Apoptotic-like programmed cell death in plants:

Caspase inhibitors affect the kinetics and

dimensions of tracheary elements in xylogenic
Zinnia (Zinnia elegans) cell cultures

Leonita Swandjaja (20611016)

Mia Fitria Utami (10408006)
Luh Putu Pitrayani Sukma (10408002)
Dewi Ayu Kencana Ungu (10408009)
Yuniar Devi Utami (10408024)

Programmed cell death (PCD)

Programmed cell death (PCD) is a cell suicidal genetically programmed developmentally and
environmentally stimulated mechanism
In vascular plants, PCD is involved in embryogenesis, developmental processes, senescence,
hypersensitive response to pathogen attacks and in the response to abiotic stress stimuli
A typical example of developmentally regulated PCD is PCD that occurs at the final stage of
cell differentiation during the formation of the xylem vascular system

Tracheary Elements (TEs)

Xylem vessels (water conducting tubes) are

composed of a number of fused vessel or tracheary
elements (TEs) that are dead, hollow cells with
patterned lignified cellulose secondary walls.

TEs originate through re-differentiation of root and

shoot pro-cambium and cambium cells and undergo
autolysis as they differentiate and mature

The development of xylogenic Zinnia (Zinnia

elegans) cell cultures, derived from leaf mesophyll
cells, has revolutionized the understanding of the
xylem differentiation process

Trans-differentiation of Zinnia cells in xyloformation

Conversion of a cell that has completed

differentiation and acquired a certain function into
another cell with a different function is called "transdifferentiation.
Stage I includes dedifferentiation of mesophyll cells
and acquisition of competence for re-differentiation
During stage II synthesis and deposition of secondary
wall material occurs
Stage III involves a progression of the PCD process
associated with (in this order) the formation of a
large vacuole, rupture of the tonoplast, DNA
fragmentation, disappearing of the nucleus, autolysis
of cell content and formation of hollow dead TEs

Xyloformation

Regulation of TE differentiation in Zinnia elegans cell culture

Tonoplast rupture
Autolysis
DNA fragmentation
Blocked PCD

Aquisition of
competence for
differentiation

Living
mesophyll
cells

PCD in Progress
Differentiation

24-48 h

Protease inhibitors
Wound- and Lightinducible genes,
PAL

Caspase-like
proteases ???
Cysteine proteases ??

Stage II Secondary
Wall Thickening

Stage III
PCD

Proteolysis
(ubiquitin-proteosome),
2+
Ca ,
MAP-kinase,
ethylene,
brassinosteroids, nucleases (ZEN1),
cycteine proteases (ZCP4), PAL, IP3,
(lignin precursors), TED genes-cell
wall
proteins,
arabinogalactans;
sucrose,
mannitol,
light,
temperature, pH, nitrogen
Stage I - dedifferentiation
Auxin +
Cytokinin

Caspase- and Cysteineprotease Inhibitors

Differentiated TE
in flourescence
staining (above)
and under light
microscope
(below)

Plant caspase-like proteases in TE development

Microarray analysis has shown upregulation of a metacaspase

9, VPEa, and xylem cysteine proteases during TE
differentiation in Arabidopsis
By immunohistochemistry and immunoelectron microscopy,
caspase-3-like protease has been detected in developing
tracheary elements in Cucurbita moschata

Caspases: The enzymes of death

Caspase : initiation and execution
phases of apoptosis.
During apoptosis, upon activation,
caspases cleave specific substrates and in
that way mediate many of the typical
biochemical and morphological changes in
apoptotic cells.
Caspases are constitutively expressed in
the majority of cells as inactive proenzymes (zymogens) that become
proteolytically processed and activated in
response to variety pro-apoptotic stimuli.

