or hazardous waste sites, but no such protocols exist for residential and public spaces suggest ing the presence of a widespread regulatory health crisis (Belluck et al., 2003) Once introduced to the soil, factors such as climate, organic and inorganic soil components and redox pot ential status impact the level and species of As in a given soil (Mandal and Suzuki, 2002) Under aerobic oxidizing conditions, arsenates predominate, strongly sorbing to clays, iron, manganese oxides/hydroxides, and organic matter (Sun and Doner, 1996) Arsenite is found in reducing anaerobic conditions, which can be methylated by microorganisms, producing monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide under oxidizing conditions (Norman, 1998) Typical mineral soils can have pH values in the range 5 to 9 with Eh values of 300 (water logged) to +900 (well aerated) millivolts (mV). In soil based studies, redox conditions and pH significantly affect the availability and consequent toxicity of arsenic (Marin et al., 1992; Meharg and Hartley Whitaker, 2002) Arsenic Toxicity Most cases of human toxicity from arsenic have been associated with exposure to inorganic arsenic (U.S. Environmental Protection Agency, 2001; ATSDR, 2009) In humans, exposure to As may lead to damages of internal organs, the respiratory, dige stive, circulatory, neural, and renal systems; the most significant hazards are skin, lung and bladder cancers (Ng et al., 2003; Tchounwou et al., 2004) Arsenate is a molecular analog of phosphate and interferes with oxidative phosphorylation and ATP

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17 synthesis (NRC, 2001) Arsenite is more broadly toxic because it binds to sulfhydryl groups or vicinal thiols in pyruvate dehydrogenase and 2 oxoglutarate dehydrogenase, impairing the fun ction of many proteins and respiration (ATSDR, 2007b) Inorganic arsenic species are highly toxic to plants. Arsenate is the dominant form of arsenic in aerobic soils and acts as a phosphate analogue, which enables transport across the plasma membrane via phosphate co tra nsport systems (Meharg and Macnair, 1992) Upon arsenic exposure, plants suffer from inhibition of root growth to death (Barrachina et al., 1995; Burlo et al., 1999; Zhao et al., 2008) Once inside the cytoplasm, arsenate competes with phosphate, replacing phosphate in ATP to form unstable ADP As, and leading to the disruption of ene rgy flows in cells (Meharg, 1994) Arsenate can be reduced to arsenite by glutathione in plant tissue, which is also highly toxic to plants as it reacts with sulfhy dryl groups ( SH) of enzymes and proteins, leading to inhibition of cellular function, generation of reactive oxygen species (ROS) and death (Ullrich Eberius et al., 1989; Abbas and Meharg, 2008) Following the reduction of arsenate to arsenite in plants, arsenic may potentially be further metabolized to methylated species leading to further oxidative stress (Zaman and Pardini, 1996) Remediation of Arsenic Contaminated Soil According to the National Priorities List (NPL), there are 578 Superfund sites where arsenic is a concern, with As contaminated soils accounti ng for ~372 (66%) of the sites (U.S. E nvironmental Protection Agency, 2004) Many engineering technologies have been developed for remediation of arsenic contaminated soils (U.S. Environmental Protection Agency, 2002) Remediation of arsenic contaminated soils is a costly endeavor. Conventional treatments often require the use of expensive equipment,

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18 which has high operational c osts Furthermore, some require excavation of the soil, greatly altering the environment, while others (solubilization and stabilization) do not remove the arsenic leaving the potential for future exposure. Alternatively, the use of phytoremediation pres erves the topsoil while reducing hazardous contaminants. It is also cost effective, requiring no special equipment or operating costs. Finally, phytoremediation can also be aesthetically pleasing, garnering more public acceptance. Phytoremediation is pla nt based technologies that degrade, extract, contain, or immobilize contaminants from soil or water. It has the potential to clean up waters, soils, slimes and sediments contaminated with pesticides, PAHs (polycyclic aromatic hydrocarbons), fuels, explosiv es, organic solvents, chemical manures, heavy metals, metalloids, and radioactive contaminants (Adams et al., 2000) In the process of phytoremediation, pollutants are taken up by plant roots and either decomposed to less harmful forms (e.g., CO 2 and H 2 O) or accumulated in the plant tissues. Thus, phytoremediation is environme ntally friendly, inexpensive (relative to other remediation techniques) and can be carried out in polluted places (in situ). The success of phytoremediation depends on several factors including the extent of soil contamination, metal availability for upta absorb, and accumulate metals in shoots (Krmer, 2005 ) Pteris vittata is one such a plant that contains the qualities necessary for successful phytoremediation, specifically due to its ability to hyperaccumulate a rsenic Pteris vittata L. D escription and Distribution Pteris vittata is a vascular fern within th e Pteridaceae family as deduced by phylogenetic studies based on rbcL nucleotide sequences (Hasebe et al., 1995) The

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19 complete taxonomic classification of P. vittata is: Kingdom Plan tae, Subkingdom Tracheobionta (vascular plants), Division Pteridophyta (True Fern), Class Filicopsida, Subclass Polypodiidae, Order Pteridales or Polypodiales, Family Pteridaceae (maidenhair), Subfamily Pteridoideae, Genus Pteris (derived f rom Greek word pteron, meaning wing or feather, for the closely spaced pinnae, which give the leaves a likeness to feathers), Species vittata (ladder), Specific epithet : vittata Linnaeus (U.S. Department of Agriculture, 2009) Pteris vittata grows in tropical and subtropical regions. Originally nat ive to Africa, China, Japan, Thailand, and Australia, it is currently found worldwide. In the U.S., P. vittata grows naturally in Hawaii, California, and the southeastern states; Texas to South Carolina and Florida (Jones, 1987) In Florida, they are often found on calcareous substrate, such as old masonry, s idewalks, building crevices, and nearly every habitat in southern Florida with exposed limestone, notably pinelands (Lellinger, 1985) Pteris vittata are hardy, fast growing perennial forbs with characteristics of mesophytes. They prefer full to partial sun while their size varies depending on conditions, can reach heights of 1.2 m. It s stipe is stout with dense brown scales, which extend along the rachis. Fronds cluster from a horizontal rhizome and blades are green to pale brown, containing numerous pinnae, which separate proximally (Lellinger, 1985) The life cycle of P vittata involves sporophytes where spores are produced on the margin of the lower side of the pinnae. The spores (used in sexual propagation) germinate and produce the heart shaped prothalli. Both male and female organs develop from the prothalli giving rise to the gametophyte stage. The most interesting characteristic about P. vittata is its extr eme tolerance and ability to hyperaccumulate

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20 arsenic making it the first embryophyte to display an affinity for arsenic (Ma et al., 2001) Arsenic H yperaccumulator Brooks et al. (1977) first coined that uptake and a dry mass, which is still in common use. Due to its high rate of arsenic accumulation, fast growth and a high production of biomass, P. vittata is ideal for phytoremediation. In both controlled and natural expe riments, arsenic reaching concentrations as high as 23000 As g 1 dry mass (Tu et al., 2002) Arsenic hyperaccumulation can also occur directly through foliage, suggesting that P. vittata foliage tissue has specific arsenic tr ansporters (Bondada et al., 2004) Even the callus, gametophyte, and sporophytes tissue of this fern were effective in accumulating arsenic (Gumaelius et al., 2004; Yang et al., 2007) Rhizosphere M odifications At an arsenic concentration of 200 mg kg 1 presence of phosphate was shown to have little effect on improving P. vittata growth ; but when arsenic concentrations in creased (>400 mg kg 1 ), P was critical for P. vittata growth (Tu and Ma, 2003) It has been suggested that minimum ratios of P/As of 1:2 in the fronds is required for normal growth of P. vittata (Tu and Ma, 2003) In ord er to achieve these ratios of P P. vittata may utilize root exudates to maximize uptake and inadvertently increase arsenic availability in soil, leading to increased hyperaccumulation of As. C ompounds released from plant roots can have a direct impact on the solubility of mineral elements or can indirectly influence turnover and availability of nutrients by interaction with soil microorganisms (Neumann, 2007) Despite their importance, little is known about the

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21 role of rhizosphere proc esses in heavy metal hyperaccumulators including P. vittata (McGrath et al., 2001; Wenzel et al., 2003; Rathinasabapathi et al., 2006) It is possible that arsenic uptake in P. vittata is a byproduct of P mobilization and acquisition. Phosphate is considered one of the least available plant nutrients in the soil. High affinity phosphate transporters are generally accepted as entry points for P, which is also the proposed route for arsenic uptake by P. vittata (Meharg and Hartley Whitaker, 2002; Wang et al., 2002) The physiological, genetic, molecular and biochemical analysis of phosphate starvation response mechanisms highlight the ability of plants to adapt and thrive under P limiting conditions. These responses, particularly root exuded carboxylates and phosphatases, help enhance the availability o f P, which increase its uptake and improve the use efficiency of P within the plant (Raghothama, 2000) Root induced mobilization o f nutrients (e.g., P, Fe and Zn) requires the release of specific stable compounds (e.g., citrate, malate, oxalate, malonate, and phytosiderophores) in the root zone with highest nutrient uptake (e.g., apical root zones) (Jones, 1998; Neumann, 2007) Anion channels have also been implicated in exudation of citrate, which are responsible for mobilization of soluble Fe and Al phosphates ( Neumann et al., 1999; Zhang et al., 2004) Porter and Peterson (1975) reported that a clear correlation exists between a rsenic and Fe in plants growing on soils heavily contaminated with arsenic with the results indicating that Fe plays a role in As accumulation in these plants. High Fe concentration in soil solutions in the rhizosphere of P. vittata have also been identified, suggesting the possible mobilization of Fe by the roots of P. vittata (Fitz et al., 2003) However, more studies are needed to explore the role of nutrient elements on the detoxification and accumulation of arsenic in P. vittata

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22 Root Induced Arsenic Mobility Changes in the rhizosphere characteristics of P vittata relevant to its use in phytoextraction have been examined. Arsenic depletio n in its rhizosphere and limited resupply from less available arsenic pools were indicated by a 19% reduction in arsenic flux (Fitz et al., 2003) Gonzaga et al. (2006) evaluated the influence of arsenic uptake by P. vittata on different fractions of arsenic in the bulk and rhizosphere soil by fractionat ing by decreasing availability P teris vittata was more efficient than Nephrolepis exaltata to access arsenic from all fractions, though most of the arsenic taken up was from the amorphous hydrous oxide bound fraction (67 77% of soil arsenic), the most abundant (61.5% of all fractions) instead of the most available (n on specifically bound fraction) (Gonzaga et al., 2006) Increased arsenic availability in the rhizosphere of P. vittata may be a byproduct of root mediated mechanisms to increase nutrient availability. Rhizosphere modifications are known to be important for the acquisition of various nutrients (Jones et al., 2004) Tu et al. (2004) examined root exudates of P vittata in a hydroponic system and found d issolved organic carbon (DOC) in the root exudates increased from 19 t o 30 mg kg 1 (root dry weight) as arsenic A similar experiment was conducted by Lou et al. (2010) who found that DOC contents from P. vittata root exudates decreased with increasing P concentrations, regardless of whether arsenic was present or not. Phosphorus deficient plan ts have been shown to enhance exudation of carboxylic acids (i.e., citric and malic acid), which are thought to change soil pH, displace P from sorption sites, and chelate metal cations that could immobilize P or to form soluble metal chelate complexes wi th P (Kirk et al., 1999) Since As and P are chemical analogues, it is reasonable to assume that root exudation may also mobilize

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23 a rsenic in the rhizosphere. This may explain the ph enomenon in P. vittata in that the presence of P decreases arsenic influx, whereas P starvation nets a arsenic influx by 2.5 fold (Wang et al., 2002) Arsenic is also known to complex with Fe oxide s urfaces (Sun and Doner, 1996) The excretion of protons and/or the release of reducing and chelating compounds by P. vittata could result in co dissolution of arsenic from Fe oxides/hydroxides, although little is known about Fe nutritional aspects and related rhizosphere processes of P. vittata (Fitz and Wenzel, 2002) However, since P. vittata are known to grown on ca lcareous soil (Jones, 1987) they may share similaritie s with other acidifuge plants, which have been reported to effectively mobilize P and Fe from limestone (Strm et al., 1994) Plant Fe deficiency normally occurs on ca lcareous soils ( pH > 7.0) (Marschner, 1995) Organic acids such as citrate and malate are known to be potent complexers of Fe in soil and induce the di ssolution of unavailable insoluble ferric oxyhydroxides (Jones, 1998) Thus, P. vittata could be a key factor determining fluxes and pool sizes of arsenic in the soil. Most root studies have been performed in solution culture, which facilitates observations in soil environments Especially because r oots gr own under hydroponic conditions may be morphologically and physiologically different from those growing in soil (e.g., no root hairs, no cortical degeneration, different branching patterns, no mechanical impedance or water stress). In addition, the aeratio n, microbial and nutrient status of these hydroponic cultures is often different from those in a typical soil environment. Phytoremediation of Arsenic Contaminated Soils by P. vittata Numerous studies have examined the use of P. vittata for phytoextrac tion (Tu et al., 2002; Salido et al., 2003; Kertulis Tartar et al., 2006; Baldwin and Butcher, 2 007;

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24 Gonzaga et al., 2008; Santos et al., 2008) However, with the exception of Kertulis Tartar et al. (2006) most of these experiments are conducted on a short time scale (months) Phytoremediation is expected to take several growing seasons (years) to effectively reduce soil arsenic concentrations. Furthermore, the use of arsenic spiked soil and hydroponic st udies do not correlate well with hyperaccumulation in a natural soil environment. Gonzaga et al. (2008) conducted a greenhouse experiment evaluating arsenic removal by P. v ittata and its effects on arsenic redistribution in six arsenic contaminated soils. T hey confirmed that it is possible to use P. vittata to remediate arsenic contaminated soils by repeated frond removal. Over the course of one year (October 2003 October 2004), three harvests were condu cted and frond biomass along with arsenic accumulation was drastically reduced compared to the first harvest (Gonzaga et al., 2008) In a similar experiments by Shelmerdine et al. (2009) and Caille et al. (2004) growth and arsenic uptake of P. vittata were significantly r educed in subsequent harvests. Problems with this experiment include: use of small pots, which limited root growth, and during the first harvest, all the fronds were cut to the base of the rhizome, greatly inhibiting plant re growth. P hosphate fertilize r amendments have been shown to reduce arsenic concentration in the fronds of P. vittata even though they increase arsenic concentration in the soluble soil fraction However, p hosphate fertilizers are known to increase arsenic uptake in othe r plant systems (Peryea, 1991, 1998) In a three month field study, Cao et al. (2003) showed that arsenic concentration in the fronds of P. vittata inc reased by 265% when 15 g kg phosphate rock was added to a contaminated

