Three studies on programmed cell death in plants are summarized: 1. A study showed that heat-induced cell death in cucumber cotyledons resulted in DNA fragmentation and the release of cytochrome c from mitochondria into the cytosol, demonstrating conserved mechanisms with animal apoptosis. 2. A study found that caspase-specific peptide inhibitors effectively inhibited chemically-induced cell death in tomato cells, indicating caspase-like proteases mediate plant apoptotic pathways. 3. A study showed that anthocyanins from black soybeans protected human skin cells from UVB-induced reactive oxygen species, apoptosis, and caspase activation by preventing pro-apoptotic signaling.

1. ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY
AGRICULTURAL COLLEGE, BAPATLA
Credit Seminar : PROGRAMMED CELL DEATH
Course no and Title : CP – 591 Master’s seminar
Degree : MSc. (Ag.) 2ndyear
Submitted by :
Rahul Chandra
BAM-19-23
1
Submitted to:
Dr. S. Ratnakumari
Professor and Head
Agricultural College, Bapatla
Department of Crop physiology

2. PROGRAMMED
CELL
DEATH
2

3. Introduction
• Cell death is a regulated process that allows a cell to
self-degrade in order for the body to eliminate
unwanted or dysfunctional cells.
• Cell-cell interactions regulate cell death in two
fundamentally different ways.
• Cells in multicellular organisms require specific
protein hormone signals to stay alive.
• In the absence of such survival signals, frequently
referred to as trophic factors, cells activate a "suicide"
program.
3

4. • Second, in some developmental contexts , including the
immune system, other specific hormone signals induce a
“murder" program that kills cells.
• Whether cells commit suicide for lack of survival signals
or are murdered by killing signals from other cells, cell
death is most often mediated by a common molecular
pathway, termed apoptosis.
• Term Apoptosis was derived from Greek language
meaning ‘shedding’.
4

5. • Macrophages and other phagocytic cells recognize apoptotic cells
and remove them by phagocytosis without inflammatory
phenomena.
5
www.onlinebiology.net
Pyknosis, karyorrhexis
Pyknosis is the irreversible condensation of chromatin in nucleus of a cell
undergoing apoptosis.
Karyorrhexis is the fragmentation of nucleus after pyknosis.

6. NECROSIS
• A different form of cell death, necrosis, occurs when cells
are subjected to injury or excessive stresses such as heat,
absence of oxygen, or infection by pathogens.
• Necrosis creates holes in the plasma membrane,
causing leakage of intracellular contents.
• The distinction whether necrosis or apoptosis took place is
found by detecting Cyt-C in the cytosol, if in addition to
Cyt-C other porteins and factors are also present then it is
due to necrosis. Eg Fumarse present in cytosol.
6

7. Apoptosis compared to necrosis
7
Feature
Regulated by organism
Apoptosis
Yes: involves a series of
enzyme-controlled reactions
Yes
No
Necrosis
No: can occur as a result
of injury
Only after cell has lysed
Yes
Yes No
DNA broken down
Cell membrane
disintegration
Nuclear membrane
broken down
Number of cells affected
Energy requirement
May be single cells
ATP dependent (active
process)
Fate of dead cells
Usually sheets of cells
Energy input not
required (passive
process)
Ingested by phagocytes
Leakage of cell contents
End point
Ingested by neighbouring
cells or phagocytes
No
Cell fragments into smaller
bodies
Yes
Lysis of whole cell
Caspase activation, chromatin
condensation

8. 8(Walker et al, 1986., Exp. pathology)

9. HISTORY AND IMPORTANT BREAKTHROUGHS
• German Scientist Carl Vogt was first to describe the
principle of apoptosis in 1842.
• In 1972 Kerr first introduced the term apoptosis in
publication. Kerr received the Paul Ehlrich and Ludwig
Darmstaedter Prize on March 14, 2000 for his description
of apoptosis.
• The 2002 Nobel Prize in Medicine was awarded to
Sydney Brenner, Horvitz and John E. Sulston for their
work in identifying genes that control apoptosis.
• The genes were identified by studies in the nematode
C. elegans and these same genes function in humans for
apoptosis.
9

