Pcd 4 th seminar

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PROGRAMMED CELL DEATH

In the mid-1960s, cell death occurring during tadpole development recognized as an actively
controlled mechanism i.e., PCD.
(Tata, 1966)
Morphogenesis of Frog
Tadpole Frog
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FLOW
OF
SEMINAR
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2
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5
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INTRODUCTION TO PROGRAMME CELL DEATH
REGULATION AND MECHANISMS OF PROGRAMMED CELL DEATH
PROGRAMMED CELL DEATH IN PLANTS
REGULATION AND MECHANISM OF PCD PLANTS
PCD IN REPRODUCTIVE DEVELOPMENT IN PLANTS
PCD IN VEGETATIVE DEVELOPMENT AND SENESCENCE IN PLANTS
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CASE STUDIES AND CONCLUSION
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 The number of cells in a body of organism is tightly regulated. Its not only the
control over cell division but also on rate of cell death.
 Controlled cell death is needed for normal development and good health
throughout life along with normal development of cells and cell cycle maturation.
INTRODUCTION
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 APOPTOSIS: Genetically programmed
process of deliberate sucide by an
unwanted cell in a multicellular
organism.
 AUTOPHAGY : Plays a housekeeping
role in removing misfold proteins,
clearing damaged organelles as well as
eliminating intracellular pathogens.
 NECROSIS : Non-programmed cell
death, death of a cell caused by external
factors such as trauma or infection.
 NECROPTOSIS : Programmed form
of necrosis.
 ONCOSIS: Series of cellular reactions
following injury that ultimately leads to
cell death.
CELL DEATH
NECROSIS NECROPTOSIS ONCOSIS
AUTOPHAGY APOPSTOSIS
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 Often occurs when the structure they form is no longer needed.
 To eliminate abnormal cells, cells that non-functional or potentially dangerous
to the animal.
 Cell death can prevent viral replication because they need a cell alive to
reproduce.
 Failure of cells to die or cells dying when they shouldn’t can lead to many
diseases.
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 Cell death occur in many ways.
1.Infection
2.Poisioning
3.Lack of oxygen
 An uncontrolled death messy. In this cells swells up and its contents leak away.
This may damage surrounding cells too.
 On the other hand, there is another tidier way to go, i.e., Programmed cell death
or apopstosis, in which cell often choose to kill themselves.
Programmed cell death
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 Programmed cell death (PCD) has been defined as a sequence of (potentially
aainterruptible) events that lead to the controlled and organized destruction of the
aacell.
 Synonyms = Chromatolysis, Karyolysis, Karyohexis, Shrinkage necrosis,
Programmed cell death, Cell suicide, Self destruction and Apoptosis.
 The term apoptosis –A Greek word originally means that “fall of the leaf or
leaf falling”. (John et al., 1972)
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Doctoral Research
 “Programmed Cell Death,”
that were published between
1964 and 1965 in the Journal
of Insect Physiology.
The Nobel Prize in Physiology or Medicine 2002 was awarded jointly to Sydney
Brenner, H. Robert Horvitz and John E. Sulston "for their discoveries concerning
genetic regulation of organ development and programmed cell death'."
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 Apoptosis refers to active, programmed cell death.
 Individual cells are induced to commit suicide
Characteristics of Apoptosis
 Cell membrane blebbing
 Cell shrinkage
 Chromatin condensation
 Chromosomal DNA fragmentation
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 During viral infections.
 During DNA damage.
 To make way for new cells.
Ex: In the womb, our fingers and toes are connected to one another by a sort of
webbing. Apopstosis is what causes that webbing to disappear, leaving us with 10
separate digits.
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 It is not only confined to animals, in plants also it occurs during development and
in the senescence of flowers and leaves.
 Programmed cell death is also necessary to prevent from effect of spurious
infection in plants.
 PCD also occurs in unicellular organisms including yeasts and bacteria.
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WHAT IS THE PURPOSE OF
THIS PCD ?
 Developmental PCD:
 Essential for successful development & growth of complex multicellular organisms.
 Regulates the rate of cell division.
 Shaping of cells, tissues & organs.
 Defensive PCD:
 Control of cell populations & defense against invading microbes.
 PCD is needed to destroy the cells that represent a threat to the integrity of the
organism.
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Huang et al., 2019
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Huang et al., 2019
Schematic representation of difference between Necrosis and Apoptosis
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 Apoptosis is a tightly regulated and
efficient cell death program involving
multiple factors.
 Caspases take major and a central role in
apoptotic mechanism.
 Contain key cysteine residue at catalytic
site.
 Cleave protein at site just C-terminal to
aspartate residue.
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Active
Caspase
Inactive
procaspase
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BCl-2 family
proteins
Pro-apoptopic
Promote apoptosis
BH123 proteins
(Bax, Bak)
BH 3-only proteins
(Bid, Bim, Bik, Bad,
PUMA, NOXA)
Anti-apoptopic
Inhibit apoptosis
(Bcl-2, Bcl-XL, Mcl-1, Bcl-1)
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Jones et al., 2001
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There are two basic PCD or apoptotic pathways
▫ Intrinsic pathway
▫ Extrinsic pathway
PCD
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Image representing the PCD events in the cell Azwali etal.,2019
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 Cell activate their apoptopic
programs from inside the cell in
response to injury or other
stresses like DNA damage or lack
of oxygen, nutrients or free
radicals, growth factors
deprivation.
 Mitochondria dependent pathway.
Azwali etal.,2019 4/6/2022 23

