Biotecnologie ambientali Phytoremediation (fitodepurazione) Parte delle slide prese da: http://courseweb.edteched.uottawa.ca/Bio4174/8%20Bioremediation%202011.ppt

PROGRAMMA Le piante coltivate e la sindrome da domesticazione: shattering e dormienza Rischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali. Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create tramite ingegneria genetica Piante per una maggiore sostenibilità ambientale (es. plastiche biodegradabili), per il risanamento (fitodepurazione) e come biosensori di contaminazione. Interazione pianta-microrganismo: le risposte di difesa delle piante e generazione di specie resistenti. Interazione simbiotiche pianta-microrganismo: fissazione dell’azoto (batteri azoto fissatori) ed efficienza nella nutrizione minerale (funghi vescicolo arbuscolari)

“Disadvantages” of plants: Phytoremediation: to remediate polluted soil and/or water with plants. An alternative to landfill disposal or physical / chemical processing. Present strategies: Bioaccumulation (transport and storage for harvest) Bioprocessing (chemical transformation to CO2, NH3, Cl- & SO42-) Advantages of plants: Plants demonstrate tolerance to toxins Photosynthesis-free energy Extensive root systems to mine soil (440 million km/h/yr**) Selective transport of materials out of water/soil Large biomass for harvesting Species adapted to different ecosystems including wetlands “Disadvantages” of plants: Transgenics & their envoronmental release **Meagher gives this value as 100 million miles/acre/yr ( = 439,767,756 km/h/y)

Tipi of fitodepurazione Phytoextraction - uptake of substances from the environment, with storage in the plant (also known as phytoaccumulation). 2. Phytostabilization - reducing the movement or transfer of substances in the environment, for example, limiting the leaching of soil contaminants. 3. Phytostimulation - enhancement of microbial activity for the degradation of contaminants, typically around plant roots. 4. Phytotransformation - uptake of substances from the environment, with degradation occurring within the plant (phytodegradation). 5. Phytovolatilization - removal of substances from the soil or water with release into the air, possibly after degradation. 6. Rhizofiltration - the removal of toxic materials from groundwater through root activity.

From Pilon-Smits (2005) Annu Rev Plant Biol 56: 15-39 Herbicides, TNT, MTBE, TCE Phytodegradation Mercury Selenium TCE, PCE Heavy metals Se, As Radionuclides TCE/PCE... Organics (PCBs, PAHs) Nonbiological remediation technologies and bio/phytoremediation are not mutually exclusive.

Pollution Heavy metals Phosphate Arsenate Nitrogen Insecticides Classification A major distinction between elemental and organic pollutants: Most organic pollutants can be mineralized, while elements cannot. Source Agriculture (pesticides, herbicides, irrigation water), mining, transport, spills (fuel, solvents), military activities (explosives, chemical weapons), industry (chemical, petrochemical), wood treatment... Heavy metals Phosphate Arsenate Nitrogen Insecticides Herbicides PCBs, TCE,... Radionuclides

Because biological processes are ultimately solar-driven, phytoremediation is on average tenfold cheaper than engineering-based remediation methods such as soil excavation, soil washing or burning, or pump-and-treat systems. Phytoremediation is usually carried out in situ contributes to its cost-effectiveness and may reduce exposure of the polluted substrate to humans, wildlife, and the environment. Examples in detail Phytoextraction (As/ H3PO4/ metals) Phytodegradation/Phytotransformation Phytodegradation of explosives Phytodetoxification of mercuric

Bioaccumulation of Arsenic the 20th most common element in the earth’s crust and the 12th most common element in the human body. Arsenic is a major worldwide contaminant that can arise through industrial activity (pesticides, mining, combustion etc.) or from soil and ground water. Associated with acute poisoning and linked to liver, lung, kidney, bladder cancer; cause skin lesions; damage to the nervous system. Physical remediation (resins etc.) Reference: http://www.who.int/mediacentre/factsheets/fs210/en/ Meyer and Verbruggen (2012) The use of the model species Arabidopsis halleri towards phytoextraction of cadmium polluted soils New Biotechnology Volume 30, Number 1 November 2012 http://www.sciencedirect.com/science/article/pii/S1871678412001422# 8

