Functional genomics of abiotic and biotic stress responses ...arquivo.ufv.br/dbg/genmelhor/Index_apresentação pdf/Genetica2005 Beth.pdfYw (MPa) Efficiency Photosynthesis 0.75 0.77

Functional genomics of abiotic and biotic stress responses

Biotic stress responses

Geminivirus-host interactions

Abiotic stress response

• ER stress response

• Water and salt stress

• Integration of signaling pathways

DOES THE MANIPULATIONOF BiP LEVELS IN THE ER

CONFER PROTECTION AGAINST STRESSES????

DBB/BIOAGRO/UFV

BiP LEVELS INCREASE IN TOBACCO PLANTS EXPRESSING A SOYBEAN BiP GENE

1 2 A5 A10 A15 A16 S1 S3 S6 S7

Control Antisense Sense

soyBiPD35S-PnptIInos-P 3’nos 3’nosLB RB

Xba I EcoRI

TransgeneXba I

SOYBEAN BiP CONFERS TOLERANCE TOWATER STRESS IN TOBACCO

Sense

Control

DBB/BIOAGRO/UFV

Plant Physiology 126: 1042-1054, 2001

Water Potetial

-2.5

-2

-1.5

-1

-0.5

0

C S AS

Treatments

Yw

(M

Pa)

Efficiency Photosynthesis

0.750.770.790.810.830.85

C I C E S I S E AS I AS E

Treatments

Fv

/Fm

Peroxidase

0

10

20

30

0 5 10 15 20 25 30 35

Duration of the experimement (days)

un

id A

PX

.m

in-1

.

mg

-1 p

rote

in

C E

S E

A S E

Superoxide Dismutase

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35

Duration of the experimement (days)u

nid

SO

D.

mg

-1 p

rote

in

C E

S E

A S E

Physiological performance of BiP-overexpressingtobacco lines under water deficit

C9-2 C9-3 C9-5 A8-7 A8-8Wt Wt

Control Sense

ECTOPIC EXPRESSION OF SOYBIPD GENE IN SOYBEAN ENHANCES TOLERANCE TO WATER STRESS

Transgenic plant Wild type

THE MOLECULAR MECHANISM UNDERLYING BIP-MEDIATED WATER

STRESS TOLERANCE?????????

Desenho experimental

H2O

H2O

AZC

PEG

TUN DMSO

Transcriptômica

Fundamentos da técnica de microarrays

PEG Cy5 X Untreated Cy3

TUN Cy3 X Untreated Cy5

Induced transcriptome by AZC, TUN and

PEG treatments.

5161 97

TUNAZC

ER stress transcriptionalresponse

9

42

7

TUNAZC

8

9053

68

PEG

Modest overlapping of ER stress andOsmotic stress transcriptional responses

Fold variation in expression levels of genes up-regulated by ER stress.