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 Extracellular signal proteins binds to
cell surface death receptors that trigger
the extrinsic pathway.
 Death receptors are transmembrane
proteins having extracellular ligand
binding domain, transmembrane
domain and an intracellular death
domain which activated the apoptopic
program.
Azwali etal.,2019
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 Fas ligand on cells binds to Fas receptor on
target cell.
 Death domains then recruits intracellular
adaptor proteins-
 FADD (Fas ligand associated death domain)
 It then recruits pro caspase 8 or 10, forming
DISC (Death Inducing Signalling Complex)
 Activation or cleavage of procaspase 8 or 10.
 Formation of Caspase 8 or 10
 Caspase 3 activation
 Execution of apoptosis.
Azwali etal.,2019
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 In plants, the vacuole is responsible for lytic activity which
leads to the removal of organelles and cell corpses.
 In plants, different types of PCD
1. ePCD ( Environment stress dependent)
It is triggered by external insults which include pathogen
attack as well as abiotic stress, such as heat stress, salinity,
drought, and flooding.
2. dPCD (vegetative and reproductive development )
It is generally autolytic, as happens in xylem tracheary
formation, where cytoplasm clearance is required for vessel
functionality.
Daneva etal.,2016
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 Plants activate PCD in senescent tissues, such as leaves, petals, sepals, in order
to recycle nutrients before eliminating the tissues that are no longer required.
 Differentiation-induced dPCD occurs as an inherent final differentiation step of
particular cell types, e.g., xylem, anther tapetum, or root cap cells. Some cell
types, however, can initiate dPCD in a facultative fashion.
 Finally, age-induced dPCD occurs in all cell types of organs or even entire
organisms as the end point of plant senescence.
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 On the other hand, plants can fail to activate defense responses against abiotic
stress and as consequence activate PCD.
 This happens when the stress-dependent metabolic impairment overwhelms the
plant’s ability to restore the physiological background and/or counteract the
derived oxidative stress.
DROUGHT STRESS FLOODING HEAT STRESS 4/6/2022 30

