Genetically Modified Crops and Food Security: an African Perspective

Some Biotechnology Success Stories from

Africa ....

Countries with Biotechnology Research Programmes

* = GM technologyEgypt* Morocco*Sudan MaliEthiopia Ghana*NigeriaKenya* Tunisia*Uganda ZambiaZimbabwe* South Africa*Madagascar Mauritius*

African Success Stories

Beans in Kenya 27 improved varieties wrtroot rot and yield 11 selectedVirus resistant sweet potato in KenyaNew Rice for Africa (NERICA) 10,000 ha in Uganda wrt nutritionClean pototo seeds bacterial wilt resistance in UgandaClean potato seeds and sugarcane virus resistance in Mauritius

Xerophyta viscosa Baker(Hydrated)

Xerophyta viscosa Baker (Dehydrated)

Xerophyta viscosa Baker (Rehydrating)

ENVIRONMENTAL STRESSES

HIGH TEMPERATURE

LOW

TEMPERATUREDROUGHT

HIGH LIGHT SALINITY

Differential Screening of X.viscosa

(85%-5% RWC) cDNA library

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

Hydrated

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

Dehydrated

Osmotic stressOsmotic stress

Membrane proteins(sensor, channels)

Second messenger(calcium, inositol

tri phosphate)

Transcription factors(DREB, EREBP, ABREBP, ICE)

Detoxification(GST, SOD)

Protein kinases(MAPK, MAPKKK)PI turnover

(phospholipase C,phosphatidic acid

phosphatase)

Protection factors(heat shock proteins,

LEA proteins)

Osmoprotectants(proline, sugars)

Membrane protein:XVSAP1

Signalling:XvEFH

XvCaM

Transcription factor XvDREB1A

DetoxificationXvPer1XvPrx2XvVTC2

Protein phosphataseXvPP

Proton ATPasesubunit: XVVATP1

Protection factors:XVT8(Dehydrin),

XvHSP90

Synthesis of osmoprotectants:XvGolS, AtGolS,

ALDRXV4, XvINO1

XvPer1 and XvPrx2, novel antioxidant enzymes

UV irradiation

High and low temperature

Dehydration

R O S

High light intensity

Pollutants, e.g. SO2,

herbicides, toxic metals.

High Salinity

Peroxiredoxins• Abundant low efficient

peroxidases

• There are four types:

• 1-cys Prx

• 2-cys Prx

• Type II Prx

• Prx Q

Peroxisome& CytosolPrxIIBPrxIICPrxIIDXvPrx2

Nucleus1-Cys PrxXvPer1

MitochondrionPrxIIF

Chloroplast2-Cys PrxA2-Cys PrxBPrxQPrxIIE

• Important antioxidants – scavenge ROS • Known to play an important role in antioxidant defense,

respiration and stress response• Likely involved in modulating redox signals?• Prxs can be regenerated by electron donors

XvPer1

Shaheen Mowla

Collaborator: Prof. Christine Foyer, Rothamsted Research Institute

Northern Blot Analysis of XvPer1A 78 63 51 44 4 4 32 42 92 (%RWC)

Dehydration/rehydration

Heat (42oC)

B 0 6 12 24 48 72 96 (h)

C 0 1 2 3 4 5 6 7 (Days)

NaCl (150 mM)

D 0 1 2 3 4 5 6 7 (Days)

Cold (4oC)

E 0 1 2 3 4 (Days)

Highlight (1500 µmol m-2 s-1)

F 0 6 12 24 48 72 (h)

ABA (100 µm)

A 95 74 51 36 11 7 6 35 80 95 (%RWC)

Dehydration / rehydration

Western Blot Analysis of XvPer1

B 0 6 12 24 48 72 96 (hrs)

Heat (42oC)

C 0 6 12 24 48 72 96 (hrs)

NaCl (150 mM)

D 0 6 12 24 48 72 (hrs)

ABA (100 µm)