10. Time line related to PCD research
(Richard et al 2001)
10

11. membrane
blebbing &
changess
mitochondrial
leakage
organelle
reduction
cell
shrinkage
nuclear
fragmentation
chromatin
condensation
(Hacker., 2000)
11

12. Stages of Apoptosis
12

13. Importance of apoptosis
• Apoptosis is needed for proper development.
• Apoptosis is needed to destroy cells such as cancer cells ,
DNA damaged cells etc.
• Important in normal physiology
–Development: Immune systems maturation,
Morphogenesis, Neural development.
–Adult: Immune privilege, DNA Damage an wound
repair.
• Excess apoptosis
–Neuro degenerative diseases
• Deficient apoptosis
–Cancer, Autoimmunity
13

14. • Sites of PCD in vascular plants
the orange spheres represent the internal dead cells and the the branched
14

15. • The functions of PCD in plants : the redd regions represent the cells that
have been targeted for PCD and the orange represent cells that have died.15

16. – Cancer cells
• Radiation and chemicals used in cancer therapy induce
apoptosis in some types of cancer cells.
SC-1 induced apoptosis in stomach
carcinoma cells
Left: Before induction
Middle: 24h after induction
Right: 48h after
induction
www.biologydiscussions.com
16

17. Jean et al , USA (2001)
17

18. Caspases
• Caspases stands for cysteine aspartate-specific protease.
• Caspases have the characteristics of high specificity for
substrates containing Asp, and use a Cys for catalyzing
peptide bond cleavage.
• Synthesized in the cell as precursors named procaspases.
18
NH2-terminal domain
Large subunit (~20kD)
Small subunit (~10kD)
www.wileyonlinelibrary.com

19. CaspaseRole in Apoptosis
• Cut off contact with surrounding cells
• Reorganize cytoskeleton
• Shut down DNA replication and repair
• Interrupt splicing
• Disrupt nuclear structure
• Induce cell to display signals marking it for
phagocytosis
• Disintegrate cells into apoptotic bodies
19

20. PATHWAYS OF APOPTOSIS
• MITOCHONDRIAL PATHWAY
INTRINSIC PATHWAY
• THE DEATH RECEPTOR PATHWAY
EXTRINSIC PATHWAY
20

21. 21

22. Intrinsic / Mitochondrial pathway
• Intrinsic pathway or mitochondrial pathway occurs due to
stimuli from within the cell such as viral infection, DNA
damage, growth factor deprivation.
• The stimuli is sensed by sensory proteins such as ATM and
CHK and activates a protein called P53.
• P53 has many roles such as
produces proteases , halts cell cycle, activates Bax
and Bak proteins and also inhibits Bcl2 protein.
22

23. • Bcl2 blocks activity of Bax and hence Bax proteins
activated , they form oligomers on the membrane of
mitochondria and produces pores and aids in release of
Cytochrome C. and IAP (Inhibitor Apoptotic Protein).
• Cyt C released into cytosol combines with a protein called
Apaf-1(apoptosis activating factor-1) and forms a
complex called as Apoptosome.
• Apoptosome combines with initiator caspase-
procaspase -9 and cleaves at Aspartate site to form
caspase-9.
• Caspase-9 cleaves procaspase-3 and forms Caspase-3.
23

24. • Caspase 3 causes-DNA fragmentation, digestion of nuclear
lamins, degrade cytoskeleton( actin, myosin and tubulin),
and releases Scramblase enzyme.
• The DNA gets fragmented and formation of apoptotic
blebs occur, Scramblase translocated Phosphatidyl serine
from inner to outer surface of the cell.
• Cells having Phosphatidyl serine on the outer surface are
recognised by Phagocytic cells and engulfed.
24

25. 25www.easybiologydiscussions.com

26. Extrinsic / Death receptor pathway
Extrinsic pathway
TNF pathway
FAS pathway
26
Based on receptor and signalling molecule extrinsic
pathway is of two types TNF pathway and FAS pathway.
• TNF stands for Tumour necrotic factor in which the
signalling molecule is TNF alpha cytokine and receptor
on the cell membrane is TNFR-1.
• While FAS stands for First Apoptotic Signal pathway,
where FAS receptor having death domain accepts FAS
ligand signalling molecule.

