PowerPoint Presentation - Abiotic stress by mql13846

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									NaCl and drought stress responses
            in plants



              MS RAFUDEEN

      DEPARTMENT OF BIOTECHNOLOGY
          PLANT BIOTECHNOLOGY
      UNIVERSITY OF THE WESTERN CAPE
                    2004
                  RESEARCH FOCUSS


Define abiotic stress responses in plants

Characterise the role of stress response proteins and second
 messengers

Analysis of abiotic stress responses at the level of the proteome
        THE PLANT PROTEOMICS APPROACH

Proteomes are chemically and spatially more complex than
genomes

Certain protein functions may have a significant epigenetic
component

Proteins transduce information within the proteome, to and
from the metabolome and into the phenotype

Intergration of metabolic profiling and proteomics

Combined large scale microarray and proteomics analyses
INTERRELATIONSHIPS OF THE PLANT PROTEOME


    Genome


      Active
      Genome
                      Metabolome

   Transcriptome



          Proteome
                     Environment
         GENETIC MODEL SYSTEMS TO STUDY
            OSMOTIC STRESS IN PLANTS


Yeast

Mesembryanthemum crystallinum- Ice plant, facultative halophyte

Craterostigma plantagineum- Resurrection plant

Arabidopsis- Glycophyte

Thellungiella halophila- Halophyte, post-Arabidopsis revolution?
THE USE OF PROTEOMICS TO STUDY OSMOTIC STRESS
  IN Arabidopsis thalania CELL SUSPENSION CULTURES

                                      Excise protein spot
Treatment     2D gel                  In-gel digestion              MALDI-TOF MS
                                      Extraction of peptide


            Positive identification                                Peptide ion mass list


                                         Genomic Database search



    - A proteomics approach to study abiotic stresses in Arabidopsis thaliana

    - Discriminate between NaCl toxicity and drought caused by non-ionic sorbitol

    - Arabidopsis genomic sequence available, large amount of micro-array and
      physiological data regarding ion transport available

    - Cell suspension cultures well characterized and reproducible biological system
            BACKGROUND TO NaCl STRESS

~20% of the world’s cultivated land and ~50% of all irrigated
land are affected by salinity

High salinity causes ionic disequilibrium and hyperosmotic
stress

Negatively on the critical biochemical processes

Ion homeostasis in saline environments is primarily dependent
 on membrane components
      EFFECTS OF Na+ TOXICITY IN PLANTS


Whole-Plant level

Plant tissue and cellular level

• Water deficit stress

• The ion specific stress results in metabolic toxicity

• Nutritional deficiencies
     Na+ UPTAKE/EXTRUSION IN THE PLANT CELL
         Plasma Membrane


                            PPi   H+                           Na+                                Na+
                                                                                                  K+
H+
                                       V-PPase                                High-affinity K+
            Na+                                       H+                        transporters
Na+/H+ antiport
                                         Vacuole

                                           Na+     Tonoplast                                     Na+
                          V-ATPase                                                               K+
                                       H+ ATP                           K+/Na+ selectiveVICs
           K+/Na+ ratio                                        ATP
                                                      H+


                                                           P-ATPase


                                                                     Adapted from Mansour et al. 2003
        ASPECTS OF SALT TOLERANCE IN PLANTS

               Ionic                                                    Ion
               stress        SOS3 SOS2            Ion transporters   homeostasis

SALT STRESS                                                                           Homeostasis


                        Osmotic        MAPK              Osmolytes      Osmotic
                         stress       cascade ?                       homeostasis
   COLD
 DROUGHT
   ABA                                                                                Detoxification




              Secondary                               Stress
                                  CBF/DREB
               stresses                              proteins
                                                                                      Cell division
                                                                                     and expansion




                                                                         Adapted from J-K Zhu 2001
MECHANISMS OF CELLULAR TOLERANCE
      TO HIGH Na+ IN PLANTS



   Sodium Extrusion

   Intracellular compartmentation

   Synthesis of osmoprotectants

   Damage response and repair
REGULATION OF ION HOMEOSTASIS BY THE
            SOS PATHWAY




                                 J-K Zhu 2003
           SECONDARY STRESS RESPONSES

                 ABIOTIC & BIOTIC STRESS

                                ROS at steady-state level

                                                  TOXICITY
                      [ROS]                     CELL DEATH

                                Signal for activation of stress-response
                                & defense pathways

