Download
1 / 76
780 likes | 905 Views
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.
E N D
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 Transgenic plants WT Western blot showing protein in transgenic plants Ced-9 Bcl-2
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. 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 Cercospora nicotianae Sclerotinia sclerotiorum
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. • 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 +BI-1 0 Bax +BI-1 Bax +Bcl-2 Bax Control
Paper 2: Under ‘normal’ conditions, the AtBI mutants look like wild-type, but they are more likely to undergo cell death when stressed Non- stressed Heat stress (550C) AtBi mutant WT
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.
pistil 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 Self-fertilisation is not always desirable, it can be prevented by self incompatibility.
pollen 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
pollen 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. 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.
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 +/- DEVD +/-SI induction - Pollen tube length + + - 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 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.
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
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
Caspase inhibitors (DEVD and YVAD) suppress cell death and DNA fragmentation
Fragmented DNA can be detected byaprocess 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.
