Understanding the Physiological and Molecular Mechanism of

May 27, 2010 - Cultivars of Cucurbita pepo ssp pepo (“Costata Romanesco”) and Cucurbita pepo ssp ovifera (“Zephyr”) (Johnny's Selected Seeds, ...
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Environ. Sci. Technol. 2010, 44, 7295–7301

Understanding the Physiological and Molecular Mechanism of Persistent Organic Pollutant Uptake and Detoxification in Cucurbit Species (Zucchini and Squash) S U D E S H C H H I K A R A , †,§ B I B I N P A U L O S E , †,§ J A S O N C . W H I T E , ‡ A N D O M P A R K A S H D H A N K H E R * ,† Department of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, Massachusetts 01002, and Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, 123 Huntington St. Box 1106, New Haven, Connecticut 06504

Received January 12, 2010. Revised manuscript received April 19, 2010. Accepted April 21, 2010.

Cucurbita pepo ssp pepo (zucchini) roots phytoextract significant amounts of persistent organic pollutants (POPs) from soil, followed by effective translocation to aboveground tissues. The closely related C. pepo ssp ovifera (squash) does not have this ability. In a DDE-contaminated field soil, zucchini roots and stems contained 3.6 and 6.6-fold greater contaminant than did squash tissues, respectively, and zucchini phytoextracted 12-times more DDE from soil than squash. In batch hydroponics, squash was significantly more sensitive to DDE (2-20 mg/ L) exposure; 4 mg/L DDE significantly reduced squash biomass (14%) whereas for zucchini, biomass reductions were observed at 20 mg/L (20%). PCR select Suppression Subtraction Hybridization was used to identify differentially expressed genes in DDE treated zucchini relative to DDE treated squash or non-treated zucchini. After differential screening to eliminate false positives, unique cDNA clones were sequenced. Out of 40 shoot cDNA sequences, 34 cDNAs have homology to parts of phloem filament protein 1 (PP1). Out of 6 cDNAs from the root tissue, two cDNAs are similar to cytochrome P450 like proteins, and one cDNA matches a putative senescence associated protein. From the DDE exposed zucchini seedlings cDNA library, out of 22 differentially expressed genes, 14 cDNAs were found to have homology with genes involved in abiotic stresses, signaling, lipid metabolism, and photosynthesis. A large number of cDNA sequences were found to encode novel unknown proteins that may be involved in uncharacterized pathways of DDE metabolism in plants. A semiquantitative RTPCR analysis of isolated genes confirmed up-regulation in response to DDE exposure.

Introduction Persistent organic pollutants (POPs) are noted for their significant resistance to degradation, toxicity, and global * Corresponding author e-mail: [email protected]; phone: 413-545-0062; fax: 413-545-3705. † University of Massachusetts. ‡ The Connecticut Agricultural Experiment Station. § Authors have contributed equally to this work. 10.1021/es100116t