GenBank Gene annotation

AW100949 photoassimilate-responsive PAR-1b protein 6,35 ± 1,54 4,15 ± 0,92

AW100917 unknown 2,18 ± 0,15 3,21 ± 0,76

AW318038 lumenal binding protein cBiPe3 2,26 ± 0,17 2,50 ± 0,94

AW101796 farnesylated protein ATFP3 1,53 ± 0,11 7,38 ± 3,95

AI748042 calnexin homolog precursor 2,78 ± 0,65 1,92 ± 0,18

AI748232 60S acidic ribossomal protein P2-B 2,24 ± 0,45 2,08 ± 0,11

AW459852 putative ATAF2 protein 5,33 ± 3,76 7,58 ± 2,88

AW185793 60S ribosomal protein L12 2,89 ± 0,93 1,99 ± 0,28

AW279462 cytosolic heat shock 70 protein 2,18 ± 0,65 14,38 ± 2,51

AW186357 unknown 3,65 ± 1,66 7,98 ± 2,26

AW471814 heat shock protein 70 36,74 ± 26,25 32,76 ± 8,42

AW508046 unknown 2,38 ± 0,60 2,81 ± 1,18

AW509037 cell division cycle protein 48 (CDC48) 10,74 ± 2,10 5,98 ± 1,68

AW508884 40S ribosomal protein S16 2,27 ± 0,05 2,24 ± 0,19

AW508375 ubiquitin-associated protein 3,45 ± 1,61 3,08 ± 0,65

AW508115 unknown 3,11 ± 0,50 19,77 ± 3,22

AW568143 merozoite surface protein-related 2,16 ± 0,90 3,27 ± 0,40

AW508946 60s ribosomal protein L21 2,32 ± 0,35 2,14 ± 0,18

AW568275 unknown 3,11 ± 1,04 4,28 ± 1,08

AW567862 soybean lumenal binding protein isoform C 23,99 ± 13,19 13,64 ± 2,13

AW397007 calnexin 16,59 ± 6,18 7,22 ± 1,10

AW459732 nam-like protein 10 3,64 ± 1,74 5,41 ± 1,45

AW432916 harpin inducing protein 1-like 9 1,97 ± 0,51 4,64 ± 0,14

AI973541 unknown 4,08 ± 1,31 5,67 ± 0,69

AW278784 heat shock protein 81-2 2,05 ± 0,40 12,71 ± 3,43

AW471739 protein phpsphatase 2C (PP2C) 2,23 ± 0,45 2,13 ± 0,10

AW308879 ferrochelatase II chloroplast precursor 5,53 ± 0,69 2,70 ± 0,26

AW472364 translation initiation factor eIF-5 1,71 ± 0,11 2,30 ± 0,06

AW509424 PP2A regulatory subunit 1,80 ± 0,09 6,95 ± 0,75

AW507857 unknown 2,19 ± 0,28 2,18 ± 0,36

AW508823 unknown 2,35 ± 0,38 2,19 ± 0,10

AW507892 luminal binding protein 2 (BiP-2) 31,53 ± 13,13 20,88 ± 2,65


AW507577 unknown 1,68 ± 0,14 1,59 ± 0,06

TUN mean AZC meanAW567594 glyceraldehyde 3-phosphate dehydrogenase 4,63 ± 2,28 6,44 ± 0,76

