Antioxidants in Plants

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Antioxidants in plants

Rio December 2007


Institute of Plant Biotechnology

For Developing Countries

Rio, December 4, 2007

Mike May

IPBO

Website: http://www.ipbo.ugent.behttp://www.psb.ugent.be

http://www.efb-central.orghttp://www.pubresreg.org

E-mail : [email protected]
http://www.ipbo.ugent.be/http://www.psb.ugent.be/http://www.efb-central.org/http://www.pubresreg.org/http://www.pubresreg.org/http://www.efb-central.org/http://www.psb.ugent.be/http://www.ipbo.ugent.be/


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Overview of seminar

Redox homeostasis:

Understanding functionthrough genetics:

Understanding thefunction of ascorbate:

Interactions with glutathioneand tocopherol:

Applications and future focus:


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New approaches for functional

analysis and crop improvement

1904, Agrobacteriumtumefaciens

1974, TiPlasmid, Gent

1990sgenomics

Overexpression& silencing
http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8http://rds.yahoo.com/_ylt=A9iby4GL3lJF7QcBxFaJzbkF;_ylu=X3oDMTBjdmNoOTVjBHBvcwMyBHNlYwNzcg--/SIG=1gd463pfr/EXP=1163145227/**http%3a//images.search.yahoo.com/search/images/view%3fback=http%253A%252F%252Fimages.search.yahoo.com%252Fsearch%252Fimages%253Fp%253DCROWN%252BGALL%2526ei%253DUTF-8%2526fr%253Dsfp%2526x%253Dwrt%26w=180%26h=143%26imgurl=soilplantlab.missouri.edu%252Fplant%252Fimages%252Fcrowngalleuonymus.jpg%26rurl=http%253A%252F%252Fsoilplantlab.missouri.edu%252Fplant%252Fdiseases%252Fcrowngall.htm%26size=12.9kB%26name=crowngalleuonymus.jpg%26p=CROWN%2bGALL%26type=jpeg%26no=2%26tt=385%26oid=2be3f4eef1bb27cc%26ei=UTF-8


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Redox homeostasis:

Reactive oxygen species

Antioxidants


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Aerobic life is based on the reduction of oxygen

via electron transfer systems: production in chloroplasts; consumption in mitochondria

This is an inefficient process; And results in the continuous production of

reactive oxygen species (ROS):

Hydrogen peroxide; Superoxide;

Hydroxyl radical

Reactive oxygen species


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Nucleus(not a source,

but ROS-mediated damageto DNA results inmutation)

Mitochondrion(ATP synthesis)

Chloroplast (water splitting, electrontransport)

PeroxisomeEndogenous sources of ROS in plants


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Pathogens(oxidative burst)

Mitochondrion(Herbicides, ozone)

Chloroplast (herbicides, highlight)

Drought, waterloggingpollutants, salinity

Stress related sources of ROS in plants

Temperatureextremes


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Unless ROS are removed from biological

systems, they cause damage to: Lipids (leakage of solutes);

Proteins (loss of function, aggregation);

Nucleic acids (mutation, nicks)

The evolution of aerobic life forms has gonehand in hand with the evolution of highlyconserved mechanisms for ROS removal andsensing of ROS.

ROS - consequences


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ROS-mediated chloroplast damage (rice)

Methyl

viologen

Salt


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Enzymatic processes: Superoxide dismutases peroxidases catalase thioredoxin/glutaredoxin

Major antioxidant compounds/cofactors: NAD(P)H ascorbate

tocopherols (vitamin E) glutathione pigments

polysaccharides

ROS - Removal


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ROS are consequently implicated in a wide variety ofdisorders:

In plants: photorespiratory damage; pathogen attack; Biotic and abiotic stresses

In animals: inflammatory disorders immune system Cancer Degenerative disorders such as Parkinsons and Alzheimer's genetic disorders eg Lou Gehrig syndrome, Downs syndrome

The study of ROS is therefore one of the most vibrant

areas of research in plant and animal science:

ROS pathology


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Fundamental: biotic and abiotic stresses; development; metabolic control; organelle dialogue;

Signalling, transcription factors, protein complexes Applied:

implication in human health (vitamin C/E);

functional foods; nutraceuticals; development of crops for climatic extremes

new growth regulators and herbicides

Study of ROS in plants


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The Ascorbate-Glutathione cycle


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Redox homeostasis

Diagram courtesyOf Christine Foyer


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Developmental control of synthesis

Environmental control of synthesis

Understanding the

function of ascorbate:


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Development-dependent and oxidative

stress-dependent Protein Carbonylformation

Source; Christine

Foyer, University ofNewcastle

A i id i l


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Ascorbate synthesis and accumulationdeclines with age: Maize

GLDH = Galactonolactone dehydrogenase, the lastenzyme in the biosynthetic pathway

Source; ChristineFoyer, University ofNewcastle

A ti id t i l t


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Ascorbate accumulates under high light inArabidopsis