 2010 American Chemical Society

Published on Web 05/27/2010

distribution (1). The half-lives of POPs such as DDT, chlordane, and PCBs in soils are typically measured in decades (2). POPs are also highly hydrophobic, readily binding to and becoming sequestered within soils. However, largely because of the activity of soil dwelling invertebrates, POPs can enter food chains. These contaminants may bioaccumulate in the lipids of organisms (3), biomagnify through food webs, and negatively impact top-level carnivores (4). POPs are also semivolatile and are readily transported globally via atmospheric processes (5); PCBs and other organochlorines are routinely detected in the tissues of arctic and Antarctic biota (6, 7). Phytoremediation relies on the inherent physiology of vegetation to alleviate contamination and can be both effective and inexpensive as long as time constraints are not significant. Moderately water-soluble organic chemicals may enter plant roots with the transpiration stream; the xenobiotics can subsequently be degraded, stored, or volatilized (8-10). Alternatively, susceptible organic contaminants may be degraded in the root zone, either by exuded plant enzymes or by the enhanced rhizosphere microbial community (9, 10). However, POP hydrophobicity, degradation-resistance, and low bioavailability in soil make successful phytoremediation unlikely (8, 11). Conversely, Hu ¨lster et al. (12) reported dioxin uptake into Cucurbita species and subsequent investigations have shown the Cucurbita pepo ssp pepo (zucchini) uniquely accumulate significant amounts of weathered POPs, including chlordane, dichlorodiphenyltrichloroethane/dichlorodiphenyldichloroethylene/dichlorodiphenyldichloroethane (DDT/DDE/DDD), and PCBs (13-16). Although the precise amount of contaminant accumulation varies with plant genotype and chemical characteristics, the pollutants are largely accumulated as unaltered parent compound (15). Stems and roots of C. pepo ssp pepo may accumulate POP concentrations that are 5-30 times greater than present in the soil, often extracting 1-5% of the contamination in a single growing season. Previous work has shown that the accumulation of weathered POPs varies significantly at the subspecies level; C. pepo ssp pepo (zucchini) accumulates up to an order of magnitude more contaminant than C. pepo ssp ovifera (squash) (13). Mechanistic investigations involving closely related Cucurbita subspecies are ongoing, and hydroponic experiments have implicated a unique transport system for hydrophobic organic chemicals (16, 17). Mattina et al. (18) used zucchini and cucumber homo- and heterografts to investigate the uptake of weathered chlordane from soil and showed that the root stock determined the extent and profile of contamination in the shoots. In the current study, the effect of DDE exposure on one cultivar each of C. pepo ssp pepo (Costata; zucchini) and C. pepo ssp ovifera (Zephyr; squash) was investigated. So as to provide a coherent and inclusive comparison, the two C. pepo subspecies were exposed to DDE under field-weathered and laboratory controlled hydroponic conditions. In addition, PCR-Select Subtractive Suppression Hybridization (SSH) was used to identify differentially expressed genes in DDE-exposed or non-exposed zucchini and squash. The subtracted candidate genes were sequenced, and their up-regulation by DDE was confirmed by semiquantitative RT-PCR. Characterization of these genes by both forward and reverse genetic approaches will shed light on their role in DDE accumulation.

Materials and Methods Field Experiment. Cultivars of Cucurbita pepo ssp pepo (“Costata Romanesco”) and Cucurbita pepo ssp ovifera VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(“Zephyr”) (Johnny’s Selected Seeds, Albion, ME) were grown at the Connecticut Agricultural Experiment Station’s Lockwood Farm in areas known to be contaminated with 50-500 ng/g weathered p,p′-DDE (DDE). The DDE is from historical DDT applications, and the residues have been present for several decades. The soil is a fine sandy loam (56% sand, 36% silt, 8% clay) with 1.4% organic carbon and a pH of 6.7. The site was covered with black polyethylene plastic sheeting, and 30 cm2 squares were cut at 3.0 m2 intervals. Each exposed area of soil served as a single replicate mound of vegetation; there were four individual plants per replicate and two replicate mounds per subspecies. Mounds were weeded and watered as necessary; fruit were harvested throughout the season. Soil Extraction. Four soil cores (2.5 cm diameter, 6-10 cm depth) were collected from each mound and were composited into a single soil sample for each replicate. The soils were air-dried, sieved to 2 mm, and 3 g portions were weighed into 20 mL amber vials with Teflon-lined caps. The vials were amended with 15 mL of hexanes containing 1 µg of o,p-DDE as an internal standard, capped, and heated at 65 °C for 4 h. Particulates were removed by passing 1 mL of the supernatant through a 0.2 µm glass micro fiber filter prior to collection in a chromatography vial that was then stored at -4 °C. Vegetation Extraction. A 1.0 m × 1.0 m × 0.25 m volume of soil containing the root system of individual mounds of vegetation was excavated. The mass of fruit, leaf, stem, and root compartments was determined. After separating plants by subspecies and tissue, vegetation was washed thoroughly, chopped, and samples were transferred to whirlpak bags for storage at -4 °C. The vegetation extraction procedure is described elsewhere (13). Briefly, 25 g (wet weight) of tissue are added to an explosion-proof blender containing 25 mL of 2-propanol, 50 mL of petroleum ether (both Ultra-ResiAnalyzed, J.T. Baker, Phillipsburg, NJ), and 1 µg of o,p-DDE as an internal standard. After blending for 5 min, the extracts are filtered through a glass-wool lined funnel, and the eluent is collected in 500 mL glass separatory funnel with Teflon stopcock. The petroleum ether is rinsed three times at 20 min intervals with distilled water and a saturated sodium sulfate solution. Four milliliter florisil cartridges (Alltech, Deerfield, IL) are preconditioned with 1.0 mL of petroleum ether, and 1.0 mL of the vegetation extract is loaded onto the cartridge. After elution with 6 mL of 6% diethyl ether in petroleum ether, the eluent is collected, and the volume is then reduced to 1 mL under nitrogen prior to storage at -4 °C. DDE Hydroponics. A batch hydroponic experiment was conducted to determine the effect of 2, 4, 8, 12, and 20 mg/L DDE on the biomass and transpiration of Costata and Zephyr. Four-day old seedlings were added to 8 mL amber vials containing 7.5 mL of 25% Hoagland’s Solution (MP Biomedicals). The seedlings were placed in a growth room at 25 °C with a 12 h photoperiod of 200 µmol m-2 s-1 photosynthetic active radiation. After 3 d, the seedlings were removed from the vials, weighed, and added to 20 mL amber vials containing 19.5 mL of 25% Hoagland’s solution. A 4000 mg/L stock of DDE in methanol was created, and 10, 20, 40, 60, or 100 µL were added to the 20 mL vials. There were 4 replicates at each DDE concentration. Control seedlings were prepared that received equivalent amounts of methanol. After an additional 7 d, all seedlings were transferred to 40 mL vials containing 25% Hoagland’s solution with equivalent levels of DDE. The amount of nutrient solution lost from each vial was monitored daily. The plants were weighed daily; the vials were also weighed and refilled with the appropriate solution. The cumulative solution amount lost was recorded as the transpiration volume. To compare the uptake of DDE (120 µg/L) by Costata and Zephyr, 3 day old seedlings were added to 250 mL amber 7296

