This document discusses programmed cell death (PCD) in plants. It provides definitions of PCD and necrosis, and describes the differences between the two processes. PCD, also called apoptosis, is an actively controlled and genetically regulated process while necrosis is unregulated cell death in response to external stressors. The document outlines the history of studying PCD/apoptosis and discusses PCD pathways, regulators like caspases, and importance in plant development and response to the environment. It also provides a case study on PCD in tomato fruit in response to heat stress.

1. Welcome
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110/09/13

2. Man,Man,
Happy ManHappy Man
210/09/13

3. Plant!!?Plant!!?
310/09/13

4. KITTUR RANI CHANNAMMA COLLEGE OF
HORTICULTURE, ARABHAVI
Programmed Cell Death in PlantsProgrammed Cell Death in Plants
410/09/13

5. TOPIC DIVISION
510/09/13

6. Definition
Programmed cell death (PCD) is a physiological cell death process
involved in the selective elimination of unwanted cells.
Ellis et al., (1991)
Necrosis is a non physiological process involving cell
swelling, lysis, and the inflammatory leakage of cell contents.
Cohen, (1993)
Programmed Cell DeathProgrammed Cell Death (PCD ……….?)Programmed Cell DeathProgrammed Cell Death (PCD ……….?)
Programmed cell death (PCD)/ apoptosis
Programmed cell death occurs via apoptosis - a Greek word
meaning “falling leaves”
- John et al., (1972)

7. 10/09/13 7

8. History of Programmed cell death / apoptosis
1800s - Numerous observation of cell death
1842 - Carl Vogt, first time describe the principles of apoptosis.
1885 - Anatomist Walther Flemming delivered a more precise
description of the process of programmed cell
death.
1908 - Mechnikov wins Nobel prize (phagocytosis).
1930-40 - Studies of metamorphosis.
1948-49 - Cell death in chick limb.
1955 - Beginning of studies of Ricinosomes.
1964-66 - Necrosis & PCD described.
1971 - Term apoptosis coined in Greece.
1977 - Cell death genes in C. elegans.
1980-82 - DNA ladder observed & ced-3 identified.
1989-91 - Apoptosis genes identified, including bcl-2,
fas/apo1 & p53, ced-3 sequenced.
2002 - Jone E Sultan, won the nobel prize in medicine for his poineering research810/09/13

9. Death by Injury vs. Death by Suicide
(Necrosis vs. Apoptosis)
(Kerr et al., 1992, Germany) 910/09/13
Fig -1 : Difference between necrosis and PCDFig -1 : Difference between necrosis and PCD

10. Necrosis vs. ApoptosisNecrosis vs. Apoptosis
• Cellular condensation
• Membranes remain intact
• Requires ATP
• Cell is phagocytosed, no
tissue reaction
• Ladder-like DNA
fragmentation
• In vivo, individual cells appear
affected
• Cellular swelling
• Membranes are broken
• ATP is depleted
• Cell lyses, eliciting an
inflammatory reaction
• DNA fragmentation is
random, or smeared
• In vivo, whole areas of
the tissue are affected
Necrosis Apoptosis
(Richerd et.al., 2001)

11. 10/09/13 11
http://science.howstuffworks.com/life/cellular-microscopic/apoptosis.htm
Fig -2 : Phases of PCD in a Vascular PlantFig -2 : Phases of PCD in a Vascular Plant

12. Why should a cell decide to commit suicide?Why should a cell decide to commit suicide?
PCD is needed for proper development
Examples:
 Deletion of suspensor cells in embryos
 Deletion of stamen primordia cells in unisexual flowers
 The resorption of the tadpole tail
 The formation of the fingers and toes of the fetus
PCD is needed to destroy cells
Examples:
– Cells infected with viruses
– Cells with DNA damage
– Cancer cells
– Galls
1210/09/13