Bioaccumulation of Arsenic In India and Bangladesh (around the Bay of Bengal) ~400 million people are at risk of arsenic poisoning, and up to 40 million people drink well water containing toxic levels of arsenic. Reference: http://www.nature.com/news/2008/080716/full/454263a.html Limite per l'arsenico proposto dall'OMS e valido per gli USA è di 0.01 mg/L (10 μg/L, 10 ppb). Limite europeo ammesso per il Glifosate (per la potabilità): 0.1 ppb 9

Bioaccumulation As/Hg: General Strategy (Meagher & Heaton, 2005) Reference: Meagher, R. and Heaton, C. (2005) Strategies for the engineered phytoremediation of toxic element pollution: mercury and arsenic. J. Ind. Microbiol. Biotechnol. 32: 502-513. 10

γ-glutamylcysteine synthetase Arabidopsis engineered to hyperaccumulate arsenic Bioaccumulation of Arsenic arsenate reductase γ-glutamylcysteine synthetase Strategy behind cloning bacterial arsenate reductase (ArsC) catalyzes reduction of arsenate to arsenite. bacterial γ-glutamylcysteine synthetase (γ-ECS) catalyzes the formation of γ-glutamylcysteine (γ-EC) from glutamate and cys for synthesis of glutathione (GSH) and phytochelatins (PCs; three arrows) reduced arsenite can bind organic thiols (RS) such as those in γ-EC, GSH, and PCs. Then transfer to vacuole. Reference: Dhankher, et al. (2002) Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and -glutamylcysteine synthetase expression. Nature Biotechnology  20: 1140 – 1145. Dhankher, et al. (2002) 11

Bioaccumulation of Arsenic The prokaryotic arsC gene has been previously shown to confer resistance in the eukaryote Saccharomyces cerevisiae

The bacterial ArsC enzyme was expressed under control of the soybean ribulose bisphosphate carboxylase small-subunit (rubisco) SRS1 gene promoter, which shows strong light-induced expression in leaves and stems. Comparative growth inhibition of three SRS1p/ArsC lines % relative concentration of free AsO43-, AsO33-, and As(III) tris-GSH in leaves

Comparative growth of two hybrid lines, the parental lines expressing either ArsC or γ-ECS alone, and (WT) grown for 3 weeks on NaAsO3 I doppi trasformanti resistono a concentrazioni di arseniato più alte

Arsenic contamination: removed by phytoextraction P. vittata, a natural arsenic hyperaccumulator, can tolerate soil concentrations of 1,500 μg arsenate/g and can accumulate up to 23 mg arsenic/g in its shoots (fronds). The striking difference between P. vittata and arsenic non-accumulators is the enormous transport of arsenic from roots to shoots in P. vittata. In most plants, only a small fraction of the arsenic taken up from soil by roots accumulates in the above-ground tissue (<20%), whereas P. vittata accumulates up to 95% of the arsenic in above-ground tissue. “The Chinese Ladder fern Pteris vittata, also known as the brake fern, is a highly efficient accumulator of arsenic.  P. vittata grows rapidly and can absorb up to 2% of its weight in arsenic.  . . When grown on soil with 100 ppm not only did it absorb more arsenic, but it grew 40% larger than normal.” Lena Q. Ma, 2001

Acrocephalus robertii Co(leaves) 1490 Psychotria douarrei Ni (roots) PLANTS METAL CONCENTRATION (mg metal /kg dry matter) Eichhornia crassipes Fe 14400 Thlapsi caerulescens Zn(roots) 25000 Minuartia verna Cu(roots) Pb(roots) Cd(leaves) 1850 26300 348 Jasione montana As (leaves) 3100 Mechovia grandiflora Mn(leaves) 7000 Acrocephalus robertii Co(leaves) 1490 Psychotria douarrei Ni (roots) 92000 Pearsonia metallifera Cr (roots) 1620 Astragalus preussi U(leaves, roots) 70 Astragalus acemosus Se(leaves) 15000 Alyssum bertholonii Ni(shoots) 13400 Miconia lutescens Al (shoots) 6800

Heavy metal hyperaccumulators Thlaspi montanum var. montanum, a Ni-hyperaccumulator plant that grows on Serpentine soils, research of Martha Palamino, IB graduate student (UC Berkeley).

Hyperaccumulator plants Alyssum serpyllifolium Brassica juncea Thlaspi caerulescens Pteris vittata

Phytoextraction of excess nutrients: Pig slurry cleanup Using duckweed (Lemna) 19

Rhizofiltration using Arundo donax • Sewage and urban wastewaters • Halogenated residues (USA) • N, P and S rich wastewaters (for nutrient stripping) • Saline waste waters (reduce volumes by transpiration thru Adx to grow biomass) Immagine da una presentazione del South Australian Research & Development Institute (Arundo-SARDI.ppt) Intensive root system (Marton and Czako, USA) 20

Song et al., (2003) Nat Biotechnol. 21:914-9.

