Transcript Cabello et al., 2009. MS in preparation

IAL (Instituto de Agrobiotecnología del Litoral)
CONICET-UNL
Laboratorio de Biotecnología Vegetal
Cátedra de Biología Celular y Molecular
Facultad de Bioquímica y Ciencias Biológicas
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Utilización de la transcriptómica para la identificación y
caracterización de sistemas de expresión coordinada de genes y
su regulación
Investigador responsable: Dr. Atilio Castagnaro
Caracterización funcional de factores de transcripción
involucrados en el desarrollo y la adaptación al medio ambiente
Investigadora responsable: Dra. Raquel Chan
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Transcription factors are clue elements in
the regulation of signal transduction
pathways in plants.
They are proteins able to recognize and
bind specific sequences in the promoter
regions of their targets and subsequently
activate or repress entire metabolic or
developmental processes.
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About 1500 TFs were informatically identified in
plants; identification mainly based in the presence of
DNA-binding domains in the translated sequences.
Several TFs seem to be powerful biotechnological
tools in order to improve agronomic crops via the
obtaining of transgenic plants or as molecular
markers. Such TFs have become the objects of
patents presentations in the whole world.
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Expression analyses (northern blot, qRT-PCR, in situ
hybridization)
Promoters isolation (bioinformatic, transgenic plants)
Heterologous transgenic plants obtaining
Phenotypic characterization (morphology,
biochemistry)
Transcriptome analysis (microarrays, qRT-PCR)
DNA-protein interaction (EMSA, SELEX, foot
printing)
Homologous transformation (transient
transformation, qPCR, reporter genes analyses)
Validation
Methodology used with plants which
genome is unknown
Real time RT-PCRs
microarrays
RNA isolation
Stable
transformants
selection
TF cDNA
cloning
Cloning of
35S+ TF cDNA
Arabidopsis
thaliana
transformation.
Methodology used with plants which
present genome available information
RNA isolation from plants in two
different conditions
microarrays
Early salt
induced
genes in
root
apexes
Early salt
repressed
genes in root
apexes
Not assigned
Secondary metabolism
Miscellaneous enzyme families
RNA (including transcription
regulation)
Stress
Hormones
Signalling
Protein
Transport
Lipid metabolism
Development
Amino acid metabolism
Cell wall
Minor carbohydrates
Nucleotide metabolism
Redox
Major carbohydrates
Cell
DNA
Photosynthesis
Tetrapyrrole synthesis
Metal handling
Gruber et al., 2009 Mol. Gen. Genet.
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Sunflower HAHB4 (HD-Zip subfamily I)
Medicago truncatula MTHB1 (HD-Zip
subfamily I)
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Sunflower HAHB10 (HD-Zip subfamily II)
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Sunflower HAFT (HD-Zip subfamily I)
Manavella 2008. Ph. D. Thesis
Ethylene perception
Ethylene biosynthesis
Manavella et al., 2006. Plant Journal
Manavella et al., 2008. J Exp. Bot.
Manavella et al., 2008. Plant Journal
100mM
NaCl 3 weeks 25mM
0
M. truncatula root growth
Medicago truncatula
Root lenght (cm)
Medicago truncatula
in response to salt stress
25
20
15
10
5
0
0mM
25mM
50mM
75mM
100mM
150mM
[NaCl]
Ariel et al., 2009 MS in preparation
35S::MTHB1
35S::GUS
Dry weight (mg/root)
Ariel et al., 2009 MS in preparation
Analysis for identifying candidate target genes
Results: 12 candidate target genes
3 very induced
1 strongly repressed
Kunitz trypsin inhibitor ST1-like
Jasmonate ZIM-domain (JAZ)
jasmonate-responsive
transcriptional repressors
LOB LATERAL ORGAN BOUNDARIES
auxin-responsive TFs unique to plants
Cytochrome P450 monooxygenase
involved in flavone biosynthesis
35S::MTHB1 35S::GUS
1,4
MTHB1 overexpressing roots
1,2
1,0
GUS S/E
ZIM
0,8
LOB
0,6
CYT
KUN
0,4
0,2
0,0
GUS S/E
ZIM
LOB
CYT
KUN
Ariel et al., 2009 MS in preparation
Rueda et al., 2005 Plant Cell Physiol.