LL IL HL

Source; ChristineFoyer, University of

Newcastle



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Understanding function

through genetics:

vtcmutants of Arabidopsis

Pathogen-mediated signalling interactions with glutathione and tocopherol



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Two pathways for biosynthesis

Source; Wolucka &Van Montagu.Phytochemistry2007

L-Galactose pathway

L-Gulose pathway



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vtc 1

vtc 2/5

vtc 4

Source: Linster et al.,(2007) JBC 282, 18879-18885)

Mapping of Arabidopsismutants on the

L-galactose pathway



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Plants:

Galactolactonedehydrogenase(GLDH)

Animals:Gulonolactoneoxidase

(GLO)

Evolutionary divergence of pathways

Glucose 6-P

AscorbateHumans,higher primates

vtc 1

vtc 2/5

vtc 4

Gulose pathway

Galactose pathway



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Analysis of vtc1 & vtc2to study the rolesof ascorbate in plants

vtcmutants wereisolated in a screenfor ozone sensitivity




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vtc1 mutants mature slower than wild type




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Low ascorbate in the vtc1 mutants:Enhances resistance to biotrophic pathogens

vtc1

Wildtype




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t o da ts p a ts

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Low ascorbate in the vtc1 mutants:Creates a lesion-mimic phenotype



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p

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Low ascorbate in the vtc1 mutants:Altered SAR signalling

NRP1 NRP1

NRP1 NRP1

Salicylic Acid

Pathogen

H2O2

Phytoalexins

NRP1 NRP1

NRP1 NRP1

GSH

NRP1SH

SH

WRKY gene expression

PR proteinsVtc1 flux

WT flux



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Interactions with

glutathione and tocopherols:

Pathogen-mediated signalling

root development & cell cycle



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Biosynthesis of tocopherol

Porfivora et al(2002)PNAS 99, 12495-12500



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Tocopherol null mutants are not highly

stress-sensitive




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Ascorbate, GSH and tocopherol

compensate for each other




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Biosynthesis of GSH

Glu + Cys Glu-Cys Glu-Cys-Gly

Gly

GSH-1 GSH-2



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cad-2:(Heavy metal screen) cadmium sensitive

otherwise normal 15-30% GSH levels

pad-1:(pathogen screen) phytoalexin deficient

pathogen sensitive

otherwise normal 22% GSH levels

rml-1:(root meristem screen) root meristemless

GSH auxotroph almost undetectable GSH

gsh-1 mutants of Arabidopsis

demonstrate the importance of GSH



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GSH biosynthesis is stimulated in response topathogen attack;

pad-1 mutants are sensitive to pathogen attack

and do not accumulate phytoalexinscad-2is more resistant than pad-1

Lowering ascorbate enhances resistancewhilst lowering GSH enhances susceptibility

GSH and pathogen signalling



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NRP1 NRP1

NRP1 NRP1

Salicylic Acid

Pathogen

H2O2

Phytoalexins

NRP1 NRP1

NRP1 NRP1

GSH

NRP1SH

SH

WRKY gene expression

PR proteins

GSH and pathogen signalling

pad-1

Biosynthesis

X



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GSH and the cell cycle

Oxidative

Stress (e.g. P53)



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Quiescent

centre (G0)

Distribution of GSH in Arabidopsisroots

Root Hair

Source: MikeMay, Gent

University



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Role of GSH in cell cycle progress

in synchronised By-2 cell suspensions

GSH is needed for the G1 S transition

+ GSH

control

+ BSO

GSHlevels

DNASynthesis(S-phase)

Cell cyclephase

Source: MikeMay, GentUniversity



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Role of GSH in root but not shoot cell

cyclerml-1

rml-1+ GSH

Wild Type

Source: MikeMay, Gent

University



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Applications and future

focus:

Functional/super foods

Nutraceuticals Metabolomics



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Fruit or vegetable Ascorbic acid content

(mg per 100 g)

Rosehip 1000

Blackcurrant 200Kale 186Green pepper 128Horse radish 120Broccoli 113

Brussels sprouts 109Watercress 79Strawberry 59Spinach 51Oranges/lemons 50

Cabbage 47New potato 30Carrot 6

A. thalianasuspension cells 8

BY-2 suspension cells 0.2-2

Vitamin C content of some common foods



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Emblica officinalis: Amla or Indian Gooseberry

Euphorbiaceae;

Used in Ayurveda for over

2000 years; A component of a patented

formula for treatment of asthmaand allergic rhinitis in the USA;

Vitamin C (700mg per 100gfruit), flavonoids, polyphenols,alkaloids;

Immune system, heart, chroniclung problems, liver, circulation;



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Lycium chinense: Goji Berry

Solanaceae;

Used in TCM for over 2000

years 54 new product introductions in

2006

China produced 95,000 tonnesin 2004

$120 million in exports

Vitamin C (163% DRI),

polysaccharides, zeaxanthin,butyric acid

Immune system, eyesight, liver,circulation



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Acai berry; the amazonianwonderfruit

Antioxidants in Plants

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