Phytoremediation of Persistent Organic Pollutants: Mechanistic studies and Field Application Jason C. White Head, Department of Analytical Chemistry The Connecticut Agricultural Experiment Station Persistent Organic Pollutants (POPs) in Soil • They persist for decades • Global distribution • Likely mutagenic, estrogenic, carcinogenic effects • Bioaccumulation, biomagnification • Other remediation strategies are ineffective due to high degree of sequestration; plants should not facilitate EPA Dirty Dozen their remediation Aldrin Chlordane DDT/DDE Dieldrin Dioxins Endrin Furans Heptachlor Lindane Mirex PCBs Toxaphene p,p'-DDE (ng/g) 1000 1500 2000 2500 3000 500 0 Soil Roots of translocation and Uptake Shoots S. squash Pumpkin Rye Clover Mustard Zucchini W. squash weathered Soil Roots species plant selected Shoots Soil Roots Shoots pepo Cucurbita Soil Roots Stems Leaf ovifera Fruit p,p’ Soil Roots Stems ssp -DDE by Leaf Cucurbita pepo Fruit Soil Roots Stems Leaf Fruit ssp Soil Roots pepo Stems Leaf Fruit Phytoextraction (% removal) of weathered by plant species. Numbers in green indicate mass ratio 2.25 of root to soil (values are 10 1.50 % Phytoextracted0.75 p,p' 0.00 system p,p' system -DDE in root -DDE in shoot 52 Rye 15 Vetch 37 Pigeonpea 0.65 Peanut 1.8 -4 Cucumber ) p,p’ 91 Mustard -DDE 0.88 9.0 W. Squash 32 Clover 9.7 Canola 40 Lupin Pumpkin UptakeUptake andand translocationtranslocation ofof chlordanechlordane fromfrom soilsoil byby selectedselected plantplant speciesspecies Soil Zucchini 24000 Cucumber Tomato Spinach Corn 21000 7500 5000 Chlordane (ng/g) 2500 0 Soil Roots Stems Leaves Fruit DDX in the xylem sap of 3 cucurbits 20.0 Zucchini DDE DDT Squash 15.0 Cucumber 10.0 DDD 5.0 1.5 1.0 0.5 Xylem sap ConcentrationXylem (ng/mL) 0.0 35 Zucchini 30 Cucumber Squash 25 20 15 10 DDE Flow (ng/h) 5 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Root mass (g, dry weight) CAES Research on the C. pepo ssp pepo system • Focused on two separate lines of investigation: 1. Optimizing remediation- Field/pot studies in an effort to further enhance contaminant uptake (cropping strategy, surfactants, organic chelators or TPAs, fungal inocula, intercropping) 2. Mechanistic studies- hydroponic, rhizotron, grafting, breeding, and molecular studies to elucidate governing processes. The C. pepo ssp pepo system • Mechanistically, you need to consider two areas • Ex planta- Release and uptake of a highly sequestered residue from soil to roots - Plant exudates are released to facilitate nutrient acquisition from soil. Species vary widely in exudation. Greater amounts of low molecular weight organic acids result in soil matrix deconstruction - This disruption in soil structure inadvertently increases the bioavailability of weathered POPs • In planta- Translocation of a hydrophobic residue from roots to shoots. - Hydrophobic contaminants must move with water through root tissue and cross into the vascular cylinder. Should be minimal but some plants are clearly more effective than others. Zucchini exude more organic acids, whether you look directly at the root surface (left) or in the rhizosphere pore water (right) 20.0 Zucchini DDE DDT Squash 15.0 Cucumber 10.0 DDD 5.0 1.5 1.0 0.5 Xylem sap ConcentrationXylem (ng/mL) 0.0 12000 8000 10000 6000 8000 6000 