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25 sandy soil, although no increase was observed f rom plants grown in soil spiked with soluble arsenic salts. It was suggested that replacement of a rsenic by P from the soil binding sites was responsible for the enhanced mobility of a r senic and subsequent increased plant uptake. Fayiga and Ma (2006) observed a slight reduction in arsenic uptake (1631 to 1530 mg kg ) by P. vittata in a 5 week pot experiment amended with slow release P fertilizer. Compost additions were found to facilitate arsenic uptake in a contaminated soil, but not an arsenic spiked soil during a 3 month pot study (Cao et al., 2003) It was suggested that the effect of compost on a rsenic uptake was likely due to changes in soil pH a nd water holding capacity; the soil had a neutral pH and compost treatments may have induced an an aerobic environment in the soil, reducing As(V) to As(III), thereby facilitating uptake by the fern (Cao et al., 2003). In contrast, arsenic adsorption onto organic matter applied in acidic soil may have decreased uptake in the spiked soil (Cao et al., 2003). During an 8 week study, P. vittata grown in rhizopots with As contaminated CCA soil (105 mg kg ) reduced water soluble arsenic and increased soil pH in the rhizosphere soil (Gonzaga et al., 2008) Most of the arsenic accumulation was associated with the rhizosphere soil (67 77%), suggesting that highe r plant density may i mprove remediation of arsenic contaminated soils by increasing root surface area per unit volume of soil The optimal planting density for P. vittata has not been explored for soil remediation. In a field trial with a planting density of 6 plants m Gray et al. (2005) reported an annual above ground biomass production of 1.03 t (dw) ha Kertulis Tartar et al. (2006) saw improved biomass (1.3 t ha yr ) using a planting density of 10 plants m Recently, Shelmerdine et al. (2009) conducted a 9 month container based

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26 experiment which equated to a planting density of 16 plants m with a estimated frond biomass of 2.5 t ha yr F ield trials with increased plant d ensity are required to assess optimal strategies for p hytoremediation with P. vittata

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27 CHAPTER 3 A NOVEL PHYTASE FROM PTERIS VITTATA RESISTANT TO ARSENATE, HIGH TEMPERATURE, AND SOIL DEACTIVATION Plant Phytases Despite being abundant in the lithosphere, phosphorus (P) is one of the most limiting nutrients affecting agricultural production around the world (Cordell et al., 2009) Plants require P to be in a soluble inorganic form to be taken up but P is often insoluble in soils due to high sorption (Richardson et al., 2009) Even with soluble inorganic P fertilizer, much of the P is quickly sorbed or transformed into forms un availabl e to plants (Snchez Caldern et al., 2010) Phosphorus fertilizers, which are mined from non renewable resources, are often required in great abundance to m aintain high crop productivity which also c ontributes to eutrophication of water bodies (Cordell et al., 2009) To protect the environment while sustaining agricultural production, improved P nutrition strategies are needed. O ne such method is to develop plants with increased ability to utilize the large pool of unavailable organic P in soils. Organic P (P o ) accounts for 30 80% of total soil P, predominantly as phytate [myo inositol 1,2,3,4,5,6 hexakisphosphate] (Richardson et al., 2005) Phytate is a stable compound resistant to bi ochemical degradation, rendering it unavailable for root uptake (Turner et al., 2002) Within the soil fraction, phytate c an make up >50% P o and >25% total P (Richardson et al., 2005) Inorganic phosphate (P i ) plays a central role in energy metabolism and regulation in plant cells. During periods of low P i availability, plants respond through changes in root morphology, secretion of organic acids into the rhizosphere, augmentation of P i uptake systems, and changes in P metabolism (Vance et al., 2003) Plants can use

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28 phosphatases to release P i from P o during seed germination, for internal remobilization, and from external mobilization in the soil (Baldwin et al., 20 01; Miller et al., 2001) Phosphatases have broad substrate specificities to various forms of P o Those that specifically cleave P i from phytate are known as phytases (Duff et al., 1994; Brinch Pedersen et al. 2002) Intracellular phytases and phosphatases are involved in utilization of P i reserves or other P i containi ng compounds (Duff et al., 1994) Root exudation of phosphatases, especially phytases, co uld be an effective mechanism to provide additional sources of P i in soil, but this biochemical strategy has not been widely used by plants. In roots, phytase enzymes can occur in the apoplast but are often localized to the cell wall, epidermal cells, and apical meristem (Duff et al., 1994; Miller et al., 2001) D espite this, most plant root phosphatases (especially agronomical ly important ones) are unable to hydrolyze sufficient P i to maintain growth owing to either poor substrate availability in soils due to sorption and precipitation, proteolytic breakdown or limited capacity to effective ly exude P i mobilizing enzymes (Findenegg and Nelemans, 1993; Rich ardson et al., 2000; George et al., 2004, 2005) Pteris vittata L. (Chinese brake fern) is native to alkaline sub/ tropical soils which are rich in P o but deficient in available P (Ramaekers et al., 2010) Furthermore, P. vittata (PV) c an hyperaccumulate arsenic (As) in the frond biomass to >1% of its dry weigh t (Ma et al., 2001) A rsenate is the most abundant form of As in soils and is a chemical and st ructural analog for P i In fact, arsenate is a competitive inhibitor of P i for uptake by plant P transporters (Meharg and Macnair, 1992) In the presence of arsenate, plant roots will readily assimilat e As leading to toxicity development P deficiency and enzyme deactivation (Meharg and Hartley Whitaker, 2002) This is not

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29 the case for PV which tolerates high concentrations of As in the soil and even higher in the frond s In addition to its natural adap ta tion to P deficien t soil, PV phosphatases including phytase may effectively hydrolyze P i from phytate and other P o while being uniquely resistant to the deleterious effects of As To test this hypothesis, phosphatase and phytase activities from P. vittata and Pteris ensiformis ( PE; a non hyperaccumulator) were assessed following exposure to As stress and P i limitatio n. Furthermore, the role of phytases in P i acquisition was examined by: 1) quantifying phytase activities in root exudates; 2) growing P. vittata on media with phytate as the sole source of P; and 3) evaluating the efficacy of P vittata phytases in soil environments. Materials and Methods Hydroponic Plant Culture Two month old ferns, P. vittata (PV) and P. ensiformis (PE; a non hyperaccumulator), were transferred to hydroponic culture in 0.2 strength Hoagland Arnon nutrient solution (HNS) for three week s. P lants were rinsed with deionized (DI) water i (KH 2 PO 4 ) and 0 As (Na 2 HAsO 4 2H 2 O) for 3 d T reatments are referred to as control (No P), P i As, and P i +As and were replicated four times. Seedling and G ametophyte C ulture Seeds from Lactuca sativa Trifolium subterraneum and Allium schoenoprasum and spores from PE, Thelypteris kunthii and PV were surface sterilized in a 2 0% bleach solution for 20 minutes followed by three washes in sterile DI water Spores were suspended in 2 mL st erile DI water Half strength modified Murashige & Skoog (MS) media was prepared with 0.8% agar without P prior to autoclaving. Phosphate, phytate,

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30 and arsenate solutions were filter sterilized and added to autoclaved MS media to obtain final concentrati ons of 0.6 mM P as P i or phytate (P 6 ; myo inositol hexapho sphoric acid dodecasodium salt ) with 0 or 0.6 mM arsenate. The MS media ( pH 6.5 ) was then poured into sterile petri dishes (100 mm 13 mm). Seeds and spores (10 L or 0.05 mg spore) were placed o n agar (5 per plate, 4 plates per treatment ) under cool/warm fluorescent lamps at 25C and 60% humidity for 15 and 40 d for seeded plants and ferns, respectively. Enzyme Collection T issues were rinsed in 10 mM Ca(NO 3 ) 2 and blotted dry, weighed, and mixed (1:2 w/v) with 10 mM acetate buffer (pH 5.0) containing 1 mM EDTA, 1 mM DTT (dithiothreitol), 0.1 mM PMSF ( phenylmethylsulfonyl fluoride) and 4% PVPP (polyvinyl polypyrrolidone). Samples were homogenized using a Magic Bullet blender (Four 15 s pulses) passed through cheesecloth and centrifuged at 10,000 g for 15 min. Supernatants were subjected to gel filtration on Sephadex G 25, pre equilibrated with 10 mM acetate buffer (pH 5.0). Root exudates were collected from media of 40 d old PV sporophyte, ana lyzing enzyme activity following gel filtration. A mmonium sulfate fractionation was performed on PV gametophyte extracts, collecting precipitates in 20% intervals from 0 80% fractions followed by gel filtration. Phytase and Phosphatase Assays P rotein co ntent was measured against bovine serum albumin (BSA) standards using the Bradford method (Walker and Kruger, 2002) Enzyme activity was analyzed by incubating ~100 g protein in 1 mL of 10 mM acetate buffer (pH 5.0) containing e ither 5 mM phytate or 5 mM p NPP ( p nitroph enylphosphate disodium salt; Sigma) at 37C for phytase and phosphatase, respectively. Reactions were terminated with equal

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31 volume s of 10% (wt/v) trichloroacetic acid after 120 min (phytase) or 25 m M NaOH after 30 min (phosphatase). Specific activities w ere calculated as the difference between P i or p NP concentration in the extracts with and without incubation, expressed as nmol of P i or p NP released per min per mg protein. Phosphate w as measured spectrophotometrically at 880 nm using the molybdenum blu e reaction at a fixed time (20 min) following addition of the color reagent (Carvalho et al., 1998) Phosphatase activity was calculated from the release of p NP as determined by measuring absorbance at 405 nm against standar d solutions. Arsenic and Phosphorus Analysis Pteris vittata tissue was dried at 60C for 96 h, weighed, and ground through a 2 mm mesh screen. Samples (0.1 g) were subjected to hot block digestion using U SEPA Method 305 0 (1983) and analyzed for total As using graphite furnace atomic absorption spectroscopy (GFAAS, Varian AA240Z, Walnut Creek, CA). Total P was calculated using the molybdenum blue method previously mentioned. To prevent interference of arsenate when using the molybdenum blue method, samples were incubated with 300 L 5% cysteine at 80C for 5 min to reduce arsenate to arsenite (Carvalho et al., 1998) Phytase Arsenic Resistance and Thermostability Arsenic tolerance of phytase and phosphatase e nzymes was analyzed by performing previously described assays in the presence of of arsenate. In addition to 5 mM P 6 or p NPP, plant extracts of PV PE and wheat phytase were incubated with 0, 0.5, 2, 2.5, and 5 mM arsenate. Thermostability of enzyme acti vities was determined by pre incubation of enzyme extracts in a water bath at 40, 60, 80, and 100C for 10 min.

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32 Phytase Stability After Mix ing W ith Soils The phytase activity in root extracts from PV and PE and wheat phytase were measured after mixing wi th soils. Briefly, 2.0 g of air dried soil was mixed with DI water and enzyme extracts (or BSA as a negative control) to a 20 mL volume containing 50 g protein per ml. Samples were placed on a rotary shaker (150 rpm) for 120 min at room temperature. Al iquots of well tip with a wide opening and centrifuged at 7,500 g for 5 min, using the supernatant (250 Activities were derived from the difference between plant enzy me mediated P i release and the amount of P i in the BSA soil suspensions. Statistical Analysis D ata are presented as the mean of all replicates with standard error. Significant differences were determined using analysis of variance and treatment means comp ared Results Pteris vittata Phytase S howed Arsenic R esistance and Thermostabilit y Partial p urification of PV phytase greatly increased its enzyme activities T he PV activities in the crude protein were 2.6 nmo l P i and 8.6 nmol p NP mg 1 protein min 1 Gel filtration tripled specific enzyme activities and ammonium sulfate precipitation increased activities by 9 to 26 fold. The the highest purification was associated with the 20 40% ammonium sulfate fractions (6 8 nmol P i and 181 nmol p NP mg 1 protein min 1 ) which were used to estimate As tolerance and thermostability. Phytase and phosphatase activities were measured by production of P i and p NP hydrolyzed by the extracts of PV PE and a crude wheat phytase in the presence of increasing concentrations of arsenate (0 5 mM). At 5 mM phytate or p NPP suspensions buffered

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33 at pH 5.0 enzyme activit ies for PV, PE, and WP were 46.7, 42.7, and 51.8 nmol P i mg 1 protein min 1 for phytase and 79.5, 149, and 163 nmol p NP mg 1 protein min 1 for phosphatase respectively ( Figure 3 1) Phytase activities in PV extracts were unaffected by arsenate up to 2 mM (46.7 to 46.1 nmol P i mg 1 protein min 1 ), with a slight decrease (~41.1 nmol P i mg 1 protein min 1 ) at concentrations above 2.5 mM, which were not significantly different than the control (p < 0.05; Figure 3 1a ). However, phytase activities from PE and wheat extracts exhibited a linear decrease (~50, 43, 34 and 24% decrease) with increasing arsenate ( Figure 3 1a ). At 5 mM, t heir activities were ~25% of the control. Phosphatase activities in extracts from PV, PE and wheat were similarly impacted by arsenate ( Figure 3 1b ). At 5 mM As their activities were 36 45% of the control. T he t hermostability of extracts w ere tested b y incubating samples for 10 min at temperatures ranging from 40C to 100C prior to the activity assays. Phytase activities of PV extracts were unaffected by all heat treatments compared to PE and wheat phytase, which lost all activities a fter incubating at 100C ( Figure 3 2a ). Unlike phytase, phosphatase activities from enzyme extracts of all three plants decreased at a similar rate with increasing temperatures ( Figure 3 2b ). Phytase and Phosphatase in P. vittata and P. ensiformis Tissues After growing i n media with P i arsenate or both for 72 h, PV and PE showed no toxicity symptoms. Phosphatase and phytase activities were detected in the frond, root, and rhizome extracts of both PV and PE ( Table 3 1 ) P hosphatase activities in all treatments were much greater in PE than PV in all tissues, with the greatest difference in the fronds (85 198 times) and smallest in the roots (1.2 2.0 times). Unlike

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34 p hosphatase phytase activities in all treatments were generally greater in PV, illustrating an inherent dif ference between the two species N either P i or As treatment had an impact on activities in the fronds or rhizomes of both PV and PE. However, some treatments reduced enzyme activities in their roots Wit h no exception, addition of P i was the most effectiv e in reducing p hosphatase and phytase activities in the root s Strangely enough, addition of As had the same effect as P i in PV, reduc ing phytase activity from 19.7 to 6.1 nmol P i mg 1 protein min 1 ( Table 3 1 ). P teris v ittat a G row th on M edia A mended w it h A rsenic and P hytate To estimate the ability of PV to utilize phytate as a sole source of P, its growth on modified MS media amended with either 0.6 mM P i and/or phytate (P 6 ) with and without 0.6 mM arsenate (P i +As, P 6 +As, and P i +P 6 +As) was compared to th ree angiosperms with known phytase activity ( Lactuca sativa Trifolium subterraneum and Allium schoenoprasum ) and two pteridophytes ( P. ensiformis and T. kunthii ). Fresh weights of plants after grow ing for 15 d for angiosperms and 40 d for ferns are list ed in Table 3 2 Germination rate for seeds were >90% and 100% for fern spores grown on modified MS media amended with 0.6 mM P i Though all three angiosperms grew on P 6 amended media, their biomass production was reduced by 2.1 3.3 times compared to th e P i treatment. The two ferns (PE and T. kunthii ) were unable to use phytate to grow. Pteris vittata was the only plant that effectively utilize d phytate and in the presence of 0.6 mM As survive beyond germination (Table 3 2 ). G row th on media amended with P i +P 6 were similar to P i treatments, verifying that the presence of phytate had no negative effect on growth. Interestingly, P i +As increased PV biomass 2 fold

