Quantitative analyses of cell division in plants
Gerrit Beemster, University Antwerpen, Belgium
EPSO: The European Plant Science Organisation
EPSO Workshop on Plant Phenotyping
November 02-03, 2009
Forschungszentrum Jülich, Germany
Forschungszentrum Jülich, Germany
ICG-3: Phytosphere
Jülich Plant Phenotyping Centre (JPPC)
Website: http://www.jppc.de
http://www.plantphenomics.com/phenotyping2009
How to analyse cell division in
plants?
Gerrit Beemster
1
Growth is a multi-level process
Genetics
Cell cycle
(Natural variation,
Transgenics, mutants)
(DNA duplication, Cytokinesis)
Molecular interactions
(Genetic network)
Source-Sink relations
(not covered)
Environment
(Kinematic analysis)
Cell expansion =
(Stress)
(Cell wall properties)
Growth
4 major stages separated in time:
- Initiation (division + expansion)
- Proliferation (division + expansion)
- Expansion (expansion)
- Mature
2
Outline of talk
•Kinematic analysis of growth in:
- Arabidopsis leaves
- Arabidopsis root tips
- maize leaves
3
High throughput
phenotyping of Arabidopsis
4
Time-lapse imaging
(continuous light)
5
Image analysis
6
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Kinematic analysis of Arabidopsis leaves
Leaf area
Cell size
Plant Cell 13: 1653-1668 (2001)
8
Acquiring high quality cell outlines
9
Acquiring high quality cell outlines
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Image analysis: Epidermal Cell Analysis
Automating kinematic analysis
Average cell area same!
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Automating kinematic analysis
Wild type
Mutant
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background levels for at least one sample. For a small
number of genes, there were duplicate probes of
different length and sequence. The expression pattern
for these probes was nearly identical in each case (data
not shown), demonstrating the reproducibility of the
hybridization. Of the genes that gave a positive signal,
2,061 (34% of all genes on the array) were significantly
modulated (P # 0.001). The largest difference in
Flow cytometry
of leaf growth. A, Leaf area. B, Relative leaf
l area. D, Number of cells per leaf. E, Cell
ex. Symbols denote averages 6 SE of three
e bottom the growth phases are indicated
and expand simultaneously), expansion
sion), and mature (no more cell growth).
A content; the remainder was of
he amount of 8C cells observed
ating nearly all cells of the leaf
tic cell cycle activity. From day
verage cell division rates were
Figure 2. Flow cytometry analysis of nuclear DNA content. The results
of a representative experiment are plotted. The bar at the bottom
denotes the growth phases based on the kinematic analysis (Fig. 1).
Plant Physoil. 138: 734-743 (2005)
Plant Physiol. Vol. 138, 2005
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Kinematic analysis of root growth
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Conceptual framework
1. Utilizing spatial organisation.
2. QC as a reference point.
3. For cell division, select one (or more) cell
types.
Division +
Elongation only
Elongation
Cap
Meristem
Elongation zone
Mature root
QC
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Velocity and expansion rates
•Time-lapse analysis
(Same for all cell types)
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(Plant) Cell Flux
•Cell lenght observations
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19
Example - expansion data
Overall parameters:
- E: V(X>Xvmax) also
from ticking (see later)
- rmax
Distance from QC, µm
(Data: Beemster&Baskin, 2000. Plant Physiology 124: 718-727)
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Cell length > division
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Additional parameters
Derived parameters:
- Mature cell length (lmat)
- Accumulative cell number from QC (N(x))
- Meristem cell production (P = Fluxmax, and E/lmat)
- Size of meristem, elongation zone and growth zone
- # cells in meristem (Ndiv), elongation zone (Nel)
- Average division rate (Davg = P/Ndiv)
- Average cell cycle duration (Tc=ln(2)/Davg)
Material parameters:
- Time in meristem (Tdiv= Log2(Ndiv)*Tc
- Time in elongation zone (Tel=P/Nel)
“Shortcut”: Indirect approach: Root elongation rate + Cell length
profile only!
“Stepwise” approach!
22
Analysis of maize leaf growth
•Longitudinal “steady-state”
system
•Enclosed by older leaves
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The kinematic analysis
ELONGATION RATE
E
CELL LENGTH
MERISTEM SIZE
l(x)/lm
Ndiv
P
r(x)
D
Tc
Complete set of parameters defining growth at the cellular level:
CELL DIVISION ! meristem size, cell production, division rate, cell cycle time
CELL EXPANSION ! mature cell size, relative expansion rate
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Effect of cold stress
Control
Stress
Difference
Leaf elongation rate (mm/h)
3.68 ± 0.23
2.93 ± 0.20
-21%
Mature cell length (um)
124 ± 10
127 ± 5
ns
Cell production rate (cells/h)
29.7 ± 2.4
23.1 ± 1.0
-22%
Meristem size (mm)
17.0 ± 5.6
16.1 ± 2.8
ns
Amount of dividing cells
821 ± 94
1060 ± 213
+29% (ns)
Cell division rate (cells/cell.h)
0.036 ± 0.004
0.022 ± 0.005
-38%
Cell cycle duration (h)
19 ± 2
32 ± 6
+65%
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Conclusions
•Kinematic analysis allows accurate quantitative
analysis of cell division rates in context of organ
growth
•Kinematic analyses can be adapted to suit the
needs of different organs
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