4000 4000 2000 2000 Total Organic Acid (picomoles) Total Organic Acid (picomoles) 0 0 Zucchini Squash Cucumber Control Zucchini Squash Cucumber Control * Organic acids summed are citric, malic, succinic, tartaric, malonic Accumulation of chlordane components from soil by shoots of intact or grafted zucchini and cucumber plants (units are raw area counts) 120 16 Zucchini * Cucumber * 12 80 * Chordanes 8 + HEPX 40 * * 4 Transnonachlor + Cis-chlordane ND*ND 0 0 + Trans-chlordane Zucchini homograft Cucumber homograft + MC5a/MC5b 60 12 40 8 20 4 0 0 ND ND Zucchini shoot on cucumber root Cucumber shoot on zucchini root 6 25 20 4 15 10 2 Mattina et al., 2007 5 ND 0 ND 0 Differential uptakeofweathered 10 12 0 2 4 BCF6 8 Average BCF Black magic ovifera- 2.4 pepo- 7.2 Black zuke Black beauty Butter scal. Costata Early Pro subspecies of Gurneys pride Goldrush (ssp Hybrid butter Hybrid crescent Hybrid jack Magda Patty pan pepo Raven Revenue Seneca Roots Starship Sunburst Sunray and Yellow Crook Zephyr Cucurbita pepo Average BCF ovifera- 0.54 pepo- 5.4 Black magic ssp Black zuke Black beauty Butter scal. Costata ovifera Early Pro Gurneys pride Goldrush Hybrid butter Hybrid crescent p,p’ Hybrid jack Magda Patty pan ) Raven Revenue -DDE by2 Stems Seneca Starship Sunburst Sunray Yellow Crook Zephyr 0 2 4 6 8 10 BCF Stem DDE bioconcentration factors and % DDE phytoextracted for 3 cultivars each of two 10 8 Zucchini ( C. pepo 6 Stem DDE BCF 4 ssp 2 pepo 0 ) Costata pepo Non-zucchini summer squash Goldrush ( C. pepo Raven ssp subspecies ovifera) Zephyr Patty Y. Crook 1.0 0.8 Zucchini ( C. pepo 0.6 % DDE0.4 phytoextracted 0.2 ssp 0.0 pepo ) Costata C. Non-zucchini summer squash Goldrush ( C. pepo Raven ssp ovifera) Zephyr Patty Y. Crook Traditional Breeding Studies- DDE-accumulating zucchini and non- accumulating squash are in the same subspecies. What will be the abilities of the hybrids? A 16 Stem 12 C bioconcentration C 8 factors (BCFs) Stem DDE BCF and % DDE 4 B phytoextracted 0 Parents F1 Hybrids by parental and Zucchini (C. pepo ssp pepo) Squash ( C. pepo ssp ovifera) A Hybrid Zucchini (ssp pepo x ssp ovifera) F1 hybrid Hybrid Squash (ssp ovifera x ssp pepo) 1.6 zucchini and 1.2 C squash 0.8 D % Phytoextracted 0.4 B 0.0 Parents F1 Hybrids 1.5 A 1.0 % Phytoextracted 0.5 (C. pepo 0.0 Zucchini B ssp Parental pepo) B F1 hybrid ( C. pepo F1 Backcross Squash ssp A ovifera Parental B ) F1 hybrid A F1 Backcross Phytoextracted and 15 parental, F1 hybrid, and F1 Backcrossed A zucchini and squash 10 Stem BCFs by Stem BCF % DDE 5 ( (C. pepo preliminary data) 0 Zucchini B ssp Parental pepo) B F1 hybrid ( C. pepo F1 Backcross Squash ssp A ovifera Parental B ) F1 hybrid C F1 Backcross Uptake of DDE by zucchini and squash exposed to 120 ng/mL DDE under batch hydroponic conditions for 96 hours 1000 A Exposed to Non-exposed DDE Controls 800 600 400 B B 200 B Stem DDE content (ng/g) 0 Zucchini Squash Zucchini Squash Zucchini and squash that were or were not exposed to DDE were subjected to differential transcript analysis and subtractive hybridization techniques. Subtracted cDNAs were cloned into a TA cloning vector and expression in E. coli via colony array analysis was employed (Fig. 1). Figure 1. A. B.. Figure 2. SHOOT ROOT Blast search on isolated differentially