MECHANISTIC PRINCIPLES PUTATIVELY INVOLVED IN THE CONTROL
OF PROGRAMMED CELL DEATH IN PLANTS
Hautegem et al., 2015
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PCD Classes EXAMPLES
AUTOLYTIC
• Rapid cytoplasm clearance after tonoplast rupture & replaced by
vacuolar volume
DevelopmentalPCD
• Similar to autophagy in animal
• For example, PCD that occurs during the formation of the male and
female zygotes, in embryonic structures, and during development of
roots and shoots.
• Mild abiotic stress, such as lack of oxygen and drought.
NON- AUTOLYTIC
• Death occurs prior to tonoplast rupture or tonoplast rupture does not
occur or tonoplast rupture is not followed by complete clearance of
the cytoplasm
• Hypersensitiveresponse(HR)-related PCD
• Endosperm in cereal seeds is an example (no tonoplast rupture)
Vandroon etal.,2011
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• During viral infection, the tonoplast is lysed with the release of lytic enzymes
of vacuoles into the cytosol.
• The vacuole tonoplast fuse with the plasmalemma to release vacuolar contents
extracellularly, or it disintegrate to release its contents to the cytosol.
• Induction of rapid cell death pathways may be an effective way of cleansing
cytoplasm from viral growth.
• Sudden release of vacuolar contents into the cytoplasm causes rapid cell death as
was also noted for the HR response to TMV in tobacco.
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 The impairment of the pathways increases the production of reactive oxygen and
nitrogen species (ROS and RNS) in the cell. When the plant fails to counteract.
 The metabolic imbalance leading to the overproduced reactive species, it activates PCD.
 Several plant hormones are also involved in the induction of both dPCD and ePCD:
salicylic acid is required in HR cell death, ethylene in aerenchyma formation and tissue
senescence, gibberellin in endosperm/aleurone development.