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bottles containing 240 mL of 25% Hoagland’s solution. The seedlings were held in place by polyurethane plugs; there were 20 replicates of each subspecies. DDE exposure began once the seedlings had two true leaves. Half of the replicates of each subspecies were amended with 144 µL from the 200 mg/L DDE stock or with 144 µL of methanol. At the end of the 4 day exposure, the mass of all tissues was recorded. The amount of solution lost from each bottle was determined as the transpiration volume. Roots, stems, and leaves of each subspecies were subjected to the vegetation extraction procedure. Chemical Analysis. Crystalline p,p′-DDE and o,p′-DDE were acquired from the EPA National Pesticide Standard Repository (Fort Meade, MD). A 1000 mg/L stock of p,p′DDE in trimethylpentane was created and diluted to calibration standards of 10, 25, 50, 100, 150, 250, and 500 ng/mL. Each calibration level was amended with 100 ng/mL o,pDDE as an internal standard. The amount of p,p′-DDE was determined on an Agilent (Avondale, PA, U.S.A.) 6890 gas chromatograph (GC) with a 63Ni microelectron capture detector (ECD). An SPB-1 film (Supelco, Bellefonte, PA) GC column (30 m × 0.53 mm × 0.5 µm) was used, and the oven program was as follows: 175 °C initial temperature ramped at 3.5 °C/min to 225 °C, then ramped at 25 °C/min to 250 °C with a hold time of 4.71 min. The injection port was maintained at 250 °C, and a 2 µL splitless injection was used. The carrier gas over the column was He. The ECD was maintained at 325 °C, and the makeup gas to the detector was 5% CH4 in Ar at 60 mL/min. RNA Isolation, cDNA Synthesis, and PCR-Select Subtraction. On the basis of the data from the above assay, 7 day old Costata (zucchini) and Zephyr (squash) seedlings were exposed to DDE at 16 mg/L for an additional 7 days. The week old pre-germinated seedlings were added to 40 mL amber vials containing 38 mL of 25% Hoagland solution amended with 160 µL of a 4000 mg/L DDE stock or with 160 µL of methanol. After 7 d, the plant tissues were washed, weighed, frozen in liquid nitrogen, and stored at -80 °C. These tissues were then used for isolating differentially expressed transcripts using PCR-Select SSH. Total RNA was extracted from control and DDE treated tissue samples using RNeasy Plant Mini kits (Qiagen) followed by mRNA isolation using NucleoTrap mRNA isolation kits. Two cDNA libraries were made from shoot and root tissue by subtracting the cDNA of DDE treated Zephyr (driver) from DDE treated Costata (tester). A third library was made by subtracting the cDNAs of DDE exposed Costata (tester) from control non-DDE exposed Costata (driver). The driver to tester ratio was 25:1. Double stranded cDNA synthesis and cDNA subtractions were performed using the PCR select cDNA subtraction kit (Clontech, CA). Briefly, cDNAs from driver and tester samples were digested with endonuclease RsaI and tester cDNAs were ligated to two different adapters at either end of the cDNAs separately (19). Costata ACT2 was amplified as an internal control to confirm efficient adapter ligation. The twin samples of tester differing in the adapters were denatured and allowed to hybridize in separate tubes with excess denatured driver cDNAs. The tester samples were then mixed without denaturation in presence of the fresh denatured driver cDNAs and allowed to anneal overnight. Subsequent PCR amplifications, first with primers corresponding to both adapters and then with nested primers, selectively amplified and enriched differentially expressed cDNAs. A reverse subtraction, by switching the tester and driver cDNAs, was also performed for differential screening (19). The subtracted and PCR amplified products were ligated to the pGEM-T-Easy T/A cloning vector (Promega) and transformed into DH5R cells. Differential Screening. A differential screening procedure eliminated false positives. This step was carried out using a

