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 animalsChromatin condenses, DNA is cleaved, apoptotic bodies are formed from the repackaging of the cell content and finally engulfed by neighbouring cells or macrophages.

PCD in animals and plants may share common cytological and molecular

biological aspects

Apoptosis-like PCD in plantsChromatin condenses, nuclear DNA is cleaved, vacuole blebs and the plasma membrane collapses away from the cell wall. The dead cell stays in situ.

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

Ced-9

Bcl-2

WTTransgenic plants

Western blot showing protein in transgenic plants

Bcl-xl

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 sclerotiorumCercospora nicotianae

Transgenic plants expressing a mutant Bcl-xl do not show resistance to S.

sclerotiorumLeaves with wild-type Bcl-xl

Bcl-xl

Leaves with mutant 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.

BaxBI-1

BI-1 orthologs have been found in plants and are able to inhibit cell death.

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

Plant BI-1 proteins share a relatively high level of identity with other BI-1

proteins

E.g. tobacco BI-1 shares, 75% identity with AtBI-1 and 42% identity with the human or rat BI-1.

humantobacco

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.

BaxBI-1

Bcl-xl

Paper1: Plant BI-1 can suppress Bax-induced apoptosis in human cells

Human cells were transfected with an expression plasmid encoding Bax together with BI-1 (from tobacco) or Bcl-2.

% dead cells

Control

0

45

Bax Bax

+Bcl-2

Bax

+BI-1

Control

Bax

Bax

+BI-1

Paper1: Plant BI-1 can suppress Bax-induced apoptosis in human cells

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

Heat stress(550C)

Non- stressed

WT AtBi mutant

BaxBI-1

Bcl-xl

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.

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

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Evidence for caspases in plants

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 --Another way to look for function is to look for a protein activity.

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

Pollination and pollen tube growth

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Self-fertilisation is not always desirable, it can be prevented by self incompatibility.

pistil

Self-incompatibility

-Sexual reproduction in many angiosperms involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding

-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

pollen

Compatibility is genetically controlled by an 'S-locus' carrying distinct specificity genes, one expressed in the pollen and the other in the pistil.

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 S-haplotype is the same as either of the two S-alleles in the diploid pistil. Any other combination is compatible.

pistil

pollen

'S-locus'

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

Time

-

+

SI induction+/- DEVD

+

-

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 self-incompatible 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

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.

Fragmented DNA can be detected by a process called TUNEL (terminal

deoxynucleotidyl transferase dUTP nick end labeling)

Caspase inhibitors (DEVD and YVAD) suppress cell death and DNA

fragmentation

UVC Cell deathCaspase-like

DEVD and YVAD

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

control

-VPE

Time after infection

O hrs 24 hrs

Silencing of VPE also prevents vacuolar collapse after TMV infection

Silencing of VPE also prevents DNA degradation after TMV infection

0 912 2424control -VPE

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

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zygotic embryo

somatic embryo

1. Inititiation of embryogenic cultures

2. Proliferation of embryos in proembryonic mass (PEM)

3. Maturation of somatic embryos

4. Germination of somatic embryos

5. Regeneration of somatic embryo plants

Somatic embryogenesis of Norway spruce

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 2Blue-dead cellsRed-live cells

Mcll-Pa is a metacaspase that is expressed in embryonic tissues that

are committed to death

Mcll-PaRNA

No Mcll-PaRNA

embryo

suspensor

Controls RNAi lines

Silencing the Mcll-Pa metacaspase in somatic cell culture with RNAi caused a reduction in caspase activity (VEIDase)

and TUNEL positive cells

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.

Fragmented DNA can be detected by a process called TUNEL (terminal

deoxynucleotidyl transferase dUTP nick end labeling)

Controls RNAi lines

Silencing the Mcll-Pa metacaspase in somatic cell culture with RNAi caused a reduction in caspase activity (VEIDase)

and TUNEL positive cells

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

Mcll-PaTUNELNuclei

suspensor

embryo

TUNEL +Mcll-Pa

Cell extractMcll-Pa

Mutated Mcll-Pa

In vitro experiments with isolated nuclei show that Mcll-Pa induces nuclear degradation in cells from the PCD-

deficient line

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.