Delineating the relationship between the chloroplast and

the vacuole during natural leaf senescence in Arabidopsis.

Ian Evans4/28/08

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

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.

Chloroplast (Cp) Senescence

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

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

avg. green avg. zone 3 avg. zone 2 avg. zone 1

To

tal C

hl/m

g

Rubisco levels• Through zoning

system Rubisco is serially degraded

• Zone 1 shows absence of intact Rubisco with increased non-specific Ab binding

Mar

ker

Gre

en

Zon

e 3

Zon

e 2

Zon

e 1

55kD

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::-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 normal growth:During senescence:

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???