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35 (115 to 225 mg) and 1.5 fold in P i +P 6 +As (119 to 151 mg) but was slightly reduced in P 6 +As (90 to 66 mg) (Table 3 2 ). Phosphorus and As U ptake by P. vittata Gametophyte Total P and As in PV gametophyte grown on MS media with 0.6 mM P i P 6 and/or As for 40 d are listed in Table 3 3 Average P concentrations in the P i treatment were 2, 208 mg/kg compared to 1,579 mg/kg in the P i +As treatment a significant decrease ( p < 0.05) It was also similar to that of the P 6 treatment (2,351 mg/kg) indicating that PV gametophyte readily hydrolyzed and accumulated P from phytate. At equal As and P concentr ations of 0.6 mM in the P i +As treatment the total P and As tissue concentrations were 1,57 9 and 1,777 mg/kg or 51 and 24 mmoles/kg respectively (Table 3 3) This indicates that PV was more effective in taking up P than As. Compared to the P i +As treatmen t concentrations of P and As in tissue from the P 6 +As treatment were both increased, which were 2 672 mg/kg and 2 630 mg/kg ( p However, this did n ot happen in the absence of As or in the presence of high P ( as total P in the P i +P 6 treatments ) w hich had similar tissue P concentrations This indicates that phytate (low P) coupled with As promoted up regulation of P transporters, helping with both P and As uptake. Phytase Activity in Pteris vittata Gametophyte and Root Exudate Given that P. vitta ta effectively utilized phytate as a sole source of P for growth, we quantified phytase activities from gametophyte and its root exudates in response to P/As stress and phytate. A ctivities from tissue extracts did not differ significantly between treatmen ts except for the As treatments, which lowered phytase activities by ~5 9 to 2 8 nmol P i mg 1 protein min 1 ( Figure 3 3a ). E xudates collected from phytate amended media exhibited the highest phytase activity However, except for P 6 enzyme

PAGE 36

36 activit ies fro m all treatments were not statistically different ( p Figure 3 3b ). Compared to phytase activities in the root tissues (5.1 to 20 nmol P i mg 1 protein min 1 ; Table 1 ), those in the root exudates were comparable or higher (9.3 to 19 nmol P i mg 1 protein min 1 ), indicating that phytase in the root exud ates plays the largest role in P acquisition Grown in a low available P media (P 6 ), PV gametophytes increased the amount of exudates. This was indicated by an increase of total protein in exudates from P 6 and P 6 +As (2.2 and 2.0 mg protein g 1 tissue) co mpared to P i media (1.0, 1.1, and 1.0 mg protein g 1 tissue for P i P i +As and P i +P 6 respectively ) P hosphatase enzymes did not appear to play a significant role in root exudates, as the activity values were only 9 to 18% of those in the PV root extracts ( Table 3 1 ) Pteris vittata Phytase Activity was not D eactivated by Soil s Root extracts from PV PE and wheat phytase were mixed with three soils for 2 h Soil 1 was an acidic (pH 5.6) sandy soil containing 2% OM with a cation exchange capacity (CEC) of 4. 2 cmol + kg 1 Soil 2 was a neutral (pH 6.5) silty clay soil with 0.8% OM and a CEC of 16.4 cmol + kg 1 Soil 3 was an acidic (pH = 5.5) clay soil with 0.4% OM and CEC of 24.8 cmol + kg 1 The effect of soils on phytase activity was analyzed by measuring t he rate of P i hydrolysis from phytate in solution following centrifugation. The amount of P i hydrolyzed represented phytase enzymes that were not sorbed to the soil matrix. For comparative analysis, enzyme samples were incubated without soil (control) an d soil samples were mixed with a non enzymatic protein, bovine serum albumin (BSA), to estimate residual soil P i released from protein soil interactions In the absence of soil, phytase enzym es from all three plants were similar, ranging from 15 to 19 nmol P i mg 1 protein min 1 ( Figure 3 4 ) After mixing with soils, PV phytase enzymes retained 66, 50 and 45% of their activity in soil 1, soil 2 and soil 3. In

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37 comparison, PE and wheat phytase retained only ~6% activity (~1 nmol P i mg 1 protein min 1 ) in all soils ( Figure 3 4 ). Discussion Arsenic Tolerance in Pteris vittata Numerous studies have demonstrated the unique ability of P. vittata to acquire, tolerate and accumulate high concentrations of As from soils and culture media (Ma et al., 2001; Tu et al., 2002) The selective factors driving the evolution of As hyperaccumulation in P. vittata is unknown, but hypotheses include a role in metal tolerance, protection against herbivores/pathogens, increased antioxidant responses, and allelopathy (Rascio and Navari Izzo, 2011) As a member of the Pteridaceae P. vittata is an advanced taxa with morphological characteristics plac ing it in the more recent portion of fern evolution (Smith et al., 2006) During its evolution, As hyperaccumulation and tolerance could be a carryover from marine algae native to As rich hot springs which had scant amount of available P i (Meharg, 2002) Thus, due to the chemical homology of arsenate and P P. vittata must be efficient in scaveng ing and maintain ing P homeostasis while preventing interferenc e from arsenate. Hence, it is no surprise that P. vittata commonly inhabits environments depleted of available P i but with abundant phytate (Jones, 1987; Turner et al., 2006) This unique characteristic was the impetus for our examination of phytase activity in P. vittata The most prevalent form of organic P in ma ny soils is phytate, contributing ov er 50% of total soil P (Turner et al., 2002) Thus, P. vittata would be expected to depend more on phytases to gain P i from phytate. Because phytase mediated hydrolysis is strongly inhibited by As (Hayes et al., 1999; Pivke and Simola, 2001) our results suggest P. vittata has e volved novel phytase enzyme s resistant to As allowing for sufficient acquisition of P i from phytate.

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38 Pteris vittata Phytase s howed A s R esistance and Thermostabilit y P hytase and phosphatase activities from ammonium sulfate precipitation of PV extracts were detected in all fractions with the highest concentration in the 20 40% fraction When PV enzyme extracts were incubated with increasing concentrations of arsenate (up to 5 mM), phyta s e enzymes, but not phosphatase, retained significant activity. Unlike PV, phytase activity in PE and wheat extracts diminished with increasing concentrations of arsenate ( Figure 3 1 ). It is well established that arsenate interferes with enzyme function including phytases (Zhao et al., 2008) This suggests P. vittata ha d a unique phytase enzyme, which was consistent with its high thermostability. Phytase activities, but not phospha tase of PV enzyme extracts were unaffected by 10 min pretreatments at 100C wh ile PE and wheat phytase activities were lost ( Figure 3 2 ). Pteris ensiformis extracts did retain some activity at 80C, suggesting a degree of commonality between the two closely related species. E nzyme incubation at pH >7 resulted in loss of activity i n PV extracts (data not shown), corroborating previously described optimum pH of 5 (Tu et al., 2010) Metal tolerance and thermostability in phytases and other enzymes have been attributed to glycosylation, hydrogen bonds, disulfide bonds, salt bridges, and presence of co factors (i.e. chaperones and heat shock proteins) (Wang et al., 2004; Guo et al., 2008) It is possible that the final folded state of PV phytases were highly stable, conferring arsenate tolerance and Phytase and Phosphatas e Activity in P. vittata and P. ensiformis T issues During periods of P limitation, plants increase their internal phosphatase and phytase production to maintain P i levels (Snchez Caldern et al., 2010) To assess the role of phosphatases in P. vittata internal P i mobilization during P limitation and

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39 arsenate exposure, their activity in the frond, root, and rhizome tissues were compared to the As sensitive fern PE When grow n in the presence of As plants often show symptoms of P deficiency because arsenate competes with P i uptake and disrupts processes involving phosphorylation and phosphate signaling pathways (Abercrombie et al., 2008) This response was observed for phytase and phosphatase activity in PE root tissues, which were significantly elevated when growing in the absence of P i or presence of As ( p < 0.05; Table 3 1 ). Interestingly, this was not the case for the enzyme activity in PV root extracts. Unlike phytase, phosphatase activity from PV root extracts increased in treatments without P i while phytase activity increased only in the con trol (No P) ( p < 0.05; Table 3 1 ). The lack of phytase activity response in As treated PV roots was unexpected. Since arsenate i s a phosphate analog, PV roots may not differentiate between them. Instead, the metabolic and regulatory systems may have per ceive d the toxic metalloid as an abundant supply of P i inhibiting the up regulation of phytase production. After 3 d of growth different treatments had no effect on enzyme activity in the frond and rhizome tissues for both ferns. Frond and rhizome enzy me activity may have been unaffected because the 3 d incubation period was not long enough to elicit sufficient P deficiency responses in those tissues. Furthermore, P. ensiformis does not translocate As to the rhizome and frond. Alternatively, enzyme ac tivity in both ferns may be associated with acquisition of P i from soil and not with internal P homeostasis, which would explain why activity responses were only observed in root tissues ( Table 3 1 ). Pteris vittata Growth on Phytate Due to the high enzymat ic phytase activity in PV roots, especially under P i limiting conditions, we investigated whether PV spores could grow on sterile media amended

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40 with P 6 as the sole source of P. Phytate has been shown to be a poor source of P for plants due to both substra te availability and enzyme activity constraints (Hayes et al., 2000; George et al., 2004) This was not the case for P. vittata which grew equally well on P i or P 6 ( Table 3 2 ) with similar total P (2 208 and 2 351 mg/kg ) after 40 d of growth Most plants lack the ability to access external phytate because their phytases are confined to the endodermal region (Hayes et al., 1999) which was supported by th e fact that other plants produc ed similar biomass in phytate treatment as the c ontrol without P ( Table 3 2 ). Even though T. subterrane um and L. sativa have been shown to increase root phytase activity in P limiting and other stressful environments (H ayes et al., 1999; Nasri et al., 2010) they were unable to hydrolyze sufficient quantities of phytate in our experiment to sustain growth. The ability of PV to grow using phytate as a sole source of P i and its lack in two other ferns suggests that phyta te utilization is an adaptive trait specifically evolved in only some fern taxa. As expected, P. vittata was the only plant to survive beyond germination in the presence of 0.6 mM arsenate. I n the presence of arsenate, PV biomass and total P concentration were affected by the source of P. A fter 40 d of growth PV grown on P i +As agar were ~2 times larger than all other treatments ( p 5 ; Table 3 2 ). Despite having the largest biomass, PV tissue from P i +As agar had the lowest P concentration, which is consistent with previous findings that arsenate stimulates growth and competes with P i for uptake (Gumaelius et al., 2004) However, arsenate had the opposite effect on gametophyte grown with phytate, r educing biomass below the P i control while significantly increasing total P ( p Table 3 3). Similar to the lack of phytase response in PV root tissue ( Table 1 ), the presence of arsenate in the growth media may

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41 be perceived as P i (due to their homol ogy) by PV gametophyte, delaying the necessary transcriptional, physiological, and morphological responses required to facilitate phytate hydrolysis. Although growth was slowed, tissues from the P 6 +As media had significantly higher concentrations of P and As compared to P i +As treatments ( p Table 3 3) With the addition of P i to the P 6 +As treatments, biomass and total P concentrations were in between the results of P i +As and P 6 +As treatments, suggesting that the presence of phytate tempers the growth promoting effect of As It shou ld be noted that after 80 d of growth the initial slow growth of P. vittata on P 6 +As treatments abated, achieving weights equivalent to the P i treatments (data now shown). Phytase Activity in P. vittata Gametophyte and Root Exudates Once it became clea r that P. vittata could effectively utilize phytate, we assessed the response of enzyme activities in gametophyte and their root exudates following 40 d of growth on modified MS media amended with 0.6 mM P i phytate, and arsenate. Phytase activity from ti ssue grown with phytate exhibited the highest phytase activities compared to P i +As treatments, which had the lowest (Figure 3 3a ). However, compared to the P i treatment, phytase activities did not differ significantly from other treatments. Similarly, ph ytase in root exudates from gametophyte grown with phytate exhibited the highest activities although not significantly different than the P i treatment. Thus, production and exudation of phytase enzymes in PV gametophyte appears to be constitutive, regardl ess of P i availability. However, total protein content in exudates of P 6 and P 6 +As treatments were double that of P i treatments, suggesting that PV responds in a low available P environment by increasing total enzyme exudation.

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42 Pteris vittata P hytases W er e Not D eactivated by S oils Pteris vittata grew with a relatively low concentration of phytate (0.6 mM) while maintaining P concentrations equal to the P i treatment ( Table 3 3 ). Phytase activity in root exudates was enhanced when grown with phytate, the li kely mechanism for the P i acquisition. Studies have shown that while plants have the capacity to exude phytases in roots, sorption and precipitation reactions in soil limit their capacity to directly obtain P i from soil phytate (Brejnholt et al., 2011) This was not the case for PV enzymes, which retained 45 66% of their phytase activities after mixing with soils compared to >90% reduction in PE and wheat extracts, further illust rating the unique properties of PV phytases ( Figure 3 4 ). As the soil cat ion exchange capacity increased (Soil 1 < Soil 2 < Soil 3) PV phytases c ould have been more strongly sorbed, explain ing the diminishing trend in increasing phytase activity fro m supernatant samples. Although othe r factors like pH would contribute to sorption. Under normal circumstances, sorption of phytase impairs the enzyme's ability to hydrolyze phosphate esters from phytate (George et al., 2005) but PV phytases remained active even when sorbed to soil particles (data not shown), indicating a high affinity for phytate. Pla nts in an environment with limited P availability and mobility have evolved tightly controlled mechanisms to maintain P homeostasis, which include acquisition of P i from soil, remobilization of stored P i as well as optimization of metabolic processes to c onserve P i (Rouached et al., 2010) Native to soils poor in P (Jones, 1987) P. vittata has adapted to environments that are depleted of available P i by utilizing a unique phytase to facilitate phytate hydrolysis, even in the presence of As which hinder s enzymatic pro cesses (Tsai et al., 2009) Furthermore, this unique phytase from P. vittata retained activity in soils which readily sorb and inactivate plant exuded phytases

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43 (George et al., 2005) This is especially significant because few plants can direct ly obtain P i from phytate in soils (Hayes et al., 2000; Richardson et al., 2005) Pteris vittata has potentially evolved a phytase that circumvents the limitations of other plant phytases which are restricted by their capacity to be active following exud ation into the soil (Hayes et al., 1999; Richardson et al., 2000)

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44 Table 3 1 Enzyme a ctivities in t issues of P vittata (PV) and P. ensiformis (PE) a Tissue Treatment phosphatase activity nmol pNP mg 1 protein min 1 phytase activity nmol P mg 1 protein min 1 PV PE PV PE Frond Control 10.9 2.8 a 1,628 299 a 2.9 1.3 a 3.2 0.9 a Frond P i 14.4 4.2 a 1,218 387 a 2.2 0.4 a 1.2 0.3 a Frond As 15.2 4.7 a 1,058 303 a 1.6 0.3 a 3.7 1.1 a Frond As+P i 7.7 1.3 a 1,530 788 a 3.4 1.7 a 1.3 0.7 a Rhizome Control 10.8 1.5 a 47.8 9.5 a 1.8 0.3 a 1.3 0.5 a Rhizome P i 9.2 2.0 a 20.3 2.6 a 2.4 0.9 a 1. 3 0.3 a Rhizome As 7.4 1.2 a 42.0 17 a 2.0 0.5 a 1.1 0.2 a Rhizome As+P i 7.9 1.5 a 20.3 6.4 a 2.3 0.8 a 0.7 0.3 a Root Control 136 29 a 158 9.5 a 19.7 4.2 a 7.8 1.9 a Root P i 74.4 22 b 101 18 b 5.1 1.4 b 2.3 0.6 b Roo t As 124 18 a 151 8.0 a 6.1 1.2 b 6.0 2.1 a Root As+P i 66.6 19 b 153 11 a 10.8 2.8 b 5.4 1.0 a a Values are the mean of four replicates with standard error and columns with the same letters are not significantly different.