13. PCD Regulators
Caspase
(Cysteinyl aspartate-specific
proteases)
Bcl-2 family Signal transduction
pathway
Caspase-8
Caspase-9
Caspase-3
Caspase-6
Caspase-7
Death
initiators
Effectors Bcl-2, Bcl-X
(antiapoptotic)
Bax, Bid and Bad
(Promoters)
Fas- ligand induced
apoptosis
(Jean et al., 1996, USA)
1310/09/13

14. 10/09/13 14
Programmed cell death

15. Programmed cell death : Pathways
Death
Ligands
Effector
Caspase 3
Death
Receptors
Initiator
Caspase 8
PCDPCD
Mitochondria/
Cytochrome C
Initiator
Caspase 9
“Extrinsic Pathway”
“Intrinsic Pathway”
DNA damage & p53DNA damage & p53
between G1between G1 →→ SS
1510/09/13

16. Fig-3 : Activation of apoptosis from outside the cell.
1610/09/13

17. APOPTOSIS: important in embryogenesis
Morphogenesis (eliminates excess cells):
Selection (eliminates non-functional cells):
(Kerr et al., 1992, Germany) 1710/09/13

18. APOPTOSIS: important in embryogenesis
Immunity (eliminates dangerous cells):
Self antigen
recognizing cell
Organ size (eliminates excess cells):
(Kerr et al., 1992, Germany)1810/09/13

19. APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):
Virgin mammary gland Late pregnancy, lactation Involution
(non-pregnant, non-lactating)
Apoptosis
Apoptosis
- Testosterone
Prostate gland
(Kerr et al., 1992, Germany)

20. Fig -4 : Sites of PCD in a Vascular Plant.
The orange spheres represent internal dead cells, and the branched structures
on the leaves represent trichomes (Roger et al., 1997, California)
Fig -4 : Sites of PCD in a Vascular Plant.
The orange spheres represent internal dead cells, and the branched structures
on the leaves represent trichomes (Roger et al., 1997, California)

21. Fig -5 : Functions of PCD in Plants. The red regions represent cells that have been targeted
for PCD and the orange regions represent cells that have died by PCD.
Fig -5 : Functions of PCD in Plants. The red regions represent cells that have been targeted
for PCD and the orange regions represent cells that have died by PCD.
Deletion of suspensor cells in embryos
Deletion of stamen primordia cells in unisexual flowers Deletion of root cap cells
Deletion of leaf cells during leaf lobing Deletion of cells during cell specialization in TEs
cells in an HR cells in uninfected leaves in
response to HR-derived signals
Deletion of aleuron cells in seeds
(Roger et al., 1997, California)10/09/13

22. Aleuron layer
In seeds , aleuron cells form a secretary tissue
that releases hydrolases to digest the endosperm
and nourish the embryo.
Unnecessary
Die
(Kuo et al., 1996)

23. Root Cap CellsRoot Cap Cells
Cell death occurs in root caps when roots are grown,Cell death occurs in root caps when roots are grown,
showing that cell death is a normal part of development andshowing that cell death is a normal part of development and
notnot a consequence ofa consequence of abrasionabrasion during soil penetration.during soil penetration.
Shrink,Shrink,
DNA staining - nuclei - becomeDNA staining - nuclei - become condensed.condensed.
(Wang(Wang et alet al., 1996)., 1996)

24. (A) Electron micrograph of a living cell.
(B) Electron micrograph of a dead cell. In the dead cell, the plasma membrane (pm) is
separated from the cell wall (cw), and the protoplast is shrunken and condensed.
Fig-6 : PCD in an Embryogenic Suspension Culture of Carrot
(Roger et al., 1997, California)

25. Senescence
 Senescence is the final phase of plant vegetative
and reproductive development, preceding the widespread
death of cells and organs.
 Involves the active turnover and recapture of cellular
material for use in other organs.
 Membrane integrity maintained until late into the
senescence process, suggesting that there is little or no
leakage of cellular contents.
(Nooden, 1988)