YCF1 confers Pb(II) and Cd(II) resistance to yeast Il mutante nullo ycf1 di lievito è più sensibile a Pb2+ e Cd2+ Il mutante sovraesprimente YCF1 è più resistente a Pb2+ e Cd2+ Il mutante sovraesprimente YCF1 presenta molto più mRNA

Fluorescence of GFP-YCF1 Pb Cd La sovraespressione di YCF1 in lievito porta un maggior accumulo di Pb e Cd YCF1 è presente nella membrana plasmatica e vacuolare brightfield Fluorescence of GFP-YCF1 FM4-64 stains plasma (top) and vacuolar (bottom) membranes

Fluorescence of GFP-YCF1 RT-PCR su Arabidopsis che esprime YCF1 Southern blot sulla RT-PCR c brightfield Fluorescence of GFP-YCF1 . GFP-YCF1 espresso in protoplasti di A. thaliana è localizzato sulla membrana plasmatica e vacuolare

Crescono meglio (maggior biomassa) Generazione T2 Arabidopsis transgenica per YCF1 mostra una maggior resistenza a a Pb2+ e Cd2+ Generazione T2 Crescono meglio (maggior biomassa)

Il contenuto espresso in μg/pianta è più efficace Il contenuto per g di peso fresco non è molto diverso, ma ci deve essere una diversa localizzazione Il contenuto espresso in μg/pianta è più efficace Vacuoli isolati dal trasformante accumulano più coniugati di GS-Cd

Phytoextraction in action Accumulatrici naturali vengono trapiantate sul terreno contaminato The location: a base-metal smelter, South Africa The problem: Ni contamination over 5ha due to Ni salt storage and spillage The solution: phytoextraction using a native nickel-accumulating species La biomassa viene poi bruciata

Phytoextraction for gold Thio-ligands can induce the solubility and uptake of gold from waste, low-grade rock Discovery made in New Zealand Proof of concept achieved and the technology is being field tested Aim is a crop of 10 t/ha biomass with 100 mg/kg gold concentration dry weight This will yield 1000 g of gold per hectare as well as other metals made soluble Current focus is on mercury (Hg) removal at the same time as gold

Biodegredation of Organomercury Methylmercury is a pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. The main targets include free cysteine in proteins and peptides leading to damage in the central nervous system. Symptoms include sensory impairment (vision, hearing, speech), disturbed sensation and a lack of coordination. Mercury occurs in deposits throughout the world and it is harmless in an insoluble form, such as mercuric sulfide, but it is poisonous as methylmercury [CH3Hg]+ due to its aqueous solubility. Sources of Mercury include burning coal and mineral extraction. Many uses of mercury are being curtailed or eliminated. Lyrra et al. (2007) Coupling two mercury resistance genes in Eastern cottonwood enhances the processing of organomercury. Plant Biotechnology Journal 5: 254-262. Omichinski, J,G, (2007) Toward methylmercury bioremediation. Science 317: 205-206. 29

Phytostabilization of mercury by willow roots Yaodong Wang, 2004

Phytodetoxification of mercuric compounds Bizily, S., Rugh, C., Meagher, R. (2000)  Phytodetoxification of hazardous organomercurials by genetically engineered plants.  Nature Biotechnology.  18:213-217. Methylmercury is found in wetlands and aquatic sediments worldwide.  Both ionic mercury and methylmercury are absorbed in the gastrointestinal tract of animals, but methylmercury is retained much longer in the body and is, therefore, is carried up through the food chain more efficiently.  Plants engineered with both the merA and merB genes should be able to extract methylmercury from contaminated environments and transpire Hg(0) into the atmosphere.   Because Hg(0) resides in the atmosphere for approximately two years, transpired Hg(0) will be diluted to much lower concentrations before being redeposited into terrestrial waters and sediments rather than being concentrated in one area.  Additionally the amount of Hg(0) emitted from sites undergoing phytovolitalization can be regulated and will most likely be small in comparison to the concentrations of Hg(0) already in the atmosphere. merB       merA/merB      merA       control        

Rhizofiltration: sunflowers after Chernobyl disaster Plants on rafts in pondwater: removed radionuclides of strontium, cesium, etc.