differential screening kit (Clontech) where the subtracted cDNAs were blotted onto nylon membranes and were hybridized with forward and reverse subtracted probes. Colonies containing plasmids with cloned cDNAs were grown in liquid LB medium overnight, and an equal amount of each culture was spotted, in duplicate, on replicate nylon membranes placed on LB plates and incubated at 37 °C overnight. Membranes containing the colonies were transferred to Whatman filters presaturated with denaturing solution (0.5 M NaOH, 1.5 M NaCl), then to Whatman filters presaturated with neutralizing solution (0.5 M Tris-HCl, 1.5 M NaCl). Each membrane was allowed to dry, and the DNA was fixed by heating at 80 °C for 2 h. These membranes were then hybridized with either 32P labeled forward or reverse subtracted probes overnight as described by Maniatis et al. (20) and washed with low and high stringency buffer prior to X-ray film exposure at -80 °C overnight. The subtracted clones showing strong hybridization signal only when probed with forward subtracted cDNA were selected. DNA Sequencing and Gene Identification. The differentially expressed cDNA clones were sequenced and searched on the NCBI GenBank database for homologous sequences and their identity using the BLAST algorithm (21). All the subtracted cDNA sequences were blasted against DNA, and protein sequences on the database and homologous gene sequences were identified. Semiquantitative RT-PCR. For RT-PCR, total RNA was extracted from 7 day old seedlings that were untreated or treated with DDE using the RNAeasy Plant mini kit (Qiagen), and cDNA was synthesized from total RNA using a Thermoscript RT-PCR kit (Invitrogen). Primers for each subtracted cDNA were designed using Primer3 (http://fokker.wi.mit. edu/primer3/input.htm). The number of PCR cycles was optimized, and the constitutively expressed actin 2, ACT2, gene was used to verify that an equal amount of cDNA template was used in each PCR reaction. A touchdown PCR was performed for all the transcripts with annealing temperature varying from 64 to 52 °C. The bands intensity of control and DDE induced samples were quantified by Kodak 1D image software limited v3.0. The mRNA expression levels were normalized to the level of the constitutively expressed actin 2, ACT2, gene (control). The relative expression levels as a difference in the expression (fold change) between untreated control and DDE treated samples are expressed as a ratio relative to the value of expression in non-induced plants.

Results and Discussion Field Experiment. The average soil DDE content was 180 ng/g ((29). The average total dry mass of Costata and Zephyr mounds of vegetation were 640 and 770 g, respectively. These values and the mass of the individual tissues were not significantly different among the two plants. The root, stem, leave, and fruit dry mass of Costata were 8.4, 290, 120, and 220 g, respectively and of Zephyr were 5.8, 140, 79, and 548 g, respectively. These biomass values are consistent with previous studies (13, 16). For Costata, the average root, stem, leaf, and fruit DDE concentrations were 3900, 1700, 33, and 160 ng/g (dry weight), respectively; for Zephyr, the values of root, stem, leaf, and fruit DDE levels were 1200, 280, 29, and 6.1 ng/g (dry weight), respectively. Tissue-to-soil bioconcentration factors (BCFs, ratio of tissue contaminant concentration to that in soil) are shown in Figure 1. Costata root and stem BCFs are 3.6 and 6.6-fold greater than Zephyr (statistically significant at p < 0.01). Costata and Zephyr contained a total of 560 and 50 µg of DDE, respectively. Assuming each replicate mound impacts 270 kg of soil (1 m × 1 m × 0.25 m volume of soil), the percent DDE phytoextracted by Costata and Zephyr was 1.1 and 0.09%, respectively (statistically significant at p < 0.01) (13, 16).