26. 10/09/13 26
Vascular plants transport water in columns of specialized dead cells termed TEs.
Differentiation of TEs involves cell elongation, the deposition of cell wall components,
including lignin, and autolysis. Autolysis begins as the cytoplasm and nuclei become
lobed, condensed, and shrunken and ends as the cytoplasm breaks into small packets
Xylogenesis- TE’sXylogenesis- TE’s
(Roger et al., 1997, California)

27. INTERACTIONS WlTH THE ENVIRONMENT
Hypoxia (O2 deprivation)
 Cell death can occur in the cortex of the root and stem base in
response to water logging and hypoxia.
 The hypoxia leads to ROS (H2O2, Ethylene)
 Ethylene – trigger apoptotic path way - eliminate some cells-
aerenchyma.
 The interna1 air spaces generated by cell death facilitate more
efficient transfer of O2 from aerial organs to waterlogged stem bases and
roots.
(Armstrong, 1979)

28. 10/09/13 28
Plant-Pathogen interactions
Plants can recognize certain pathogens and activate defenses
(called the resistance response) that result in the limitation of
pathogen growth at the site of infection. One dramatic hallmark of
the resistance response is the induction of a localized cell death
response (the hypersensitive response or HR) at the site of the
infection.
The HR is likely to be important for limiting a pathogen's
nutrient supply, since the dying tissue rapidly becomes dehydrated.
The HR appears to be a form of PCD in plants. since
apoptotic features such as DNA breaks with 3'OH ends, blebbing of
the plasma membrane as well as nuclear and cytoplasmic
condensation are present in some cells undergoing the HR.
(Jean et al., 1996, USA)

29. Case studies
2910/09/13

30. Tomato: Lycopersicon esculantum cv. Zhonghusihao
Grown in GH 260
C / 180
C , day/ night and natural day length and stored @ 250
C .
Devided in to groups (5 in each).
In the heat shock treatment, fruit bag sub merged in water bath for 20 min @ 450
C,
the control was placed @ 250
C for 20 min.
Objectives: To identify which caspase- like enzymes are induced in the tomato fruit heat
stress response and to monitor the effect of caspase like –proteases on heat stress- induced
cell death.

31. Fig-7: Measurement of relative ion leakage from pericarp discs of tomato fruit as
an indicator of cell death by measuring ion leakage from pericarp discs.
(Gui et al., 2009,U.S.A.)
Fig-7: Measurement of relative ion leakage from pericarp discs of tomato fruit as
an indicator of cell death by measuring ion leakage from pericarp discs.
(Gui et al., 2009,U.S.A.)

32. Fig-8: In-situ detection of DNA fragmentation by TUNEL analysis in
tomato fruit. (Gui et al., 2009, U.S.A)
a–c Pericarp sample from
control tomato fruit.
d–f Pericarp sample from
tomato fruit that was treated at
45°C for 20 min
g–i Pericarp
sample from tomato fruit 6 h
after treatment at 45°C for
20 min.
j–l Pericarp sample
from tomato fruit 12 h after
treatment at 45°C for 20 min.
Samples were
stained using the
TUNEL method
(a, d, g, j),
DAPI (b, e, h,
k)
bright-field (c, f, i, l).
Arrows
indicate TUNEL-positive nuclei.

33. Fig-9 : In-situ detection of DNA fragmentation by TUNEL analysis in tomato fruit.
(Gui et al., 2009, U.S.A)10/09/13

34. Caspase-1 (Ac-YVAD-AMC), caspase-3 (Ac-DEVD-AMC), caspase-8 (Ac-IETD-
AMC)
and caspase-9 (Ac-LEHD-AMC) , HS - Treated at 45°C for 20 min,
Fig-10 : Substrate specificity of HS-induced caspase-like activities in tomato fruit.
(Gui et al., 2009, U.S.A)10/09/13

35. 3510/09/13
Fig -11 : Effect of Z-VAD-FMK pretreatment on cell death in heat treated tomato
pericarp discs. . (Gui et al., 2009, U.S.A)
Fig -11 : Effect of Z-VAD-FMK pretreatment on cell death in heat treated tomato
pericarp discs. . (Gui et al., 2009, U.S.A)
caspase-3 (Ac-DEVD-AMC), caspase-9 (Ac-LEHD-AMC) , Heat - Treated at 45°C for
20 min and Z-VAD-FMK (Z-Val-Ala-Asp Flouromethylkitone).