Subcellular Immunofluorescence localisation of XvPer1 in dehydrated X. viscosa leaf cells

(a) (b)

(c) (d)

Regeneration of Maize Transformants

High light stress on transgenic maize

Control Transgenic ControlTransgenic

Before high light stress Under high light stress

XvPrx2

Kershini Govender

Collaborator: Prof. Karl Josef Dietz, Univ. of Bielefeld

Sequence alignment and homology tree♦ Type II Prxs contain 2 cysteines (1 in catalytic centre)

♦ XvPrx2 possesses a single cysteine residue at position 51

♦ Displays greatest homology with the O. sativa Prx

Southern blot analysisB

glBgl

IIII

Eco

Eco R

IRI

Hin

dH

ind I

IIIII

Xba

Xba II

Eco

Eco R

VRV

Eco

Eco R

I/RI/ E

coEco R

VRV

Eco

Eco R

I/RI/ P

vuPvu II

Eco

Eco R

I/RI/ X

baXba II

-- veve

cont

rol

cont

rol

++ veve

cont

rol

cont

rol

♦ Confirmed the presence of XvPrx2 in the X. viscosa genome

♦ XvPrx2 appears to belong to a small gene family in the X. viscosa genome

♦ O. sativa and A. thaliana, have also been shown to express multiple type II Prxs

Northern blot analysesHigh temperature (42High temperature (42ººC)C)

00 66 1212 2424 4848 7272 9696 120 h120 h150 150 mMmM NaClNaCl

00 66 1212 2424 4848 7272 9696 120 h120 h

100 100 µµM ABAM ABA00 66 1212 2424 4848 72 h72 h Dehydration/Dehydration/rehydrationrehydration

DehydrationDehydration RehydrationRehydration

00 88 1515 1616 1717 2020 2727 2828 2929 3030 3131 dd

Low temperature (4Low temperature (4ººC)C)00 66 1212 2424 4848 7272 9696 120 h120 h 1500 1500 µµmol/mmol/m22/s high light/s high light

00 66 1212 2424 4848 7272 9696 120120 144144 168 h168 h

Protein purification♦Purified XvPrx2 was observed as a monomer of ca. 22 kDa

and as a dimer of ca. 44 kDa

22 22 kDakDa22 22 kDakDa

44 44 kDakDa

Denaturing conditionDenaturing condition NonNon--denaturing conditiondenaturing condition

In vitro DNA protection assay

In vitro mechanism

Reaction 1:

2RSH + O2 => RSSR + H2O2

Reacton 2:

2H2O2 => 2H2O + O2

Fe3=>Fe2DTT

Prx

In vitro DNA protection assay

1 2 3 4 5 6 7

DTT

FeCl3

pBSK

BSA

XvPrx2

-

-

+

-

-

+

+

+

-

+

+

+

+

+

-

+

-

+

-

-

+

+

+

-

-

+

+

+

+

-

-

+

+

-

- ♦Lane 4 = protection by XvPrx2

♦Lane 5 = no protection in absence (sc and oc)

Enzyme assays

H2O2

H2O

PRXred

PRXox

DTTox

DTTred

DTT dependent Prx activity

0

50

100

150

200

250

300

350

LOOH POOH BOOH H2O2 Cumene

Substrates

umol

per

oxid

e us

ed/m

in/u

mol

prx

WT-DTTV76C-DTT

♦XvPrx2 activity was maximal when DTT served as electron donor.♦Highest level of XvPrx2 activity was observed using H2O2 as substrate

with BOOH being the next preferred♦The mutant (V76C) displayed significantly lower activity compared to

the WT for all substrates assessed

Fluorescent vs Confocal microscopy: GFP localisationGFP only ABI5:GFP XvPrx2:GFP

♦ ABI5 (control) localised to the nucleus, XvPrx2 localised to the cytosol

•XvPer1 codes for a 1-Cys peroxiredoxin, a novel antioxidant enzyme in vegetative tissue