AW508203 lipoxygenase 6,18 ± 0,60 3,65 ± 1,01

AI736109 60S ribosomal protein L2 2,45 ± 0,17 2,09 ± 0,58

AI748112 60S ribosomal protein L9 2,19 ± 0,40 1,78 ± 0,21


AI960794 translation elongation factor 1-gamma 2,27 ± 0,41 2,04 ± 0,20

AW471972 unknown 3,85 ± 0,77 13,00 ± 4,65

AW432994 protein disulfide isomerase 6,29 ± 1,58 2,71 ± 0,18

AW471731 unknown 2,09 ± 0,27 2,14 ± 0,12

AW509482 luminal binding protein isoform D 39,33 ± 14,28 32,06 ± 7,90

AW508776 40S ribossomal protein S18 2,81 ± 0,59 1,86 ± 0,11

AW569111 BiPD 26,23 ± 6,24 24,46 ± 6,92

AW508885 ribosomal protein L14-like protein 1,93 ± 0,33 1,63 ± 0,01

AW569128 calnexin 13,03 ± 2,53 6,10 ± 0,39

AW567808 unknown 1,94 ± 0,39 2,56 ± 0,15

AW317679 eukaryotic translation initiation factor 3(10) 2,10 ± 0,53 1,59 ± 0,05

AW570334 unknown 2,04 ± 0,12 3,21 ± 0,83

AI748065 calnexin 2,51 ± 0,40 1,89 ± 0,45

AI940871 ribosomal protein L28 1,67 ± 0,13 1,56 ± 0,14

AW186110 unknown 3,25 ± 0,78 9,63 ± 1,30

AW184865 unknown 2,46 ± 0,25 2,52 ± 0,64

AW277892 putative ABC transporter protein 1,84 ± 0,21 2,62 ± 1,12

AW472128 histone H3 1,76 ± 0,16 1,69 ± 0,21

AW472161 2 4-D inducible glutathione S-transferase 3,19 ± 0,27 2,79 ± 0,55

AW471959 unknown 3,24 ± 0,42 2,60 ± 0,24

AW508528 putative UDP-galactose transporter 4,54 ± 0,54 4,08 ± 0,29

AW508066 calnexin 23,20 ± 4,96 15,42 ± 3,93

AW568451 acidic ribosomal protein P0 2,66 ± 0,47 2,21 ± 0,18

AW507612 der1-like protein 4,49 ± 0,75 6,11 ± 2,32

AW508978 polyubiquitin (UBQ10) (SEN3) 2,05 ± 0,70 7,41 ± 2,13

AW508125 GYF domain-containing protein 1,66 ± 0,15 4,89 ± 1,84

AW508980 20S proteasome alpha subunit D 3,35 ± 0,59 1,84 ± 0,34

AW507594 chloroplast RNA binding protein 2,24 ± 0,39 6,29 ± 0,99

AW569267 protein phosphatase 2C 3,58 ± 1,49 2,98 ± 0,39

AW567827 protein disulfide isomerase 7,83 ± 4,40 5,18 ± 1,55

GenBank Gene annotation TUN mean AZC mean

Fold variation in expression levels of genes up-regulated by osmotic stress.

