Metacaspase-8 Modulates Programmed Cell Death Induced by Ultraviolet Light and H2O2 in Arabidopsis

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H2O2, or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H2O2 no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H2O2, and knocking out AtMC8 reduced cell death triggered by UVC and H2O2 in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress. Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H2O2, or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H2O2 no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H2O2, and knocking out AtMC8 reduced cell death triggered by UVC and H2O2 in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress. In some instances, programmed cell death (PCD) 4The abbreviations used are:PCDprogrammed cell deathKOknock outWSWassilewskijaLerLandsberg erectaMSMurashige and SkoogMVmethyl viologenORFopen reading frameRTreverse transcriptionCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidQ-PCRquantitative-PCRAMCaminomethylcoumarinMES4-morpholineethanesulfonic acid. 4The abbreviations used are:PCDprogrammed cell deathKOknock outWSWassilewskijaLerLandsberg erectaMSMurashige and SkoogMVmethyl viologenORFopen reading frameRTreverse transcriptionCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidQ-PCRquantitative-PCRAMCaminomethylcoumarinMES4-morpholineethanesulfonic acid. in plants is comparable with animal apoptosis at the cellular level. However, sequencing the Arabidopsis genome revealed that very few of the animal PCD regulators are conserved in plants. This suggests a greater divergence of the PCD pathways across kingdoms than thought. Initial reports seemed to provide indirect evidence supporting the existence of caspase orthologues in plants, with several caspase-like activities detected in plant extracts and inhibitor studies that show them to be required for PCD (for review, see Ref. 1Rotari V.I. Rui H. Gallois P. Physiol. Plant. 2005; 123: 376-385Crossref Scopus (51) Google Scholar). Although several research groups reported the absence of orthologous caspase sequences in plant genomes, a more in depth analysis revealed a greater diversity of caspase-related proteases than previously suspected (2Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). In particular, two families of predicted proteases were identified that are more closely related to animal caspases than to other proteases: the paracaspases and metacaspases. Paracaspases and caspases appear animal specific, whereas metacaspases are present in other eukaryotes, including plants. Plant metacaspases are subdivided in type I and type II on the basis of their structure; type I have an N-terminal prodomain that is not present in type II. A role for metacaspases in plant PCD was proposed (3Koonin E. Aravind L. Cell Death Differ. 2002; 9: 394-404Crossref PubMed Scopus (311) Google Scholar) for four reasons; 1) a common origin with caspases, 2) the absence of closer caspase homologues in plants, 3) the proliferation of the genes coding for metacaspases in plant genomes mirrors the pattern of the proliferation and specialization of caspases in animal genomes, and 4) the fusion of type I plant metacaspases with a zinc-finger domain also present in LSD1, a regulator of the hypersensitive response in plants (4Dietrich R.A. Richberg M.H. Schmidt R. Dean C. Dangl J.L. Cell. 1997; 88: 685-694Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Early observations suggested that metacaspases could be responsible for caspase-like activities detected in plants (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar, 6Bozhkov P.V. Filonova L.H. Suarez M.F. Helmersson A. Smertenko A.P. Zhivotovsky B. von Arnold S. Cell Death Differ. 2004; 11: 175-182Crossref PubMed Scopus (124) Google Scholar); however, three reports have now demonstrated that metacaspases are unable to cleave caspase substrates. So far, all four recombinant Arabidopsis metacaspases tested have arginine as the preferred P1 amino acid in the cleavage site instead of caspase aspartic acid (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). A recombinant metacaspase from pine showed the same preferred cleavage site with Arg in P1 (8Bozhkov P.V. Suarez M.F. Filonova L.H. Daniel S. Zhivotovsky B. Smertenko A. S. A. 2005; PubMed Scopus Google Scholar). activities are not by caspase programmed cell death out and methyl reading acid acid. programmed cell death out and methyl reading acid acid. Although metacaspases not have caspase-like is for a role in PCD. The yeast metacaspase in the of H2O2 (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google and the of a metacaspase PCD in the of M.F. Filonova L.H. Smertenko A. von Arnold S. Zhivotovsky B. P.V. