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Kale Ravindra Ramrao
Outline

 Stress

 Function of drought inducible genes

 Signal transduction

 Introduction to transcription factors

 TFs Motifs

 Families of plant TFs
 What is stress ? Types?



 Stress resistance mechanisms

       • Avoidance mechanisms

       • Tolerance mechanisms

       • Acclimatization
                                 (Bartels and Ramanjulu ., 2005)
Functions of Drought inducible genes

                        Detoxification
                           enzymes
                          (catalase)


Protection factors of
                                                        Transcription
macromolecules(LEA
proteins, chaperons                                     factors (MYB
        etc.)                Drought                     , NAC etc.)




                                                        Water channel
            Protein                                        protein
                            Key enzymes for
            kinase       osmolyte biosynthesis
                          (proline , sugar etc.)

                                                   ( Agarwal et al.,2006)
Introduction to Transcription Factors

 TFs are proteins that binds to specific sequence of DNA(cis-

  acting)elements in promoter region and regulate transcription




 Number of transcription factors depends on genome size




 Use different mechanism to up or down regulate gene expression
Transcription Factor Motifs




bZIP            Zinc finger    Basic Helix-loop-helix
Structure of TFs

TFs are modular in structure and contain
  3 – domains:
 DNA binding domain-
       DNA sequence binds to TFs is
       known as responsive elements
 Trans-activation domain –
       Contain binding sites for other
       proteins
 Signal-sensing domain –
       Sense external signals and transmits
       these to rest of transcription complex
Model of Hormone-dependent Gene Activation
         by Homodimeric Receptors




                                             8
Transcriptional factors in stress tolerance
Different Families of plant stress TFs


 ERF / AP2

 WRKY

 bZIP

 NAC

 MYB
                            (Agarwal et al., 2006)
AP2 / ERF Family


 It include DREB or CBF for DRE or C repeats
                                                          (Agarwal et al., 2006)


 Arabidopsis AP2 proteins classified on the basis of similarities in their
   DNA-binding domains



 CBF genes coordinate both activation or repression of stress responsive
   genes.                                              (Bartels and Sulumar., 2005)
Contd…



 Phenotype for loss-of-function mutants have not yet been reported for any
  ERF genes



 Tsi 1( tobacco stress-inducible gene ) provides direct evidence of regulation
  of responses t biotic and abiotic stresses through single ERF gene



                                                        (Singh et al., 2002)
Contd…
Stress Response Related to ERF




                            (Singh et al., 2002)
Contd…




(Yamaguchi and Shinozaki ., 2006)
WRKY Family

 Presence of zinc fingure motif within WRKY domain

 Classification done on the basis of number of WRKY domains and the
  feature of their zinc-fingure-motif

 Group I and II have same fingure motif(C₂H₂) while group III have fingure
  motif(C₂HC)

 cis-acting element for WRKY is W box having sequence (T)
  (T)TGAC(C/T)which contain the invariant TGAC core


                                                      (Thomas et al., 2000)
Classification of WRKY




                         (Thomas et al., 2000)
MeJA

                                                    wounding                                                 auxin
                                                    light
                                                              SA                   Mangnaportae grisea
                                                              BTH

                                          OsWRKY45                                          OsWRKY31
      OsWRKY03


      NH1




       bZIP
                                                            GST
                                                                           RCl-1   PBZ1
 ZB8                                                                                          OsSci2     Os1AA4      Arl1/Crl1
              POX22.3                  PR1b
PAL           peroxidase


      Resistance to bacterial blight          Resistance to fungal blast
      x.oryza                                                                                     Root development
                                              M.grisea


                                                                                                  (Khong et al., 2008)
Contd….

 OsWRKY03 act upstream of the the NPR1/NH1 regulation pathway.

 OsWRKY45 acts independent of NPR1/NH1

 Expression of gene RCL-1 in response to BTH is independent of

  OsWRKY45 and NH1

 OsWRKY31 links activation and inhibition of plant growth
bZIP Family


 Contain DNA-binding domain rich in basic residues adjacent to
  leucin zipper dimerization domain
                                         (Bartels and Ramanjulu., 2005)

 Binds to ACGT (G –box motif ) core sequence

 One class of it is TGA/OBF binds to activation sequence-1 / ocs
  element

 TGA/OBF interact with NPR1 a key component of SA defense
  signaling pathway
                                                    (Singh et al., 2002)
Contd….