AW309137 hydroxyproline-rich protein 3,34 ± 1,58

AW471785 unknown 2,95 ± 1,55

AW471733 50S ribosomal protein L21 3,34 ± 0,64

AW509471 seed maturation protein PM39 2,14 ± 0,03


AW568621 unknown 2,26 ± 0,65


AW509480 glycine-rich protein 1,93 ± 0,20

AW568784 protein disulfide isomerase 2,53 ± 0,51

AW509036 plastid-lipid-associated protein 1,72 ± 0,23

AI759650 senescence-associated protein 1,94 ± 0,23

AI930841 proline-rich protein 4,66 ± 0,85

AW185926 unknown 2,75 ± 0,61

AW185914 lipid transfer protein 3,07 ± 1,12

AW186144 unknown 1,77 ± 0,23

AW186103 unknown 1,86 ± 0,23

AW186110 unknown 2,08 ± 0,40

AW277787 epoxide hydrolase 2,40 ± 0,69

AW186337 ferritin precursor 2,24 ± 0,90

AW279531 cellulose synthase 2,73 ± 0,66

AI959838 PP1/PP2A phosphatases pleiotropic regulator 2,21 ± 0,31

AW279500 Cys/Met metabolism protein 1,97 ± 0,88

AW164630 sucrose synthase 2,26 ± 0,16

AW433077 ATAF1 protein 3,82 ± 1,98

AW472214 RanBP1-domain protein 1,62 ± 0,21

AW278792 translation initiation factor 5A 4,76 ± 0,50

AW308920 sucrose-phosphatase 1,88 ± 0,33

AW472161 glutathione S-transferase 3,52 ± 1,48

AW279562 putatice cyclin 4,05 ± 0,20

AW279019 flavin-containing monooxygenase protein 4,18 ± 1,35

AW508125 GYF domain-containing protein 3,95 ± 0,98

AW509040 protein phosphatase 2C 3,06 ± 1,39


±

PEG meanGenBank Gene annotation

AI735868 glucose and ribitol dehydrogenase 5,83 ± 2,93

AW397327 gibberellin-regulated protein 3,32 ± 1,87

AI748083 dehydrin 2,79 ± 0,72

AI748086 dehydrin 1,87 ± 0,11

AW459852 ATAF2 protein 3,08 ± 0,81

AI930875 O-Methyltransferase 4,64 ± 2,77

AW277660 PDI-like protein 2,77 ± 0,71

AW164308 unknown 2,54 ± 0,44

AW568531 unknown 2,68 ± 0,58

AW508375 ubiquitin-associated protein 2,67 ± 0,92

AW508115 unknown 1,76 ± 0,22

AW568142 seed maturation protein 4,54 ± 1,73

AW508192 unknown 3,78 ± 0,71

AW459732 nam-like protein 10 2,40 ± 0,82

AI973541 N-rich protein 2,50 ± 0,48

AW164480 SNF1 like protein kinase 4,35 ± 2,16

AW460045 cysteine proteinase inhibitor 2,73 ± 0,81

AW472364 translation initiation factor eIF-5 3,03 ± 0,82


AW569110 unknown 3,24 ± 0,90

AW569197 unknown 1,87 ± 0,51


AW507587 inositol 1 3 4-Trisphosphate 5/6 kinase 2,16 ± 0,66

AW508084 polygalacturonase-like protein 4,12 ± 0,94

AW568173 peripheral-type benzodiazepine receptor 2,20 ± 0,79

AW567877 glutathione peroxidase 3,54 ± 1,91

AW317783 beta-conglycinin alpha chain precursor 1,93 ± 0,34


AI930894 unknown 2,70 ± 0,93

AW457909 NAC3 protein 4,28 ± 2,29

AI940815 seed maturation protein PM29 1,77 ± 0,11

AW185778 Ca+2-binding EF hand protein 3,84 ± 0,10

±

PEG mean

chromosome organization 1%

protein biosynthesis 19%

unknown 19%

protein folding 14%

calcium ion binding 7%

Proteolysis 6%

Transport 6%

protein phosphorylation 4%

response to pathogen 4%

transcription regulation 4%

response to antibiotic 3%

translation initiation 3%

ATPase activity 1%

cell wall organization 1% ferrochelatase activity 1%

glucose metabolism 1%

metal ion binding 1%

RNA binding 1%

translation elongation 1%

Funcional categorization of the ER-stress (A) and osmotic-stress

(B) responsive genes.

unknown 36%

oxidoreductase activity 8%


hydrolase activity 5%

response to water 5%

kinase activity 3%

phosphatase activity 3%

protein folding 3%


Aging 2%

amino acid metabolism 2%


carbohydrate metabolism 2%

cellulose biosynthesis 2%

iron ion binding 2%

lipid transport 2%

methyltransferase activity 2%

nucleotide binding activity 2%

peroxidase activity 2%

protease inhibition 2%


protein translocation 2%

proteolysis 2%

regulation of cell cycle 2%

response to gibberellic acid 2%


sucrose biosynthesis 2%

Co-regulated genes by ER stress and osmotic stress

reveal a modest overlapping of ER stress and

osmotic stress transcriptional responses


AW459852 ATAF2 protein 5.33 ± 3.76 7.58 ± 2.88 2.94 ± 0.86

AW508115 unknow n 3.11 ± 0.50 19.77 ± 3.22 1.76 ± 0.22

AW459732 nam-like protein 10 3.64 ± 1.74 5.41 ± 1.45 2.26 ± 0.89

AW472364 translation initiation factor eIF-5 1.71 ± 0.11 2.32 ± 0.06 3.03 ± 0.82

AW186110 unknow n 3.25 ± 0.78 9.63 ± 1.30 2.08 ± 0.40

AW472161 2 4-D inducible glutathione S-transferase 3.19 ± 0.27 2.79 ± 0.55 3.52 ± 1.48

AW508375 ubiquitin-associated protein 3.45 ± 1.61 3.08 ± 0.65 2.20 ± 0.74

AI973541 unknow n 4.08 ± 1.31 5.67 ± 0.69 2.50 ± 0.48

AW508125 GYF domain-containing protein 1.66 ± 0.15 4.89 ± 1.84 3.95 ± 0.98

TUN mean AZC mean PEG mean

(N-rich protein)

(Dual oxidase)

-4

-2

0

2

4

6

8

10

TUN

4h

AZC4h

PEG4h

TUN

10h

AZC10

h

PEG10

h

TUN

16h

AZC16

h

PEG16

hAZC

+PEG

16h

Gene

SMP

CALN

qRT-PCR confirmed microarray results

-4

-2

0

2

4

6

8

TUN

4h

AZC4h

PEG4h

TUN

10h

AZC10

h

PEG10

h

TUN

16h

AZC16

h

PEG16

hAZC

+PEG

16h

Gene

PDI1

PDI2

PDI3

The PDI Gene Family is Differentially Regulated by

Osmotic and ER stresses

-4

-2

0

2

4

6

8

10

SMP

CALN

GST

HSP

70D

NAM

NH

II

NR

ICH

NR

ICH

2

PDI1

PDI2

PDI3

ATAF2

UBIQ

Gene

TUN16h AZC16h PEG16h AZC+PEG16h

Synergistic effect of co-regulated gene induction by thecombination of AZC and PEG treatments

-2

-1

0

1

2

3

4

5

6

7

8

9

TUN

4h

AZC4h

PEG4h

TUN

10h

AZC10

h

PEG10

h

TUN

16h

AZC16

h

PEG16

hAZC

+PEG

16h

Gene

GST

NAM

ATAF2

GST may be NAN and ATF2 dowstream target

ER StressOsmotic stress

ATF2 NAN

TATA

GSTNH1NH2

CalnexinBiP, PDICalreticulin

BiP

XUPR

AREB

DREB

ABA

?