Dezar et al., 2009 MS in preparation
Dezar et al., 2009 MS in preparation
Adapted from Blázquez 2005
Dezar et al., 2009 MS in preparation
HAFT is involved in cold stress tolerance
Membrane stability
100
Conductividad relativa
90
80
70
60
wt
50
TG-A
40
TG-B
30
20
10
0
Control
30´
2 hs
Tiempo de exposición a -8 °C
WT
TG-A
TG-B
TG-C
TG-D
TG-E
promedio
22 %
85 %
75 %
70 %
65 %
60 %
desviació
n
3
2
4
3
2
5
7 hs a -8 °C
Cabello et al., 2009. MS in preparation
Promoter HAFT+ GUS
Promoter HAFT + HAFT
WT
TG-A
A
B
C
D
TG-C
TGB
Longitud del tallo ppal. (mm)
Life cycle
400.0
350.0
300.0
wt
TG-A
250.0
TG-B
200.0
TG-C
150.0
100.0
29
32
38
44
49
Time days
E
F
G
H
Survivors to freezing treatments
WT
TG-A
TG-B
TG-C
Average
35 %
85 %
80%
70 %
Deviatio
n
5
8
6
10
WT
TG-C
TG-A
Cabello et al., 2009. MS in preparation
Microarray assay validation
At4g02380 SAG21
3
TG-B
2
TG-C
1
TG-D
3
TG-C
2
TG-D
4
TG-A
3
TG-B
2
TG-C
TG-D
TG-B
TG-C
TG-D
Fold change
TG-A
4
3
2
1
0
TG-A
3
TG-B
2
TG-C
TG-A
TG-B
TG-C
TG-D
4
3
2
1
0
WT
4
TG-A
3
TG-B
2
TG-C
1
TG-D
TG-D
0
At5g56110 ATMYB103
5
WT
TG-D
5
WT
At4g25490 DREB 1a
5
WT
TG-C
At1g62440 DREB1b
0
Fold change
5
TG-B
2
0
4
At3g04720 PR4
TG-A
3
1
TG-D
1
0
At3g07450 LTP
TG-C
1
5
WT
1
0
TG-B
WT
4
At1g06160 ERF
Fold change
TG-B
2
Fold change
Fold change
TG-A
3
TG-A
0
5
1
Fold change
TG-D
4
At1g27730 ZAT10
WT
4
0
TG-C
0
5
1
TG-B
2
At3g23240 ERF
2
TG-A
3
1
0
3
WT
4
5
WT
Fold change
TG-A
5
Fold change
4
Fold change
Fold change
WT
At1g62440 Extensina LRRP
Fold change
5
5
4
At3g12500 Quitinase
5
WT
TG-A
TG-B
TG-C
TG-D
Fold change
At5g44420 PDF1.2
4
3
2
1
WT
TG-A
TG-B
TG-C
TG-D
0
Cabello et al., 2009. MS in preparation
Arabidopsis apoplastic proteins
Acclimated 10 days at 4 °C
MW (Kda)
WT
WT
TG-A
TG-B
97,0
66,0
45,0
At3g57260.1 (PR2)
30,0
At1g75040.1 (PR5)
20,1
At1g67040.1 (unknown protein)
14,4
Cabello et al., 2009. MS in preparation
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Transgenic plants phenotype discovery
Signal transduction pathways in which TFs are
involved
Molecular mechanisms involved in phenotypic
features
Cross-talk between different pathways
mediated by TFs
Use of the information in Biotechnology
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Delay in senescence
Insensitivity to ethylene
Jasmonic acid biosynthesis (defense against
herbivores)
Green volatiles biosynthesis (systemic
response)
Energy saving in darkness
Biosynthesis of protease inhibitors
HAHB4 as a biotechnological tool: enhancer of
drought and insect tolerance
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Involved in salt stress response
Particiapates in root architecture
Inhibits LOB and concomitantely lateral roots
formation
Enhances central root growth
MTHB1 as a biotechnological tool: putative salt
tolerance, future perspective
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Accelerates plant development
Promotes avanced flowering
Related to the cross talk between SA signalling
and flowering as a defence response mechanism
Involved in oxidative stress response
HAHB10 as a biotechnological tool: life cycle
shortening without loss in yield in response to
pathogen infections, oxidative stress tolerance.
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Involved in cold stress tolerance
Involved in salt stress tolerance
Involved in drought tolerance
Induces PR2 and PR4 expression
Induces protein trasport to the apoplast in
order to generate antifreeze tolerance
HAFT as a biotechnological tool: enhancer of
cold stress, salt and drought tolerances
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Identification of TFs involved in biotic and abiotic
stress response
Characterization of sunflower WRKY encoding genes
Sunflower transcriptome analysis (PAE-sunflowerANPCyT)
a- detection of TFs regulated by stress conditions
b- Target genes of previousy characterized TFs
Muchas gracias
Thank you very much for your attention