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This lecture:
What genes are involved in regulating PCD in
plants? Are they the same as in other
organisms?
-look for genes that function in different organisms and
seeing if they work in plants
-look for a protein activity
In plants PD is necessary for growth and
survival and can occur on a local or large scale
Apoptosis in animals
Chromatin condenses, DNA is cleaved, apoptotic bodies
are formed from the repackaging of the cell content and
finally engulfed by neighbouring cells or macrophages.
Apoptosis-like PCD in plants
Chromatin condenses, nuclear DNA is cleaved, vacuole
blebs and the plasma membrane collapses away from the
cell wall. The dead cell stays in situ.
PCD in animals and plants may share common
cytological and molecular biological aspects
However, the processes seen during PCD in
plants vary between tissues
-It appears that isn’t one set of processes
occurring during every case of PCD.
In metazoans (from humans to C. elegans)
apoptosis is regulated by three classes of
conserved regulators
BCL-2/CED9-binary switch regulates cell life/death
APAF1/CED4-protease activating factor
Caspases-degrade cellular organelles
Caspases can be divided into two
catagories
initiator which cleave inactive pro-forms of
effector caspases, thereby activating them
initiator
effector
Is apoptosis in animals and PCD in
plants regulated by similar
mechanisms?
On a molecular level much less is known
about PCD processes in plants, however
there does seem to be some conservation
between plants and other organisms.
Are the genes involved in regulating the
disassembly of animal cells conserved in
plants?
One way to look for analogy is to look for genes
that function in different organisms e.g. taking
animal PCD genes and seeing if they work in plants
Three repressors of cell death, Ced-9,
Bcl-xl and Bcl-2, were expressed in
transgenic tobacco plants
Bcl-xl
WT
Ced-9
Bcl-2
Transgenic plants
Western blot
showing protein
in transgenic
plants
The transgenic plants containing Ced-9, Bcl-2
and Bcl-xl were tested to see whether
necrotrophic fungi were able to grow
If expression of Ced-9 or Bcl-2 prevents plant
cell death, the necrotrophs will not be able to
grow.
Bcl-xl
Necrotrophic pathogens
Necrotrophs are pathogens that require host
cell death to grow, colonise and reproduce in
the plant. They probably derive nutrients from
dead or dying cells.
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Botrytis cinerea
"grapes like ashes"
The transgenic plants containing Ced-9, Bcl-2
and Bcl-xl were tested to see whether
necrotrophic fungi were able to grow
If expression of Ced-9 or Bcl-2 prevents plant
cell death, the necrotrophs will not be able to
grow.
Bcl-xl
Bcl2 and Ced-9 transgenic plant leaves
infected with necrotrophic fungi do not show
typical symptoms of cell death
Sclerotinia sclerotiorum
Cercospora nicotianae
Transgenic plants expressing a mutant Bcl-xl
do not show resistance to S. sclerotiorum
Leaves with wild-type Bcl-xl
Leaves with mutant Bcl-xl
Bcl-xl
Therefore Bcl, Ced-9 and Bcl-xl are
functional in plants.
If metazoan repressors (Bcl and Ced-9 ) of
apoptosis are functional in plants, there
should be analogous plant genes which can
suppress apoptosis.
Do plants have orthologs (same gene,
different organism) of the apoptosis
regulators, Bcl and CED-9?
There are no obvious orthologs at the
primary sequence level of Bcl-related
proteins.
However, apoptosis is also positively-regulated
in metazoan cells by the product of a gene
called Bax.
Bax is negatively-regulated by Bax-inhibitor 1
(BI-1)
Bcl-xl
BI-1
Bax
BI-1 orthologs have been found in plants and
are able to inhibit cell death.
Plant BI-1 proteins share a relatively high
level of identity with other BI-1 proteins
tobacco
human
E.g. tobacco BI-1 shares, 75% identity with AtBI-1
and 42% identity with the human or rat BI-1.
Two papers showing that plant BI-1 orthologs
are able to inhibit cell death
•
Planta. 2003 Jan;216(3):377-86.
Molecular characterization of two plant BI-1
homologues which suppress Bax-induced
apoptosis in human 293 cells.
Bolduc N, Ouellet M, Pitre F, Brisson LF.
2. Plant J. 2006 Mar;45(6):884-94.
Arabidopsis Bax inhibitor-1 functions as an
attenuator of biotic and abiotic types of cell death.
Watanabe N, Lam E.
Paper1: Plant BI-1 can suppress Bax-induced
apoptosis in human cells
Bcl-xl
BI-1
Bax
Human cells were transfected with an
expression plasmid encoding Bax together with
BI-1 (from tobacco) or Bcl-2.
Paper1: Plant BI-1 can suppress Bax-induced
apoptosis in human cells
45
Control
% dead
cells
Bax
Bax
0
+BI-1
Control Bax
Bax
Bax
+Bcl-2 +BI-1
Paper2:Two T-DNA insertion mutants in AtBI
Paper 2: Under ‘normal’ conditions, the AtBI
mutants look like wild-type, but they are more
likely to undergo cell death when stressed
Nonstressed
Heat
stress
(550C)
WT
AtBi mutant
Summary:
Cell death regulators (Bcl and Ced-9) from other
organisms are functional in plants
However there are no obvious orthologs for these
genes in plants.