PCD PHASES IN PLANTS
Daneva etal.,2016
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• Plant proteases: Metacaspases
Type 1: Metacaspases (AtMC1,AtMC2)
Type 2: Metacaspases (AtMC4,AtMC 9)
• Subtilisin- like serine proteases, VPE (vacuolar processing enzyme) family of
proteases.
• Bcl-2 associated athanogene (BAG) family: It is an evolutionary conserved family of
co-chaperons in plants and animals distinguished by a characteristics BAG domain
that mediates direct interaction with HSP70.
• Hypersensitive mediated PCD: Rapid localised plant cell death upon contact with
avirulent pathogens.
• Necrotrophic pathogen mediated cell death: Death of cell caused by hydrolytic
enzymes and host selective toxins.
Vartapetian etal.,2011
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• Proteolytic enzymes that are involved in
PCD are localized in different
compartments of plant cells:
• Cytoplasm (metacaspases)
• Vacuoles (VPE)
• Intercellular fluid (phytaspases).
Vartapetian etal.,2011
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Jones etal., 2001
Azwali etal.,2019
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PCD FEATURES ANIMALS PLANTS
Cell shrinkage Yes Yes
Chromatin condensation Yes Yes
Phosphotidylserine externalization Yes Yes
DNA laddering Yes Yes
TUNELS-DNA cleavage Yes Yes
Caspases Yes No
Mitochondria permabilization Yes Yes
Mitochondria depolarization Yes Yes
Cytochrome c release Yes Yes
Cytochrome c–dependent activation of cell death Yes No
Apoptotic bodies Yes Yes
Vacuole leakage and fusion with plasmalemma No Yes
Cell shrinkage Yes Yes
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REPRODUCTIVE STAGE
 Gametophyte development.
 Megaspore cell death
 Cell death of the nucellar tissue
 Antipodal cell death
 Tapetum cell death
 Cell death in sex determination
 Self-incompatibility-induced cell
death.
VEGETATIVE STAGE
 Xylogenesis
 Lateral Root Cap Differentiation
 Aerenchyma Formation
 Leaf Morphogenesis
 Organ Abscission and Dehiscence
 Leaf Senescence
 Floral Organ Senescence
Pennel etal.,1997
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PCD IN REPRODUCTIVE DEVELOPMENT IN PLANTS
Megaspore cell death Cell death of the nucellar
tissue
Tapetum cell death.
• The selection of the functional megaspore is position-
dependentand varies between species.
• The deletion of the functional megaspores is important
for optimal seed development.
• In Arabidopsis (Arabidopsis thaliana), the arabinogalactan
protein AGP18is expressed during meiosis in the abaxial
pole of the ovule.
• Callose deposition.
GAMETOPHYTE DEVELOPMENT
Daneva etal., 2016
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Cell death of the nucellar
tissue
• In the degenerating nucellar tissue of castor bean
(Ricinus communis L.), proteomic analyses
identified multiple proteases and protease
inhibitors (Nogueira et al. 2012).
• Nuclear condensation and DNA fragmentation
indicative of PCD have been observed in the
nucellus of wheat (Triticum aestivum L.)
Tapetum cell death.
• Precise timing of tapetum degeneration is crucial for pollen
maturation.
• The phytohormone gibberellic acid (GA) regulates tapetum
differentiation and dPCD through GA-regulated
myeloblastosis (GAMYB) TFs in rice and their homologues
MYB33 and MYB65 in Arabidopsis.
Kurusu etal.,2017
GAMETOPHYTE DEVELOPMENT
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Maize produces separate unisexual flowers through programed abortion of preformed organ primordia. In
the male inflorescence (tassel), stamen primordia develop to sexual maturity, while gynoecia (pistil
primordia) are aborted.
• TS2 encodes a dehydrogenase/reductase domain–containing, NAD(P)- binding Rossmann-fold protein. Shortly
before abortion of the gynoecium, TS2 mRNA is expressed subepidermally in that primordium and thus gynoecium is
aborted through PCD.
• In tasselseed2 (Ts2) mutant plants, floral structures in the tassel adopt a female developmental program.
• Dependent on the lipoxygenase-encodingTS1 gene, TS2 mRNA accumulates in all pistils, but in the female
inflorescence pistils survive protected from PCD by the action of the SILKLESS1 gene.
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Self-incompatibility-induced cell
death
Transmitting tract cell
death.
Synergid cell death and pollen tube
rupture
PCD IN FERTILIZATION
Daneva etal., 2016
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death
death.
rupture
• SI in poppy (Papaver rhoeas) is controlled by a small, secreted
stigma protein, PrsS, and a pollen membrane protein, PrpS, the
interaction of which induces a calcium-triggered signaling network
that leads to growth arrest and PCD of the incompatible PTs.
(Eaves et al., 2014).
• Actin depolymerization, ROS signaling, caspase-like protease
activities and DNA fragmentation.
• A drop of cytoplasmic pH in incompatible pollen.
• The poppy SI system was successfully transferred to the self-
compatible A. thaliana, suggesting that PrsS and PrpS factors can
signal to a conserved PCD network in A. thaliana pollen.
( Lin et al., 2015).
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death
death.
rupture
death.
• The transmitting tract (TT) is a specialized
tissue that guides and supports PT growth toward the ovule.
• In A. thaliana, TT cell death starts before anthesis and can
be accelerated by pollination.
• It has been proposed that pollination-induced TT PCD is
activated by ethylene signaling and mRNA poly(A)
tail shortening.
(Crawford et al., 2007)
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death
death.
rupture
rupture
• The Rsyn undergoes cell death upon arrival of the PT. The
subsequent rupture of the PT, releasing two sperm cells
into the female gametophyte, represents a second controlled
cell death event during PT and Rsyn interaction.
• In A. thaliana, Rsyn cell death is thought to be elicited by
short-range signaling between PTs and Rsyn.
• In the female gametophyte mutant gametophytic factor 2
(gfa2), ovules remain unfertilized because PT penetration
and Rsyn cell death do not occur.
(Leydon et al., 2015)
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PCD IN SEED DEVELOPMENT
 In the large suspensor of the runner bean (Phaseolus coccineus), tissue degeneration in
the suspensor is marked by DNA fragmentation, by activation of caspase-like proteases,
and by cytochrome c release from mitochondria.
Embryonic suspensor cell
death
Endosperm cell
death
Seed coat cell
death
Aleurone PCD
 In many species, the embryo-surrounding region (ESR) breaks down during mid-seed
development to provide space for the growing embryo. In cereals, the starchy endosperm
(SE) undergoes a PCD after seed filling.
 Integument PCD is by cysteine protease which is specifically expressed in two
integument layers.
 In the innermost integument layer, PCD is characterized by vacuolar collapse and nuclear
fragmentation and is regulated by PROTEIN DISULFIDE ISOMERASE 5 (PDI5).
 Cereal aleurone PCD is controlled by phytohormones: the PCD-promoting GA and the
antagonistically acting ABA. Downstream of GA and ABA, a number of TFs, proteases,
nucleases, and vesicular trafficking regulators have been implicated in aleurone PCD
Daneva etal., 2016
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PCD IN VEGETATIVE
DEVELOPMENT
Xylogenesis and Root cap
differentiation
Aerenchyma formation Leaf Morphogenesis
Daneva etal., 20164/6/2022 48