•The active site of the enzyme is highly conserved and contains a nuclear localization signal

• Enzyme localized in nucleus of dehydrated X. viscosaleaf cells

• XvPer1 might function as a protector of nucleic acids against damage by ROS

• XvPer1 is stress inducible

SUMMARY

•XvPrx2 is stress inducible (highly induced by ABA)

• XvPrx2 is localised to the cytosol

• Plays a role as an antioxidant since protection of DNA from ROS is observed in the presence of XvPrx2

• Antisense and overexpressor Arabidopsis plants expressing XvPer1 and XvPrx2, respectively have been generated

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

Hydrated

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

Dehydrated

Complementation by Functional

Sufficiency in E. coli (srl:: Tn10)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40 50

Time (h)

OD

600

Growth analysis of E.coli (srl::Tn10)expressing XVSAP1

Aldose reductase = ALDRXV4

Alice T Maredza

Northern BlotsTranscript levels in Arabidopsis thaliana sub-lines 8 and 12 (Panels A & B ),

respectivelyTranscript levels in Digitaria sanguinalis lines (Panels C & D)

AWT 8-1 8-2 8-3 8-4 8-5 8-6

ALDRXV4

18s rRNA

BWT 12-1 12-2 12-3 12-4 12-5 12-6 12-7 12-8 VT7

ALDRXV4

18s rRNA

CWT VT tr1 tr2 tr5 tr6 tr10 tr13 tr14 tr17 tr18

ALDRXV4

18s rRNA

DWT VT tr21 tr22 tr45 tr48 tr50 tr53 tr62 tr64 tr72 tr73

ALDRXV4

18s rRNA

Western blotsLevels of aldose reductase protein in transgenic Arabidopsis (A) and D. sanguinalis (B)

A WT VT7 M TR7 TR6 TR8 TR12 TR34 +ve +ve

45kDa

36kDa

B

36kDa45kDa

M WT VT25 TR1 TR2 TR5 TR6 Tr10 +ve

Dehydration stress4 week old Arabidopsis plants dehydrated for 7 days

WT

Tr8

Tr12

WT

Tr8

Tr12

Before Dehydration Day 8 of Dehydration

a) PNA (Control) b) 50 mM Mannitol c) 75 mM MannitolWT Tr7 Tr8 Tr12 WT Tr7 Tr8 Tr12 WT Tr7 Tr8 Tr12

d) 150 mM Mannitol e) 75 mM NaCl f) 100 mM NaClWT Tr7 Tr8 Tr12 WT Tr7 Tr8 Tr12 WT Tr7 Tr8 Tr12

Arabidopsis plants expressing the X. viscosa AR are more tolerant to stress

XVSAP1

944 bp long, ORF 867 bp264 aaHydrophobic protein Multiple sequence alignment:

56 % identity with ATCAP and 49% identity with WCOR413

XVSAP1 - a cold regulated protein ?