(Singh et al., 2002)
Contd….


 Different TGA / OBF members may involved in different stress
  responses

 Other class include ABF/ AREB that work thorough ABA dependent
  signal transduction pathway

 bZIP shows complexity in regulation that shows pathogen responses
  override UV protection
NAC Family Of TF


 Plant specific NAC (NAM , ATAF1/2) TFs play diverse roles in plant
   development and stress response

 NAC family is essential to the activation of the ERD1 gene

 OsNAC 1 involved in stress resistance in rice

 Expression study of GFP placed under the control of OsNAC1 promoter in
   plant , shows that this gene is normally expressed in ligulae , stamen and
   in guard cells of stomata

                                                           (Khong et al., 2008)
Contd…




Network of rice tolerance to water deficit
                                             (Khong et al., 2008)
MYB Family Of TF

 MYB proteins from animal contain 3 repeats while plant MYB contain 2
   repeats
 It carry out expression of ABA-related gene and jasmonic acid-related
   genes which shows cross talk pathway between abiotic and biotic stress
                                                            (Abe et al,.2003)

 Shows conserved tryptophan residues that form core motif


 In plants first tryptophan replaced by phenylalanine or isoleucine
Functions of MYB

1. Phenylpropanoid metabolism

2. Cell shape –

     MIXTA and it’s orthologous PhMYB1 gene.

     GL1 gene from Arabidopsis


3. Response to hormones –

     GA application induce expression of barely MYB protein (GAMYB)


                                                        (Cathie and Javier ., 1997)
Results :-




 Analysis of AtMYB60 expression
Light induced stomatal Opening is impaired in the atmyb60-1 Mutant
bHLH Family Of TF

 In A.thalinia a Myb transcription factor encoded by PHR1 participate in Pi
  starvation signalling pathway

 Os PTF1 from rice has been isolated from a cDNA library obtained from
  roots of plants cultured in normal or Pi-deficient condition
                                                                 (Yi et al., 2005)

 Os PTF1 acts on the cues of Pi uptake by stimulation root growth resulting
  in an increase in root surface area

 Os PTF1 regulate genes that play ole in Pi starvation rescue but no high
  affinity Pi transporter genes found
                                                         (Schachtmn et al., 1998)
Contd….




Networks involving bHLH transcription factors for tolerance to
mineral nutrition
CONCLUSION
DISCUSSION
THANK YOU

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Transcriptional factors in stress tolerance