There IS a plant homologue of BI-1, a cell death
inhibitor, in plants and it is functional in other
organisms and may be important in plants.
Bcl-xl
BI-1
Bax
Are there plant activities that resemble
other elements involved in metazoan
apoptosis?
caspases
What are caspases?
-family of cysteine proteases, conserved
evolutionarily from nematodes to humans
-activated from dormant precursors during
apoptosis
-induce breaks after specific amino acid
residues in a few key cellular protein
substrates
-among the most specific proteases known
-caspase-3 is responsible for most of the
effects orchestrating cell death
-caspase inhibitors often prevent apoptosis
Are the genes involved in regulating the
disassembly of animal cells conserved in
plants?
Evidence for caspases in plants
-One way to look for analogy is to look for genes
that function in different organisms e.g. taking
animal PCD genes and seeing if they work in plants -Another way to look for function is to look for a
protein activity.
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Pollination and pollen tube growth
Pollen from the anther lands on the stigma where it hydrates,
germinates and extends a tube through the style to reach the
transmitting tract. The tube emerges out of the tract and into the
ovule through the micropile
pistil
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Self-fertilisation is not always desirable, it can be prevented by self
incompatibility.
Self-incompatibility
-Sexual reproduction in many angiosperms
involves self-incompatibility (SI), which is one
of the most important mechanisms to prevent
inbreeding
pollen
-there are highly specific interactions between
pollen and the pistil on which it lands
-incompatible (self) pollen is rejected,
compatible (non-self) pollen is allowed to
fertilise the plant
-rejection involves PCD of pollen tube cells
pistil
'S-locus'
Compatibility is genetically controlled
by an 'S-locus' carrying distinct
specificity genes, one expressed in
the pollen and the other in the pistil.
pollen
The S-locus is extremely polymorphic;
even small populations may have
dozens of different S-alleles (S1-,
S2-, and so on).
Pollen is rejected when its Shaplotype is the same as either of
the two S-alleles in the diploid pistil.
Any other combination is compatible.
pistil
Pollen germination on the stigma
Pollen can also be grown on a solid germination medium
Pollen was grown on a solid germination
medium and then recombinant stigmatic S
proteins (that determine SI) were added to
induce PCD
S
PCD in SI pollen tubes in Papaver rhoeas is
accompanied by DNA fragmentation
The DNA fragmentation seen in SI pollen
tubes can be inhibited by DEVD, an inhibitor
of caspase-3
Caspase-3 is instrumental in the cleavage of DNA
Incompatible pollen tubes cease growth after
SI induction. Pretreatment with DEVD, an
inhibitor of caspase-3, allows the pollen
tubes to keep growing.
+/-SI induction
Pollen
tube
length
-
SI induction
+/- DEVD
+
+
Time
One of the functions of Caspase-3 in
metazoan cells is to degrade poly(ADP) ribose
polymerase (PARP*)
Plants have at least two PARP genes so PARP
could be a substrate for the caspase-3 like
activity observed.
*Poly (ADP-ribose) polymerase (PARP) is a protein involved in a number of
cellular processes involving mainly DNA repair and PCD.
SI-stimulated incompatible pollen has an
activity that can cleave bovine PARP
Neither compatible pollen nor untreated pollen have
the PARP cleavage activity suggesting that they do
not have caspase-3 activity.
Summary:
PCD of pollen tubes in at least some selfincompatible responses is regulated by a
caspase-3-like activity that can be
inhibited by DEVD, a caspase-3 inhibitor.
Plants also have orthologues of PARP genes,
one of the caspase-3 targets in other
organisms.
Another example of caspase activity in plants
The hypersensitive response
The hypersensitive response (HR) is a mechanism, used by
plants, to prevent the spread of infections by microbial
pathogens. The HR is characterized by the rapid death of
cells in the local region surrounding an infection. The HR
serves to restrict the growth and spread of pathogens to
other parts of the plant
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Caspase activity is induced in tobacco plants
undergoing the hypersensitive response after
infection with the TMV virus
VirD2
The research was carried out using VirD2,
(a protein from a plant pathogen) that was
predicted to be a possible substrate for
caspases in a computer-assisted search for
novel nuclear proteins that might be
cleaved by caspases.
Human caspase-3 can cleave the VirD2 protein
(this cleavage is inhibited by the caspase-3
inhibitor DEVD)
Plant extracts have a caspase-like activity
which can specifically cleave the VirD2 protein
Intact VirD
Cleaved VirD
Final example of caspase activity in plants
Death of plants treated with UVC
Treatment with 30kJ/m2 UVC also kills
protoplasts
Caspase inhibitors (DEVD and YVAD) suppress
cell death and DNA fragmentation
Fragmented DNA can be detected by a process
called TUNEL (terminal deoxynucleotidyl
transferase dUTP nick end labeling)
The fragmentation of DNA
during apoptosis generates
exposed 3’-hydroxyl ends in
the nuclear DNA. These
DNA breaks can be
detected in situ using
terminal deoxynucleotidyl
transferase (TdT), which
covalently adds labeled
nucleotides to the 3'hydroxyl ends of these
DNA fragments.