Dahlia Garwe

J.Exp.Bot. 54(381): 1-11

hydrophobic

hydrophilic

I

II

III IV

VVI

I

XVSAP1 1 -------MRNEGFLKMKTDVGVADEVISGDLKQLGDAAKRLAKHAIKLGASFGVGSTIVQ WCOR413 1 ---MAKSFLAMKTGPAAGASEASQALLESDLRELTMAARKLANHAIVLGGGIGFIGTFLQ ATCAP1 1 -------MGRMDYLAMKTDDVDTVALVNSDMEELKVAAKKLFSDVSKLGG-LGFGVSFLK ATCAP2 1 ----------MPMKSLRNDHGTLKAMIGSDFNELTIAAKNLATHAFTLTG-LGFGTSVLE ATCAP3 1 -------MGRGEFLAMKTEEN-AANLINSDMNEFVAAAKKLVKDVGMLGG-VGFGTSVLQ ATCAP4 1 ----------------------------MENIEYLNEIQAVAGKLIHSYGVPVMITLFLR RCAP 1 MGKGFMSYLAMKT-DAAGGEAAQAALIDADLQELGVAARKLANHALVLGGGLGFGTTFLK consensus 1 . .. . ... ... .. ....... ... ... ........ XVSAP1 54 AIASIAAIYLLILDRTNWRTN--ILTSLLIPYVYLSLPSVIFNLFRGDLGRWLSFIGVVM WCOR413 58 WLAFAAAVYLLVLDKTNWKTN--MLTGLLVPYIFFTMPGLLFGFIRGEIGAWIAFVVVVL ATCAP1 53 FLASFAAIYLLILDRTNWKTK--MLTSLLIPYIFLSLPSVIFNFLSGDVGKWIAFVAVVL ATCAP2 50 WVASIAAIYLLVLDRTNWKTN--MLTSLLIPYIFFSLPSLIFGIFRGEIGKWIAFVAVVV ATCAP3 52 WAASIFAIYLLILDRTNWKTK--MLTTLLVPYIFFTLPSVIFQFFSGDFGKWIALIAIIV ATCAP4 33 WLASIVAVFLMILDQTKWKYSNNIMASLLAPYLFSSLPIVIFQVLRNGVGKWIALLTVIL RCAP 60 WLAFFAAVYLLILDRTNWKTN--MLTALLVPYIFFTLPGGLFSLLRGEIGKWIAIIAVIL consensus 61 ..*...*..*..**.*.*... ....**.**.....*...*.......*.*........ XVSAP1 112 KLFFHRHFPVTLELLVSLILLIVVSPTFIAHTIRGS--LIGVFIFLVIACYLLQEHIRSA WCOR413 116 RLFFPRHFPDWLELPGSLILLTVVAPAIFADTFRGSWLIIGVGVCLVIGCYLLHEHIKAS ATCAP1 111 RLFFPKHFPDWLEMPGSLILLLVVSPHFLAHHIRGT--WIGTVISLFIGCYLLQEHIRAS ATCAP2 108 QLFFPKHAREYLELPVALVLLAVVAPNLIAGTFRDS--WIGLAICLGIGCYLLQEHIRAS ATCAP3 110 RLFFPKEFPEWLEIPVALILIVVVSPSLIAWTLRES--WVGAVICLVIACYLFHEHIKAS ATCAP4 93 RLFLPNHFHESLEIPGATILLIVVTPSDIGAIFRDDLRYTGGDVCLLTSFYLINKHTKAC RCAP 118 RLFFPRHFPDWLELPGAVILLIAVAPNLFASTFRG--DLVGIFICLIIGCYLLQEHIRAS consensus 121 .**........**......*...*.* ... ..*.. ..*. ..*....**...*.... XVSAP1 170 GGFKNAFTKSNGISNSVGIIILLIHPIWSLVYFLYTSLLQLLAYSPSPCCCILYNKWFNF WCOR413 176 GGLKEAFQKPNGWSNTIGILLLFIYPVWAVVMWFL------------------------- ATCAP1 169 GGFRNSFTQPRGVSNTLGIILLLVYPVWALIVRVM------------------------- ATCAP2 166 GGFRNAFTKANGISNTVGIICLVVFPVWALIF---------------------------- ATCAP3 168 GGFKNSFTQKNGISNTIGIVALLVYPVWTIFFHIF------------------------- ATCAP4 153 GGIKNSFTQKDKVTYSICLWILFVYPILSSFAALFYL----------------------- RCAP 176 GGFRNAFRKGNGVSNSIGILLLFIYPVWALVLNFL------------------------- consensus 181 **....*.. ...........*...*..... . XVSAP1 230 MHVCKCVSLHMYSQSIGSCVSIFFVQFVFIYEAEF WCOR413 -----------------------------------49% ATCAP1 -----------------------------------56% ATCAP2 -----------------------------------55% ATCAP3 -----------------------------------51% ATCAP4 -----------------------------------38% RCAP -----------------------------------53% consensus 241

