Hörtensteiner, Stefan

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PROGRESS REPORTStefan Ho¨rtensteinerThe loss of green color during chlorophyll degradation—a prerequisite to prevent cell death?Received: 15 December 2003 / Accepted: 10 January 2004 / Published online: 8 April 2004 Springer-Verlag 2004Keywords Cell death Æ Chlorophyll degradation ÆNon-ﬂuorescent chlorophyll catabolite ÆPheophorbide a oxygenase Æ Red chlorophyll catabolitereductase Æ SenescenceDuring plant senescence, chlorophyll (Chl) is degradedto non-ﬂuorescent Chl catabolites (NCCs; Fig. 1a).These linear tetrapyrroles accumulate in the vacuoles ofsenescing cells and, in many plant species, represent theﬁnal products of Chl catabolism (Matile et al. 1988;Kra¨utler 2003). Despite the billions of tons of Chl dis-appearing this way every year and the fascinatingautumnal color change of deciduous trees resulting fromit, most reactions underlying conversion of Chl to NCChave only recently been elucidated (for recent reviews,see Ho¨rtensteiner 1999; Takamiya et al. 2000; Kra¨utler2003). Chl breakdown is a multi-step pathway (Fig. 1a)aiming to increase pigment solubility and to abolish thephotodynamic properties of Chl by complete disruptionof the conjugated p-electron system. Taking this intoaccount, Chl breakdown can be apostrophized as Chldetoxiﬁcation (Ho¨rtensteiner 1999).As inferred from the structures of NCCs (Kra¨utleret al. 1991), the most remarkable structural change is theoxygenolytic opening of the porphyrin macrocycle ofChl. This reaction is catalyzed by the joint action of twoenzymes, pheophorbide a oxygenase (PaO) and red Chlcatabolite reductase (RCCR) converting pheophorbide(pheide) a to a primary ﬂuorescent Chl catabolite(pFCC; Fig. 1a; Rodoni et al. 1997a). Several lines ofevidence suggest that the two enzymes interact duringcatalysis, thereby channeling the ﬁrst porphyrin cleavageproduct, red Chl catabolite (RCC). Thus, in vitro, RCCdoes not accumulate in the absence of PaO. In addition,RCCR is sensitive to oxygen, although PaO requires O2for incorporation into pheide a (Rodoni et al. 1997a;Wu¨thrich et al. 2000). PaO has been demonstrated to bea non-heme iron-containing monooxygenase, that spe-ciﬁcally introduces one oxygen atom of O2 at the a-methine bridge of pheide a (Ho¨rtensteiner et al. 1995,1998). In addition, PaO is speciﬁc for pheide a withpheide b inhibiting in a competitive manner (Ho¨rten-steiner et al. 1995). PaO is located at the inner envelopemembrane of senescing chloroplasts (Matile and Schel-lenberg 1996). In contrast, RCCR is a soluble chloro-plast protein (Wu¨thrich et al. 2000), suggesting that thesite of conversion of pheide a to pFCC is at the stromalperiphery of the envelope. RCCR stereospeciﬁcally re-duces the C1/C20 double bond of RCC, thereby formingtwo possible C1 stereoisomers of pFCC. The source ofRCCR determines which one is formed; thus, forexample, RCCR of Arabidopsis thaliana produces adiﬀerent isomer than RCCR isolated from tomato(Ho¨rtensteiner et al. 2000b).Exploiting the biochemical characteristics of PaO, 21candidate genes for PaO in Arabidopsis were identiﬁedrecently by using functional genomics (Pruzˇinska´ et al.2003). One of them, Accelerated cell death 1 (Acd1), wassubsequently shown to exhibit PaO activity in vitro afterexpression in Escherichia coli. The properties of theheterologously expressed protein were identical to nativePaO. In contrast, a homologue of ACD1, At4g25650,did not exhibit PaO activity (Pruzˇinska´ et al. 2003).Thus, Acd1 encodes Arabidopsis PaO (AtPaO). AtPaO isa Rieske-type iron–sulfur cluster-containing oxygenase.In Arabidopsis, ﬁve Rieske-type oxygenases are presentwhich have rather diverse functions (Gray et al. 2002).Besides PaO and At4g25650, the function of which isunknown, chlorophyll a oxygenase (CAO; Tanaka et al.1998) and choline monooxygenase (CMO; Rathinas-abapathi et al. 1997) contain Rieske centers. In addition,Tic55, a component of the protein import machinery atthe inner envelope (TIC) also belongs to this group ofS. Ho¨rtensteinerInstitute of Plant Sciences,University of Bern, Altenbergrain21, 3013 Bern, SwitzerlandE-mail: shorten@ips.unibe.chTel.: +41-31-6313156Fax: +41-31-6314942Planta (2004) 219: 191–194DOI 10.1007/s00425-004-1231-8oxygenases (Calibe et al. 1997), although an enzymatic(oxygenase) activity has not been demonstrated in thiscase. Rieske-type oxygenases are widely distributed inpro- and eukaryotes. In all cases, electrons necessary todrive the redox cycle of the Rieske center irons areprovided from reduced ferredoxin (Fd; Schmidt andShaw 2001). Accordingly, PaO, CAO and CMO are Fd-dependent enzymes (Schellenberg et al. 1993; Rathinas-abapathi et al. 1997; Tanaka et al. 1998). Fd is kept inthe reduced