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45 Table 3 2 Plant g rowth on m odified MS m edia a Treatment and Fresh Weight (mg) P lant C ontrol As P i P i +As P 6 P 6 +As P i +P 6 P i +P 6 +As L. Sativa b 37 3b 40.2c 12517a 51c 383b 40.3c 11523a 6 1c A. schoenoprasum b 15 1b ng d 311a ng 152b ng 322a ng T. subterra neum b 43 8b ng 8926a ng 4312b ng 9619a ng P. ensiformis c 2 0.2b ng 951a ng 20.1b ng 961a ng T. kunthii c 2 0.3b ng 831a ng 20.1b ng 814a ng P. vittata c 5 1d 7 1 d 115 5bc 225 27a 904cd 66 4 c 119 5 bc 151 5 b a Values are the mean of 20 r eplicates with standard error and rows with the same letters are not significantly different. b Average biomass from one seed after 15 d. c Biomass from 0.05 mg spore after 40 d. d ng = no germination

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46 Table 3 3 Concentration of P and As (mg/kg) in P. vittata g ametophyte a Treatment Phosphorus Arseni c P i 2208 222 ab nd b P i +As 1579 307 c 1777 175 b P i +P 6 2012 117 bc nd P 6 2351 201 ab nd P 6 +As 2672 181 a 2630 229 a P i +P 6 +As 2138 93 abc 2206 340 a a Values are the mean of six replicates, bars representing standard error and columns with the same letters are not significantly different. b nd = none

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47 Figure 3 1. Pteris vittata phytase was resistant to arsenate. Phytase (A) and phosphatase (B) activities from the extracts of P. vittata (PV), P. ensiformis (PE) and purified wheat phytase (WP) wer e determined by incubating samples in 5 mM phytate or pNPP suspensions buffered at pH 5.0 with increasing concentrations of arsenate. Specific activity values for PV, PE, and WP were 46.7, 42.7, and 51.8 nmol P i mg 1 protein min 1 for phytase and 79.5, 14 9, and 163 nmol pNP mg 1 protein min 1 for phosphatase respectively. Data are the means of ten replicates with bars representing standard error.

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48 Figure 3 2. Pteris vittata phytase was resistant to heat shock. Phytase (A) and phosphatase (B) ac tivities from extracts of P. vittata (PV), P. ensiformis (PE) and wheat phytase were determined by incubating 5 mM phytate or p NPP suspensions buffered at pH 5.0 following 10 min pretreatments in a water bath held at 40, 60, 80, or 100 C. Data are the me ans of ten replicates with bars representing standard error.

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49 Figure 3 3. Presence of phytate increased phytase activity in P. vittata Phytase activities determined from gametophyte (A) and root exudates (B) determined from P vittata grown with p hosphate (P i ), phytate (P 6 ), and arsenate (As). Data represent the mean of eight replicates with standard error and b ars with the same letters are not significantly different.

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50 Figure 3 4 Pteris vittata phytase activity was resistant to soil i nactiv ation Phytase specific activities (nmol P i mg 1 protein min 1 ) in the supernatant of soil suspensions after mixed with enzyme extracts of P. vittata (PV), P. ensiformis (PE), and wheat phytase (WP). Enzyme extracts were added to soil suspensions, mixed for 2 h and centrifuged prior to activity measurement. Data are the means of four replicates with bars representing standard error.

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51 CHAPTER 4 IMPROVED HUSBANDRY AND PHOSPHATE ROCK AMENDMENTS SIGNIFICANTLY IMPROVE SOIL ARSENIC PHYTOREMEDIATION BY PT ERIS VITTATA : A TWO YEAR STUDY Phytoremediation Using Pteris vittata Due to its toxicity and carcinogenicity, arsenic is ranked by the Agency for Toxic Substances & Disease Registry as the #1 contaminant in the environment (ATSDR, 2007a) For near ly five decades (1930 to 1980), the application of arsenic al pesticides (e.g., PbHAsO 4 and CaHAsO 4 ) amounted to soil As additions of ~ 1 300 metric tons yr 1 (Brooks, 2012) The USEPA r egional s creening l evel for soil As under residential use averages 0.39 mg kg 1 while t he Florida direct exposure Soil Cleanup Target Level (SCTL) is 2.1 mg kg 1 (Teaf et al., 2010) Despite being a known issue, As accumulation in soils is a continual pro blem. Natural leaching and i nappropriate disposal of As treated products pose a threat to public health and the environment. By 2002, m ore than 90% of all outdoor wooden structures in the U .S. were treated with copper chrome arsenate (CCA) pesticide (Gray and Houlihan, 2002) With high concentrations of As ( ~1,200 mg kg 1 ), CCA treated wood has a long life span (20 50 years) and acts as a source of As contamination in the vicinity (Stook et al., 2004) Even though CCA wood was banned f or residential use in 2004, ~6.1 10 6 kg of As is used annually for wood treatments in the U.S. (Brooks, 2012) Normally, soil As concentrations exceeding the limit result s in regulatory actions at industrial or haz ardous waste sites, but no such protocols exist for residential and public spaces suggest ing the presence of a widespread regulatory health crisis (Belluck et al., 2003) M any engineeri ng technologies have been developed for remediation of As contaminated soils but they are costly and invasive. Methods of removal either disturb

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52 the environment (excavation) or do not remove the As (solubilization and stabilization), allowing potential fo r future exposure. Alternatively, the use of phytoremediation preserves the topsoil while reducing hazardous contaminants. This technique requires no special equipment or high operating costs and can be aesthetically pleasing, garnering more public accep tance. Arsenic hyperaccumulator Pteris vittata L. (Chinese brake f ern) (Ma et al., 2001) can acc umulate up to 22,630 mg arsenic kg (dry weight) in the aboveground biomass, indicating its capacity for high tolerance and detoxification of As. Numerous studies have demonstrated the unique ability of P. vittata to tolerate and accumulate high concentrations of As, but are based on sho rt growing periods (~12 weeks) in conditions un representative of soil environment s (i.e., hydroponic, small pots under glasshouse conditions) (Salido et al., 2003; Caille et al., 2004; Cao and Ma, 2004; Kertulis Tartar et al., 2006; Baldwin and Butcher, 2007; Gonzaga et al., 2008; Shelmerdine et al., 2009) To achieve the As SCTL in li ght to moderately contaminated soils, phytoremediation can require years to decades to achieve. Thus, a long term stud y to elucidate conditions conducive to maximizing As uptake and biomass production is required to evaluate P. vittata 's full potential fo r phytoremediation. A rsenate the most prevalent form of As in soil, uses phosphate t ransport ers in higher plants, acting as a competitive inhibitor (Meharg and Macnair, 1992) Due to shared homology of arsenate and phosphate, p lant roots will readily assimilate arsenate, leading to P deficiency (Meharg and Hartley W hitaker, 2002) During P starvation, many plants respond by increasing root length, density of root hairs and by exud ing organic compounds to mobilize different types of P associated compounds (Raghothama and Karthikeyan, 2005) Pteris vittata is native to soils w hich are

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53 characterized by low available P in addition to tolerat ing high concentrations of arsenic (Jones, 1987) These properties make P. vittata unique in its ability to scavenge for P, even in the presence of high concentrations of As. Arsenic concentrations in fronds and roots of P. vittata were shown to increase with decreasing P i in nutrient solution (Lou et al., 2010) To maximize the remediative capacity of P. vittata soil should be supplied with a source of P with minimal availability. P hosphate rock (PR) which is the raw material used to manufacture phosphatic fertilizers could provide a long term source of P with limited plant availability. Phosphate rock is typically not suitable as a direct substit ute for soluble P fertilizers because the rate of dissolution is not adequate to meet plants demands. In a study comparing PR and mono ammonium phosphate amendments in 16 Brassica species, PR treatments reduced plant biomass >2.5 times and P concentration ~1.5 times in all of the cultivars (Aziz et al., 2011) There are factors that influenc e rate of PR dissolution like soil characteristics and plant interactions In a PR study with white clover ( Trifolium repens ) and ryegrass ( Lolium perenne ), dry matter yields at pH 5.3 were equivalent to mono calcium phosphate treatments, but decreased 24% at p H 5.6 and 28% at pH 6.4 (Rajan et al., 1991) Some plants influence the rate of PR dissolution by altering the rhizosphere pH, uptake of Ca, and through producti on of chelating organic acids (citric, malic and 2 ketogluconic acid) which complex Ca and deplete P in the soil solution (Ramaekers et al., 2010) In a PR amendment study with white lupin ( Lupinus albus ), initial limited P availability stimulated root growth and exudation allowing for increased root induced dissol ution of PR (Hinsinger and Gilkes, 1995)

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54 In P. vittata P limitation has been shown to promote root growth (Santos et al., 2008) and increase As uptake which positively influences the plant's biomass (Tu and Ma, 2003) These unique responses by P. vittata could be taken advantage of to improve lo ng term phytoremediation. Use of a slow release P fertilizer (13 g plant 1 ) in a two year field experiment by Kertulis Tartar et al. (2006) led to relatively small P. vittata biomass in the first year (12.1 g plant 1 ) with a slight reduction (11.7 g plant 1 ) in the 2nd. In addition to maintaining low available P, appropriate clipping techniques need to be determi ned to maximize plant re growth. In a sixteen month pot study with three harvests by Gonzaga et al. (2008) clipping of P. vittata fronds at the rhizome base hindered re grow th, leading to biomass declines of 74 and 40% in the 2nd and 3rd harvests. W e hypothesize that P. vittata can remove As on a long term basis, over multiple harvests by maintaining low available P to increase P scavenging responses which will enhance remed iation efficiency. The objectives were to: 1) demonstrate the feasibility of using P. vittata to remediate As contaminated soils over a long period, 2) study the effect of PR amendments on P/As uptake, and 3) determine if proper husbandry practices impro ve re growth of biomass between harvests. Materials and Methods Soil Collection Three soils were collected from As contaminated areas in central Florida. Two soils (A o and B t horizons; Arenic Albaqualfs) were collected from abandoned cattle dipping vat s ( DVA and DVB), contaminated with an arsenical tickicide, and a third soil (A horizon; Grossarenic Paleudult) from an abandoned wood treatment facility which used copper chromate arsen ate (CCA). Soils were air dried, sieved through a 2 mm mesh screen and an alyzed for pH (1:2 soil to water), organic matter content (Walkley

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55 Black method), cation exchange capacity (ammonium acetate method) and particle size (pipette method) (Tan, 2005) S oil samples were subjected to HNO 3 /H 2 O 2 digestion (USEPA Method 3051) on a hot block (Environmental Express, Ventura, CA). The digested samples were analyzed for total As concentration using graphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer SIMMA 6000, Perkin Elmer Corp., Norwalk, CT) and total P was measured spectrophotometrically (UVI1800U, Shimadzu Corp., Columbia, MD) at 880 nm using the molybdenum blue reaction. Due to arsenate interf erence with the molybdenum reaction, samples were first incubated with cysteine at 80C for 5 min to reduce arsenate (AsV) to arsenite (AsIII). Water soluble P and exchangeable fractions of As were analyzed using extracts of deionized wate r (1:2 soil to s olution ratio) and 0.05 M (NH 4 ) 2 S O 4 /(NH 4 ) H 2 PO 4 (1:4 soil to solution ratio) respectively The digests were used to determine total Fe, Al, Ca, and Mg by ICP AES. Selected physico chemical properties of the soils are shown in Table 4 1. Experimental Setup Raised beds were constructed (0.36 m 2 to a 35 cm depth) and filled with s oil (four beds per soil) mixed with 15 g 1 kg 1 phosphate rock [PR, Ca 10 (PO 4 ) 6 F 2 (CaCO 3 )x, <1 mm; PCS Pho sphate, White Springs, Florida] and without PR as a control. The beds were w atered to field capacity whi ch was maintained for two weeks and had a final soil depth of 30 cm. In December 2009, three month old P. vittata (3 4 fronds ~15 cm in length) purchased from Milestone Agriculture (Apopka, Florida) were washed clean of potting mix and transplanted 15 cm apart (9 per bed ) in hand dug holes ~5 cm deep. Containers (20 L) with PR amended soil without plants were maintained as a negative control. At time of transplant, P free granulated fertilizer ( N:P:K ratio of 10:0:10; 2 8 g 1 b ed; 7 g 1 container, Rite Green; Sunniland Corporation, Sanford Florida) was surface

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56 applied to PR amended soils while a granulated fertilizer with P ( N:P:K ratio of 6:4:6; 35 g 1 bed ) was used on control soils which was repeated bimonthly. Overhead and drip irrigation were employed to maintain soil moisture ( 6 0 8 0% field capacity) which was measured with a Kelway HB 2 Acidity and Moisture tester (Kel Instruments, Wyckoff, New Jersey). One application of h ydrated lime was spread (28 g 1 quadrant; 7 g 1 c ontainer) to the surface of the dipping vat s oils (A and B). Plant Harvest Four harvests were conducted in six month intervals (July 2010, January 2011, July 2011, and January 2012). Frond biomass was collected by cutting mature fronds ~ 20 cm above the rh izome, ensuring at a few leaflets remained and leaving young fiddleheads intact to expedite re growth. Samples were oven dried at 60 C for 96 h, weighed, and ground through a 2 mm mesh screen in a Wiley Mill (Thomas Scientific, Swedesboro, NJ). Frond ( 0. 1 g ) samples were subjected to HNO 3 /H 2 O 2 digestion and analyzed for As and P as previously described. Soil and Root Sampling At planting and each harvest, 30 cm soil cores were taking with an auger (3 cm diameter) approximately 7.5 cm from the base of the ferns. Samples were taken before subsequent fertilizer applications to minimize influence of soluble P in controls. Two cores were taken from each bed, separated by depth (top 0 15 cm and bottom 15 30 cm) and composited. Samples were immediately sieved through a 2 mm screen to separate root tissue which was then weighed. Soil samples dried at 60C for 48 h and analyzed for elemental analysis as previously described.