Aim
Invistigated the potency of specific peptide caspase inhibitors
(the broad range irreversible aspase inhibitor Z-Asp-CH2-DCB,
irreversible caspase 1 inhibitor Ac-YVAD-CMK and reversible
caspase 3 inhibitor Ac-DEVD-CHO) to modulate the
development of TEs in xylogenic Zinnia cell cultures

14-day old
zinnia seedling

A
Zinnia mesophyll
cell culture

Hormone free
control
No differentiation

B
1 mg/L BA +
0,1 mg/L NAA
First true pair of
leaves, 3rd pair
just emerging

120 h after
addition of
hormones ;
CFW staining of
diferentiated TE

Materials dan Methods

Kontrol negatif : bebas hormon
Perlakuan : penambahan hormon untuk induksi diferensiasi
1 mg/L BA + 0,1 mg/L NAA

Penambahan inhibitor kaspase ke kontrol negatif dan perlakuan:

irreversible broad-ranged human caspase-3 inhibitor : Z-Asp-CH2-DCB
irreversible caspase-1 inhibitor : Ac-YVAD-CMK
reversible caspase-3 inhibitor : Ac-DEVD-CHO

Disiapkan juga kontrol lain : penambahan hormon tanpa inhibitor kaspase

A : Kultur sel
+ inhibitor
- hormon

B : Kultur sel
+ inhibitor
+ hormon

C : Kultur sel
- inhibitor
+ hormon

A. Effect of inhibitor
concentrations on the
percent of formed TEs
=> The caspase inhibitors
suppressed TE formation in
a concentration dependent
way
B. Effect of inhibitors on
cell viability
=> In the presence of
caspase inhibitors cell
viability did not differ
significantly from nonhormone control

Plant proteases with substrate

specificity comparable to
animal caspases are involved
in the trans-differentiation
process in Zinnia xylogenic cell
cultures

Effect of Z-Asp-CH2-DCB on DNA fragmentation in xylogenic Zinnia cell

cultures

Lane 2: No DNA fragmentation was

detected in control
Lane 3: Oligonucleosomal DNA
fragments of around 160-180 bp
were clearly visible in xylogenic cell
cultures
Lane 4: DNA fragmentation was
greatly inhibited in the
presence of 100 nM Z-Asp-CH2-DCB

TE trans-differentiation is associated with DNA fragmentation and that the

caspase inhibitor Z-Asp-CH2-DCB efficiently prevents this phenomenon

Confocal images of Zinnia suspension cultured cells,

representing the cell morphology during TEs differentiation

Effect of Z-Asp-CH2-DCB on morphology, kinetics and yield of TEs generation and

interference with the two waves of TE differentiation in xylogenic Zinnia cell
cultures

Percentage of formed TEs to the initial number of living cells in 24 h intervals after addition of 1 mg/L
NAA and 1 mg/L BA (hormones) and a combination of the hormones with Z-Asp-CH2-DCB (H+1 nM; H+10 nM;
H+100 nM), until completion of TE differentiation.

Effect of Z-Asp-CH2-DCB on morphology, kinetics and yield of TEs

generation and interference with the two waves of TE
differentiation in xylogenic Zinnia cell cultures

A. Control (without
induction)
B. Induced culture
treated with 1 mg/L
NAA and 1 mg/L BA
C. Hormones + 10 nm ZAsp-CH2-DCB;
D. Hormones + 100 nM
Z-Asp-CH2-DCB.

Structural changes were delayed and the TEs are longer, in the presence
of larger caspase inhibitor concentration

Conclusions
This results proved that plant proteases with functional
similarity to animal caspases participate in TE formation,
presumably through their effect on PCD

References

Iakimova, E. T. And E. J. Woltering. 2009. Modulation of programmed cell

death in a model system of xylogenic Zinnia (Zinnia elegans) cell culture.
BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE

Twumasi, P. 2007. Hidraulic properties of Zinnia elegans from cellular

development in vitro to performance in planta . PhD Dissertation, Wageningen
University, The Netherlands .

Twumasi, P., E. T. Iakimova, T. Qian, W. van leperen, J. H. N. Schel, A. M. C.

Emons, O. Van Kooten, E. J. Woltering. 2010. Caspase inhibitors affect the
kinetics and dimensions of tracheary elements in xylogenic Zinnia (Zinnia
elegans) cell cultures. BMC Plant Biology, 10:162