Tolerance mechanisms for inorganic and organic pollutants in plant cells. Detoxification generally involves conjugation followed by active sequestration in the vacuole and apoplast, where the pollutant can do the least harm. Chelators shown are GSH: glutathione, Glu: glucose, MT: metallothioneins, NA: nicotianamine, OA: organic acids, PC: phytochelatins. Active transporters are shown as boxes with arrows. I contaminanti organici sono spesso biodegradati

Phytodegradation/Phytotransformation Breakdown of contaminants taken up by plants through metabolic processes within or external to plant through effect of compound produced by plants Oxidoreductases, dehalogenases, nitroreductases, peroxidases, nitrilases & laccases may involved Phytodegradation/Phytotransformation

Phytodegradation of pesticides by a notorious pest plant The potential of water hyacinth (Eichhornia crassipes) to remove a phosphorus pesticide ethion were investigated. The disappearance rate constants of ethion . . . implied that plant uptake and phytodegradation contributed 69% and that of microbial degradation took up 12% to the removal of the applied ethion. The accumulated ethion in live water hyacinth plant decreased by 55-91% in shoots and 74-81% in roots after the plant growing 1 week in ethion free culture solutions, suggesting that plant uptake and phytodegradation might be the dominant process for ethion removal by the plant. This plant might be utilized as an efficient, economical and ecological alternative to accelerate the removal and degradation of agro-industrial wastewater polluted with ethion. Xia H, Ma X. (2006) Phytoremediation of ethion by water hyacinth (Eichhornia crassipes) from water. Bioresour Technol. 2006 May;97(8):1050-4.

Michael Dossett1, and Xiaoping Wang2, and Stuart E. Strand3 Phytodegradation of TCE, other chlorinated hydrocarbons by hybrid and/or transgenic poplar A MASS BALANCE FIELD TRIAL OF CARBON TETRACHLORIDE PHYTOREMEDIATION USING POPLAR: PHYTODEGRADATION IS THE LIKELY FATE Michael Dossett1, and Xiaoping Wang2, and Stuart E. Strand3

- Trichloroethylene Chloroacetate Trinitrotoluene Triaminotoulene Cl Cl Cl O C=C C C CO2 + Cl2 H Cl Cl2 H O Trichloroethylene Chloroacetate CH3 NO2 CH3 NH3+ NO2 NH3+ CO2 + NH4+ NO2 NH3+ Trinitrotoluene Triaminotoulene

Biodegradation of explosives The Problem: Contamination to explosives TNT, RDX and glycerol trinitrate. “Exposure to TNT and RDX, and their degradation products causes symptoms such as anemia and liver damage. These chemicals can be lethal and are suspected carcinogens. Hundreds of tons of these compounds are found in sediments at innumerable manufacturing sites and storage sites for unexploded ordnance around the world. Tens of thousands of acres of land and water resources are unsafe because of RDX and TNT contamination.” The “Solution”: Engineer plants that are able to degrade these compounds in situ. References: French et al. (1999) Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase Nature Biotechnology 17: 491-494. Meagher, R.B. (2001) Pink water, green plants, and pink elephants. Nature Biotechnology 19: 1120-1122. Rylott et al. (2006) An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX. Nature Biotechnology 24: 216-219. Meagher, R.B. (2006) Plants tackle explosive contamination. Nature Biotechnology 24: 161-163. 38

Biodegradation of explosives These are the targets Breakdown of TNT to ADNT (monoaminodinitrotoluene) can create “sterile” pink water lagoons. 39

Biodegradation of explosives: The general strategy is to isolate catabolic genes and through standard cloning technologies convert them into plant genes expressed via a constitutive promoter such as the CaMV 35S promoter for expression throughout the plant. tissue-specific promoter for expression to leaves, stems etc. Following transformation and plant regeneration, the properties of the plant are evaluated by standard tests. Bacteria are the usual source of the genes, bacteria such as Enterobacter cloacae or Rhodococcus rhodochrous that can grow on these contaminants. 40

Biodegradation of explosives: Summary from Meagher (2006). Key: RDX, TNT, ADNT: See Fig 6 NR: nitroreductase XplA: RDX-degrading cytochrome P450 NDAB: 4-nitro-2,4-diazabutanal. These rhodococcal strains degrade RDX by initial denitration, followed by ring cleavage, yielding nitrite, formaldehyde and 4-nitro-2,4-diazabutanal7, 9 (Fig. 1a). 41

Proposed RDX breakdown pathway Protein purification on 10% SDS-PAGE. Lane 1, molecular weight marker; Lane 2, solubilized recombinant protein; Lane 3, affinity purified XplA. (c) UV-visible absorbance spectra of purified XplA: oxidized; sodium dithionite reduced; reduced bubbled with carbon monoxide. (d) UV-visible absorbance spectra of extracted flavin cofactor. (e) Activity of purified protein under anaerobic conditions. Solid shapes, active XplA; open shapes, boiled XplA.