27. • As varied from intrinsic pathway , extrinsic pathway
consists of signals for cell death produced externally in
other cells to fight against an infection or damage.
• The cell which produces the signalling molecule which
initiates apoptosis after binding with receptor located on
target cell are called Signalling cells commonly – cells
associated with immune system such as CytotoxicT-
lymphocytes.
• The receptor on target cell contains an associated
adapter molecules called death domain which undergoes
changes to initiate apoptosis.
27

28. FAS pathway
• FAS ligand produced by signalling cell binds with the FAS
receptor on the target cell .
• The associated death domain called FADD ( FAS
associated Death Domain) gets activated and aggregate
to form a DISC ( Death Induced Signalling Cascade).
• Procaspase -8 binds to DISC and activated into Caspase-
8, which further cleaves Procaspase-3 to form effector
executioner Caspase-3.
• Caspase-3 degrade nuclease inhibitor and activate
nucleases which enter nucleus and cause DNA
fragmentation and finally undergoes phagocytosis.
28

29. • Here Procaspase-8 plays a major role in initiating cross
talk between two pathways.
• Procaspase-8 converted as Caspase-8 , activate a protein
BID into TBID which further activates Bax/ Bak proteins
which forms pores on mitochondrial membrane to release
Cyt-C and trigger Intrinsic pathway.
• Thus extrinsic pathway also triggers intrinsic development
of Apoptosis.
29

30. 30www.researchgate.com

31. CASE STUDIES
31

32. FEBS Letters 463 (1999), 151-154
Translocation of cytochrome c from the mitochondria to the cytosol
occurs during heat-induced programmed cell death in cucumber plants
Janneke Balk, Christopher J. Leaver*, Paul F. McCabe
University of Oxford, Department of Plant Sciences, South Parks Road, Oxford OX1 3RB, UK
• Objectives:To investigate the activation mechanism of plant PCD by induced
cell death in cucumber cotyledons to ascertain if mitochondrial components
were released during the cell death programme
• Materials and methods- PCD was triggered in cucumber cotyledons by
subjecting them to a short 55³C heat treatment. And DNA was extracted and
southern blot analysis was done to determine inter nucleosomal cleavage in
DNA due to PCD.
• Immunodetection of cytochrome distribution in cytosol and mitochondria
was done by western blot method using antibodies against Cyt-C and
fumarase.
32
Case study 1

33. Southern blot analysis results on X ray film ( after hybridisation with radioactive
prime labelling) showing displacement of Cucumber DNA .
Inference: cucumber cotyledons were exposed to 55°C for 10 min and then
incubated at 25°C. The cotyledons were frozen in liquid nitrogen at various time
points over 12 h after heat treatment prior to extraction of genomic DNA.
Clear evidence of DNA laddering was observed at 12 hours after heat treatment.

34. 34
Immuno detection of mitochondrial proteins in mitochondrial and cytosolic
fractions of cucumber cotyledons following induction of PCD.
Western blot was performed and revealed that the mitochondria began to
release cytochrome C to the cytosol during the 10-min heat treatment , and
that cytochrome C could no longer be detected in mitochondria after 3 hours. (
fumarase not transferred as it is not mere heat damage to mitochondrial walls
that cause release of Cyt C).

