Background Phytoremediation potential of the novel Atrazine, 2 - PDF document

1/25/2012 Background Phytoremediation potential of the novel • Atrazine, 2 ‐ chloro ‐ 4 ‐ (ethylamino) ‐ 6 ‐ atrazine tolerant Lolium multiflorum (isopropylamino) ‐ s ‐ and studies on the mechanisms involved triazine (ATZ) • Cyanuric chloride treated Cyanuric chloride treated Merini et al., 2009 Merini et al 2009 with ethylamine and Presented by: isopropyl amine Ellen Sherck & Rhiannon Fox • Inhibits photosynthesis by binding to the plastoquinone ‐ binding protein in Photosystem II Applications and Extent Environmental and Health Risks • 76 million pounds • Most common • Effects on wildlife applied annually in U.S. herbicide or pesticide – Contributes to • 2 nd ‐ largest selling contaminant of ground amphibian declines pesticide worldwide (Rohr et al., 2004) and surface water – Endocrine disruptor in E d i di t i (Capel & Larson, 2001) • Over 6,000 tons applied fish and amphibians annually in Argentinian • Highly mobile and can – Neural damage and Pampa region travel in precipitation pregnancy loss in (Thurman & Cromwell, • Other uses: rodents landscaping, forestry, 2000) • Effects on humans golf courses, etc. • Half life up to 120 days – Fertility loss in men (Swan et al., 2003) Other Remediation Strategies Phytoremediation! • Bioaugmentation ‐ inoculating soils with a • Challenges microorganism that can catabolize ATZ – Atrazine inhibits photosynthesis – Remediating plant cannot interfere with main crop • Addition of killed and stabilized suspensions of • Objectives: • Objectives: Escherichia coli cells engineered to Escherichia coli cells engineered to overexpress the enzyme atrazine – Assess the tolerance of annual ryegrass ( Lolium multiflorum ) to agronomic doses of atrazine chlorohyrolase (Strong et al. 2000) – Examine the mechanisms involved in Atrazine • All are effective, but expensive and often slow tolerance – Evaluate its potential for phytoremediation 1

1/25/2012 Lolium mutiflorum Atrazine Tolerance in Plants • Annual ryegrass that • Genetic mechanisms grows in Argentina – Point mutation in psbA gene  changes in D1 protein of PSII  A to G transition confers resistance • Already used for • Enzymatic mechanisms intercropping with corn – The cytochrome P450 detoxification pathway  detoxes (Carruthers et al., 2000) and eliminates endogen metabolites & xenobiotics  • One of the best adapted commonly confers Atrazine resistance ryegrass species • Non ‐ enzymatic mechanisms • Ability to acquire – Benzoxazinone hydroxylation of atrazine  confers tolerance to herbicides Atrazine resistance in some plants Methods – Tolerance Assay Results: Atrazine Tolerance • Seeds were sterilized and germinated in semisolid • Able to grow and sterile media amended with a 2,000 mg / kg atrazine germinate in presence solution of 1 mg kg ⁻ ¹ of Atrazine • Atrazine concentration ranged from 1 mg / kg (equal (more than 2X heavy to 2x the agronomic application rate) to 50 mg / kg to 2x the agronomic application rate) to 50 mg / kg field application) (heavy industrial contamination) with two • Able to germinate intermediate concentrations without external source • Triplicates of each seed type sown and incubated in a of carbon making it a culture chamber good candidate for • Growth of plants were recorded regularly – phytoremediation harvested at 21 days Methods – Phytoremediation Assay Results: Phytoremediation • Microcosms of clean (i.e. no atrazine) soils placed in • After 21 days atrazine growing chambers. degradation in soil – Control = no seeds microcosms was 20% higher with L. multiflorum plants – Experimental = 10 seeds each • During first week During first week • At 3 rd leaf stage, atrazine added  concentration of degradation rate was 2.5X 1 mg / kg of soil higher than natural soil attenuation capacity • Triplicates sampled at 0, 7, 14, and 21 days after • Higher degradation rates herbicide application than studies using other • Plants harvested, tested for chlorophyll a and b plants (Singh et al., 2004) content and enzymatic activity • Soil metabolites tested by HPLC 2

