Genetic Engineering of Plant Selenium Metabolism for Phytoremediation

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1 Genetic Engineering of Plant Selenium Metabolism for Phytoremediation Elizabeth Pilon-Smits Douglas Van Hoewyk Miriam Loeffler Marinus Pilon Colorado State University Norman Terry Danika LeDuc UC Berkeley Gary Banuelos USDA-ARS, Fresno, CA

2 Introduction of plant Selenium metabolism Genetic engineering of plant Se metabolism Testing transgenics for Se phytoremediation Ecological aspects of plant Se accumulation

3 Selenium Nutrient Toxin Pollutant

4 Selenium deficiency and toxicity are both problems in the U.S. Kubota and Allaway, 1972

5 Can plants be used to clean up Se, or as source What can of Se plants in animal do with diet? selenium? selenate selenite Se-cysteine proteins sulfate sulfite cysteine accumulation volatilization In some plants: hyperaccumulation Se-methionine methionine dimethylselenide dimethylsulfide Stanleya pinnata

6 Genetic engineering of plant Se metabolism proteins selenate selenite ATP sulfurylase(aps) alanine + Se Se-cysteine lyase (SL) Se-cysteine Cystathionine γ synthase (CγS) Se-methionine Brassica juncea dimethylselenide

7 APS overexpression leads to: enhanced Se accumulation as organic Se enhanced Se tolerance no difference in Se volatilization Shoot Se concentration (mg Se g -1 DW) APS9 APS8 WT External selenate concentration (µm)

8 proteins selenate selenite ATP sulfurylase(aps) alanine + Se Se-cysteine lyase (SL) Se-cysteine Cystathionine γ synthase (CγS) Se-methionine Kerry Hale dimethylselenide Tiffany van Huysen

9 CgS transgenics: volatilize more Se, accumulate less Se Se volatilization (µg Se g -1 FW) Volatilization.25 SeO3 4 µm WT B3 B11 B12 B13 B15 CgS Shoot Se conc. (mg Se kg -1 DW) Accumulation SeO 3 2 µm WT B3 B11 B12 B13 B15 CgS

10 proteins selenate selenite ATP sulfurylase(aps) alanine + Se Se-cysteine lyase (SL) Se-cysteine Cystathionine γ synthase (CγS) Se-methionine Jennifer Owen dimethylselenide Gulnara Garifullin

11 WT cytsl13 cpsl1 5 Control 25 5 µmsecys CytSL Lines: Enhanced Se tolerance Control Root length (mm) Arabidopsis CpSL lines: Reduced Se tolerance SeCys 5 µm SeO µm SeO µm WT cytsl13 cpsl1 Root length (mm) WT 25µM SeO cytsl 1 9 cpsl WT 5 µm SeO cytsl 1 9 cpsl

12 Arabidopsis cytsl & cpsl transgenics: have less Se in protein accumulate more Se µg Se. g protein WT cytsl cpsl 9 Shoot Se conc. (µg g -1 DW) WT cytsl cpsl

13 Genetic engineering of plant Se metabolism alanine + Se proteins accumulation tolerance cyt cp Se-cysteine lyase (SL) selenate ATP sulfurylase(aps) selenite accumulation tolerance Se-cysteine cystathionine γ-synthase (CγS) Se-methionine accumulation tolerance volatilization dimethylselenide

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15 Greenhouse pot experiment using Se-rich CO soil Tissue Se concentration (µg Se g -1 DW) Se concentration Shoot FW (g) 9 Biomass WT APS 8 CGS Stanleya pinnata APS transgenics accumulated 3-fold more Se than wildtype plants CgS transgenics accumulated ~4% less Se than wildtype plants (due to higher Se volatilization?)