 PCD of xylem tracheids and tracheary elements (TEs) culminates
in protoplast clearance, producing interconnected hollow cell
corpses with reinforced cell walls.
 TE differentiation encompasses the subsequent phases of
secondary cell wall (SCW) deposition, PCD, protoplast
autolysis, and SCW lignification.
 Various hormones, including brassinosteroids and ethylene,
initiate TE differentiation and dPCD.
 The root cap consists of a central columella and a
peripheral lateral root cap (LRC).
 The constant size and location of the LRC at the growing
root tip are maintained by rapid cellular turnover.
 The continuous generation of new LRC layers by specific
stem cells is counterbalanced by tightly controlled dPCD
that eliminates cells at the root cap edge
LATERAL ROOT CAP
DIFFERENTIATION
Daneva etal., 2016 4/6/2022 49

AERENCHYMA FORMATION
 The formation of lysigenous aerenchyma mediated by PCD occurs constitutively in
wetland species and can be induced by oxygen deprivation in waterlogged plants.
• Hypoxia (Oxygen deprivation)
• Cell death can occur in the cortex of the root and stem base
in response to water logging and hypoxia.
• It leads to ROS (H2O2, ethylene).
• Ethylene triggers apoptotic pathway- eliminate some cells-
aerenchyma.
• The internal air spaces generated by cell death facilitate
more efficient transfer of oxygen from aerial organs to
waterlogged stem base and roots.
Daneva etal., 2016 4/6/2022 50

LEAF MORPHOGENESIS
 Perforation PCD occurs between the leaf veins, creating the fenestrate
pattern of mature lace plant leaves. Tonoplast rupture is the first evident
PCD feature to occur, followed by DNA fragmentation, changes in
nuclear morphology, cytoplasmic shrinkage, and organelle dismantling.
 Ethylene Response Sensor 1 (ERS1)-mediated ethylene sensitivity.
 Downstream of ethylene, caspase-1-like activities triggered by the
release of a mitochondrial signal have been detected.
(Rantong et al., 2015).
Leaf morphogenesis in
monstera plant
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PCD IN LACE PLANT
Aponogeton madagascariensis which is
known as the Madagascar Lace Plant ,
decorative aquatic plant – skeleton
like appearance
• PCD process starts in young leaves between leaf veins
• Alterations in the actin cytoskeleton
• Chloroplast accumulation around the nucleus
• Organelles are taken up into the vacuole in membrane
bound vesicles - autophagy
– Loss of mitochondrial membrane potential
– DNAfragmentation,
– Activation of caspase-like proteases
– Before vacuolar collapse, plasma membrane rupture
and cell wall degradation occurs
• Perforation PCD occurs between the leaf veins, creating
the fenestrate pattern of mature lace plant leaves.
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PCD AS THE END POINT OF
SENESCENCE
Plant senescence controls nutrient remobilization during stress or age induced
degeneration of tissues, organs, or entire organisms, concluding the life cycle of
plant species.
Leaf senescence Floral organ senescence 4/6/2022 53