DETOXIFICATION                                         AVOIDANCE
                                                            Anatomical adaptation
                                                            Physiological adaptation
                                                            Alternative channeling
   SCAVENGING                    ELIMINATION                Molecular mechanisms
ROS scavenging enzymes        Antioxidant compounds
  Superoxide dimutase             Ascorbic acid
  Glutathione peroxidase          Glutathione
  Catalase                        Thioredoxin
                                  Carotenoids
INVESTIGATION OF THE ROLE OF PLANT NATRIURETIC
        PEPTIDES IN NaCl STRESS RESPONSES


     Higher plants contain natriuretic peptides (PNPs)
     associated with the conductive tissue

     PNPs affect ion fluxes, protoplast volume regulation and
     stomatal guard cell responses

     Exact role in homeostasis is undefined

     PNPs belong to the superfamily of expansins
    STRUCTURE OF PNP MOLECULES

     PNP-like molecules

        Intron


                                 Cellulose-binding
N                                     domain
                                                     C


        Intron    Intron     Intron



                 Expansins
MODEL OF PNP DEPENDANT PROCESSES
              SPECIFIC AIMS


Study the effect of NaCl and sorbitol on PNP levels in
Erucastrum strigosum plants and Arabidopsis thaliana
cell suspension cultures

Proteomic identification of proteins induced by NaCl,
sorbitol and PNPs in Arabidopsis cell suspension
cultures
NaCl-DEPENDENT CHANGES IN Erucastrum Strigosum
            WET AND DRY MASS

                         16                                                    3
                                      a
                         14
                                                                               2.5
                                          ab
                         12
        Fresh mass (g)




                                                                                     Dry mass (g)
                                                          b                    2
                         10       a
                          8                                          b         1.5
                                          b
                          6                           b
                                                                     b         1
                          4
                                                                               0.5
                          2

                          0                                                    0
                              0           100         200                300

                                               NaCl (mM)


                                                              Wet mass changes
                                                              Dry mass changes
EFFECT OF NaCl ON ION LEVELS IN
       Erucastrum Strigosum

                    120
                    100                           c
                                                        : Na
   Element (mg) /                           bc
                     80
   dry mass (g)                        b
                     60           a
                     40                a     a    a
                                                        :K
                     20       a
                      0
                          0           100   200   300     NaCl
                                                          (mM)
                    16                 a          a
                    14        a              a          : Ca
  Element (mg) /




                    12
  dry mass (g)




                    10        a
                     8
                     6                b
                                             c    bc
                     4                                  : Mg
                     2
                     0                                  NaCl
                          0           100   200   300
                                                        (mM)
ISOLATION OF irPNP AND BIOCAD IMMUNOAFFINITY
  PURIFICATION OF irPNP FROM Erucastrum Strigosum




              Absorbance at 280nm
          A                         0.020                                               1
                                                               M TP




                                                                                              NaCl (M)
                                    0.016               kD:                             0.8
                                                         30
                                    0.012               21.1                            0.6
                                                        14.3
                                    0.008                                               0.4




                                                                       
                                    0.004                                               0.2
                                    0.000                                               0
                                               0   10    20 30 40 50 60 70         80
                                                        Fraction number (1 mL)

                                    kD:
                                          45
          B                               30
                                       21.1
                                                                      C
                                       14.3

                                                                 
                                        6.5




                                       irPNP-A (Erucastrum strigosum)
          D                                                            
                                       MAVKFVVVMI       VFAQILAPIA    EAAQGKAVYY
                                       DPPYTRSACY       GTQRETLVVG    VKNNLWQNGR
                                       ACGRRYRVRC       IGATYNFDRA    CTGRTVDVKV
                                       VDFCREPCNG       DLNLSRDAFR    VVANTDAGNI
                                       RVVYTP                         
RELATIVE AMOUNTS OF PNP IN Erucastrum strigosum
  PLANTS AFTER DIFFERENT NaCl EXPOSURES


                                 300

                                                                      b
                                 250
         Rel. irPNP conc. in %


                                 200

                                               C       1   2
                                 150
                                           a                   a

                                 100               a

                                  50


                                       0
                                           0       100         200   300


                                               NaCl (mM)
EFFECT OF NaCl ON Arabidopsis Thalania CELLS
        IN SUSPENSION CULTURE

                                 A                                          B

          40                                                                         200
                                             Na (mM):     0                     c
          35