Caspase inhibitors (DEVD and YVAD) suppress
cell death and DNA fragmentation
UVC
Caspase
-like
DEVD and YVAD
Cell death
Summary:
There is considerable evidence that
plants, therefore appear to have a
caspase-like activity.
However, there are, as yet, no known
caspase genes in plants.
If there are no caspase genes in plants,
what proteins are responsible for the
caspase-like activity?
There are two groups of proteases
that have caspase-like activities in
plants
1. Vacuolar processing enzymes - VPEs
-has caspase-1 activity
2. Metacaspases
-have conserved caspase-like secondary structure
-have cysteine protease functions
Vacuolar processing enzymes (VPEs) are
involved in PCD during the hypersensitve
reponse in tobacco
Silencing of VPE causes a decrease in
hypersensitive cell death after TMV infection
control
-VPE
Silencing of VPE also prevents vacuolar
collapse after TMV infection
control
-VPE
O hrs
24 hrs
Time after infection
Silencing of VPE also prevents DNA
degradation after TMV infection
control -VPE
0 9 12 24 24
However, plants with null mutations in
all 4 VPE genes develop normally - so
VPEs do not seem to have a role in
development.
There are two groups of proteases
that have caspase-like activities in
plants
1. Vacuolar processing enzymes - VPEs
-has caspase-1 activity
2. Metacaspases
-have conserved caspase-like secondary structure
-have cysteine protease functions but cleave at a
different site (adjacent to arginine and lysine
not aspartate)
Used somatic embryogenesis
of Norway spruce (Picea
abies to examine the role of
metacaspases in PCD
Somatic embryos
-Morphologically similar to a zygotic embryo
-Initiated from somatic plant cells e.g. leaf or
embryo
-Under in vitro conditions, somatic embryos go
through developmental processes similar to embryos
of zygotic origin.
-Each somatic embryo is potentially capable of
developing into a normal plantlet
Somatic embryos are larger than zygotic
embryos, but show similar development
despite the lack of maternal tissue
zygotic
embryo
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somatic
embryo
Somatic embryogenesis of Norway spruce
1. Inititiation
of
embryogenic
cultures
5. Regeneration of
somatic embryo plants
2. Proliferation
of embryos in
proembryonic
mass (PEM)
4. Germination of
somatic embryos
3. Maturation
of somatic
embryos
PCD has two roles in somatic embryogenesis
of Norway spruce
1. Removal of PEMs when they differentiate
into embryos.
2. Removal of embryo suspensors
1
Blue-dead cells
Red-live cells
2
Mcll-Pa is a metacaspase that is
expressed in embryonic tissues that are
committed to death
embryo
suspensor
No Mcll-Pa
RNA
Mcll-Pa
RNA
Silencing the Mcll-Pa metacaspase in somatic
cell culture with RNAi caused a reduction in
caspase activity (VEIDase) and TUNEL positive
cells
Controls
RNAi lines
Fragmented DNA can be detected by a process
called TUNEL (terminal deoxynucleotidyl
transferase dUTP nick end labeling)
The fragmentation of DNA
during apoptosis generates
exposed 3’-hydroxyl ends in
the nuclear DNA. These
DNA breaks can be
detected in situ using
terminal deoxynucleotidyl
transferase (TdT), which
covalently adds labeled
nucleotides to the 3'hydroxyl ends of these
DNA fragments.
Silencing the Mcll-Pa metacaspase in somatic
cell culture with RNAi caused a reduction in
caspase activity (VEIDase) and TUNEL positive
cells
Controls
RNAi lines
Different mammalian substrates of caspases
were tested to see which were cleaved best
by Norway spruce embryo extracts
YVAD-AMC (for caspase-1)
VDVAD-AMC (for caspase-2)
DEVD-AMC (for caspases-3 and -7)
VEID-AMC (for caspase-6)
IETD-AMC (for caspases-8 and –6)
LEHD-AMC (for caspase-9)
Silencing the metacaspase (RNAi) also caused
the somatic cells to proliferate without
forming embryos
Controls
RNAi line
Mcll-Pa co-localizes with nuclei containing
fragmented DNA in suspensor cells
embryo
suspensor
Nuclei
TUNEL Mcll-Pa TUNEL +
Mcll-Pa
In vitro experiments with isolated nuclei show
that Mcll-Pa induces nuclear degradation in cells
from the PCD-deficient line
Cell extract
Mcll-Pa
Mutated Mcll-Pa
Summary;
Metacaspases and vacuolar processing
enzymes (VPEs) might be the plant
equivalents of caspases.
Summary
-In plants, many tissues and organs undergo PCD
-The mechanisms of PCD in plants is less well
understood than in animals.
-There are several morphological and biochemical
similarities between PCD in animals and plants. e.g.
condensation and shrinkage of nucleus and cytoplasm
and DNA laddering.
-Although however, as yet, there are no plant
orthologs of most animal PCD genes. there is evidence
that the animal regulators of PCD have counterparts
in plants, e.g. metacaspases and VEPs.