FIGURE 1. Root, stem, leaf, and fruit bioconcentration factors (BCFs) for Costata (C. pepo ssp pepo) and Zephyr (C. pepo ssp ovifera) grown in DDE-contaminated soil under field conditions. Across plant type but within a specific tissue, bars with different letters are significantly different (Student t test). DDE Hydroponics. The effect of DDE exposure (2-20 mg/L) on seedling biomass under hydroponic conditions is shown in Supporting Information, Table 1. Exposure to DDE did not significantly impact the biomass of either subspecies during the first 14 d; at 20 d exposure, 20 mg/L DDE reduced Costata biomass by up to 25% (significantly different p < 0.01). Zephyr appeared to be more sensitive to DDE exposure, with 4 mg/L significant significantly reducing biomass by day 17. The transpiration of replicate plants was monitored during days 4-14 (Supporting Information, Figure 1). Again, Zephyr was significantly more sensitive to DDE exposure than Costata. By 3 days, DDE at 20 and 8 mg/L had significantly reduced Zephyr transpiration; by 6 days, all exposure levels significantly reduced Zephyr transpiration. For Costata at day 3, transpiration was significantly reduced at 20, 12, and 4 mg/L, but by day 7, these differences had disappeared. By day 10, transpiration at 12 and 20 mg/L were significantly less than at 8 mg/L but no other significant differences were evident. Phytotoxicity data for DDT/DDE is quite limited. Chung et al. (22) compared DDT/DDE phytotoxicity in ryegrass and algae. While ryegrass germination assays showed no toxicity (EC50 > 1000 mg/kg), several measures of algal physiology were affected. Costata and Zephyr that were exposed to DDE at 120 µg/L for 96 h contained 290 µg/g DDE (dry weight) in the roots. The root DDE content of the control plants were 200 ng/g. Costata stems contained nearly 6-times the amount of DDE as Zephyr; the levels were 950 and 160 ng/g, respectively (significantly different at p < 0.05). The stems of non-exposed plants contained 120 ng/g. The DDE content of the leaves were statistically equivalent across plant type and DDE treatment and averaged 50 ng/g. The shoot systems of Costata and Zephyr plants contained 270 and 57 total ng of DDE, respectively (significantly different at p < 0.05). Unexposed Costata and Zephyr shoots contained approximately 50 ng DDE. Zephyr transpiration was not impacted by DDE exposure, but Costata transpiration was reduced by 10% (statistically significant; p < 0.05). The observation of minimal DDE in control plants is not unexpected given that all seedlings were incubated in a small growth room with moderate air exchange. Minimal air contamination via DDE volatilization from solution is likely, and subsequent deposition onto the leaves is a well-known exposure pathway (12). DDE content in shoots growing in contaminant free-solution represents 72-79% of the total plant burden, but in DDE contaminated solution, the shoots contain 0.27-1.2% of the total plant levels, respectively. The presence of high DDE in the roots of exposed plants is due to extensive sorption from solution to plant surfaces. VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. List of Subtracted cDNAs Isolated from DDE Treated Zucchini Subtracted from Non-DDE Exposed Zucchini Seedling gene description

accession number

E value

max % identitya

2-oxoisovalerate dehydrogenase (2-OVD) aspartate aminotransferase (AAT) brassinosteroid Insensitive 1 associated receptor kinase (BAK1) chlorophyll a/b-binding protein-1 (chl a/b 1) chlorophyll a/b-binding protein-2 (chl a/b 2) cystatin like protein (Cystatin) cysteine Proteinase (Cys Pro) DNA binding protein with bHLH domain (bHLH) glucosylceramidase (Gluc. Cera.) protein phsophatase-2a 50S ribosomal protein L5 (L5 Ribo 50S) rubisco activase (RAC) putative senescence-associated protein (SNAP) unknown protein 1 (UK 1) unknown protein 2 (UK 2) unknown protein 3 (UK 3) unknown protein 4 (UK 4) unknown protein (predicted protein mahagunin like) (UK 5) unknown protein (predicted protein with chlorophyll a/b-binding) (UK 6) unknown protein (Predicted protein with DNA binding domain (UK 7) unkown predicted protein (UK 8) zeaxanthin epoxidase (ZEP)