36. Materials and methods:
Tomato ( Lycopersicon esculentum Mill.) cell suspension cultures, line Msk 8
(Koornneef et al., 1987)
Murashige and Skoog liquid medium (5µMNAA, 1µM BA and vitamins)
Cells were sub-cultured every 7 days and used for experiments 5 d after sub
culture.
Cell death inducers (Camptothecin-5µM, Staurosporin-2µM, Fumonisin-20µM)
.
5% percent buffer formaline for detection of nuclear changes and DNA
fragmentation. 36
Objectives: To study the PCD by treatment with known inducers of apoptosis in mammalian cells.

37. Fig -12 : Induction of cell death in tomato suspension cells by Fluorescine
diacetate (FDA )staining method.
□ - Staurosporin-2µM
∆ - Camptothecin-5µM
X - Fumonisin-20µM
O –Untreated control
□ - Staurosporin-2µM
∆ - Camptothecin-5µM
X - Fumonisin-20µM
O –Untreated control
(Anke et al., 2000, Netherland )

38. STAU- Staurosporin-2µM
CPT - Camptothesin-5µM
FUM - Fumonisin-20µM
FeSo4 -100mM
STAU- Staurosporin-2µM
CPT - Camptothesin-5µM
FUM - Fumonisin-20µM
FeSo4 -100mM
Fig-13: Chemical induced DNA fragmentation in tomato suspension cells by
using DNA laddering pattern. (Anke et al., 2000, Netherland )10/09/13

39. 5µM -Camptothecin (CPT),
50µM- N-ethylmaleimide (NEM),
5µM -iodoacetamide (IA),
200µM- Nm Nα-p-tosyl- L-lysine
chloromethylekitone (TLCK),
1mM -4,2,amiloethyle
benzensulfonyl fluoride (AEBSF)
20µM -Fumonisin BI (FUM)
2µM -Staurosporine (STAU)
5µM -Camptothecin (CPT),
50µM- N-ethylmaleimide (NEM),
5µM -iodoacetamide (IA),
200µM- Nm Nα-p-tosyl- L-lysine
chloromethylekitone (TLCK),
1mM -4,2,amiloethyle
benzensulfonyl fluoride (AEBSF)
20µM -Fumonisin BI (FUM)
2µM -Staurosporine (STAU)
Fig -14: Inhibition of chemical induced cell death in tomato suspension cells by
protease inhibitors. (Anke et al., 2000, Netherland )39

40. CPT - Camptothesin-5µM
(CPT+ YVAD) – 100nM Caspase
specific peptide inhibitor- Acyl -Tyr-
Val-Ala-Asp- chloromethylketone .
(CPT+ AAPV) – 100nM control
peptide - MethoxySuccinyl-Ala-Ala-
Pro-Val-chloromethylketone .
CPT - Camptothesin-5µM
(CPT+ YVAD) – 100nM Caspase
specific peptide inhibitor- Acyl -Tyr-
Val-Ala-Asp- chloromethylketone .
(CPT+ AAPV) – 100nM control
peptide - MethoxySuccinyl-Ala-Ala-
Pro-Val-chloromethylketone .
Fig-15: Inhibition of DNA fragmentation by caspase specific peptide inhibitors
by DNA laddering pattern.
(Anke et al., 2000, Netherland )10/09/13

41. 4110/09/13
Tomato seeds were incubated in the dark @ 250
c prior to use. Isolation of
endosperm tissue was performed aseptically and were surface sterilized for 10 s with
70% ethanol. Then endosperm halves were made and used for the experiment.