LEAF SENESCENCE
 Ethylene promotes senescence
 ETHYLENE-INSENSITIVE2 (EIN2) activates EIN3
expression
 EIN3 upregulates the NAC TF ORESARA1/ANAC092
(ORE1) and other TFs and concurrently represses
miR164.
 ORE1 activates the expression of senescence-promoting
TFs
 ORE1 inhibits the activity of the chloroplast-supporting
TFs GOLDEN-LIKE1 (GLK1) and GLK2.
 Promotes the expression of dPCD-associated genes such
as BFN1.
 BFN1 localization has been associated with DNA
degradation in aging leaf protoplasts
Leaf senescence
Leaf senescence and dPCD are linked by the trifurcate pathway downstream of ethylene
Kim et al., 2014
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FLORAL ORGAN SENESCENCE
 In rose flowers, miR164 expression decreased with age and upon
ethylene treatment, suggesting that the trifurcate ethylene pathway
is also functional in floral organ senescence.
 The A. thaliana MADS-box TF FOREVER YOUNG FLOWER
(FYF) negatively regulates the expression of several senescence-
promoting ethylene response DNA-binding factors (EDFs) in both
pollinated and senescing flowers.
 Plants with constitutive expression of EDF manifest significant
upregulation of senescence-associated genes, PCD associated
VPEs, and metacaspase genes.
Floral organ senescence and dPCD are also linked by the trifurcate pathway downstream of ethylene
Floral organ senescence
Pei et al., 2013
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OVERVIEW OF MOLECULAR REGULATORS DIRECTLY OR INDIRECTLY INVOLVED IN PLANT DPCD
Hautegem etal., 2015
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 It is commonly known that animal pathogens often target and
suppress programmed cell death (PCD) pathway components to
manipulate their hosts.
 In contrast, plant pathogens often trigger PCD. In cases in which
plant PCD accompanies disease resistance, an event called the
hypersensitive response.
 HR cell death is an active process in which the accumulation of 02
-
and H202 leads to an elevation in cytosolic Ca2+ and triggers a
protein kinase-mediated cell death process that is similar
physiologically to PCD.
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Taiz and Zeiger, 1991
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 The first line of defense in both plants and
animals is provided by pattern recognition
receptors (PRRs)
 They recognize microbe or danger associated
molecular patterns (MAMPs and DAMPs,
respectively).
 Trigger immune signaling.
Representation of Programmed cell death in the plant immune system
Coll et al., 2011 4/6/2022 59

