Arsenic phytoremediation

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Credit seminar (Minor)
Phytoremediation of Heavy Metal Arsenic stress
and detoxification in plants
Rowndel Khwairakpam 2013-ADJ-21 Department of
Crop Physiology
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Contents

• Introduction
• Uptake and transport
• Mechanisms of As Tolerance and Detoxification
• Different types of arsenic phytoremediation
• Case study
• Conclusion

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Introduction

• Arsenic is the most ubiquitous environmental
  toxin and carcinogen, endangering human health
• EPA and ATSDR rank arsenic at the top of the US
  Priority List of Hazardous Substances
• Bangladesh and West Bengal, India, has been
  called the largest mass poisoning of a population
  in history
• Arsenic affected streches to Ganga-Meghana-Brahmap
  utra plains
• In the flood plains of Brahmaputra and Barack

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Stocks and fluxes of arsenic in various Earth
components.
Annu. Rev. Earth Planet. Sci. 2014
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ARSENIC OCCURENCE

• Oxidation states 5, 3, 0 and -3
• In living organisms arsenic is found in the
  pentavalent and trivalent oxidation states
• Phytoavailable Arsenate As(V) and arsenite
  As(III)
• Hyperaccumulator and Non hyperaccumulator
• Distribution of arsenic between roots and shoots
  is a dynamic process
• Phosphate transporters and NIP subfamily of
  aquaporins

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Arsenic Uptake in form of Arsenate in Plants
Mol Biophys Biochem, 2016
Fig. Arsenate transport pathway in higher plants

• the Pi transporter (PHT) proteins have a higher
  affinity for phosphate than for arsenate
• As(V) may outcompete Pi for entry into the plant

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Arsenic Uptake in form of Arsenate in Plants
Mol Biophys Biochem, 2016

• arsenite enters plant root cells through nodulin
  26-like intrinsic proteins (NIPs)
• rapidly transported through the xylem

Continued..
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Uptake in form of Methylated Arsenic in Plants

• through the aquaporin channel OsLsi1
• MMA(V) and DMA(V) enter rice roots through the
  aquaporin channel OsLsi1
• rate of uptake is much slower than that of
  As(III) or As(V)
• arsenocholine, arsenobetaine, and arseno-sugars

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Reduction of Arsenate to Arsenite

• The reduction of As(V) to As(III) occurs both
  non- enzymatically and enzymatically
• As(V) can be directly reduced to As(III) by
  arsenate reductase (ACR)
• Yeast Arsenate Reductase gene ScAcr2
• HlAsr, AtAsr/AtACR2, PvACR2, OsACR2.1

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Mechanisms of As toxicity

Biologia (2016)

• Exposure of crop plants to inorganic As results
  in uncontrolled production of reactive oxygen
  species (ROS)

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Mechanisms of As Tolerance and Detoxification

• Complexation and Sequestration of As
• Antioxidative Defense System
• Osmolyte Accumulation

Complexation and Sequestration of As

• High affinity to the sulfhydryl (SH) groups of
  peptides such as Phytochelatins and GSH
• Complexation of arsenite by PCs is an important
  mechanism of As detoxification

• (?E-C)2-G
• (?E-C)3-G

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• As preferentially binds to PC3, forming the
  AsPC3 complex
• The AsPC3 complex is the dominant complex formed
  in the As-tolerant H.lanatus
• In the As-tolerant Cysticus striatus, PC4 was the
  major species
• Helianthus annuus contained up to 14 different As
  species including monomethylarsonic
  phytochelatins-2

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• GSH can bind to several metals and metalloids and
  is also a key metabolite in cellular redox balance

Plant, Cell and Environment (2012)

• Increasing GSH synthesis is considered a means of
  increasing metal(loid) binding capacity
• a way to increase cellular defense against
  oxidative stress

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Osmolyte Accumulation

• plants tend to accumulate certain metabolites of
  low molecular weight known as compatible solutes.
• These differ among plant species

• phytopolyhydroxylated sugar alcohols- Sorbitol
• amino acids and their derivatives- Proline,
  Polyamines
• tertiary sulfonium and quaternary ammonium
  compounds- glycine betaine

Antioxidative Defense System

• (SOD),(CAT),(APX),(GPX),(GR), Ascorbate (Ascorbic
  acid), Glutathione, Carotenoids

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• ROS, are constantly produced as by-products of
  distinct metabolic pathways operating chiefly in
  chloroplasts and mitochondria
• Photosynthesis generate O2 in the chloroplasts,
  which accepts electrons passing through the
  photosystem-I and photosystem-II resulting in
  theformation of O2.-

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Metal containing FeSOD, MnSOD, CuZnSOD

• SOD constitutes first line of defense against

Heme containing Chloroplast, mitochondria,
peroxisome, apoplast

• CAT scavenges H2O2 produced in peroxisomes

Tetrameric heme containing Peroxisomes

• plays an essential role in the control of
  intracellular ROS by reducing H2O2 into water

Chloroplast, mitochondria, Cytosol
POX catalyzes lignin biosynthesis and
organogenesis via degradation of auxin or
synthesis of ethylene, and plays a role in plant
defense
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Different types of arsenic phytoremediation
Phytoextraction Absorb contaminant along with
other nutrients and water

• Hyperaccumulator-upto 1 of their dry weight
• Indicators accumulators-
• Actively accumulate trace element in their aerial
  shoot
• the ultimate goal can be achieved in less time
  owing to their high biomass
• Excluders- Restrict metalloid uptake and
  translocation of arsenic to shoot
• breakthrough step for As phytoremediation program
  was the discovery of the As hyperaccumulator fern
  P. vittata

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• Pteris vittata collect 2.3 As in its biomass and
  stores 93 of it in the fronds
• defensive strategy to kill pathogens and keep
  away herbivores
• phytoremediation by P. vittata markedly reduces
  As concentration in rice from As contaminated
  paddy soil

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• Phytostabilization
• use of the extensive root system of plants
• target of phytostabilization is stabilization and
  reduce the risk to exposure.
• Ideal As tolerant plants immobilizing the
  contaminant and poor translocation.
• Selected candidate plants should be easy to grow
  and establish dense canopies and root systems
  quickly.