35. Conclusion
• Thus the experiment proves that triggering cell death with a
known PCD-inducing abiotic stress have shown that the release
of cytochrome occurs from the mitochondria into the cytosol, is
an early event in plant cell death.
35

36. CASE STUDY 2
36

37. 37

38. 38

39. 39

40. 40

41. Conclusion
• It is seen that proteolysis plays a crucial role in apoptosis
in plants .
• Caspase specific peptide inhibitors were found to be potent
inhibitors of chemically induced cell death in tomato cells.
• This indicated that as in animal system caspase like
proteases are involved in apoptotic cell death pathway in
plants.
41

42. • Materials and methods
Tomato cell suspension culture was grown in liquid MS medium
supplemented with 5 micro molar NAA and 1 Micro molar BA and 3%
sucrose.
Cell death inducers CdSO4 and CdCl2 were added to 5 ml of culture.
Inducer effects tested for potency by measured indirectly by H2O2 and
ethylene production.
Cell death determination using fluorescence microscope. 42
CASE STUDY 3

43. 43

44. 44

45. 45

46. 46

47. • Chronic exposure to 50–100 μM CdSO4-induced loss of
cell viability associated with typical apoptosis-like
morphological changes and DNA laddering.
• Max. cell death% observed at CdSO4 10mM and CdCl2
100 microM which indicates CdCl2 is more potent
chemical apoptosis inducer.
47

48. CASE STUDY 5
Journal. Agric. Food Chem. 2008, 56, 10600–10605
Protective Effect of Anthocyanins from Black Soybean Seed Coats on
UVB-Induced Apoptotic Cell Death in Vitro and in Vivo
Konstantin Tsoyi, Hyung Bin Bark, Young Min Kim,Jong il Chung, Sung Chul Shin.
• Objectives: s are not yet known. Thus, the aims of this study were
to investigate the protective effect of anthocyanins from black
soybean (Glycine max (L.) Merr) on UVB-induced apoptosis.
• Materials and methods: Anthocyanins was extracted from black
soyabean seed coat.
• Uv-B irradiation was done on immortalized human keratinocyte
cell line, HaCaT. Anthocyanin application and its effect on UVB
induced apoptosis and ROS production was studied.
48

49. Effect of anthocyanins on ROS production induced by UVB in HaCaT.
Cells were pretreated with anthocyanins (10 or 100 µg/mL) for 24 h and
then irradiated with UVB (100 J/m2). After 1 h of incubation with/without
anthocyanins, ROS production by confocal microscopy (bright green)
analysis was performed.

50. 50
Protective effect of anthocyanins on UVB-induced apoptotic cell death in HaCaT by
TUNEL assay.
UVB irradiation increased TUNEL positive cells (dying cells) about 25% at 100 J/m2,
which was reduced to 6% by anthocyanins at 10 µg/mL .
Again increasing the amount of Anthocyanin to 100 µg/mL reduces TUNEL positive cells
by 25%.

51. Conclusion
• Pretreatment with anthocyanins reduced UVB-induced
reactive oxygen species levels and inhibited UVB-induced
apoptotic cell death through the prevention of caspase-3
pathway activation and reduction of proapoptotic Bax
protein levels.
• It is concluded that anthocyanins from the seed coat of
black soy beans may be useful compounds to modulate
UVB-induced photoaging.
51

52. • Balk. J., Leaver, C.J. and McCabe, P.J. Translocation of cytochrome c
from the mitochondria to the cytosol occurs during heat-induced
programmed cell death in cucumber plants. FEBS Letters 463 (1999)
.University of Oxford, Department of Plant Sciences, UK. 151-154.
• De jong. A.J., Hoeberichts. F.A., Yakimova. E.T. and Maximova.E.
Chemical induced programmed cell death in tomato cells , involving
caspase like proteases. Planta.(2000) 211.656-662
• Prasad. S.H., Amit. J., Anita .S. (2014). Apoptosis and programmed
cell death – A review. World Journal of Pharmaceutical Research. 3.
1854-1872.
• Tsoyi.K., Hyung. B. P. Young .M. K., Jong I.C and Hye. J. K.
Protective Effect of Anthocyanins from Black Soybean Seed Coats on
UVB-Induced Apoptotic Cell Death in Vitro and in Vivo. J. Agric.
Food Chem. 2008, 56, 10600–10605.
• Yakimovaa E.T., Kapchina-Totevab. V.M., Laarhovenc .F.M. and
Harrence .E.J. Cadmium-induced programmed cell death in tomato
suspension cells. Plant Physiology and Biochemistry 44 (2006) 581–589.
52
References

53. 53