NM_203027 XM_002515409 XM_002532844

3E-73 5E-108 1E-104

79% 85% 80%

oxidative stress Amino acid metabolism defense

DQ471302 XM_002284820 ABF94587 AJ415386 XP_002521010 NM_124368 XM_002513094 XM_002511867 EF506513 BAB33421 XP_002527893 BT009458 AY942801 XM_002532526 XM_002519497

1E-76 4E-97 3E-07 3E-59 3E-32 1E-09 2E-60 7E-51 1E-122 4E-44 0.004 8E-27 4E-33 3E-48 1E-52

84% 83% 53% 82% 45% 81% 84% 79% 82% 79% 48% 100% 77% 80% 82%

photosynthesis photosynthesis signaling and abiotic stresses signaling and abiotic stresses signaling lipid metabolism involved in phosphorylation unknown photosynthesis Stress tolerance unknown unknown unknown unknown unknown

U01964

1E-23

83%

unknown

XP_002531567

4E-09

49%

unknown

XM_002309035 DQ641126

1E-31 2E-98

78% 90%

unknown abiotic stress tolerance

functions

a The max. % identity column in Table 1 is the percentage identity shown on protein alignment using basic local alignment search tool (BLAST) from NCBI.

In a continuous flow hydroponic system, cucurbit roots exposed to 2 µg/L DDE for several weeks retained or sorbed more than 90% supplied contaminant (17). At 120 µg/L in 240 mL, each bottle contains 29 µg of DDE. Zephyr and Costata plants contained 21 and 23 µg or 72 and 80%, respectively (not significantly different). The finding of significantly greater DDE root-to-shoot translocation by zucchini also agrees with Gent et al. (17). The current study found that DDE accumulation in zucchini shoots was 6-fold that of squash. Gent et al. (17) found 18-fold greater levels of DDE in zucchini shoots as compared to cucumber (Cucumis sativus). In a fugacity-based model describing DDE movement in plants, modeling contaminant root-to-shoot translocation in cucumber was straightforward; however, to describe contaminant content in zucchini stems, the apparent partitioning of DDE to xylem had to be 25-times greater than that for water (23). Isolation of Differentially Expressed mRNA Transcripts from Zucchini. A PCR-Select SSH approach was used to isolate the differentially expressed transcripts from zucchini in response to DDE exposure. During cDNA subtraction, RNA extraction/quality, RsaI digestion, and adapter ligation were monitored to ensure subtraction quality. As the driver to tester amounts affect subtraction stringency, two parallel subtractions were performed varying the ratios. From the shoot subtraction library made by subtracting DDE treated zucchini cDNA (driver) from DDE treated squash (tester), a total of 540 subtracted cDNA clones were obtained. In the root subtraction cDNA library, we obtained 370 cDNA clones. From the library made by subtracting DDE treated zucchini cDNA (driver) from untreated zucchini controls (tester), we obtained 500 subtracted cDNA. After differential screening of the first two libraries, 46 cDNAs clones showing strong hybridization signals on membrane probed only with forward subtracted cDNA but not with reverse subtracted cDNA were sequenced; 40 cDNAs isolated from shoots and 6 from roots. Further details on individual genes can be found below, but of 40 shoot sequences, 34 cDNAs were similar to different parts of phloem 7298