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57 Statistical Analysis Data are presented as the mean of all replicates and error bars ( where shown) represent one standard error either side of the mean Significant differences were multiple range test, at p 0.05. Results and Discussion Soil C haracteristics Select physico chemical properties of soils at time of collection and following the two year experiment are listed in Tables 4 1 and 4 2. The textural classes of the CCA, DVA and DVB soils were loamy sa nd, sand, and sandy loam respectively. Dipping vat soils (A and B) were acidic (~pH 5.2) and lightly contaminated (26 to 30 mg kg 1 As) The calcareous CCA soil was slightly alkaline (pH 7.2) and moderately contaminated with As at 129 mg kg 1 These con centrations substantially exceed the SCTL of 2.1 mg kg 1 for Florida, indicating they are a potential health risk and require remediation. The fraction of As in the soil that is more available for plant uptake needs to be considered when employing phytore mediation. Plant available As depends on adsorbing soil constituents (i.e. Fe, Al, Ca ) pH, organic matter, and clay minerals (Zhang and Selim, 2008) Iron and Al oxides and hydroxides have particularly high affinity to As and adsorption i ncreases in presence of Ca (Smith et al., 2002) The CCA soil had the highest total exchangeable As (9.5 mg kg 1 ) and DVA/B had 3.2 and 4.0 mg kg 1 respectively. However, in the CCA soil, exchangeable As accounted for 7% of total As compared to ~13% in DVA and DVB soils The smaller fraction of exchangeable As in CCA soil might be associated with the relatively high concentrations of available Ca (1541 mg kg 1 ) along with amorphous Fe (20 04 mg kg 1 ) and Al (854 mg kg 1 ), which

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58 more strongly bond As, making them less available. In the DVA/B soils, exchangeable As was likely controlled by amorphous Al (~512 mg kg 1 ) as the available Ca (~194 mg kg 1 ) and amorphous Fe (~56 mg kg 1 ) were low, explaining the higher percentage of exchangeable As relative to CCA soil. P hosphate rock amendments contained 9 % P, 2 4% Ca, 3% K and 2% Mg, and did not significantly alter soil pH, soluble P, or available Ca, K and Mg. However, control soils were slight ly more acidic after two years, possibly due to buffering effects of PR ( Table 4 2 ). Initial total P concentrations were 382, 166 and 500 mg kg 1 in CCA, DVA and DVB soils respectively. Initial water soluble P concentrations were <0.4 mg kg 1 in all soil s and did not increase with addition of PR amendments, despite total P concentrations increasing to ~2300 mg kg 1 (data not shown). S oil related factors that increase dissolution of PR are low pH, high cation exchange and high P sorption (Robinson et al., 1994) The soluble P concentration remained stable in PR amended plant less soils (data not shown), suggesting that soil proper ties had little effect on PR dissolution. This was not entirely unexpected because the PR was very course (size fraction: 0.05 2.0 mm) and soils were not highly acidic with relatively low cation exchange ( Table 4 1 ). When PR is used to supply P to field crops, it is often ground to a fine powder or partially acidulated to expedite dissolution (Robinson et al., 1994) S oluble P in control soils, which were supplied with P fertilizer, ranged between 4 to 10 mg kg 1 throughout the two year experiment, a ~20 fold increase over the PR amended soils (data not shown). Minimizing soluble P in soils is advantageous when nutrient leachin g is a concern, which is an added benefit of using PR in lieu of soluble P fertilizer.

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59 H arvest S cheme I mproved R e growth of P. vittata Hyperaccumulation based phytoremediation is influenced by rate and amount of biomass production. Pteris vittata is a p erennial fern requiring repeated frond removal to facilitate the remediation process. After six months of growth, regardless of treatment, harvested frond biomass averaged 17 g plant 1 increasing to 19, 28, and 34 g plant 1 in the 12, 18, and 24 month ha rvests, respectively ( Figure 4 1 ). A six month harvest interval was used because it coincides with peak frond maturity which minimizes As loss via frond senescence (Kertulis Tartar et al., 2006) and spore dispersal (Lombi et al., 2002) Furthermore, at harvest, fronds were clipped ~15 cm from the rhizome base and fiddleheads were left intact to promote faster re growth. Overall, frond clippings yielded an average 100 g (or 50 g year 1 ) of biomass over four harv ests, with an increase of 26% in between each harvest ( Figure 4 1 ). In similar long term phytoremediation experiments with multiple harvests, yearly average biomass yields were 12, 6, 32, 40, and 8 g plant 1 (Caille et al., 2004 ; Li et al., 2005; Kertulis Tartar et al., 2006; Gonzaga et al., 2008; Shelmerdine et al., 2009) In each experiment, re growth was slowed, reducing harvestable biomass in subsequent harvests. For example, in separate pot studies, initial harvests of 13 and 29 g plant 1 were reduced to 4 and 8 g plant 1 in the 2nd harvest, an average reduction of 73% (Caille et al., 2004; Gonzaga et al., 2008) These declines were attributed to cutting fronds at the rhizome, which severely hinders the plant due to the lack of photosynthesis, forcing the plant to rely on carbon stores in the roots and rhizome for re growth (Wade and Westerfield, 2009) Furthermore, through the use of containers,

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60 plant and root environments are restricted due to small reservoirs for water and short substrate columns that adversely affect drainage (Fonteno, 1993) The results in this study represent the first report of increasing frond biomass of P vittata over subsequent harvests. Average increase in frond biomass between harvests exhibited a linear increase (R 2 = 0.95) which is likely attributed to increased soil volume (compared to containers), modified harvesting practices, and a consistent fe rtilizing regime. Phosphate R ock A mendments I ncreased P. vittata B iomass Pteris vittata which is native to soils with limited P availability, responded to PR amendments by increasing frond and root biomass relative to controls. In four harvests over two years, frond biomass of P. vittata in PR amended soils averaged 115 g plant 1 compared to 82 g plant 1 in control soils, a 40% increase ( Figure 4 1 ). Similarly, root density of two year old P. vittata plants (fresh weight) in PR amended soils averaged 64 g kg 1 soil compared to 42 g kg 1 soil in control treatments, a 52% increase. In addition to having larger root biomass, P. vittata roots in PR amended soils had extensive root hair and adventitious root growth which were absent in control roots ( Figure 4 2 ). This increase in root mass maximizes interactions at the root soil interface, allowing for more nutrient acquisition, including PR mineralization (Pret et al., 2011) Similar observations have been made in Lupinus albus where limiting P availability increased root biomass and proteoid root development facilitating dissolution of PR in the rhizosphere (Hinsinger and Gilkes, 1995) This reaction is very species specific however, as many plants are not capable of mineralizing PR at a rate that provides enough P to meet the plant demands (Rajan and Watkinson, 1992) In a study with 15 wheat cultivars, average shoot biomass in PR treatments was reduced ~1.5 times

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61 compared to ammonium phosphate treatments (Yaseen and Malhi, 2009) The observed increase in growth suggests that P. vittata may be uniquely adapted to P limiting environments, especially due to its novel ability to hyperaccumulate and tolerate As. Phosphate R ock I mproved A rsenic U ptake in P. vittata Frond arsenic concentrations ranged from 764 3480 mg kg 1 in the first harvest to 264 1813 mg kg 1 in the fourth harvest. The addition of PR amendments increased average frond As concentrations ~78% in the first year and ~43% in the second in all soils ( Table 4 3 ). In CCA soil amended with PR, frond As concentrations averaged 3150 mg kg 1 in the first year and 2290 mg kg 1 in the second. C omparatively, CCA control fronds averaged 1500 mg kg 1 As in the first year and 1900 mg kg 1 in the second. The increased P. vittata biomass ( Figure 4 1 ) and As uptake in PR amendments doubled As accumulation from CCA soil, increasing from 172 in controls to 345 mg plant 1 over four harvests ( Table 4 3 ). This trend also extended to DVA and DVB soils, with PR amendments increasing As uptake in P. vittata to ~93 mg plant 1 from ~40 mg plant 1 in controls. The increased As uptake in PR amended soils can be partly attributed to the limited soluble P (<0.4 mg kg 1 ) maintained throughout the two year study. Arsenic and P exert antagonistic effect s on each other du ring plant uptake and transport (Meharg and Hartley Whitaker, 2002) Thus, even though soluble P fertilizers increas e As availability, root uptake is negatively impacted, resulting in lower frond As accumulation ( T able 4 3 ). The use of PR circumvents this problem by supplying the plant with a source of P without elevating soluble P concentrations in the soil. This encourages greater root exploration to increase PR mineralization, which has the added benefit of

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62 inc reasing As uptake. Furthermore, As is known to stimulate biomass growth in P. vittata In a study by Gumaelius et al. (2004) addition of 75 mg kg 1 As to growth media improved total P. vittata biomass by 20% while 375 mg kg 1 As increased growth by an additional 55%. Plants in fertilized control soils lacked these growth promoting benefi ts ( Figure 4 1 ) due to increased P availability which decreased plant affinity for As relative to PR amended plants ( Table 4 3 ). The approximate two fold increase in As uptake from harvested P. vittata biomass in PR amended soils is best illustrated by c omparing bioconcentration ratios. The bioconcentration ratio (BC), which is the ratio of frond to soil As concentration; showed plants in PR amendments were more efficient in extracting As in all soils ( Table 4 3 ). The sustained high BC rates demonstrate d by P. vittata in PR amendments are critical to facilitating more rapid As removal. Average BC ratio for total soil As increased from 21 in the control to 40 in PR amendments and from 169 to 327 for available soil As. Even though exchangeable As concentr ations were ~53% higher in control soils, BC rates were half of PR. The effect of PR amendments on BC was significant, which is impressive considering that BC ratios in our controls were very high. For comparison, in similar long term phytoremediation st udies using P. vittata BC rates for total As were reported to be 3, 6, 18 and 10 (Caille et al., 2004; Cao and Ma, 2004; Kertulis Tartar et al., 2006; Gonzaga et al., 2008) The lower BC's are likely attributed to poor re growth from severe clipping and absence of su fficient fertilization schemes. Pteris vittata P A cquisition During periods of the low P availability, p lants respond morphologically to facilitate acquisitio n of P from previously unavailable sources (Vance et al., 2003) Since P. vittata is native to calcareous soil s (Jones, 1987) they may share similarities with other

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63 acidifuge plants known to effectively mobilize P from previously unavailable sources (Strm et a l., 1994) In the PR treatments, P. vittata responded to low soluble P by increasing root biomass which would enhance P uptake. Total P concentration in fronds grown in PR treated soils averaged 1 975 mg kg 1 compared to 2 26 0 mg kg 1 in the controls ( T able 4 5 ). Since PR is highly insoluble and the control plants were supplied with a continuous supply of P, it was not surprising that frond biomass from PR treatments had slightly lower concentrations of P. However, considering the increased frond bioma ss in PR treatments, total P uptake over four harvests increased from 181 mg plant 1 in controls to 224 mg plant 1 a 23% increase in the PR amended soil ( figure 4 3 ). The P limiting environment in the PR treatments induced greater root biomass in P. vitt ata containing more adventitious roots and root hairs ( Figure 4 2 ) which are more metabolically efficient in acquiring P due to the large absorptive surface area relative to the root volume (Ramaekers et al., 2010) Furthermore, P. vittata roots increase exudation of dissolved organic carbon content including oxali c and malic acid in P limiting conditions (Lou et al., 2010) In the PR amended soil, these exudates would facilitate P acquisition by increasing dissolution of PR. Frond tissue in PR amended soils averaged 2480, 1680, and 1720 mg kg 1 P in CCA, DVA and DVB soils respectively. Frond biomass from CCA soils had the highest P concentrations despite the alkaline soil conditions, suggesting that mineralization of PR was root mediated. Similar results have been observed in L. albus whose larger root biomass and proteoid root development was shown to dissolve PR in an alkaline soil (Hinsinger and Gilkes, 1995) Furthermore, soluble P in plan t less PR amended soils did not increase over time (data not shown), indicating PR solubilization was not influenced by soil properties.

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64 Soil Arsenic R emoval Regardless of treatment, P. vittata significantly lowered As concentrations ( p < 0.05) in a ll three soils, exhibiting a linear decline over two years (R 2 > 0.97; Figure 4 4 ). Phosphate rock amendments improved As removal, decreasing concentrations in all soils ~41% compared to ~27% in controls. The largest decline after two years was observed i n the CCA soil, which began at 130 mg kg 1 lowering to 98 mg kg 1 in control and 88 mg kg 1 in PR treatments. A similar trend was observed in DVA and DVB soils, with average As reductions of 28% in control and 46% in PR amendments ( Figure 4 4 B and C). C ompared to controls, PR amendments removed 7% (10 mg kg 1 ), 12% (3 mg kg 1 ), and 24% (7.1 mg kg 1 ) more As from CCA, DVA and DVB soils respectively. Following two years of growth with P. vittata depletion of exchangeable As in CCA soil was reduced from 9.5 to 4.3 mg kg 1 in PR amendments and to 6.4 mg kg 1 in control treatments ( Table 4 2 ). In DVA and DVB soils, exchangeable As was reduced 18 and 36% in PR amendments, respectively while increasing ~11% in control fractions. Higher concentrations of ex changeable As in control soils can be attributed to the 21 fold increase (0.19 to 4.0 mg kg 1 ) in soluble P from fertilizers which displace As due to competition at soil sorption sites (Smith et al., 2002) In our experiment, the exchangeable As concentrations did not correlate with of As uptake by P. vittata After two years, control soils contained 34% more exchangeable As than PR amended soils, but As uptake by P. vittata remained more e fficient in PR amended soils ( Table 4 4 ). Depth of As removal is another important factor to consider in successful phytoremediation. Regardless of soil or treatment, approximately 16% more As was removed from the top 15 cm of soil than the bottom 15 30 cm ( Figure 4 5 ). The root densities were similarly structured, with slightly less biomass associated with the 15 30

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65 cm fraction (data not shown). This shows that P. vittata can readily access the top 30 cm of soil, further exemplifying its ability to rem ediate As. In both treatments, P. vittata removed equal or more As in the 2nd year compared to the 1st, showing that remediation efficiency increased over time. This observation is unique compared to other phytoremediation experiments where As removal de clined after the first harvest, increasing estimated time length for remediation (Caille et al., 2004; Gonzaga et al., 2008; Kertulis Tartar et al., 2006; Shelmerdine et al., 2009) The combination maintaini ng low soluble P with PR, increased soil volume, and improved husbandry practices were shown to drastically improve rate and efficiency of soil As remediation using P. vittata ( Table 4 6 ). Based on the rate of As removal, we estimate that the SCTL of 2.1 mg kg 1 could be achieved in approximately 6, 5, and 4 years for CCA, DVA, and DVB soils respectively. Based on As removal from similar experiments, we have shown that P. vittata can remediate moderately contaminated soils 5 10 times faster than previousl y reported ( Table 4 6 ). Conclusion The majority of outdoor wooden structures in the U.S. were treated with arsenical pesticide which readily leaches into soil. Due to the abundance and relatively small areas affected, phytoremediation using P. vittata is ideal to clean and protect public health. In this study, we show that maintaining low soluble P concentrations in natural soil environments with improved husbandry practices dramatically improves the remediation capacity of P. vittata Furthermore, we sh ow that P. vittata can accumulate As more efficiently over time, suggesting it is a viable option to achieve SCTL's. Pteris vittata provides a plausible, sustainable, and affordable solution to the pervasive soil As

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66 contamination in the U.S. and around th e world, especially for residential areas, where problems need to be addressed

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67 Table 4 1 Selected physiochemical properties of soils used in this study Soil characteristic CCA DVA DVB pH 7.2 5.3 5.1 Total As (mg kg 1 ) 129.4 25.5 29.9 Exchangeabl e As (mg kg 1 ) a 9.5 4.0 5.5 Water soluble P (mg kg 1 ) 0.38 0.09 0.09 Amorphous Al (mg kg 1 ) b 854 5 43 481 Amorphous Fe (mg kg 1 ) 2004 83 29 Available Ca (mg kg 1 ) c 1541 132 256 Available Mg (mg kg 1 ) 115 18 54 Available K (mg kg 1 ) 27 12 23 Organic m atter (%) 1.1 2.2 0.4 CEC (cmol + kg 1 ) 7.8 3.3 12.4 Sand (%) 86.3 95.5 80.7 Silt (%) 9.9 2.7 6.6 Clay (%) 3.8 1.8 12.7 Textural class Loamy sand Sand Sandy loam a Ammonium phosphate, 0.05 mM b Oxalic acid + ammonium oxalate, 0.2 M c Mehlich III

