Download Ian-Evans

Delineating the relationship
between the chloroplast and
the vacuole during natural leaf
senescence in Arabidopsis.
Ian Evans
4/28/08
Biol 466H
Defining Senescence in Plants
• Foliar senescence is the final stage of
leaf development in which nutrients are
remobilized to younger tissues
Lim et al. (2007) Annual Review of
Plant Biology 58:122.
Senescence is tightly regulated
Chloroplast (Cp) Senescence
Chlorophyll degradation is well
characterized, but degradation
of the LHC proteins that bind
Chl is not well understood.
Hörtensteiner, S (2006) Annual Review of Plant
Biology 57:55.
Spatial Relationships in
Senescence
Evidence for Vacuolar
Degradation
• Endopetidases are present in vacuole and
their activity is upregulated in senescence
• TEMs show Cps in the center of the cell
during senescence but not during normal
growth
Wittenbach et al. Plant Physiol. 1982 69:98
Wheat Mesophyll Cell
Minamikawa et al. Protoplasma. 2001 218:144
Bean Mesophyll Cell
Evidence for Internal
Degradation
• Upregulation of Cp-localized proteases
• Initial Chl catabolism occurs in the
stroma
• Early literature shows internal changes
in Cps
Barton et al. Planta. 1966 71:314
Bean Mesophyll Cell senescing Cps
Freeman et al. 1978 Protoplasma 94:221
Citrus Mesophyl Cell senescing Cp
Aims of my research
• Aim 1: Characterize the ultrastructure of
the Cp in senescence
• Aim 2: Define the relationship between the
Cp and the vacuole in senescence
Rationale for Approach
to Aim 1
• TEM will allow subcellular structure
to be analyzed within senescent cells
• Morphological changes in Cp will be
evident
Zoning an Arabidopsis leaf
Green
Zone 3
Zone 2
Zone 1
Chlorophyll levels
• Chl levels decrease
as leaf senescence
progresses
3.50
Total Chl/mg
3.00
2.50
2.00
1.50
1.00
0.50
0.00
avg. green avg. zone 3 avg. zone 2 avg. zone 1
Rubisco levels
Zone 1
Zone 2
Zone 3
Green
55kD
Marker
• Through zoning
system Rubisco is
serially degraded
• Zone 1 shows
absence of intact
Rubisco with
increased nonspecific Ab binding
Ultrastructure of Leaves
•
Use Transmission
Electron Microscope
(TEM) to visualize
subcellular structure
within each zone
Healthy Green Cp
Green 15,000X
Plastoglobuli formation
Zone 3
15,000X
Separation of Thylakoids
Zone 2 15,000X
Dismantling of Thylakoid
System
Zone 1 20,000X
Circularization and loss of
distinguishable grana
Zone 1 40,000X
Late Stage Separation from
Plasma Membrane
Zone 1 40,000X
Aim 1 Conclusions
• While Chl and Rubisco are degraded, Cps
remain intact along the plasma membrane
• Ultrastructural changes include separation
of thylakoids, formation of plastoglobuli,
and circularization of Cps.
Rationale for Approach
to Aim 2
• TEM is impractical even with serial
sectioning or immunogold staining
• Newer techniques are looked upon
favorably
• Confocal has less artifacts and
preparation problems
Structure of the Vacuole
•The
tonoplast
surrounds
the central
vacuole in
plant cells
Confocal Microscopy
• To visualize the tonoplast
- use a 35S-GFP::d-TIP line.
To visualize the chloroplast
- use autofluorescence.
Avila et al. 2003. Plant Phys. 133:1674
• GFP lines treated
with EMS to induce
single nt mutations
•Above, tvs mutant’s
vacuoles transected by
transvacuolar strands
•To the right, bub
mutants have many small
vacuoles
Eggink, et al. 2004. BMC Plant Biology, 4:5
http://icecube.berkeley.edu/~bramall/work/astrobiology/images/chlorophyllspectra.jpg
What would results look like?
During senescence:
normal growth:
During
Conclusions and Significance
• Degradation of chloroplast will be
characterized in Arabidopsis
• Relevance of vacuole in senescence of
the chloroplast will be assessed
• Areas of high impact:
- plant molecular biology
- agriculture
- CSULB community
THANK YOU
Questions???