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 "trans-differentiation.“

• 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

Living mesophy

ll cells

Blocked PCD

Aquisition of competence

for differentiation

Protease inhibitors

Wound- and Light- inducible

genes, PAL

24-48 h

Auxin + Cytokini

n

Stage II – Secondary Wall

Thickening

Stage III – PCD

Proteolysis (ubiquitin-proteosome), Ca2+, 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

PCD in Progress

Differentiation

Caspase-like proteases ???

Cysteine proteases ??

Tonoplast rupture Autolysis

DNA fragmentation

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

Stage I - dedifferentiation Caspase- and Cysteine-protease Inhibitors

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 executionphases 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 pro-enzymes (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

First true pair of leaves, 3rd pair just emerging

14-day old zinnia

seedling Zinnia mesophyll cell culture

1 mg/L BA + 0,1 mg/L NAA

A

B

Hormone free control

No differentiation

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 inhibitorconcentrations on the percent of formed TEs => The caspase inhibitors suppressed TE formation in a concentration dependent wayB. Effect of inhibitors on cell viability => In the presence of caspase inhibitors cell viability did not differ significantly from non-hormone 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 thepresence 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 Z-Asp-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