42. Fig -16 : Post-germinative tomato endosperm cells display features of reserve mobilization
and PCD including SlCysEP-containing ricinosomes.
(Christoper et al.,2013,
Fig -16 : Post-germinative tomato endosperm cells display features of reserve mobilization
and PCD including SlCysEP-containing ricinosomes.
(Christoper et al.,2013,
a) Cells farthest from the
embryo show less reserve
mobilization and no signs of
PCD. Cells closer to the embryo
contain fewer reserves; cells next
to the embryo are crushed or
have recently died
b)The top cell farthest from the
embryo appears disorganized
suggesting it is undergoing PCD;
cells closer to the embryo (below
frame) appear dead with large lipid
aggregates
c) A cell showing signs of
reserve mobilization, but not
PCD, contains a developing
ricinosomes like
structure
4210/09/13

43. Conti….
d)Localization of SlCysEP within ricinosomes is
evidenced by the white arrowheads lying within
the ricinosomes,
e) A labelled-ricinosome in a
cell with few reserves next to a dead cell
(top left) illustrating the
asynchronous nature of endosperm PCD
Fig -17 : Post-germinative tomato endosperm cells display features of reserve
mobilization and PCD, including SlCysEP-containing ricinosomes.
(Christoper et al.,2013, Heidelberg)
Fig -17 : Post-germinative tomato endosperm cells display features of reserve
mobilization and PCD, including SlCysEP-containing ricinosomes.
(Christoper et al.,2013, Heidelberg)4310/09/13

44. Fig-18 : SlCysEp transcript and enzyme accumulation in tomato seeds.
(Christoper et al.,2013, Heidelberg)
Fig-18 : SlCysEp transcript and enzyme accumulation in tomato seeds.
(Christoper et al.,2013, Heidelberg) 4410/09/13
HAI
34 k BP

45. Fig -19 : Tissue specific SlCysEP expression and enzyme accumulation using
Northern-blot analysis
(Christoper et al.,2013, Heidelberg)
Fig -19 : Tissue specific SlCysEP expression and enzyme accumulation using
Northern-blot analysis
(Christoper et al.,2013, Heidelberg)4510/09/13

46. a) Non-germinated (NG), b) short seedling (SS), c) long seedling (LS)
Endosperm halves from NG (d), SS , LS (f) seeds. (*) the micropylar endosperm collar
Fig-20: SlCysEP accumulation pattern change in response to development @144HAI.
(Christoper et al.,2013, Heidelberg)
Fig-20: SlCysEP accumulation pattern change in response to development @144HAI.
(Christoper et al.,2013, Heidelberg)4610/09/13

47. 4710/09/13
Fig-21 :Quantification of protien and SlCysEP profile isolated on en equal tissue basis.
(Christoper et al.,2013,
Fig-21 :Quantification of protien and SlCysEP profile isolated on en equal tissue basis.
(Christoper et al.,2013,

48. Tomato (Lycopersicon esculentum Mill.) cell suspension culture,
line Msk8, was grown in a liquid MS medium + vitamins and minerals, supplemented with
5 μM a-NAA, 1 μM 6-BA and 3% (w/v) sucrose .
Cell death inducers CdSO4 and CdCl2 were added to 5 ml of suspension culture,
Inhibitors were applied 30 min before CdSO4. In general,
Inhibitors were tested in a range of concentrations (from nM to mM) with and without the
addition of 100 μM CdSO4. Lowest concentrations of inhibitors giving significant reduction of
CdSO4-induced cell death are presented. The next chemicals have been tested for their
potency to suppress Cd-induced cell death:
Cell death determination by fluorescence microscopy.
Hydrogen peroxide assay
Ethylene production measurement
4810/09/13
Biotechnology & Biotechnological Equipment, 2009 23(2): 538-541

49. Fig -22: Effect of cadmium salts on cell death in tomato suspension cells 24 h after
the treatments.
(Iakimova et al., 2009, Bulgaria )
Fig -22: Effect of cadmium salts on cell death in tomato suspension cells 24 h after
the treatments.
(Iakimova et al., 2009, Bulgaria )
4910/09/13