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68 Table 4 2. P hysicochemical properties of soils amended with phosphate rock (PR) or control treatments following two years of growth with P. vittata CCA DVA DVB Soil characteristic PR Control PR Control PR Control pH 7.3 0.1 7.0 0.1 5.8 0.1 5.5 0. 2 5.8 0.2 5.0 0.1 Total As (mg kg 1 ) 88.3 3.2 98.2 3.8 15.2 0.5 18.2 0.6 14.5 1.0 21.6 0.9 Exchangeable As (mg kg 1 ) a 4.3 0.2 6.4 0.8 3.3 0.2 4.4 0.1 3.5 0.6 6.2 0.3 Water soluble P (mg kg 1 ) 0.38 0.01 4.2 0.2 0.15 0.01 4.0 0.3 0.15 0 .01 4.0 0.4 Amorphous Al (mg kg 1 ) b 870 26 947 34 484 16 358 48 281 15 400 12 Amorphous Fe (mg kg 1 ) 1958 29 2165 54 63 1.3 53 14 28 1 31 3 Available Ca (mg kg 1 ) c 1680 96 1878 153 192 8 239 65 334 80 274 77 Available Mg (mg kg 1 ) 23 5 9 298 6 73 7 92 6 156 37 131 4 Available K (mg kg 1 ) 83 17 127 41 37 8 30 5 37 6 151 15 a Ammonium phosphate, 0.05 mM b Oxalic acid + ammonium oxalate, 0.2 M c Mehlich III

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69 Table 4 3. Arsenic concentration and uptake in P. vittata frond biomass Frond As concentration, mg kg 1 Frond As uptake, mg plant 1 Soil Treatment Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 1 Harvest 2 Harvest 3 Harvest 4 CCA PR 3480 328 2826 203 2762 112 1813 65 70 11 82 8 107 9 86 6 Contr ol 1489 360 1222 58 2764 199 1665 99 21 6 26 2 71 9 54 10 DVA PR 1387 192 909 14 767 110 524 40 32 6 17 1 23 3 18 1 Control 771 13 385 36 843 110 523 26 11 2 6 1 21 4 17 3 DVB PR 1798 261 869 64 724 80 681 97 33 5 17 1 22 4 23 3 Control 764 43 439 164 251 38 264 20 10 1 5 2 4 1 6 1

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70 Table 4 4. Bioconcentration f actor a Exchangeable S oil As b Total S oil As Soil Treatment Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 1 Harvest 2 Harvest 3 Harvest 4 CCA PR 518 49 380 27 607 31 421 10 29 3 25 2 27 2 21 1 Control 242 59 140 7 441 32 260 16 12 3 10 1 26 2 17 1 DVA PR 392 54 331 5 210 20 158 8 61 8 48 1 45 5 35 2 Control 203 4 121 11 165 19 118 6 31 1 16 2 42 5 27 1 DVB PR 281 41 376 28 149 11 194 18 70 9 38 3 31 2 50 9 Control 175 10 94 35 52 9 43 3 27 2 17 6 12 2 12 1 a Concentration ratio of arsenic in fronds to soil b Ammonium phosphate, 0.05 mM

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71 Table 4 5. Frond P concentration, mg kg 1 Soil Treatment Harvest 1 Harvest 2 Harvest 3 Harvest 4 CCA PR 2569 188 2519 110 2774 60 2066 74 Control 3052 343 2735 397 3293 237 2424 118 DVA PR 1901 23 1825 48 1524 41 1557 35 Control 1743 45 2009 291 2134 73 2764 83 DVB PR 2133 58 1778 75 1523 16 1530 42 Control 1579 21 1688 21 1701 28 1829 66

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72 Table 4 6. Multi harvest phytoremediation studies with P. vittata grown in moderately a contaminated soils Reference Frond biomass b g year 1 As uptake mg plant 1 yea r 1 BC Soil As removed c kg ha 1 year 1 Time d to remediate, years Gonzaga et al. (2008) 40 32 10 14 ~17 30 Kertulis Tartar et al. (2006) 12 40 18 18 ~18 28 Caille et al. (2004) 24 18 3 8 ~25 63 Shelmerdine et al. (2009) 6 1 <1 <1 >100 Li et al. (2 005) 6 15 18 7 ~67 70 This study 76 172 26 76 ~6 7 a Soil As concentration range was 100 to 360 mg kg 1 b Average harvested biomass normalized to one year c Assuming 15 cm plant spacing on a soil containing 125 mg kg 1 As d Time estimates are based on the highest and average plant As uptake values reported and assumes a final soil As concentration < 2 mg kg 1

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73 Figure 4 1. Harvested frond biomass from P. vittata (g plant 1 dry weight) increased at each six month harvest in CCA, DVA and DVB s oils with phosphate rock amendments significantly improving biomass over the control ( p 0 5 10 15 20 25 30 35 40 45 Harvest 1 Harvest 2 Harvest 3 Harvest 4 Frond biomass (g plant 1 DW) Phosphate Rock Control

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74 Figure 4 2 Two year old P. vittata roots growing in phosphate rock amended CCA soils contained abundant adventitious roots and root hairs. Photo courtesy of Jason Lessl.

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75 Figure 4 3 Total P uptake in P. vittata frond biomass collected at each six month harvest from CCA, DVA and DVB soils with phosphate rock (PR) and control (C) amendments from July 2009 to Jan 2012. Values are the mean of 4 replicates with standard error. 0 20 40 60 80 100 120 CCA PR CCA C DVA PR DVA C DVB PR DVB C Frond P uptake (mg plant 1 ) Soil and treatment Harvest 1 Harvest 2 Harvest 3 Harvest 4

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76 Figure 4 4 Soil As concentrations (mg kg 1 ) declined at a linear rate in CCA (A), DVA (B) and DVB (C) soils over two years. Phosphate rock amendments significantly improved As removal in all soils compared to controls ( p The soil line represents the plant less control. Values are the mean of 4 replicates, bars representing one standard error.

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77 Figure 4 5 Soil As removed from the top 0 15 and bottom 15 30 cm fractions of CCA, DVA and DVB soils with phosphate rock and control amendments after two years of P. vittata growth. Values are the mean of 4 replicates, bars representing one standard error. 0 10 20 30 40 50 60 0 15 cm 15 30 cm 0 15 cm 15 30 cm 0 15 cm 15 30 cm CCA DVA DVB Arsenic removed (%) Soil and sampling depth Phosphate Rock Control

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78 CHAPTER 5 A RSENIC DISTRIBUTION IN THE SO IL AND FROND OF PTERIS VITTATA L. DURING P HYTOEXTRACTION HARVESTED OVER TWO YEARS Arsenic Soil Distribution Human exposure to arsenic increase s mortality through acute toxicity and multiple internal organ cancers (liv er, kidney, lung, and bladder) (ATS DR, 2009) ATSDR ranks arsenic number one on the 2001 CERCLA Priority List of Hazardous Substances (ATSDR, 2007a) The soil arsenic chronic Reference Dose Media Evaluation Guide (RMEG) is 20 mg kg 1 for non carcinogenic effects and 0.5 mg kg 1 f or the Cancer Risk Evaluation Guide (CREG) (MPCA, 1999) One of the most pervasive sources of soil As contamination comes from arsenical insecticides in wood treatments. Until 2002, m ore than 90% of all outdoo r wooden structures in the United States were made with As treated lumber which averages 1200 mg kg 1 As, accounting for an estimated 550 million pounds of arsenic from 1964 to 2001 (Gray and Houlihan, 2002) Arsenic continually leaches from t reated wood acting as a large reservoir for environmental contami nation (Belluck et al., 2003) Although a number of techniques exist to remove arsenic from soils, m ost sites remain contaminated due to high economic and environmental costs. Owing to the large number of potentially contaminated areas (i.e., residential decks, fencing, playgrounds), methods of environmental restoration using plant based technology offers a viable alternative. P hytoremediation is a cost effective and environmentally friend ly remediation method for contaminated soils that is especially well suited for As remediation associated from treated lumber. Due to its high rate of As accumulation, fast growth, and high production of biomass, Pteris vittata L. (Chinese brake Fe rn) is ideal for phytoremediation (Ma et al.

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79 2001). The capacity to remove high concentrations of arsenic from soil offers an easy, non invasive, and cost effective remediation method compared to traditional clean up techniques (U.S. Environmental Protection Agency, 2002) Successful phytoremediation depends on several factors including the e xtent of soil contamination, As arsenic into shoots. Understanding the mechanisms and processes that govern As uptake will help improve the utility of P. vittata as a viable clean up option. Arsenic is slowly mobile in s oils and exists predominantly in its most stable form, As(V), a deprotonated oxyanion includ ing the arsenate anion, AsO 3 4 A rsenic sorption largely depends on the amount of amorphous Al and Fe oxyhydroxides whic h exist as colloidal precipitates, surface coatings and along edges of clay minerals (Bissen and Frimmel, 2003) The continual physical, chemical and biological processes in soil effect As redistribution among solid phase components. Using the sequential extraction procedure developed by Wenzel et al. (2001) arsenic soil distribution can be operationally defined as being soluble exch angeable amorphous hydrous oxide bound, crystalline hy drous oxide bound, and residual. The soluble and exchangeable fractions represent the most e nvironmentally important forms of As due to their increased bioavailability Furthermore, these fractions a re a good indicator of bioavailability for plant uptake since plants preferentially take up their nutrients from the soil solution (Linehan et al., 1985) (McBride, 1994). In a phytoremediation experiment by Fitz et al., (2003) soil As concentrations associated with the bioavailable fraction did not decrease during the 41 d experiment despite s ubstantial As removal by P. vittata This was attributed to the large buffer ing

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80 capacity of the soil with As from amorphous and crystalline bound fractions replenishing the bioavailable fractions. This was also observed by Gonzaga et al (2006) during a 56 d phytoremediation experiment with P. vittata who found most As removed from soil was associated with the the amorphous bound fracti on. T he capacity of the soil to replenish the soluble and exchangeable forms of metals depend on the diffusion rates (Hinsinger et al., 2005) F ractionation provides an understanding of the relative mobility and bioavailability of metals in soils (Fitz and Wenzel, 2002; Gonzaga et al., 2006) This is because plant met al uptake or metal toxicity is related to those fractions (Gulz et al. 2005) Soluble and exchangeable forms of any nutrient or metal are considered to be the most availab le to plants (Jungk, 2001) Al l t hese factors influence the ability of P. vittata to access As from soils, but little is understood about the As redistribution over several years. Monitoring changes of bi oavailable As fractions and its r edistribution from less available pools during long periods of phytoextraction is essential to evaluate the efficiency of phyto remediation (Fitz and Wenzel, 2002; Wenzel et al., 2003) In order to understand how different fractions change As lability during r emediation over a long period we used an operationally defined sequential fractionation method to evaluate the effect of As distribution in soils by P. vittata over several growing seasons. Materials and Methods Soil Collection Three soils were collected from As contaminated areas in central Florida. Two soils (A o and B t horizons; Arenic Albaqualfs) were collected from abandoned cattle dipping vat s (DVA and DVB), contaminated with an arsenic al tickicide, and a third soil (A horizon; Grossarenic Paleudult) from an abandoned wood treatment facility which

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81 used copper chromate arsen ate (CCA). Soils were air dried, sieved through a 2 mm mesh screen and analyzed for pH (1:2 soil to water), organic matter content (Walkley Black method), cation exchange capacity (ammonium acetate method) and particle size (pipette method) (Tan, 2005 ) Plant and s oil samples were subjected to HNO 3 /H 2 O 2 digestion (USEPA Method 3051) on a hot block (Environmental Express, Ventura, CA). The digested samples were analyzed for total As concentration using graphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer SIMMA 6000, Perkin Elmer Corp., Norwalk, CT) The digests were also used to determine total Fe, Al, Ca, and Mg by ICP AES. T he improved sequential extraction procedure developed by Wenzel et al. (2001) was followed to fractionate arsenic into five operationally defined fractions, including soluble ( S ), exchangeable ( E ), amorphous hydrous oxide bound (A), c rystalline hydrous oxide bound (C), and residual (R). Experimental Setup Raised beds were constructed (0.36 m 2 to a 35 cm depth) and filled with s oil (four beds per soil) mixed with 15 g 1 kg 1 phosphate rock [PR, Ca 10 (PO 4 ) 6 F 2 (CaCO 3 )x, <1 mm; PCS Pho sphat e, White Springs, Florida]. In December 2009, three month old P. vittata (3 4 fronds ~15 cm in length) purchased from Milestone Agriculture (Apopka, Florida) transplanted 15 cm apart (9 per bed ). Containers (20 L) with PR amended soil without plants were maintained as a control. G ranulated fertilizer ( N:P:K ratio of 10:0:10; 2 8 g 1 bed; 7 g 1 container, Rite Green; Sunniland Corporation, Sanford Florida) was applied bimonthly. A roof was constructed over the beds using c lear corrugated plastic roofing material with a light transmission rating of 93% Overhead and drip irrigation were employed to maintain soil moisture ( 6 0 8 0% field capacity) which was measured with a Kelway HB 2 Acidity and Moisture tester (Kel Instruments, Wyckoff,

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82 New Jersey). An application of lime was spread (28 g 1 quadrant; 7 g 1 container) to the surface of the dipping vat s oils (A and B). Soil Sampling and Plant Harvest F our harvests were conducted in six month intervals (July 2010, January 2011, July 2011, and January 2012). Fronds were collected by cutting stems ~ 20 cm above the rhizome and were immediately separated by age. Young and mature fronds were differentiated by the development of sori (green/absent or brown) while senescent tissues were characterized by a browning of the leaflets. Fronds were further separated by stem (rachis and stipe) and leaflets. At planting and each harvest, 30 cm so il cores were taking with an auger (3 cm diameter) approximately 7.5 cm from the base of ferns. Samples were oven dried at 60 C for 96 h, weighed, and sieved through a 2 mm mesh screen. Soil and plant digests were subjected to HNO 3 /H 2 O 2 digestion for As analysis. Soil and mature frond digests were used for elemental analysis Statistical Analysis Data are presented as the mean of all replicates and error bars (where shown) represent one standard error either side of the mean Significant differences wer e multiple range test, at p 0.05. Results and Discussion Soil Characteristics Select physico chemical properties of soils at time of planting are listed in Table 1. The calcareous CCA soil was slightly alkaline (pH 7.2) and moderately contaminated with As at 129 mg kg 1 Dipping vat soils (A and B) were acidic (~pH 5.2) and lightly

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83 contaminated with As (26 to 30 mg kg 1 ). These As concentrations exceed the RMEG and CREG, indicating they are a potential health risk and require remediation. P hosphate rock amendments were very cour se (size fraction: 0.05 2.0 mm) and contained 9 % P, 2 4% Ca, 3% K and 2% Mg which did not significantly alter soil pH, soluble P, or available Ca. Normally, PR is ground to a fine powder or partially acidulated to expedite dissolution (Robinson et al., 1994) However, to more accurately assess the effect P. vittata has on As distribution, it was important to minimize soil P i concen trations as it displaces As in the exchangeable fraction. Water soluble P concentrations were <0.4 mg kg 1 in all soils and did not increase with PR amendments (Table 5 1) Furthermore, low soluble P has the advantage of lowering nutrient leaching, which is an added benefit of using PR in lieu of soluble P fertilizer. Biomass and A rsenic A ccumulation in Pteris vittata Pteris vittata is a perennial fern, so for phytoremediation, numerous frond harvests need to be employed to expedite the clean up process. A six month interval was used because it coincided with peak frond maturity. After four harvests in two years, P. vittata grown in CCA, DVA, and DVB soils produced 4.54, 3.45, and 3.49 kg of frond biomass (dry weight) with average As concentrations of 27 20, 900, and 1020 mg kg 1 respectively. Arsenic concentrations in the frond declined in subsequent harvests, from 3480 to 1813 mg kg 1 in CCA, 1387 to 524 in DVA, and 1800 to 681 in DVB soil s Each plant yielded an average 60 g of frond biomass per year, increasing 18% between six month harvests. In similar studies with P. vittata using multiple harvests, yearly average biomass yields were 12, 6, 32, 40, and 8 g plant 1 (Caille et al., 2004; Li et al., 2005; Kertulis Tartar et al., 2006; Gonzaga et al., 2008; Shelmerdine et al., 2009) These declines were attributed to cutting fronds at the rhizome, which severely hinders the