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Arabidopsis detected in metacaspase of Arabidopsis type II metacaspases had a reduced cell whereas of type I metacaspases had an increased cell death by P. M. Mol. Plant PubMed Scopus Google Scholar). A of in the that type II metacaspases cell death in response to as pathogen led to type II metacaspase up-regulation A. PubMed Scopus Google Scholar). In is no for a metacaspase role in PCD a of stresses in A. P. L. J. Cell PubMed Scopus Google Scholar) metacaspases in cell proliferation and A. M. 2002; PubMed Scopus Google Scholar). A metacaspase in metacaspases required for a cellular of PCD not for other as of caspase-like activities and of R. Mol. PubMed Scopus Google Scholar). it has reported that Arabidopsis plants members of the metacaspase gene family had no cell phenotype B. B. R. A. J.L. F. J. Mol. Biol. PubMed Scopus Google Scholar). evidence is that some metacaspases a role in and some the proliferation of metacaspase genes in plants some of Arabidopsis thaliana has nine metacaspase genes, and it be that some members of the family are involved in the regulation of and some be involved in other an of two metacaspases and are up-regulated in Arabidopsis plants with whereas type II metacaspases are not (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In metacaspases have pH B. P. K. J. F. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). to the of member of gene family in In PCD pathways activated in plants be or This hypothesis is in the yeast metacaspase PCD induced by H2O2 or acid (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google by J. Cell Biol. 2005; PubMed Scopus Google and by stress R. B. P. F. M.T. M. Mol. 2005; PubMed Scopus Google in it is not required for PCD during S. A. E. Mol. Biol. Cell. PubMed Scopus Google triggered by L. J. Cell Biol. 2005; PubMed Scopus Google or induced by P. C. L.A. Frohlich F. L. Mol. PubMed Scopus Google Scholar). We show here that Arabidopsis a type II is up-regulated by the oxidative stress caused by UVC and to and up-regulation is We have evidence that metacaspase is part of a PCD pathway induced by UVC, H2O2, or methyl viologen. This to of the of the metacaspase family in Arabidopsis and the origin of PCD. Plant A. thaliana and were metacaspase-8 were in a The in a was by and in a was from and were from The was by R. of were at in or in In seedlings were in a at AtMC8 was and from seedlings with and The was the which a at and an at the to A was the The AtMC8 was from with the and and and M. PubMed Scopus Google Scholar) and to a and The was with II to the AtMC8 coding with the and This was the P. P. Plant Mol. Biol. PubMed Scopus Google Scholar) and to was the same as a The type and the AtMC8 were the yeast and to and in and were were to an of and induced with for at were in with a of were a with of by of on A was by at for at a cell was a at Cell were by at for was yeast in and the type were by and were in and The was out as (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar). In yeast were with or to the same and or not with H2O2 for were a cell and on yeast for The was the of after at Plant and were were with and and used to Arabidopsis by the N. J. C. R. Scholar) in with of seeds were and on and was from seedlings in Plant to the and with for The was used to the with the The were used as for with the the were studies the were and and and and A and and and and and and and C. was as in the and used as in to the and an the and were the and were AtMC8 the and were A of Arabidopsis was used to the of in H2O2 was used to H2O2 in protoplasts. a with the were in the for and the was used to the was pH the pH was for pH for pH and for pH extracts were out in and plant the was yeast was from yeast cell by and with a activities were by of of plant of yeast of E. or of AtMC8 to with of at and and for for and for for for and and for were used at The of was at with a an of and an of were by the and activities were in or of was the used a recombinant AtMC8 were and a analysis was out to J. P. A Scholar) and were were in for and the to to in of was in of a were out from and were in the of the in was and an were by UVC a or the of H2O2 to the to the and the were in at for The of the in was and the and after was to the of cell death of were used for and in for were with and including and pH in the for at were by for at and in of and pH The was in including and pH at a of of was used of a were by a or of H2O2 to in and the were in for at The was to to the of of an of the was on a of at four of were The of of cell was the was a to a on were for in and on with pH were with or methyl were for at and at in was a were with a was out in with at AtMC8 by and with Cell have previously that a UVC PCD in Arabidopsis A. Gallois P. PubMed Scopus Google Scholar). and the during PCD of the nine metacaspases that are present in the Arabidopsis seedlings were with and in the was or after We that the was the metacaspase strongly with a after UVC We no for and and were present and by the was at a and up-regulated with at was The of AtMC8 was showed that a of is required to the of AtMC8 as a as a gene instead of in a of not UVC A of was and a in a that of the AtMC8 is to the of stress. AtMC8 could be to the of PCD or to the stress caused by the and stresses to be with a stress and not the of have that PCD is A. V.I. A. N. Gallois P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and showed that was required after UVC to AtMC8 a the of AtMC8 and the of PCD. A suggested a with oxidative stress. H2O2 was with UVC and was to be after was to after seedlings were on with a with oxidative stress and cell the of methyl an herbicide that an PCD S. J. 2004; PubMed Scopus Google Scholar) and the of in the which is was to AtMC8 after seedlings were on with This