1/25/2012 Methods ‐ Genetics Results: Genetic Mechanisms • PCR DNA extraction performed on aerial parts • Sequencing of psbA gene of the plants – Mutation in psbA encoding the substitution of amino acids signifies resistance to photosynthesis • psbA gene had not been previously sequenced inhibitors herbicides in L. multiflorum in L multiflorum – No substitution in amino acid serine in D1 protein • Used taxonomically similar species to design • If replaced by glycine confers resistance to triazinic primers for amplification of psbA gene in L. herbicides multiflorum – In the other 16 amino acids there was only one substitution of tyrosine by threonine • PCR fragments were purified and sequenced • herbicide tolerance is under investigation Methods – Enzymatic Mechanisms Results: Enzymatic Mechanisms • L. multiflorum response to atrazine + 1 ‐ • Atrazine and 1 ‐ aminobenzotriazole (ABT) aminobenzotriazole (ABT) – What was the significance of cytocrome P ₄₅₀ to • Sterilized seeds sown in nutrient solution. atrazine resistance in L. multiflorum? • At day 14, 4 treatments: control, ABT, atrazine, and atrazine + ABT • After 7 days of treatment, solutions replaced by fresh nutrient solution • After another 21 days, survival index and shoot length were recorded for each treatment Methods – Non ‐ Enzymatic Results: Enzymatic Mechanisms Mechanisms • No significant • Benzoxazinones (Bx) were extracted from difference in seedlings (roots and shoots) plants with • Extract was dissolved in a buffer containing 1 exposure to ABT mg / L atrazine mg / L atrazine or ATZ compared to control • Control solution without extract was • Significant processed the same way difference • Samples collected at 0, 30 min, 1, 6, and 24 h between ATZ and and analyzed by HPLC to determine atrazine ATZ + ABT degradation and presence of metabolites 3

1/25/2012 Results: Non ‐ enzymatic Conclusions mechanisms • Benzoxainones (Bx) extraction and analysis • Atrazine tolerance in L. multiflorum is conferred by the P ₄₅₀ enzymatic capacity to – After plant material extraction analysis of Bx was oxidatively deactivate the herbicide (increased performed metabolism via P ₄₅₀ ) metabolism via P ₄₅₀ ) – HPLC analysis on the samples from atrazine HPLC analysis on the samples from atrazine hydrolysis tests showed no difference with respect • Benzoxazinones were found in L. multiflorum to control after 24hrs of incubation at 120 r.p.m. but a correlation between these and 25° C alleochemicals and atrazine tolerance was not – Bx presence in plant tissues does not necessarily observed signify Atrazine tolerance Conclusions References • Ackerman, F., 2007. The economics of Atrazine. International Journal of • L. multiflorum is a promising candidate for Occupational and Environmental Health, 13:441 ‐ 449 • Capel, P. and S. Larson, 2001. Effect of scale on the behavior of atrazine in surface field scale phytoremediation! waters. Environ. Sci. Tech., 35(4): 648 ‐ 657. • Carruthers, K., Prithiviraj, B., Fe, Q., Cloutier, D., Martin, R.C., Smith, D.L., 2000. – Ability to germinate & grow in above agronomic Intercropping corn with soybean, lupin and forages: yield component responses. European Journal of Agronomy 12, 103–115 doses of Atrazine doses of Atrazine Rohr, J. R., A. A. Elskus, B. S. Shepherd, P. H. Crowley, T. M. McCarthy, J. H. Rohr J R A A Elskus B S Shepherd P H Crowley T M McCarthy J H • • Niedzwiecki, T. Sager, A. Sih and B. D. Palmer. Multiple Stressors and Salamanders: – Elevated herbicide removal capacity Effects of an herbicide, food limitation, and hydroperiod. Ecol. Appl. 14*4) • Strong, L. C., H. McTavish, M. J. Sadowsky, and L. P. Wackett. 2000. Field ‐ scale – High initial degradation rate (Important for remediation of atrazine ‐ contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase. Environmental Microbiology 2 :91 ‐ 98. avoiding Atrazine run ‐ off) • Thurman, E. and A. Cromwell, 2000. Atmospheric transport, deposition, and fate of triazine herbicides and their metabolites in pristine areas at Isle Royale National – Already proven agronomic benefits from Park. Environ. Sci. Tech., 34: 3079 ‐ 3085. • USDA Forest Service Species Index. Available: intercropping http://www.fs.fed.us/database/feis/plants/graminoid/lolmul/all.html Accessed: 01/22/2012 4