• Phytochemicals exuded into the rhizosphere causes
  precipitation of the contaminants in the root
  zone.
• Binding to it on the root surfaces.
• Facilitate transfer and safe storage of As into
  the root vacuoles

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Phytovolatilization

• involves the uptake of contaminants by plant
  roots and its conversion to a gaseous state
• This process is driven by the evapotranspiration
  of plants
• The vapor released from the frond of P. vittata
  was found to contain Dimethylarsine and
  trimethylarsine are about 100 times more potent
  genotoxic

• Phytofiltration
• Several aquatic plants have the ability to remove
  As from water
• most aquatic plants have inherent drawbacks in
  achieving high BM
• small, slow-growing roots and high water content
  in their cells complicating the disposal process
  of drying, composting, or incineration.
• Natural attenuation of As mainly involves
  immobilization method through sorption

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Biotechnological Approach

• Natural hyperaccumulators of heavy metals are
  available
• Lack the critical biomass required for effective
  phytoremediation
• Biotechnological approaches
• manipulating metal/metalloid transporter genes
  and uptake systems
• Enhancing metal and metalloid ligand production
• Conversion of metals and metalloids to less toxic
  and volatile forms

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Identification of candidate genes/ regulators
Genetic and metabolic engineering approaches
Frontiers in Plant Science (2016)

• studies employing omics can elucidate the
  genetic determinants and pathways involved in
  heavy metal and metalloid tolerance in plants

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Case Study
Environ. Sci. Technol. (2013)
Yanshan Chen, Wenzhong Xu,Hongling Shen, Huili
Yan, Wenxiu Xu,Zhenyan He and Mi Ma
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Introduction

• Major interest, the plant-based cleanup of
  contaminated soils,
• One-gene transgenic approach for As tolerance and
  accumulation in Arabidopsis thaliana.
• In 2010, PvACR3 was identified in P. vittata ,
  localizes to the vacuole
• This plant is highly efficient in terms of
  extraction of As from soil and its translocation
  to above-ground biomass
• This species has great potential as a low-cost
  remediation method for As-contaminated soils

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Materials and Methods
Synthesis of the PvACR3 Gene from Pteris vittata
Generation and Selection of Transgenic Arabidopsis
BamHI and KpnI strain C58 Dip floral
transformation
Adapters 35S promoter cassette of pSN1301
Plant Growth Conditions and Arsenic-Tolerance
Analysis
22 C 16-h light/8-h dark
3 days ½ MS agar medium /- sodium
arsenite/arsenate
Total Arsenic Determination

• inductively coupled plasma optical emission
  spectrometer (ICP-OES)

Arsenite Efflux Assays and Arsenic Speciation
Analyses

• anion-exchange chromatograms from HPLC/ICP-MS

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RESULTS
PvACR3 Increases Arsenite Efflux in Yeast.
functional As(III) antiporter that played an
important role in As(III) efflux
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PvACR3 Confers Arsenic Tolerance on Transgenic
Plants

• enhanced As resistance and promoted growth,
  regardless of the presence of As(III) or As(V)

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PvACR3 Localizes to the Plasma Membrane in
Arabidopsis

• These results confirmed that PvACR3 localized to
  the plasma membrane.

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Heterologous Expression of PvACR3 Increases
Arsenite Efflux

• suggests that PvACR3 increases As(III) efflux
  into external medium in Arabidopsis roots.

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Expressing PvACR3 Decreases Root Arsenic
Accumulation and Alters the Partitioning of
Arsenic in Arabidopsis
PvACR3 increases As(III) efflux into external
medium in Arabidopsis roots.
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Increased Movement of Arsenic to Shoots Is Not
Detrimental to Overall Tolerance in Arabidopsis

• transgenic plants retained higher As
  concentrations and much higher amounts of biomass

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Expression of PvACR3 Enhances Shoot Arsenic
Accumulation in Arabidopsis after Long-Term Soil
Cultivation.
Higher AsshootAssoil ratios, Suggest that
transgenic plants would be more suitable for As
phytoremediation.
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Conclusion

• Arsenite Efflux Mediated by PvACR3 Is Critical
  for Arsenic Detoxification in Arabidopsis
• Expression of PvACR3 Enhances Shoot Arsenic
  Accumulation in Arabidopsis

• Increased As translocation to plant shoots under
  As(V)
• Effective in Arabidopsis and can be used to
  engineer As tolerance and accumulation in plants
• Additionally, efforts should be made to develop
  breeding programs to improve the biomass and
  growth habits of natural
• hyperaccumulators and breed those traits into
  non-food, high biomass, fast growing plants for
  commercial phytoremediation of heavy metals and
  metalloids.

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Thank you