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filament protein 1 (PP1). Of 6 root cDNAs, two were similar to cytochrome P450 like proteins, and one cDNA matched a putative senescence associated protein (SNAP) (Supporting Information, Table 2). From the third cDNA library made from DDE exposed and non-exposed zucchini, 22 cDNA clones were identified after differential screening (Table 1). These 22 cDNAs encode genes involved in abiotic stresses, signaling, lipid metabolism, and photosynthesis. In addition, a large number of the transcripts encoded novel proteins that may be involved in uncharacterized pathways of DDE metabolism in plants. The shoot cDNA library contained numerous transcripts containing cysteine proteinase inhibitor domains similar to repeating units of phloem protein 1 (PP1). PP1 is the largest of a family of proteins common in dicotyledon phloem sap (24) and is present at higher concentrations in cucurbits (25). These proteins are involved in long distance transport of macromolecules through phloem by both passive and active transport (26). The role of PP1 in DDE transport and/or metabolism is unclear but could include controlling phloem permeability and maintenance of sieve element integrity, as well as potential direct involvement in long distance DDE transport. The zucchini root cDNA subtraction library contained sequences encoding cytochrome P450 (CYP450), a large gene family critical for xenobiotic degradation in plants and animals. The upregulation of CYP450 by DDE was also observed in rat liver (27). In wheat microsomes, CYP450 hydroxylated the aryl group on the herbicide diclofop (28). A similar aryl group hydroxylation was observed in Terrabacter sp. during DDE metabolism (29). In zucchini, CYP450 may facilitate similar enzymatic attack of the DDE ring, leading to further degradation or subsequent conjugation and vacuolar sequestration. 2-Oxoisovalerate dehydrogenase (2OVD) is a part of branched-chain R-keto acid dehydrogenase complex that catalyzes the NAD+ dependent oxidative decarboxylation during the amino acid degradation (30). In the PCB- degrading bacteria Burkholderia xenovorans, the genes for branched-

chain amino acid metabolism and transport were clustered with aromatic degradation operons (31). In zucchini, the enzymes in this pathway could be involved directly or indirectly in DDE metabolism. Aspartate amino transferases (AAT) play a major role in nitrogen assimilation and transport in plants. However, the sequence in our library is homologous to the chloroplast isoform in Arabidopsis thaliana, which is predicted to be involved in shuttling NADH between subcellular compartments (32). It is possible that AAT has an indirect role in DDE metabolism by influencing energy transfer reactions in plants. Members of basic helix-loop-helix (bHLH) superfamily are transcription factors that regulate diverse biological processes. This super family is well characterized in animals, and the members are divided among six groups (33). One group contains the Per-ARNT-Sim (PAS)-bHLH domain and regulates toxin metabolism (34). The aryl hydrocarbon receptor (AhR), a member of the PAS-bHLH group of proteins, interacts with halogenated aromatic hydrocarbons and activates genes involved in xenobiotic metabolism (35). Moreover, AhR can induce CYP450 (36). In zucchini, the DNA binding proteins with bHLH domains may induce genes involved in xenobiotic degradation. The subtracted cDNA library contains 3 different chlorophyll a/b binding proteins and one Rubisco activase (RCA), indicating impaired photosynthesis upon DDE exposure. Photosynthetic electron transport in isolated spinach and barley chloroplasts was significantly inhibited by DDT/DDE (37) and by other pesticides in Lemna minor (38). RCA is a chloroplast protein that regulates rubisco (ribulose 1,5bisphosphate carboxylase/oxygenase) activity, an important enzyme of photosynthesis (39); reductions in photosynthesis may be linked to rubisco deactivation due to RCA inhibition. Higher amounts of these proteins suggests the plants are compensating for reductions in photosynthesis caused by DDE. Another DNA binding protein in our library belongs to the Myb transcription factor superfamily. Many of these factors are involved in cell cycle regulation, as well as being induced by abiotic (40) and biotic stress (41). Wang et al. (42) reported on a Myb-family transcription factor that regulates the light harvesting chlorophyll a/b protein. Additional members of this superfamily may be involved in photosynthesis regulation. Brassinosteroids (BRs) are polyhedroxy steroids, which regulate physiological responses such as cell division, seed germination, vegetative growth (43), and stress responses (44). Brassinosteroids are a protein complex with two transmembrane receptor kinases, Brassinosteroid insensitive 1 (BRI1) and BRI1- associated receptor kinase 1 (BAK1) (45). BRI1 encodes a leucine-rich repeat receptor-like kinase (LRRRLK) that mediates plant-pathogen interaction for plant immunity (46). In A. thaliana, BAK1 regulates the containment of microbial infection-induced cell death (47), as well as a functional role in plant defense (48). Cysteine proteinases are endopeptidyl hydrolases that play an essential role in plant growth/development, senescence/ programmed cell death (PCD), signaling pathways, and in response to biotic and abiotic stresses. Cysteine proteinases were induced in response to stress, such as drought in A. thaliana (49) and wounding in tobacco (50). Cystatins are inhibitors of cysteine proteinases and are involved in defense mechanisms, regulation of catabolism, PCD and abiotic stress responses. Gaddour et al (51) found barley cDNA encoding cysteine proteinase inhibitor (Icy gene) mRNA level increased in roots and leaves upon anaerobiosis and cold shock (6 °C). In cold- and saline-shocks, Pernas et al (52) observed strong induction of CsC gene in roots and leaves of chestnut seedlings.