16 Field experiment in CA central valley Accumulated Se Se Concentration 14 8 APS transgenics accumulated 5-fold more Se than wildtype plants Accumulated Se (mg m -2 ) Leaf Se Concentration (mg kg -1 ) WT APS

17 Identification of new genes involved in Se metabolism selenate proteins selenite alanine + Se Se-cysteine lyase (SL) Se-cysteine Se-methionine SL activity found in Arabidopsis dimethylselenide

18 Arabidopsis thaliana cpnifs In chloroplast SeCys Lyase activity and Cys desulfurase act 3-fold higher Functions?

19 CpNifS overexpression WT Root length (mm) Doug van Hoewyk enhances Se tolerance Control Se (Se 4 ) WT WT NifS NifS µg Se g protein a b b Wt Shoot conc. (mg/kg DW) Expression (Western blot) and Se & S accumulation Se WT NifS + Se (Se 4 ) S WT NifS Less Se incorporation into protein Function in Se tolerance?

20 Microarray analysis Material Wildtype & CpNifS overexpressors Treatments +/- 4 µm selenate Findings Takahashi lab, RIKEN General: Effect of Se on transcriptome indicative of stress and S deficiency Effects less/more pronounced in overexpressors: - Genes related to S-deficiency less upregulated, but sulfur transporter sultr2;1 (root xylem loading) more upregulated explains higher S, Se in shoot - General stress proteins (e.g. hsp) expressed, induced more in overexpressors

21 Conclusions Part I It is possible to genetically manipulate plant Se metabolism This may be used to: - enhance phytoremediation efficiency - create fortified foods - promote basic scientific knowledge

22 Ecological Aspects of Plant Se Accumulation How does Se accumulation affect other organisms? herbivores microbes plants Brassica juncea Why do some plants hyperaccumulate Se? Astragalus.5% Se Does Se protect against biotic or abiotic stress? Stanleya.1% Se

23 -Se +Se -Se +Se.1% Pieris rapae caterpillars prefer Brassica leaves Se 6h -Se Brassica juncea +Se Feeding rate (g FW leaf d -1 g FW animal -1 ) Se +Se %FW d -1 9d old caterpillars Se +Se -Se +Se plants are toxic to the caterpillars Caterpillar FW (g) +Se Se+Se

24 Se in leaves deters colonization of Brassica by green peach aphids 5 +Se (a) Aphids per plant (a) Leaf Se conc. (mg kg -1 DW) (b) Supplied Se concentration (µm) Leaf Se conc. (mg kg -1 DW) Se -Se -Se No Choice No Choice (b) Supplied Se concentration (µm)

25 Aphid population growth (% week -1 ) Aphids per leaf (a) (b) (c) Supplied Se conc. (µm) Leaf Se conc. (mg kg -1 DW) Supplied Se concentration (µm) Leaf Se conc. (mg kg -1 DW) Se in leaves is toxic to aphids Aphids per leaf can be used as a pesticide

26 Se protects B. juncea from fungal infection Alternaria Fusarium Se +Se 1 5 -Se +Se -Se +Se Number of lesions leaf -1 Seedling growth (%FW gain week -1 ) control +Fusarium

27 Field study using plants +/- Se Se may protect plants from prairie dog herbivory # leaves eaten/plant Se -Se Se conc. (ppm) 7 6 R 2 = No. leaves eaten B. juncea

28 Se hyperaccumulators in the field contain fewer insects per leaf area than nonaccumulator relatives.1.5 Insects / cm 2 leaf.15 p <.1 Miriam Loeffler. A. bisulcatus A. control S. pinnata S. control Hyperaccumulator species appear to harbor specialist herbivores

29 Conclusions Part II Se can protect plants from herbivory Se can protect plants from fungal infection sheds light on possible selection pressures for evolution of hyperaccumulation may have implications for phytoremediation and applications in agriculture

30 Brady Hanso n Tim Anders on Hong Ye Marinus Pilon Doug Van Hoewyk Jennifer Owen Hale Tufan Salah Abdel-Ghany LiHong Zhang Tiffany Van Huysen Thank you! Kerry Hale Stormy Dawn Lindblom Gulnara Garifullina This research was made possible by funds from NSF, EPA, USDA Brady Hanson Ashley Ackley