caused and cell death of seedlings the oxidative stress AtMC8 from the the of AtMC8 in the is proposed to genes, and is to by the R. S. K. P. M. A. H. H. E. M. J. Plant Cell. 2004; PubMed Scopus Google Scholar). A UVC was to an of the of AtMC8 in the than an to the of UVC on and members of metacaspase gene was a of seedlings to with This suggested that in the absence of an by oxidative AtMC8 is to a of by stress. This is with some members of the metacaspase gene family and that most are after and P. of by H2O2 and to a for a functional suggested that an oxidative stress at the AtMC8 In these show a AtMC8 oxidative and cell for studies in that a at the is not of a metacaspase-8 and PCD. Recombinant AtMC8 with in and at pH AtMC8 was and from a type II is a with a predicted of studies have is a conservation of the in the for proteases (2Uren A.G. O'Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). was required for the of AtMC8 in cell the in was to a AtMC8 and were in the which and of the recombinant a or an analysis that the was an AtMC8 the of a to the The and the evidence of by a at as for other metacaspases (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, P.V. Suarez M.F. Filonova L.H. Daniel S. Zhivotovsky B. Smertenko A. S. A. 2005; PubMed Scopus Google Scholar, B. P. K. J. F. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). on E. extracts and recombinant that AtMC8 has no the caspase or the AtMC8 was to cleave the and This was with the of the predicted in the recombinant The optimum pH was to be and with no pH AtMC8 in yeast the yeast showed the same as the recombinant AtMC8 in E. not AtMC8 a was to the of AtMC8 in yeast PCD. The AtMC8 was the yeast The and the were two type or metacaspase was used to the of the recombinant and H2O2 was to the yeast for of AtMC8 in the was by analysis The the the H2O2 and by the type and the AtMC8 were to have a of by and AtMC8 is to the cell death of the yeast metacaspase and to if AtMC8 was involved in the regulation of PCD induced by H2O2 in AtMC8 to PCD by H2O2 or of AtMC8 on PCD in Arabidopsis was by of AtMC8 or the of a AtMC8 and two were for at type AtMC8 and were in an after in the plants AtMC8 was it a of in of a in the increased was from the two of The increased with the in The in type is the activities of metacaspases and of was no of caspase-like activities not was no phenotype during the development of the were to two death and was to PCD by A. Gallois P. PubMed Scopus Google Scholar, A. V.I. A. N. Gallois P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). H2O2 has to PCD in Arabidopsis R. A. J. S. J. PubMed Scopus Google and the metacaspase in yeast is required for PCD (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar). The of AtMC8 on PCD in Arabidopsis was from is of a of and has used in hypersensitive response studies in it with cell see Ref. K. H. R. C. C. H. J. Plant Cell. 2000; PubMed Scopus Google In the used here has that Arabidopsis the PCD have a reduced during PCD induced by H. S. A. N. H. M. M. H. R. Plant J. PubMed Scopus Google that with PCD. from same were from plants in in and on The to be used were to be or to on that were with H2O2 for was to a of in plants AtMC8 with This was in three The the of the to H2O2 and showed an to the type A of increased in the the The increased in in was not across and as with a were from AtMC8 or PCD was induced by were after A of H2O2 not cell death in type by in was a in cell from to Overexpressing the not cell death with H2O2 The of was also and were from plants. A of was used in no cell death in from type to PCD by H2O2, cell death increased with from and showed a cell death to that of type AtMC8 increased the of Arabidopsis to the two PCD used in these the increased or cell death with the at the an of in protoplasts. In with H2O2 or the increased death in the as the with type is reduced not AtMC8 caused an increased at the of the AtMC8 a Cell Death by H2O2 or in the AtMC8 gene and identified two and the the and are of the and is of the not show a reduced that the were to The has an in the and was The be predicted to the of a that The of the was not in not The has an in the of the showed that had no that the was not with the at a not This a and is In if also in a that the of the site was no phenotype during development of the with their The type and were to or H2O2 in or protoplasts. The two type and were to have of cell death than and were and H2O2 that knocking out AtMC8 had on cell death as a the in the showed a of with and were very In and H2O2 or knocking the gene in a or in a of cell death by and and of AtMC8 a to of cell death in as metacaspase-8 in a PCD pathway activated by an oxidative stress caused by or The herbicide is a that oxidative stress by and H2O2 in and AtMC8 has to PCD with the of a S. J. 2004; PubMed Scopus Google Scholar). with a of from to to with H2O2 and not any in the not Cell death and were in to a seeds of type and were on with of on was after the two type seeds in the of the the two were to at for and for The seeds a of a few not seeds were on the the type seeds of and at and whereas the seeds of and at The more type and was that of seeds a that at whereas of seedlings and of seedlings not and or evidence that AtMC8 is a member of the Arabidopsis metacaspase family that is involved in the regulation of PCD induced by oxidative stress. So far no other has demonstrated a role for an Arabidopsis metacaspase in PCD. the during development that metacaspases have a functional role in plant PCD M.F. Filonova L.H. Smertenko A. von Arnold S. Zhivotovsky B. P.V. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). a metacaspase from was reported involved in the cell death of and is most to be orthologous to which at present has to a role in PCD. AtMC8 has not previously was no evidence of (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, B. P. K. J. F. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Although was so far no cell phenotype in Arabidopsis plants some of the members of the metacaspase family B. B. R. A. J.L. F. J. Mol. Biol. PubMed Scopus Google suggest at This the that member of the Arabidopsis metacaspase family have their is that metacaspases be to out in some to cell death and some to other This divergence of be for to the divergence in the caspase family members apoptosis or of M. Wesselborg S. K. Full Text Full Text PDF PubMed Scopus Google Scholar). In to out that some of PCD are and some analysis of for the of that AtMC8 is induced by of with pathogen This that UVC and pathogen response common response genes C. S. N. Plant Cell. 2002; PubMed Scopus Google Scholar). is a pattern of AtMC8 during PCD induced by which show with a PCD This is by the that H2O2 and cell death and AtMC8 This gene is the in the metacaspase family that is induced strongly by the is present at very in and it is induced as as after a to PCD. This of an in at AtMC8 is dependent of a functional RCD1, and AtMC8 in an oxidative stress pathway activated by in after by This is with methyl and H2O2 in that in AtMC8 AtMC8 could be of as has to with E. E. M. S. 2000; PubMed Scopus Google Scholar) that in with the AtMC8 is that the has reported to H. K. N. Plant Physiol. 2004; PubMed Scopus Google and AtMC8 is not induced in the by UVC or is to R. S. K. P. M. A. H. H. E. M. J. Plant Cell. 2004; PubMed Scopus Google the that and AtMC8 are in the same AtMC8 is dependent of the seedlings in the after the as is cell This pattern suggests that AtMC8 is involved in the of a PCD induced here by has in several of cell death as cell death in K. P. 2005; PubMed Scopus Google P. J. Plant Cell. 2000; PubMed Scopus Google cell death A. K. S. A. PubMed Scopus Google and hypersensitive response M. S. R. P. Plant J. PubMed Scopus Google Scholar). is yet as to is in plant PCD suggest here that AtMC8 is part of The showed increased cell death in and This a functional The the increased in plants and cell death suggests that cell death in in to metacaspase-8 with the very and phenotype the two are more and appear to with the on the showed a of the with a to in the of cell death is in some however, the induced cell death is not The is that AtMC8 is not the Arabidopsis metacaspase to be involved in the PCD induced by or that after to metacaspases are at with and the most at the level. We are that these other members of the metacaspase family that also to the oxidative stress PCD pathway at the level. and and the show a The absence or phenotype in suggests that the cell in components that for the of AtMC8 in the PCD in metacaspase-8 be required for of by not required for a cell death be of This is of in A. metacaspases are required for and not for of R. Mol. PubMed Scopus Google Scholar). Although suggest a PCD and AtMC8 in Arabidopsis is not plants more to or The of plants at a that the metacaspase-8 pathway in the absence of a cell death are components of the PCD that are of AtMC8 and in plants. AtMC8 is to two of the at the of and the at the of the of the family are for a has to B. B. R. A. J.L. F. J. Mol. Biol. PubMed Scopus Google Scholar). In has as a for metacaspase in B. M. A. M. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This suggests that a of activated metacaspase to be to with is at of the This of be during it be that metacaspase-8 be involved in the PCD than in the of We show here that AtMC8 cell death induced by of H2O2, and is to the in yeast (5Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar). This suggests that H2O2 an pathway present in yeast and This is by the that AtMC8 in a yeast was to the phenotype in the of be that and are also to the same yeast (7Watanabe N. Lam E. J. Biol. Chem. 2005; 280: 14691-14699Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). in E. showed here that AtMC8 has an for the or and is unable to cleave the caspase This is by in yeast and of the nine Arabidopsis and have also to be proteases and unable to cleave the that metacaspases are not plant We AtMC8 to be more at pH to that AtMC8 most in the or with observations AtMC8 from which has an optimum pH of and is in the cell B. B. R. A. J.L. F. J. Mol. Biol. PubMed Scopus Google Scholar). of it that AtMC8 is in the cell In or AtMC8 cell death in that have of their cell This phenotype in also suggests that is not as be or during In have that in Arabidopsis metacaspase-8 cell death induced by This is an to be the pathway involved and the of AtMC8 in the in to and The that are cleaved by metacaspase-8 and the that to at the gene or to be identified and the We for with the We and for