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84 plant due to the lack of photosynthesis (Wade and Westerfield, 2009) Our results showed that P. vittata proliferated with raised beds, PR amendments and the use of a regimented fertilizing schedule. Furthermore, plant re growth between harvests was promoted by leaving fiddleheads and a few leaflets from mature fronds. Frond As distribution was highest in P. vittata leaflets followed by spores and then stems. Tissue As concentrations correlated with total soil As levels (Table 5 1), with fronds from CCA soil co ntaining ~4 times more As than from DVA and DVB soils (Table 5 2). This also extended to spores, which contained ~1170 mg kg 1 As compared to ~146 mg kg 1 from plants in dipping vat soils. Arsenic concentrations declined in leaflets and stems at maturity However, mature tissues accounted for ~56% of the harvested biomass compared to ~20% in young leaflets, equating to substantially more As uptake. The As decline between young and mature tissues is likely attributed to a diluting effect due to plant gro wth. Of the leaflets, the senescent tissue had the smallest As concentrations while senescent stem concentrations were similar to mature. The continued As decline in senescent fronds can be attributed to As leaching and volatilization as they dry. Tu et al. (2003) found that during air drying of P. vittata fronds, As concentrations dropped ~ 15% In a field study with P. vittata As concentrations were 49 and 25% less in the senescent fronds over two harvests (Kertulis Tartar et al., 2006) Frond elemental concentrations fell into normal ranges for mature plants and were similar to a previous report on nutrient uptake by P. vitt ata (Tu and Ma, 2005) indicating that soil type or phosphate rock amendments had no apparent negative impact on overall plant health (Table 5 3). Except for P, As and Mn, elemental analysis of fronds

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85 showed little difference between all three soils. The higher P and As in fronds from CCA soil is due to the higher As concentrations, which have been shown to be correlate with each other in tissues. In a hydroponic study, the addition of As up to 30 mg L 1 induced higher P uptake by P. vittata (Tu and Ma, 2005) Fronds from the dipping vats contained more Mn because of the lower soil pH (Table 5 1), which increases mobility and uptake of Mn. Soil As Fractionation Arsenic sorption depends on soil constituents (i.e. Fe, Al, Ca ) pH, organic matter, and clay minerals (Zhang and Selim, 2008) Iron and Al oxides and hydroxides have particularly high affinity to As which increases in presence of Ca (Smith et al., 2002) To differentiate between As fractions, a five step sequential extraction procedure was used The first extraction step, 0.05M (NH 4 ) 2 SO 4 represents the most soluble (labile) As. In t he second extractio n, 0.05M NH 4 H 2 PO 4 was used to assess exchangeable As in that it can be specifically replaced by phosphate. Although not as easily released as the first fraction, the exchangeable fraction is also considered labile. The next three steps target As b ound t o amorphous and crystalline hydrous oxides of Fe and Al along with the residual fraction, which are all c onsidered non labile. Of the three soils, CCA had the highest amorphous Al, Fe and available Ca (Table 5 1). This was reflected in the As distribut ion of the CCA soil before planting, where 87% of the As (113.2 mg kg 1 ) was associated with the amorphous and crystalline fractions compared to 5% (6.8 mg kg 1 ) in the soluble and exchangeable fractions (Figure 5 1A). Comparatively, the DVA (Figure 5 1B) and DVB (Figure 5 1C) had a higher proportion of soluble and exchangeable As at 13 and 18% (3.3 and 5.4 mg kg 1 ) with ~75% (19.3 and 21.9 mg kg 1 ) associated with amorphous and crystalline

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86 fractions, respectively. The higher bioavailability of As in the dipping vat soils is likely due to low concentrations of amorphous Fe and available Ca compared to the CCA soil (Table 5 1). The residual fractions of all three soils were similarly proportioned at ~9.1%. Overall, the As frac tions in our soils were consi stent with data repo rted by Wenzel et al. (2001) who conducted As fractionations in twenty different As contaminated soils (96 2183 mg kg 1 ). Arsenic in their soils were primarily associated with the amorphous (~42%) and crystalline (~29%) fractions (Wenzel et al., 2001) which was similar to our soils at ~50% and ~30%, respectively. Following two years of phytoremediation with P. vittata As concentrations were significantly reduced in CCA (130 to 88 mg kg 1 ), DVA (26 to 15 mg kg 1 ) and DV B (30 to 14 mg kg 1 ) soils ( p P. vittata arises from the available fractions, after two years, a small decline was observed in the soluble with little change or slight increase in exchangeable fractions in the three soils (Figure 5 2). The lack of depletion from the exchangeable fraction can be attributed to As replenishment from the amorphous and crystalline fractions. In the CCA soil, amorphously bound As was reduced from 75.1 to 44.4 mg kg 1 while crystalline bound As was reduced from 38.1 to 30.5 mg kg 1 As (Figure 5 2A). A similar trend was observed in the dipping vat soils, with the greatest reduction of As associated with the amorphously bound As after two years followed by the crystalline fraction (Figure 5 2B an d C). In all three soils, ~22% of the total soil As reduction was associated with the amorphous fraction followed by ~13% in the crystalline. There was little change in the residual fraction of CCA soil (9.7 to 9.1 mg kg 1 ) and slight declines in the dip ping vat

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87 soils, accounting for 2.5 and 3.6% reductions in DVA (2.9 to 0.9 mg kg 1 ) and DVB (2.5 to 1.8 mg kg 1 ) soils respectively. Predicting A r s enic U ptake in P. vittata U sing S equential E xtraction D ata Regression analysis was used to assess potential c orrelation between frond and soil As concentrations. We compared the As in sequentially extracted fractions of soil during two years of phytoextraction to develop a model predicting As accumulation by P. vittata For most plants, a linear correlation exi sts between total As concentrations and plant growth inhibition (Sheppard, 1992) F urther investigation by Gulz et al. (2005) found that the As solubility, P availability and P de mand should be collectively considered to p redict As uptake in common plants. However, these models may not apply to P. vittata due to its unique ability to hyperaccumulate and tolerate high concentrations of As In a nine month pot study with P. vittata by Shelmerdine et al. (2009) a model was developed from soluble As data to predict the length of remediation needed to clean up a variety of As contaminat ed soils. While the model was found to correlate well with frond As concentrations in the study (R 2 = 0.71), it predicted that after 30 years, a soil contaminated with ~24 mg kg 1 would be reduced to ~15 mg kg 1 (Shelmerdine et al., 2009) In our study, a soil containing 30 mg kg 1 As (DVB) was reduced to 14 mg kg 1 after two years of remediation with P. vittata This highlights one of the benefits of a long term study, which could allow for more realistic predictions of remediation length. Frond bioconcentration, which is the ratio of As in fronds to soil, was most useful in predicting As uptake. The use of bioconcentration rates also normalizes rate of As uptake, allowing for com parisons of soils with wide ranges contamination. The pH was also considered because it affects the lability of As is in soil. Considering individual As

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88 fractions for model development, the best to worst in predicting frond As uptake were: amorphous > to tal > residual > crystalline > exchangeable > soluble. Despite plant mediated As uptake originating from the available pools, the soluble (R 2 = 0.46) and exchangeable (R 2 = 0.09) fractions did not accurately predict frond As accumulation. Normally, c once ntration s of soluble nutrients are a good indicator of bioavailability for plant uptake in soil since plants preferentially take up their nutrients from the soil solution (Neumann, 2007) However, t he supply of elements such as P a nd As at the root soil interface is limited by diffusion. If the rate of plant facilitated As uptake exceeds rate of soil As redistribution to the available fractions, concentrations will remain low and static while total As declines. This suggests that for more accurate predictions of As uptake, the pool responsible for replenishing the available fractions should be used. In our study, the greatest decline in As in all three soils was associated with the amorphously bound fractions (Figure 5 2). Regres sion analysis indicated that bioconcentration rates based on the amorphously bound As had good correlation (R 2 = 0.77) between frond As concentrations throughout the two year study. This model was further improved (R 2 = 0.94) when using the natural logar ithm of the ratio between available (S+E) and amorphously bound fractions (Figure 5 3). This model accounts for the relative difference between pools of As which are available for plant uptake and the source of primary replenishment, which was shown to de crease over time (Table 5 4). When the As in the amorphous fraction approaches equilibrium with the available, the rate of diffusion will slow, which accounts for the declining frond As concentrations i n subsequent harvests

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89 Conclusions It is important to be able to predi ct the time frame required to remediate contaminated soil. Inclusion of the amorphously bound fraction of As will aid in accessing the length of remediation required in achieving soil As cleanup levels. Phosphate rock amendments maintained low soluble P without negatively impacting plant health. Our results show that soil with moderate to low concentrations of Arsenic can be efficiently remediated using P. vittata Furthermore, by sequentially extracting As from soils, a reasonable time frame of clean u p can be predicted based on the ratio of available to amorphously bound As concentrations.

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90 Table 5 1. Select characteristics of soils used in this study Soil characteristic CCA DVA DVB Total As (mg kg 1 ) 129.4 25.5 29.9 Total Al (mg kg 1 ) 3661 1710 7455 Amorphous Al (mg kg 1 ) a 780 417 470 Total Fe (mg kg 1 ) 2227 324 1298 Amorphous Fe (mg kg 1 ) 1309 46 60 Total Ca (mg kg 1 ) 7300 356 700 Available Ca (mg kg 1 ) b 1540 129 244 Total P (mg kg 1 ) 382 166 516 Water soluble P (mg kg 1 ) 0.35 0.18 0.33 pH 7.2 5.3 5.1 Organic matter (%) 1.1 2.2 0.4 CEC (cmol + kg 1 ) 7.8 3.3 12.4 Sand (%) 86.3 95.5 80.7 Silt (%) 9.9 2.7 6.6 Clay (%) 3.8 1.8 12.7 Textural class Loamy sand Sand Sandy loam a Oxalic acid + ammonium oxal ate, 0.2 M b Mehlich III

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91 Table 5 2. Arsenic distribution in harvested P. vittata fronds Tissue Arsenic mg kg 1 Frond weight CCA DVA DVB (%) Y. leaflet 3472 1488 1735 16.7 M. leaflet 3246 1057 1297 47.3 S. leaflet 3053 611 864 18.1 Y stem 478 70 112 2.9 M. stem 227 40 43 6.3 S. stem 201 55 56 6.5 Spore 1116 149 143 2.3

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92 Table 5 3. Elemental analysis of harvested P. vittata fronds Element (mg kg 1) CCA DVA DVB As 2720 900 1020 P 2266 1806 1784 K 22314 20984 21475 Ca 5153 5132 5251 Mg 3217 3600 3621 Al 193 171 228 Fe 123 116 151 Cu 77 66 67 Mn 29 112 55

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93 Table 5 4 The ratio of amorphous:available arsenic in soil over two years Sample CCA DVA DVB 1 22.4 6.0 5 .3 2 17.5 3.3 3.1 3 17.3 2.6 2.8 4 13.2 2. 4 1.9

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94 Figure 5 1. Relative distribution of arsenic in the soluble (S), exchangeable (E), amorphous (A), crystalline (C) and residual (R) fractions at time of planting. 0 10 20 30 40 50 60 70 S E A C R Soil As (%) Extraction step CCA DVA DVB

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95 Figure 5 2. Arsenic in the soluble (S), exchangeable (E), amorphous (A), c rystalline (C) and residual (R) fractions of CCA (A), DVA (B), and DVB (C) soils during two years of growth with P. vittata

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96 Figure 5 3 Predictive model based on the natural logarithm of measured bioconcentration (BC) ratios between amorphous and ava ilable soil As fractions (A) to predict As uptake (B) in P. vittata

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97 CHAPTER 6 ROLE OF ARSENIC HYPERACCUMULATION IN PTERIS VITTATA Role of M etal A ccumulation in P lants E nvironmental stress plays a significant role in the evolution of biological systems, from gene to entire ecosystems. Many areas of the world are contaminated by metals, either naturally or by anthropogenic activity. All heavy metals, and metalloi ds such as arsenic and selenium are toxic to plants when concentrations exceed trace quantiti es (Macnair, 1997) The proper ties that make some metal ions essential for life can also be toxic in elevated concentrations The formation of hydroxyl radicals or high affinity binding to S, N, and O containing functional groups in biological molecules can cause inactivation and/or d amage. Furthermore, there are elements which can interfere with essential elements of the same group in clud ing Cd (for Zn), As (for P) and Se (for S), making them extremely toxic. (Clemens, 2006) The physiological range for essential metals between de ficiency and toxicity is narrow, requiring tightly controlled mechanisms to adapt to changes in micronutrient availability (Nelson, 1999) Some plant s can grow on soil containing metal concentrations that would normally inhibit growth. They possess naturally selected higher levels of tolerance, which is typically specific for ce rtain metals (Schat and Vooijs, 1997) Metal tolerance in natural plant populations is largely due to selective pressure from growing on soils enriched with metals Plants have a range of cellular mechanisms that might be involved in the detoxification and thus tolerance to heavy metal stress. Most plant tolerance is achieved by avoiding the build up of metal concentrations. Comparatively, there also exist plants with ubiquitous basal tolerance (e.g. Pteris vittata )

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98 Heavy metal phytotoxicity results from alterations of numerous physiological processes by inactivating enzymes, blocking functional groups, displacing or substituting for essential elements or disrupting membrane integrity (Rascio and Navari Izzo 2011) A common consequence of heavy metal poisoning is the enhanced production of reactive oxygen species (ROS) due to interference with electron transport activities (Pagliano et al., 2006) Increased ROS induces oxidative stress leading to lipid peroxidation, macromolecule deterioration, membrane dismantling, ion leakage, and DNA strand cleavage (Quartacci et al., 2001) Plants responses include a series of defense mec hanisms that control uptake, accumulation and translocation of metals and detoxify them by excluding the free ionic forms from the cytoplasm. A common plant response is to hinder entrance of metals into the root by binding them to exuded organic acids or to anionic groups of cell walls (Rascio et al., 2008) If heavy metals enter the plant, tolerant plants will often store them in root cells, where they can be detoxified by complexation with amino acids, org anic acids or metal binding peptides (Rascio and Navari Izzo, 2011) This restricts translocation to the above gr ound organs, protecting the metabolically active photosynthetic cells from damage. Another defense mechanism used by tolerant plants is enhancement of the antioxidant system to counteract oxidative stress (Sgherri et al., 2003) Typically, metal tolerant species rely on strategies which restrict metal translocation. However, many hyper tolerant (hyperaccumulators) species exhibit the opposite behavior as far as metal uptake and distribution in the pl ant (Hall, 2002) Genetic analysis of in tra and inter specific crosses determined that tolerance and hyperaccumulation are independent characters. For example, tolerance of Cu, Cd,