FIGURE 2. Semiquantitative RT-PCR analysis of zucchini PPI and CYP450 cDNA clones from shoots and roots, respectively. Zucchini actin 2, ACT2, was used used as a control for equal loading of cDNA template. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin into violaxanthin, is an important enzyme of abscisic acid (ABA) biosynthesis and in the xanthophyll cycle. ABA regulates plant responses to abiotic stresses and is required for stress tolerance. In a rice mutant of ZEP, low ABA levels and little upregulation were observed upon water stress (53). Light-stress induced modification of ZEP caused the downregulation of Zeaxanthin epoxidation in A. thaliana (54). Overexpression of A. thaliana ZEP gene (AtZEP) enhanced ABA biosynthesis, resulting in vigorous growth under salt and drought stress (55). In zucchini, differential ZEP regulation may facilitate adaptation to stress caused by DDE exposure. Our subtraction library also contains mahogunin like protein, penta peptide repeat like protein (PPR), protein phosphatase, and 50S ribosomal protein. In addition, there were many cDNA sequences in our library that lacked significant homology with known proteins and are named unknowns (UK). Functional characterization of these proteins may reveal novel pathways of xenobiotic degradation in plants. Validation of PCR-Select Subtraction Data by RT-PCR. To confirm upregulation of the subtracted cDNA clones in response to DDE exposure, semiquantitative RT-PCR was performed for all genes present in the subtracted libraries. RT-PCR analysis revealed that most of the subtracted cDNA clones showed induction of the corresponding mRNA transcripts upon DDE exposure. The PP1 and CYP450 showed more than 2-fold induction in DDE-exposed zucchini shoot and roots, respectively, as compared to DDE-exposed squash or non DDE-exposed zucchini and squash controls (Figure 2; Supporting Information, Figure 2). Similarly, genes corresponding to subtracted cDNAs from DDE exposed zucchini seedlings, such as 2OVD, AAT, chl a/b 1 and 2, cysteine proteinase, cystatin, bHLH, SNAP, phosphatase 2A, L5 Ribo 50S, RCA, and several unknowns showed approximately 2-fold induction in DDE exposed zucchini tissues as compared to controls (Figure 3; Supporting Information, Figure 3). ZEP showed a 3-fold induction, whereas, BAK1 and UK1 showed highest levels (four and 5-fold, respectively) of upregulation in response to DDE exposure (Figure 3; Supporting Information, Figure 3). The strong induction of these subtracted cDNA clones in zucchini suggests a significant role in DDE metabolism and transport. Persistent organic pollutants such as DDT/DDE/DDD are ubiquitous environmental contaminants but remedial options are limited and expensive. Cucurbita pepo ssp pepo has a unique ability not only to accumulate significant amounts of weathered POPs from soil into the root system but also to translocate large quantities of the pollutants to the shoots. Under field conditions, percent level contaminant removal from soil has been observed. The current work on subtractive hybridization and differential transcript analysis demonstrates significant upregulation of a number of known and novel unknown genes in zucchini upon DDE exposure. Elucidation of the cellular mechanisms and governing molecular processes for this phytoextraction ability will VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Semiquantitative RT-PCR analysis of zucchini subtracted cDNA clones from seedlings. Zucchini actin 2, ACT2, was used as a control for equal loading of cDNA template. Number of PCR cycles are given for each gene. Genes full names are given in Table 1. enable optimization of the zucchini system in the field through either cultivation practices or through manipulating gene expression, as well as potentially gene transfer to an alternative plant.

Acknowledgments We thank Terri Arsenault and Joseph Hawthorne for technical assistance. We thank Drs. John Lopez and Klaus Nusselin of the Microbiology Department (UMass Amherst) for permission to use their radioactive facility. B.P. is thankful to the Department of Plant, Soil, and Insect Sciences for partial TA support.

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Supporting Information Available Two tables on the effect of DDE on the biomass of zucchini and squash under batch hydroponic conditions and a list of subtracted cDNAs isolated from DDE treated zucchini. Three figures on the impact of DDE on the transpiration volume of zucchini and squash and on Relative Expression levels. This material is available free of charge via the Internet at http://pubs.acs.org.

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