  • 2. Outline  Stress  Function of drought inducible genes  Signal transduction  Introduction to transcription factors  TFs Motifs  Families of plant TFs
  • 3.  What is stress ? Types?  Stress resistance mechanisms • Avoidance mechanisms • Tolerance mechanisms • Acclimatization (Bartels and Ramanjulu ., 2005)
  • 4. Functions of Drought inducible genes Detoxification enzymes (catalase) Protection factors of Transcription macromolecules(LEA proteins, chaperons factors (MYB etc.) Drought , NAC etc.) Water channel Protein protein Key enzymes for kinase osmolyte biosynthesis (proline , sugar etc.) ( Agarwal et al.,2006)
  • 5. Introduction to Transcription Factors  TFs are proteins that binds to specific sequence of DNA(cis- acting)elements in promoter region and regulate transcription  Number of transcription factors depends on genome size  Use different mechanism to up or down regulate gene expression
  • 6. Transcription Factor Motifs bZIP Zinc finger Basic Helix-loop-helix
  • 7. Structure of TFs TFs are modular in structure and contain 3 – domains:  DNA binding domain- DNA sequence binds to TFs is known as responsive elements  Trans-activation domain – Contain binding sites for other proteins  Signal-sensing domain – Sense external signals and transmits these to rest of transcription complex
  • 8. Model of Hormone-dependent Gene Activation by Homodimeric Receptors 8
  • 10. Different Families of plant stress TFs  ERF / AP2  WRKY  bZIP  NAC  MYB (Agarwal et al., 2006)
  • 11. AP2 / ERF Family  It include DREB or CBF for DRE or C repeats (Agarwal et al., 2006)  Arabidopsis AP2 proteins classified on the basis of similarities in their DNA-binding domains  CBF genes coordinate both activation or repression of stress responsive genes. (Bartels and Sulumar., 2005)
  • 12. Contd…  Phenotype for loss-of-function mutants have not yet been reported for any ERF genes  Tsi 1( tobacco stress-inducible gene ) provides direct evidence of regulation of responses t biotic and abiotic stresses through single ERF gene (Singh et al., 2002)
  • 13. Contd… Stress Response Related to ERF (Singh et al., 2002)
  • 15. WRKY Family  Presence of zinc fingure motif within WRKY domain  Classification done on the basis of number of WRKY domains and the feature of their zinc-fingure-motif  Group I and II have same fingure motif(C₂H₂) while group III have fingure motif(C₂HC)  cis-acting element for WRKY is W box having sequence (T) (T)TGAC(C/T)which contain the invariant TGAC core (Thomas et al., 2000)
  • 16. Classification of WRKY (Thomas et al., 2000)
  • 17. MeJA wounding auxin light SA Mangnaportae grisea BTH OsWRKY45 OsWRKY31 OsWRKY03 NH1 bZIP GST RCl-1 PBZ1 ZB8 OsSci2 Os1AA4 Arl1/Crl1 POX22.3 PR1b PAL peroxidase Resistance to bacterial blight Resistance to fungal blast x.oryza Root development M.grisea (Khong et al., 2008)
  • 18. Contd….  OsWRKY03 act upstream of the the NPR1/NH1 regulation pathway.  OsWRKY45 acts independent of NPR1/NH1  Expression of gene RCL-1 in response to BTH is independent of OsWRKY45 and NH1  OsWRKY31 links activation and inhibition of plant growth
  • 19. bZIP Family  Contain DNA-binding domain rich in basic residues adjacent to leucin zipper dimerization domain (Bartels and Ramanjulu., 2005)  Binds to ACGT (G –box motif ) core sequence  One class of it is TGA/OBF binds to activation sequence-1 / ocs element  TGA/OBF interact with NPR1 a key component of SA defense signaling pathway (Singh et al., 2002)
  • 21. Contd….  Different TGA / OBF members may involved in different stress responses  Other class include ABF/ AREB that work thorough ABA dependent signal transduction pathway  bZIP shows complexity in regulation that shows pathogen responses override UV protection
  • 22. NAC Family Of TF  Plant specific NAC (NAM , ATAF1/2) TFs play diverse roles in plant development and stress response  NAC family is essential to the activation of the ERD1 gene  OsNAC 1 involved in stress resistance in rice  Expression study of GFP placed under the control of OsNAC1 promoter in plant , shows that this gene is normally expressed in ligulae , stamen and in guard cells of stomata (Khong et al., 2008)
  • 23. Contd… Network of rice tolerance to water deficit (Khong et al., 2008)
  • 24. MYB Family Of TF  MYB proteins from animal contain 3 repeats while plant MYB contain 2 repeats  It carry out expression of ABA-related gene and jasmonic acid-related genes which shows cross talk pathway between abiotic and biotic stress (Abe et al,.2003)  Shows conserved tryptophan residues that form core motif  In plants first tryptophan replaced by phenylalanine or isoleucine
  • 25. Functions of MYB 1. Phenylpropanoid metabolism 2. Cell shape –  MIXTA and it’s orthologous PhMYB1 gene.  GL1 gene from Arabidopsis 3. Response to hormones –  GA application induce expression of barely MYB protein (GAMYB) (Cathie and Javier ., 1997)
  • 26. Results :- Analysis of AtMYB60 expression
  • 27. Light induced stomatal Opening is impaired in the atmyb60-1 Mutant
  • 28. bHLH Family Of TF  In A.thalinia a Myb transcription factor encoded by PHR1 participate in Pi starvation signalling pathway  Os PTF1 from rice has been isolated from a cDNA library obtained from roots of plants cultured in normal or Pi-deficient condition (Yi et al., 2005)  Os PTF1 acts on the cues of Pi uptake by stimulation root growth resulting in an increase in root surface area  Os PTF1 regulate genes that play ole in Pi starvation rescue but no high affinity Pi transporter genes found (Schachtmn et al., 1998)
  • 29. Contd…. Networks involving bHLH transcription factors for tolerance to mineral nutrition