                                                                                           Rel. irPNP conc. in %
          30                                             50                          150
                Prot.                                                       b
%Growth




          25                                                    ab
                extracted
           20                                           100          a               100

          15       

          10                                                                          50
                                                        150
           5

           0                                                                           0
             0     24       48   72    96   120   144     168   0    50    100 150
             
          NaCl added             Time (h)                                NaCl (mM)
EFFECT OF SORBITOL ON Arabidopsis thalania CELLS
           IN SUSPENSION CULTURE

                                        A                                               B

             40                                                                                 c   300
                                                 Sorbitol (mM):     0
             35
                                                                  100




                                                                                                          Rel. irPNP conc. in %
             30                                                   300
                                                                                                    200
% Growth




             25                                                                             b
                       Prot.                                      200
             20        extracted
                                                                              ab
             15             
                                                                                                    100
                                                                                    a
             10

              5
              0                                                                                     0
                   0        24     48       72     96      120          144    0   100 200 300
                  
           Sorbitol added                   Time (h)                               Sorbitol (mM)
EFFECT OF RECOMBINANT AtPNP-A ON PROTOPLAST
               WATER UP-TAKE
                                                                                          A
                                    0.010                                                                               100
       Absorbance at 280nm
                                                           Ind. C         M                                AtPNP-A
                                                                                                                         90




                                                                                                                                Conductivity (mS)
                                                                              kD:
                                                                                                                         80
                                                                              45
                                                                                                                         70
                                                                              30                                         60
                                    0.005                                     20.1                                       50
                                                                             14.3                                 *     40
                                                                                          *                              30
                                                                                                                         20
                                                                                                                         10
                                    0.000                                                                                0
                                               0                20                   40           60          80

                                                       Fraction number (1 mL/fraction)
     Mean Protoplast Volume (m3)




                                                                B                                                       C
                                     15000                                                    10000
                                     13500
                                                                                 d
                                                                                               9000
                                                                                                                   b    Cycloheximide:                   ■
                                     12000                           c                         8000
                                     10500                                                     7000
                                      9000                                                     6000
                                                                                                                                                    a
                                      7500                 b                                   5000
                                                                                                                            a
                                      6000                                                     4000    a
                                      4500                                                     3000
                                                   a                                           2000
                                      3000
                                      1500                                                     1000
                                          0                                                       0
                                    AtPNP-A:       0       10        50         100                    0           50       0                       50
                                     (ng/mL)
 PROTEOMIC IDENTIFICATION OF A Hsp70.1 IN A. thaliana
SUSPENSION CELL CULTURES STRESSED WITH NaCl AND
                   SORBITOL

     A
                            Control                 0.2 M NaCl             0.4 M Sorbitol
          pH: 4.0                     6.0 4.0                    6.0 4.0                    6.0
         kD:
         66         1        2                          2
                                                1                          1    2
         45
         36

         29

     B
          pH: 4.0                     6.0 4.0                    6.0 4.0                    6.0
         kD:
         66             1        2                  1       2                       2
                                                                            1
         45
         36
         29
                    SUMMARY OF RESULTS

The change in PNP levels in response to water and salt balance support
PNPs role in plant homeostasis

PNP up-regulation may be a response to counter-act increasing osmotic stress

Protein synthesis is required for PNP function- synthesis of compatible solutes?

Osmotic stress may lead to processing of the peptide to allow the PNP to act as
a extracellular, mobile signalling molecule

Proteomic studies identified a post-translational modified heat shock protein Hsp70.1

Previously shown that Hsp70.1 plays a osmoprotective role in animal and microbial
systems and in micro-array data of abiotically stressed plants
                    OUT-LOOK

Investigate AtPNP-A and Osmotic Stress responses on the Plant
Proteome


2D DIGE, identification, mass spectrometry and molecular
characterization of AtPNP-A/osmotic stress responsive proteins


Identification of regulatory elements, protein-protein interaction
and signaling mechanisms


Proteomic comparison of wild-type A. thaliana with stress
sensitive mutants


Selection of novel or responsive proteins for further
characterization in A. thaliana systems
             ACKNOWLEDGEMENTS


Professor Chris Gehring, Dr Graeme Bradley and the
UWC Plant Biotechnology laboratory.
Dr B.K Ndimba, School of Biological and Biomedical
Sciences, University of Durham.
Claude Leon Harris Foundation.
The work was supported by the South African NRF and
Royal Society-NRF exchange program

								
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