50. Fig -23: Morphological features of living (A) and dead (B) tomato cells 24 h after
treatment with 100 μM CdSO4.
( Iakimova et al., 2009, Bulgaria )
Fig -23: Morphological features of living (A) and dead (B) tomato cells 24 h after
treatment with 100 μM CdSO4.
( Iakimova et al., 2009, Bulgaria )
A - living cell with diffused nucleus, preserved cytoskeleton and intact
cytoplasm;
B – dead cell showing compacted nucleus and shrunken cytoplasm
separated from the cell wall.
cyt-cytoplasm, cw- cell wall, nu- nucleus.
5010/09/13

51. Fig-24: Kinetic of hydrogen peroxide production in tomato suspension cells
exposed to 100 μM CdSO4. ( Iakimova et al., 2009, Bulgaria )
Fig-24: Kinetic of hydrogen peroxide production in tomato suspension cells
exposed to 100 μM CdSO4. ( Iakimova et al., 2009, Bulgaria )
5110/09/13

52. Fig -25: Kinetic of ethylene production in tomato suspension cells exposed
to 100 μM CdSO4.
( Iakimova et al., 2009, Bulgaria )
Fig -25: Kinetic of ethylene production in tomato suspension cells exposed
to 100 μM CdSO4.
( Iakimova et al., 2009, Bulgaria )
5210/09/13

53. Objectives : Elicitors of different origin (fumonisin B1, fungal toxin), camptothecin
(alkaloid from Camptotheca acuminata), mastoparan (wasp venom)
and the heavy metal (cadmium) were tested for their ability to induce
programmed cell death (PCD) in a model system of tomato cell
culture, line MsK8.
5310/09/13

54. Fig-26 : Effect of 20 μM Fumonisin B1 (FumB1), 5 μM camptothecin (CPT),
100 μM CdSO4 or 5 μM mastoparan (MP) on cell death induction in
tomato suspension cells. (Elena et al., 2007, Bulgaria)
Fig-26 : Effect of 20 μM Fumonisin B1 (FumB1), 5 μM camptothecin (CPT),
100 μM CdSO4 or 5 μM mastoparan (MP) on cell death induction in
tomato suspension cells. (Elena et al., 2007, Bulgaria)
5410/09/13

55. Fig -27 : Effect of 100 nM Ac-YVAD-CMK, 100 nM Z-Asp-CH2-DCB and 1mM (AEBSF) on
cell death in tomato suspension cells induced by 20 μM FumB1, 5 μM CPT, 100 μM
CdSO4 or 5 μM MP. Cell death was calculated after 24 h of treatment followed by
FDA staining of the living cells and is expressed as per cent of inhibition.
Fig -27 : Effect of 100 nM Ac-YVAD-CMK, 100 nM Z-Asp-CH2-DCB and 1mM (AEBSF) on
cell death in tomato suspension cells induced by 20 μM FumB1, 5 μM CPT, 100 μM
CdSO4 or 5 μM MP. Cell death was calculated after 24 h of treatment followed by
FDA staining of the living cells and is expressed as per cent of inhibition.
5510/09/13

56. 5610/09/13
 PCD is essential for ensuring the proper
development of plant
 Interaction with environment which results robust
defence response against biotic and abiotic stresses

57. 10/09/13 57
GERMAN SCIENTIST CARL VOGT WAS FIRST TO
DESCRIBE THE PRINCIPLE OF APOPTOSIS IN 1842.
GERMAN SCIENTIST CARL VOGT WAS FIRST TO
DESCRIBE THE PRINCIPLE OF APOPTOSIS IN 1842.
IN 1885, ANATOMIST WALTHER FLEMMING
DELIVERED A MORE PRECISE DESCRIPTION OF THE
PROCESS OF PROGRAMMED CELL DEATH.
JONE E. SULSTON WON THE NOBEL PRIZE IN
MEDICIN IN 2002 FOR HIS POINERING RESRACH ON
APOPTOSIS
JONE E. SULSTON WON THE NOBEL PRIZE IN
MEDICIN IN 2002 FOR HIS POINERING RESRACH ON
APOPTOSIS