90% 78% 63 % 51% 44% 4% RWCbp

900

320

Dehydration

4% 32% 42% 85% 92% RWC Bp

900

320

Rehydration

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18

Time (d)

RW

C

Relative Water Content for duration of treatment

bp

900

320

900

320

900

320

900

320

Days 0 1 2 3 4 5 6 7 8 M

Days 0 1 2 3 4 5 6 7 M

Days 0 1 2 3 4 M

h 0 6 12 18 24 30 36 42 48 54 60 M

Heat (42°C)

Cold (4°C)

NaCl (100 mM)

High light (1500 µmol m-2 s-1)

LBRB

Xho IEco RI

1 2 3 4 5 6 7 8kb

14

5.1

2.8

35S -XVSAP1-nos cos ocs-BAR-nos

Arabidopsis

1 2 3 4 5 6 7 8kb

14

5.1

2.8

Tobacco

kb

1.1

0.9

0.5

M WT 6K 10C 21G

Arabidopsis

M WT AZ A2 A5 A7kb

1.1

0.9

0.5

Tobacco

RT-PCR

Control 150 mM Mannitol

21J AZ WT 6K 10C AZ 21J 6K 10C WT

Arabidopsis

21J AZ WT 6K 10CControl 50 mM NaCl

WT 6K 1 0C 21J AZ

100 mM NaCl150 mM NaCl21J AZ WT 6K 10C

21J AZ WT 6K 10C

Arabidopsis

- Water

Controls Transgenics

21J AZ WT 6K 10CControl 50 mM NaCl

WT 6K 1 0C 21J AZ

100 mM NaCl150 mM NaCl21J AZ WT 6K 10C

21J AZ WT 6K 10C

Arabidopsis

Wild type 6K

21J 10C

Arabidopsis

Transgenic, WT and azygous tobacco plants

7-14 WT AC

7-14 WT AC

A

B

A = controlB = 200mM NaClC = 9% PEG

C

7-14 WT AC

- Water

Controls Transgenics

SUMMARY

• XVSAP1 is likely to be a membrane protein with a prokaryotic membrane lipoprotein lipid attachment site and myristoylation sites.

• XVSAP1 is induced significantly by dehydration, salt, high light intensity, and low and high temperatures.

• Possible roles of XVSAP1 would include: cell wall-plasma membrane linker, a transporter protein involved in the transport of small molecules or conferring stability to the plasma membrane.

Promoters of XvPer1 and XvSAP1

Rob Ingle

Collaborator: Dr Mel Oliver, USDA, Texas

Isolate promoter by splinkerette PCR

Clone promoter:luc construct into pTFH15.3

KpnI

luc

Promoter

HindIII

EcoRV

NotI

Pphb7

NPTII

Linearise vector

PEG-mediated protoplast transformation

Regenerate plants and select on kanamycin

Conclusions

Two X. viscosa peroxiredoxins cloned– both induced by stress– XvPer1 → tolerance to high light

intensity?Aldose reductase → dehydration and salt

tolerance?Membrane protein XvSap1 →

dehydration, salt and heat tolerance?

Acknowledgements

• Rockefeller Foundation

• International Foundation for Science

• National Research Foundation (South Africa)

• Maize Trust of South Africa

• University of Cape Town

• DACST Innovation (South Africa)

Countries with Biotechnology Research Programmes

* = GM technologyEgypt* Morocco*Sudan MaliEthiopia Ghana*NigeriaKenya* Tunisia*Uganda ZambiaZimbabwe* South Africa*Madagascar Mauritius*

African Success Stories

Beans in Kenya 27 improved varieties wrtroot rot and yield 11 selectedVirus resistant sweet potato in KenyaNew Rice for Africa (NERICA) 10,000 ha in Uganda wrt nutritionClean pototo seeds bacterial wilt resistance in UgandaClean potato seeds and sugarcane virus resistance in Mauritius