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99 or Zn in different Silene vulgaris accessions from normal soil and metalliferous sites are controlled by one or t wo major genes (pleiotropic) with additional modifiers controlling the level of tolerance (Schat, 1999) The selective factors driving th e evolution of hyperaccumulation are unknown and difficult to identify. The different hypotheses in the literature include: increased metal tolerance, protection against herbivores or pathogens, inadvertent uptake, drought tolerance, and allelopathy (Rascio and Navari Izzo, 2011) There is wide variability between populations of hyperaccumulator species in their capa city to tolerate and accumulate metals. For example, numerous arsenic hyperaccumulators have been identified in the Pteridaceae family, including Pteris vittata P. cretica P. longifolia and P. umbrosa and Pityrogramma calomelanos (Meharg, 2002) Though the number of ferns tested for arsenic hyperaccumulation is small, the Pteridaceae contain over 400 species, which means less than 1.25% can accumulate arsenic. Ferns are among the most primitive plants and morphological characteristics place the Pteridaceae in the latter portion of fern evolution. During the evolution of land plants, those that evolved in arsenic rich environments would hav e required mechanisms for coping with this element, with hyperaccumulation being one strategy. Arsenic tolerance could be a carryover from marine algae that contain arsenate in high amounts due to the scarcity of phosphate in seawater. Another theory sug gests the evolution of early terrestrial life began around arsenic rich hot springs (Meharg and Hartley Whitaker, 200 2) Tolerance mechanisms may have been lost as plants spread out into non arsenic contaminated environments, with members of the Pteridaceae retaining these mechanisms, either as evolutionary baggage or because this trait conferred them with some advanta ge, arsenic related or not (Meharg, 2002)

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100 Alternatively, the hyperaccumulation trait could have evolved later in response to selective pres sure, or perhaps the ferns were confined to arsenic rich habitats throughout their evolutionary development (Meharg 2002) It is also possible that arsenic had little to do with arsenic hyperaccumulation. The physiological mechanisms may have evolved for other reasons, conferring arsenic hyperaccumulation to the ferns as a side effect. This is the case for arsenic resistance in angiosperms where resistance is a consequence of suppressed high affinity phosphate transport (Lou et al., 20 10) as arsenate and phosphate are chemical analogues. Arsenic hyperaccumulation may therefore be a consequence of unusual phosphorus metabolism in ferns, although there is little evidence to suggest this. The most interesting aspects of metal toleranc e pertains to the plants that hyperaccumulate toxic elements (e.g. As, Pb, Cd), especially since the reasons for this unusual behavior remain elusive. Why do some plants do it, what functions does hyperaccumulation perform and what are the benefits and th e adaptive values of metal hyperaccumulation? Clemens (2006) hypothesizes three possible explanations: (i) the detoxification pathways for non essential metals play an important role in essential metal homeostasis; (ii) molecular determinants of toxic metal tolerance serve addit ional, as yet unknown, essential functions; (iii) toxic metals are actually essential elements and the detoxification pathways are part of the homeostatic network for these elements. In marine ecosystems, Cd has been observed to behave like a micronutrient Its distribution is similar to phosphate, in that concentrations remain low in surface water due to uptake by photosynthetically active algae and concentration increases with depth (Butler, 1998) The marine diatom Thalassiosira weissflogii shows improved growth

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101 under conditions of low Zn availability and low CO 2 when Cd ions are added to the medium. The algal cells were found to use Cd(II) as a co factor for a carboni c anhydrase that is expressed only under these conditions to replace the regular Zn requiring enzyme (Lane et al., 2005) In plants, accessions of the Zn/Cd hyperaccumulator T. caerulescens grow better in nutrient solution containing low concentrations of Cd than in nutrient solution without Cd (Clemens, 2006) In addition to translocation, the accumulation of toxic metals in plants remains an interesting puzzle. A variety of hypotheses have been proposed to explain the role of toxic metal accumulation in aboveground biomass, including: metal tolerance, metal disposal, improved stress responses, interference with neighborin g plants, and defense against herbivory. According to the tolerance/disposal hypothesis, the hyperaccumulation pattern allows plants to displace metals away from the roots and eliminating them from the plant body by shedding the high metal tissues. Heavy metals may increase plant drought resistance, with a water conserving role in the cell walls or acting as osmolytes inside the cells. The interference hypothesis (or elemental allelopathy), suggests that perennial hyperaccumulator plants interfere with n eighboring plants through enrichment of metal in the surface soil. This would be an addition al benefit to the tolerance/disposal hypothesis. The high metal leaf litter would potentially prevent the establishment of less metal tolerant species. High Ni l evels in the surface soil under the canopy of hyperaccumulator S. acuminata has been observed when compared to surface soil in non hyperaccumulator species (Boyd, 2001) Another hypothesis suggests that the toxic metal concentrations in aboveground biomass function as a self defe nse strategy against natural enemies, such as

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102 herbivores and pathogens. Recent studies confirm the defensive function of Ni (Jhee et al., 2006) Cd (Jiang et al., 2005) Zn (Behmer et al., 2005) As (Rathinasabapathi et al., 2007) and Se (Galeas et al., 2008) in plant defense. In spite of important progress made in recent years by the numerous studies accomplished, the complexity of hyperaccumulation is far from being understood. Metal tolerant and metal hyperaccumulator plants, which a re widespread on metal soils in both tropical and temperate zones of all the continents, belong to several unrelated families. This shows that tolerance has evolved more than once, further complicating the questions pertaining to evolutionary fitness. Mo re elements and tolerant species require examination in order to validate any one particular hypothesis of the defensive effects of heavy metals. In this study, we observed some of the beneficial roles that arsenic hyperaccumulation potentially play in P. vittata Specifically we looked at how arsenic effects P. vittata 1) growth in low nutrient environments ; 2) defense against herbivory; and 3) enrichment of arsenic at the soil surface Materials and Methods Fern and Gametophyte Setup Topsoil was air dried, sieved through a 2 mm mesh screen and analyzed for pH (1:2 soil to water), organic matter content (Walkley Black method) and particle size (pipette method) (Tan, 2005) Soils were separated into separate buckets and mixed with suspensions of sodium arsenate (Na 2 HAsO 4 2H 2 O) to achieve final concentrations of 25, 50 and 100 mg kg 1 As. Samples were subjected to HNO 3 /H 2 O 2 digestion ( USEPA Method 3051) on a hot block (Environmental Express, Ventura, CA). The digested samples were analyzed for total As concentration using graphite furnace

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103 atomic absorption spectroscopy (GFAAS, Perkin Elmer SIMMA 6000, Perkin Elmer Corp., Norwalk, CT). Three month old P. vittata (3 4 fronds ~15 cm in length) purchased from Milestone Agriculture (Apopka, Florida) were transplanted 15 cm apart ( 6 per bed). Growth was observed for one year with no fertilizer applications. Drought stress was simulated in the first month by allowing soil moisture levels to decline until visible wilt symptoms were observed, at which time, soil moisture was brought back to field capacity where it was maintained for the duration of the experiment. Spores from P. vittata were collected from plants growing in arsenic contaminated soils with varying concentrations (25 130 mg kg 1 As) and surface sterilized in a 20% bleach solution for 20 minutes followed by three washes in sterile DI water. Spores were suspended in 2 mL sterile DI water. Half strength modified Murashige & Skoog (MS) media was prepared with 0.8% agar without P prior to autoclaving. Phosphate, phytate, and arsenate solutions were filter sterilized and added to autoclaved MS media to obtain final concentrations of 0.6 mM P as P i (KH 2 PO 4 ) or phytate (P 6 ; myo inositol hexaphosphoric acid dodecasodium salt) with 0 or 0.6 mM arsenate. The MS media (pH 6.5) was then poured into sterile petri dishes (100 mm 13 mm). Spores (10 L or 0.05 mg spore) were placed on agar ( 1 0 per plate, 4 plates per treatment) under cool/warm fluorescent lamps at 25C and 60% humidity for 3 0 d. Herbivory Setup During the summer of 2009, caterpillars were observed eating P. vittata leaflets growing in a campus greenhouse. The caterpillars were cap tured and sent to the Florida department of Agriculture and Consumer Sciences who identified them as Spodoptera latifascia (ID# E2009 7753 1). To gauge the role of arsenic accumulation in herbivory prevention, f ive month old P. vittata ferns were transfer red to hydroponic

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104 culture in 0.2 strength Hoagland Arnon nutrient solution (HNS) containing As. The caterpillars identified as S. latifascia were transferred onto ferns to observe their herbivory behavior. Frass and leaflet samples were taken after one week to analyze As concentration by digestion analysis as previously described. Allelopathy Experiment Pteris vittata ferns were observed growing naturally at an apartment complex in Gainesville Florida built in 1980. The ferns were isolated to areas underneath stairwells which were constructed from arsenic treated wood. According to the management, the ferns, which were not planted, had been left unattended for at least 6 years. Samples were taken from 25 different stairwells containing actively growing P. vittata Soil cores (20 cm deep) were collected at the base of P. vittata plants and a distance of 50 cm directly away. Soil cores were separated by top (0 2 cm) and bottom (18 20 cm) to analyze total arsenic as previously described. Statistical Analysis Data are presented as the mean of all replicates with standard error. Sig nificant dif ferences were determined using analysis of variance and treatment means compared Results and Discussion The Effect of Arsenic on P. vittata Growth The soil was 89% sand, 6% silt and 5% clay with a pH of 7.5. At the beginning of the experiment, average fresh weight of transplanted P. vittata plants was 31 g. There was no observable difference between treatments during the first three weeks of growth (Figure 6 1) During the fourth week, the ferns were subjected to drought stress, which was characterized by a wilting of the fronds. Pteris vittata is sensitive to drought, which

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105 was reflected by the wilting tissues immediately senescing. One week following the drought stress, and one month into the experiment, the ferns had lost all photosynthetic tissues. At this point, new frond growth is required in order for plants to survive. New frond growth was observed one week after the drought stress in plants growing in the As treated soils while one plant in the control soil be gan producing frond growth. For the next four months, plants in the arsenic treated soil continued to proliferate while the remaining plant in the control soil remained stunted (Figure 6 1) After six months, average frond biomass (dry weight) was 2, 18, 36, and 32 g plant 1 with arsenic concentrations of 70, 3700, 8700 and 9600 mg kg 1 in soil with 0, 25, 50 and 100 mg kg 1 As, respectively. Despite the toxicity associated with arsenic, P. vittata is able to survive high concentrations (Ma et al., 2001). Many heavy metals stimulate the formation of free radicals and reactive oxygen species (ROS), lead ing to uncontrolled oxidation and radical chain reactions, which stress the plant (Zaman and Pardin i, 1996) Drought stress has been shown to induce the same responses in plants (Mittler, 2002) In P. vittata the presence of As has been shown to stimulate the production of anti oxidants like superoxide dismutase, catalase, and ascorbate peroxidase (Srivastava et al., 2005) Thus, in the presence of arsenic, an increase in anti oxidant enzymes may improve survivability of P. vittata during dro ught stress To gauge the effect of arsenic on spore germination and growth, spores were germinated on MS media. Although the presence of arsenic in the media significantly improved biomass, pre exposure to arsenic had little impact on rate of biomass g rowth over a 30 day period (Table 6 1) Spores containing no arsenic prior to germination

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106 generally exhibited the highest biomass, indicating that pre exposure of arsenic may actually slow growth, even though presence of arsenic in the media improves growth. Phytate was used because it requires a plant produced enzymatic reaction to cleave phosphate for uptake by the growing gametophyte. Pre exposure to arsenic did not inhibit the activity of this enzyme, as growth was not impeded on media amended with phytate. Arse nic as a Defense against Herbivory The S. latifascia observed eating the P. vittata plants in our greenhouse was the first report of herbivory by the caterpillar (Figure 6 2) The plants did not however, contain arsenic, as they were growing in un contaminated pottin g mix. Upon moving the caterpillars to P. vittata growing in arsenic amended media, they continued to eat, unperturbed by arsenic in the tissues. However, upon analysis of the frond tissue, arsenic concentrations were relatively low at ~200 mg kg 1 As (f resh weight). The arsenic concentrations in the collected frass reflected this, with approximately 10 mg kg 1 As (dry weight). The caterpillars consumed approximately ~10 g of tissue on both control and arsenic contaminated plants, with roughly all of th e arsenic passing through their system. We were unable to rear a second generation of the caterpillars and therefore could not test the effect of higher arsenic concentrations in the frond. It should be noted that the caterpillars were only found on plan ts free of arsenic in the greenhouse, as there were several experimental plants they could have eaten, but did not. Allelopathy Arsenic readily leaches from arsenic treated wood, contaminated the soil around it. This was the the case pertaining to the so il underneath the decking of an apartment

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107 complex built with arsenic treated wood. Pteris vittata were found to grow naturally underneath the stairwells, likely because the elevated arsenic concentrations are toxic to most other plants, providing them wit h a unique niche. To maintain such a niche, it has been theorized that metal hyperaccumulation could be used to enrich toxic metals at the soil surface to deter competitive plants. At the apartment complex, all soils sampled around structures built with arsenic treated structures site were extensively contaminated with arsenic, ranging from 20 to 100 mg kg 1 As. Soil samples taken at the base of plants was slightly elevated compared to concentrations just outside the canopy (50 cm from plant) (Figure 6 3) The avera ge soil arsenic concentration at the base of plants was 44 mg kg 1 compared to 33 mg kg 1 in areas with no P. vittata At a depth of 20 cm, arsenic concentrations were significantly lower, averaging 10 mg kg 1 underneath P. vittata and 13 mg kg 1 at a 50 cm distance away (Figure 6 3) The higher concentration at the surface is likely due to arsenic leaching from wood, not necessarily plant mediated. A point by point comparison of surface soils with and without P. vittata showed that the difference in arsenic concentra tions was not significantly different. This suggests that P. vittata may be enriching surface soil with arsenic, but contamination at this site was not homogenous enough to accurately assess this theory.

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108 Table 6 1. Pteris vittata s pore g rowth Treatmen t and Fresh Weight (g) Spore As mg kg 1 P P+As P 6 P+P 6 P 6 +As 0 0.34 0.46 0.23 0.44 0.25 88 0.26 0.28 0.14 0.25 0.15 138 0.18 0.25 0.17 0.30 0.14 177 0.19 0.37 0.12 0.15 0.16 186 0.25 0.28 0.16 0.31 0.22 282 0.18 0.26 0.23 0.25 0.11 819 0.19 0.28 0 .20 0.34 0.10 996 0.22 0.25 0.22 0.18 0.10 1717 0.20 0.24 0.18 0.28 0.13

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109 Figure 6 1. Pteris vittata growing in soil spiked with 0, 25, 50 and 100 mg kg 1 arsenic. Plants after transplant (top) and after six months (bottom) of growth. Photos cou rtesy of Jason Lessl.

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110 Figure 6 2. Spodoptera latifascia caterpillar (top) and adult moth (bottom). P hotos courtesy of Lyle Buss.

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111 Figure 6 3. The natural logarithm of soil arsenic concentrations at the surface soil (top) and at a 20 cm depth ta ken at the base of P. vittata (PV) and at a distance 50 cm away from plants.

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125 BIOGRAPHICAL SKETCH Jason T. Lessl, was born in Milwaukee Wisconsin to Tom and Ruth Lessl. After his high school education, he attended the University of Georgia for his bachel or's degree in b i ology and a master's degree in p lant p athology. The title of his master's thesis was The role of bacterial motility in watermelon blossom colonization and seed infestation by acidovorax avenae subsp. citrulli causal agent of bacterial fruit blotch ." Awarded an alumni graduate fellowship at the University of Florida, he joined the department of Soil and Water Science in 2008 to pursue a PhD studying the arsenic hyperaccumulator Pteris vittata