 The morphology of cells undergoing the HR at late stages suggest that it is a form
of PCD with some apoptotic features.
 Membrane dysfunction (loss of ability to be plasmolysed) and progressive
vacuolization of the cytoplasm.
 There was a gap in the time course in which these events may have occurred.
Thus, the HR in this system has a subset of apoptotic features.
 Apoptosis-related events such as mitochondrial swelling, chromatin condensation
and endonucleolytic cleavage occur before general organelle dysfunction.
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 This would be expected if the HR occurs by an apoptotic-like
mechanism.
 In particular, apoptotic like bodies with avirulent Pseudomonas
syringae infections were observed.
(Levine et al., 1996).
 It was found early changes in mitochondrial morphology
(swelling and cristae disorganization) in avirulent P. syringae-
infected lettuce, similar to what occurs in animal cells
undergoing apoptosis.
(Wakabaashi et al., 2001).
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AIM :
 To provide evidence for involvement of chloroplast as alternate organelle for initiating
PCD in plants under light and abiotic stress.
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 Materials and methods:
 Plant material and growth conditions:
Pusa basmati-1 which has been certified as salt sensitive cultivar.
Seeds were soaked were incubated in dark another 3 days for germination.
1. 30 °C and light period 16 h and 8h dark
2. Kept in dark at same temperature
 Isolation of protoplasts and salt treatment
Protoplasts were isolated from the leaves of green and etiolated rice seedlings grown for
7d on respective growth conditions. Isolated protoplasts were subjected to 150 mM NaCl
for different time points.
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Fig 1: Evans blue staining of the protoplasts to count viability
Protoplast isolated from seddlings in light (A,B) Protoplast isolated from etiolated seedlings (C,D)
Salt stress induces cell death in protoplasts isolated from green and
etiolated rice seedlings
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Fig 2: Light and protease dependency of salt induced cell death
Salt stress induced cell death is light dependent and Cystein protease mediated
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SALINITY-INDUCED CELL DEATH IS MEDIATED BY CELLULAR ROS
 Results demonstrate that untreated protoplasts isolated from green leaves generated H2O2 that was
almost double of that generated from cells prepared from etiolated leaves.
Fig 3: ROS generation during salt induced cell death.
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SALT STRESS-INDUCED CELL DEATH IS APOPTOSIS LIKE CELL DEATH (AL-PCD)
Fig 4: Identification of cell membrane blebbing and nuclear loabing during salt stress.
A) Green protoplasts untreated B) 2h salt treated green protoplasts. C) 4h salt treated green protoplasts. D) 6h salt treated green
protoplasts.
A) bright field image B) Acridine orange C) ethidium bromide staining D) merge of b and c
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EVIDENCE FROM ULTRA-STRUCTURE STUDIES
CHLOROPLASTS ARE PREFERRED TARGET DURING SALT STRESS INDUCED PCD
Fig 5: Ultra structural changes in chloroplast and mitochondria during salt induced cell death.
 As this study attempt has been made to understand the mechanism of salt-stress-
induced PCD and involvement of chloroplasts in the execution of the termination
event.
 The results clearly demonstrate that in salt-treated etiolated and green protoplasts,
cell death is accentuated by light. Thus, suggesting the involvement of etio
chloroplasts and chloroplasts in cell death that is modulated by light.
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AIM :
 To investigate the role of CEP1 in anther development and to identify whether it is the
crucial executor during tapetal PCD which is necessary for timely degeneration of tapetal
cells and functional pollen formation.
Papain-like cysteine proteases, CEP1, CEP2, and CEP3, are expressed in Arabidopsis
roots, flowers, and green siliques where the cells are about to collapse.
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 Materials and methods:
 Plant material and growth conditions:
 Arabidopsis thaliana accession Columbia-0
 Arabidopsis thaliana (Wild, Control)
 Plants were grown on a soil mixture (3:1:1 mixture of peat moss-enriched soil:
vermiculite: perlite) with a 14-h-light/10-h-dark photoperiod at 23°C.
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Identification of the cep1 Mutant
Fig 1: image representing SALK_094934 and SALK_013036 T-DNA insertion positions in
At5g50260. Filled boxes represent exons.
Fig 2. Comparison of the Wild Type and the cep1 Mutant
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EXPRESSION CHARACTERIZATION OF CEP1 IN ARABIDOPSIS
Fig 3. CEP1 spatial and temporal expression analyses by RT-PCR. fl-flower; le-leaf; ro-root and st-stem.
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ANTHER DEVELOPMENT IN THE CEP1 MUTANT
 Figure 4. Transverse Section Comparison of Anther Development in the Wild Type and cep1 Mutant
Anther maturation was abnormal in the cep1 mutant, particularly tapetal degeneration and pollen development.
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Figure 5. Transmission Electron Micrographs of Microspores from the Wild Type and cep1 Mutant
In cep1 mutant, pollen development was impaired, resulting in defective pollen exine, abnormal pollen shape, and
collapsed pollen grains. Thus CEP1 is important for proper pollen development.
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 Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in
angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant
PCD.
 CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development.
 As they characterized the phenotype of the cep1 mutant, CEP1 expression in Arabidopsis,
enzymatic characteristics of CEP1 in vitro, between the wild type and cep1 mutant showed
there is a normal pollen development in the wild than the mutant .
 Results indicate that CEP1 acts as an irreplaceable executor during tapetal cell PCD,
which regulates pollen development.
4/6/2022 75

THE BUCKET LIST:
THINGS TO DO BEFORE YOU
DIE
4/6/2022 76

PCD is like a SOLDIERS who can
sacrifice their LIFE for others
GROWTH and DEVELOPMENT..,
4/6/2022 77

Pcd 4 th seminar

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