Osmotic and stress response pathways convergeat the the level of gene activation

unknown 36%

oxidoreductase activity 18%


hydrolase activity 5%

response to water 5%kinase activity 3%

phosphatase activity 3%

protein folding 3%


Aging 2%

amino acid metabolism 2%


carbohydrate metabolism 2%

cellulose biosynthesis 2%

iron ion binding 2%

lipid transport 2%

methyltransferase activity 2%

nucleotide binding activity 2%

peroxidase activity 2%

protease inhibition 2%


protein translocation 2%

proteolysis 2%

regulation of cell cycle 2%

response to gibberellic acid 2%


sucrose biosynthesis 2%

Overexpression of the soybean ALDH7 antiquitin-like gene in tobacco transgenic lines

Incorporation of the transgene(PCR)

ALDH7 mRNA accumulation(PCR)

ALDH7 protein accumulation(PCR)

Ectopic expression of the soybean antiquitinGmTP55 gene in tobacco confers tolerance to

drought

Water-stressedplants

Irrigatedcounterpart

0 100 150 200

Salt concentration [mM NaCl]

35

50

65

80

95

110

0 100 150 200

NaCl (mM)

Germ

ination (%

) ControlTP55-S2TP55-S3* * *

*

*

*

GmTP55-overexpressing tobacco lines are more tolerant to

salt stress during germination

0

10

20

30

40

50

60

70

80

90

100

0 10 15 20

H2O2 (µM)

Germ

ination (%)

Control

TP55-S2

TP55-S3*

*

*

* *

*

Performance of GmTP55-overexpressing plants under oxidative stresses

The stress protective role of GmTP55 is associated

with an aldehyde detoxification pathway

Lipid peroxidation(accumulation of MDA)

Paraquat treatment

0

20

40

60

80

100

120

0 100 150 200

NaCl (mM)

Germ

ination (%

)

Control

ATQ8

ATQ12*

*

*

*

*

0

20

40

60

80

100

120

0 10 15 20

H2O2 (µM)

Germ

ination (%

)

Control

ATQ8

ATQ12*

*

*

*

*

*

Ectopic expression of GmTP55 in Arabidopsis confirmsthe ALDH7-mediated stress tolerance

Salt stress

Oxidative stress

1. A função de ALDH7 está associada à detoxificação de aldeídos e não à manutenção de homeostase osmótica

• ALDH7 conferiu proteção contra estresses abióticos- estresse oxidativo

como componente comum.

• Superexpressao de ALDH7 foi correlacionada com um decréscimo em

peroxidação de lipídeos -diminuição no acúmulo de MDA

2. GmALDH7 –excelente alvo para melhorar resistência a estresses do meio ambiente em culturas agronomicamente importantes

• A expressão ectópica de GmTP55 tanto em Arabidopsis quanto em tabaco

resultou em tolerância cruzada a múltiplos estresses abióticos

• As plantas transgênicas foram fenotipicamente idênticas aos controles sob

condições normais.

• Superexpressâo do gene GmTP55 nao deve afetar o desempenho global

no campo das plantas transgênicas sob condições normais.

AbioticAbiotic StressStress

SoniaSonia

AndrAndréé, , AneteAnete, , DirceDirce

Leonardo, Leonardo, RejaneRejane

Max, Max, BrenoBreno

Pedro, Elder, Pedro, Elder, JussaraJussara

Biotic StressBiotic Stress

Alessandro, Alessandro,

AnAnéésiasia, , LilianLilian, Mariana, Mariana

SilvanaSilvana, Ana Paula, , Ana Paula,

Carolina, Carolina, GuilhermeGuilherme

Collaboration: Collaboration:

Dr Maria Cristina Dr Maria Cristina BaractBaract

Dr Marcelo E. Dr Marcelo E. LoureiroLoureiro

DraDra MarliMarli L. de OliveiraL. de Oliveira

Dr Dr MuriloMurilo ZerbiniZerbini

DraDra AndreaAndrea AlmeidaAlmeida

MUITO OBRIGADA

Dr Francisco Aragão

Documents

Functional genomics of abiotic and biotic stress responses ...arquivo.ufv.br/dbg/genmelhor/Index_apresentação pdf/Genetica2005 Beth.pdfYw (MPa) Efficiency Photosynthesis 0.75 0.77