expressed cDNAs Zucchini Squash Zucchini Squash DDE DDE DDE DDE includes sequence homology - + - + - + - + match to “phloem protein I” (PP1) and a cytochrome P450. PP1 Cyt P450 Semi-quantitative RT PCR Actin Actin confirmed differential regulation (Fig. 2). List of DDE induced subtracted cDNA clones from zucchini Gene Description Accession E value Max % Functions Number Identity Confirmation of upregulation of zucchini 2-Oxoisovalerate dehydrogenase (2 NM_203027 3e-73 79% oxidative stress subtracted cDNAs by RT-PCR analysis OVD) Aspartate aminotransferase (AAT) XM_002515409 5e-108 85% Amino acid DDE metabolism Brassinosteroid Insensitive 1 associated XM_002532844 1e-104 80% defense – + receptor kinase (Brassino. Inh) UK 1 Chlorophyll a/b-binding protein -1 DQ471302 1e-76 84% photosynthesis chlorophyll a/b-binding protein -2 XM_002284820 4e-97 83% photosynthesis UK 3 Cystatin (Cystatin like) ABF94587 3e-07 53% cystein UK 4 proteinase inhibitor UK 5 Cysteine Proteinase (Cys Pro) AJ415386 3e-59 82% signaling and abiotic stresses Brassino. Insensitive DNA binding protein with bHLH XP_002521010 3e-32 45% unknown domain (bHLH) Glucosylceramidase ( Gluc. Cera.) NM_124368 1e-09 81% Lipid Ze Epo. metabolism Protein phsophatase -2a XM_002513094 2e-60 84% Involved in Rub. Activase phosphorylation 50S ribosomal protein L5 (L5 Ribo 50S) XM_002511867 7e-51 79% Unknown Cystatin like Ribulose activase EF506513 1e-122 82% photosynthesis Phosphatase 2A Putative senescence-associated protein BAB33421 4e-44 79% Stress tolerance AAT Unknown protein 1 (UK 1) XP_002527893 0.004 48% Unknown bHLH Unknown protein 2 (UK 2) BT009458 8e-27 100% Unknown Chl a/b 1 Unknown protein 3 (UK 3) AY942801 4e-33 77% Unknown Unknown protein 4 (UK 4) XM_002532526 3e-48 80% Unknown Chl a/b 2 Unknown protein (predicted protein XM_002519497 1e-52 82% Unknown Cys Pro. mahagunin like) (UK 5) Unknown protein (predicted protein with U01964 1e-23 83% Unknown chlorophyll a/b-binding) (UK 6) 2 OVD Unknown protein (Predicted protein XP_002531567 4e-09 49% Unknown with DNA binding domain (UK 7) Unkown predicted protein (UK 8) XM_002309035 1e-31 78% Unknown L5 Ribo 50S Zeaxanthin epoxidase (Ze. Epo.) DQ641126 2e-98 90% abiotic stress ACT2 control tolerance Conclusions • C. pepo ssp pepo uniquely accumulates significant quantities of weathered POPs from soil • POP accumulation is dependent on both contaminant properties and plant genotype • Soil amendments such as organic acids, surfactants, and fungal inocula can increase POP extraction but effects are cultivar- and treatment-specific • Although C. pepo ssp pepo has a unique organic acid exudation profile (as a nutrient acquisition strategy), the differences do not explain the observed POP accumulation • Based on hydroponic, grafting, and molecular data, it appears that a unique transport protein in C. pepo ssp pepo tissue permits POP movement into the vascular cylinder • Investigations elucidating the nature of this transport system are underway Acknowledgements • Dr. Om Parkash (UMass), Dr. MaryJane Mattina, Dr. Brian Eitzer, Dr. Martin Gent, Dr. Neil Schultes • Dr. Zakia Parrish, Dr. Mehmet Isleyen, Dr. Xiaoping Wang, Dr. Wen-Yee Lee • Lydia Wagner, Bill Iannucci-Berger, Craig Musante, Terri Arsenault • Funding from US EPA STAR, USDA CSREES.