Biodegradation of explosives: from Rylott et al. (2006) Conclusion from these experiments: decontamination works in a model system TNT can be catabolized RDX is catabolized and the nitrogen used for growth (win-win!!) 43

(Hexahydro-1,3,5-trinitro-1,3,5-triazine) Transcriptional Profiling: Arabidopsis Thaliana Root Responses to Explosives SAGE—Serial Analysis of Gene Expression—30 000 tags Very different metabolism RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) TNT (Trinitrotoluene) I geni indotti sono ottimi candidati per la sovraespressione NPR1-like protein putative glutathione transferase DnaJ-like protein Le reazioni di detossificazione seguono il modello del “green liver” di Sandermann MYB like protein gamma-VPE (vacuolar processing enzyme) Carbamoyl-P synthetase small subunit putative transcription factor transporter-like protein putative peroxidase putative ser/thr-protein kinase putative 3-dehydroquinate synthase monodehydroascorbate reductase-like vacuolar H+-ATPase subunit H (VHA-H) NAM, no apical meristem, - like protein vacuolar H+-transporting ATPase 16K chain P2 similar to bacterial tolB proteins putative transcription factor alpha-hydroxynitrile lyase-like protein cytochrome P450, putative (TCCCCTATTA) no matches in genome

Jackson et al., (2007) Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B. PNAS 104:16822–16827. E’ possibile sfruttare i sistemi degradativi scoperti nei batteri o altri microrgansmi e introdurli nelle piante

Uptake and metabolic use of cyanide by willows Transport and metabolism of free cyanide and iron cyanide complexes by willow S. EBBS1, J. BUSHEY2, S. POSTON1, D. KOSMA1, M. SAMIOTAKIS1 & D. DZOMBAK

Remediation of saline soils Salicornia (pickleweed) accumulates salt in vacuole. A form of table salt can then be extracted from plant.

Aluminium detoxification: Gene for citrate synthase used for production of transgenic plants produce higher level of citrate & secrete it into the soil through roots ,which bind with Aluminium which is incapable of entering roots E.g tobacco, papaya, rice & corn TNT detoxification: Transgenic tobacco produced by transformation with nitroreductase gene nfs1 isolated from Enterobacter cloacae can tolerate high TNT conc.

The most important single act of phytoremediation? 6 CO2 + 6 H2O C6H12O6 + 6 O2 In sintesi: tanta ricerca, tanti risultati interessanti ma per ora non ci sono prodotti commerciali e vista l’attuale situazione normativa difficilmente se ne vedranno

Bibliografia Song et al. (2003) Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat. Biotech. Ghosh Moyukh , Singh S.P ,Trivedy R.K, Sharma Sadhana(Eds) 2005 Phytoremediation of heavy metal contaminated soils:role of natural & synthetic chelatins,Biotechnological applications in environmental management,B.S.publication. Sharma J., Saini V., Singh A., Singh N., Trivedy R.K, Sharma S. (Eds) (2005) Phytoremediation of organic pollutants , Biotechnological applications in environmental management, B.S.publication. Kumar R., Sharma J., Gaur P.,Trivedy R.K, Sharma Sadhana (Eds) 2005 Phytoremediation of soil contaminated with heavy metals,Biotechnological applications in environmental management ,B.S.publication. Singh B.D Biotechnology Expanding horizon , Kalyani Publishers 2008 Mc Grath S.P , Zhao F.J (2003) Phytoextraction of metal and metalloid from contaminated soil , current opinion in Biotechnology 14:277-282. Richard B Meagher (2000) Phytoremediation of toxic elemental and organic pollutants. Current opinion in plant biology 20003:435. Singh J.S , Singh S.P , Gupta S.R , Ecology Environment and Resources Conservation , Anamaya publishers 2006. Rao et al., (2009) Phytoremediation and phytosensing of chemical contaminants, RDX and TNT: identification of the required target genes. Funct Integr Genomics. ???