state by the activity of a reductase. In higherplants, the nature of this reductase is unknown so far,but possibly Fd-NADPH oxidoreductase (FNR) is in-volved. It has been shown that another protein of theprotein import machinery at the inner envelope, Tic62, isable to bind FNR (Ku¨chler et al. 2002).The RCCR gene has been identiﬁed using classicalprotein puriﬁcation and PCR-based cloning strategies(Rodoni et al. 1997b; Wu¨thrich et al. 2000). RCCR is anovel protein that does not have high homology to otherreductases, but is distantly related to a family of Fd-dependent bilin reductases, necessary for the biosyn-thesis of phycobilins and the phytochrome chromophore(Frankenberg et al. 2001). In contrast to other Fd-dependent enzymes, RCCR and the bilin reductasesappear to lack a metal or ﬂavin cofactor. Thus, electrontransfer is believed to occur directly from reduced Fd tothe respective substrates (Frankenberg and Lagarias2003). In this respect, RCCR would rather be active as a‘‘chaperone’’, mediating the interaction of Fd and RCC(at this stage still bound to PaO), and controlling theregio- and stereoselective reduction (Kra¨utler 2003).When comparing the amino acid sequences of diﬀerentRCCRs, no obvious domains can be identiﬁed thatcould be responsible for the stereospeciﬁcity. Therefore,chimeric proteins that were composed of portions ofRCCR from tomato and Arabidopsis were produced inE. coli and their stereospeciﬁcity analyzed. It turned outthat a Phe-to-Val exchange at position 218 was suﬃcientto change the stereospeciﬁcity of the Arabidopsis RCCR(I. Anders and S. Ho¨rtensteiner, unpublished). Inter-estingly, Phe218 lies adjacent to a stretch of four aminoacids that is absent in the bilin reductases (Frankenberget al. 2001).The rather complex PaO/RCCR reaction represents akey step of the entire Chl catabolic pathway. Thus, PaOactivity is restricted to senescence (Schellenberg et al.1993; Ho¨rtensteiner et al. 1995), whereas activities ofother enzymes of the pathway, such as chlorophyllase(catalyzing the initial removal of phytol from Chl) orRCCR are constitutive (Trebitsh et al. 1993; Rodoniet al. 1997a; Jakob-Wilk et al. 1999). Surprisingly, bothPaO mRNA and protein are present in non-senescentleaf tissue (Gray et al. 2002; Pruzˇinska´ et al. 2003). Al-though PaO expression is up-regulated to some extentupon senescence induction, at the same time the increasein activity is a magnitude higher (Pruzˇinska´ et al. 2003).From this it is concluded that PaO is regulated on theposttranscriptional level as well. So far, the nature ofthis proposed regulation has not been elucidated.The joint reaction of PaO and RCCR is responsiblefor the loss of green pigment color. In this respect, it ismost important for Chl detoxiﬁcation during senes-cence. The importance of Chl catabolism for plant sur-vival can be inferred from the analysis of Chl catabolicmutants. Diﬀerent mutants have been identiﬁed that aredefective in either PaO or RCCR. These include Ara-Fig. 1 a Pathway of chlorophyll degradation during senescence.Depicted are the structures of the Chl catabolites pheophorbide a(pheide a), red Chl catabolite (RCC), primary ﬂuorescent Chlcatabolite (pFCC), and non-ﬂuorescent Chl catabolite (NCC). Thekey reaction is catalyzed by the joint action of pheide a oxygenase(PaO) and RCC reductase (RCCR) without release of theintermediate, RCC. Relevant carbon atoms and the a-methinebridge that is cleaved by PaO are labeled. R1, R2 and R3 in theNCCs indicate species-speciﬁc diﬀerences (Kra¨utler 2003). b Leavesfrom wild-type maize at a senescent stage (left) and from lls1showing a lesion mimic phenotype (right)192bidopsis accelerated cell death 1 (acd1; Greenberg andAusubel 1993) and maize lethal leaf spot 1 (lls1; Grayet al. 1997), in which the maize homologue of AtPaO isaﬀected (Fig. 1b), and Arabidopsis acd2, which isdefective in RCCR (Greenberg et al. 1994; Mach et al.2001). All of these mutants develop cell death lesions ontheir leaves in an age-dependent fashion. The phenotypeis similar to the induction of defense reactions in path-ogen resistance; thus, respective mutants are termed le-sion mimic mutants (Mach et al. 2001). The aﬀectedgenes were believed to be involved in a cell death sup-pression mechanism either directly (aﬀecting a signalcascade?) or through, for example, the removal of toxicmolecules (Greenberg and Ausubel 1993; Gray et al.1997). In favor of the latter was the ﬁnding that in lls1 acell death-inducing signal was derived from plastids andlesion formation was light dependent (Gray et al. 2002).Indeed, both PaO and RCCR mutants accumulate Chlcatabolites (pheide a in lls1 and acd1, and RCC in acd2)upon dark-induced senescence. In addition, the contentof these catabolites positively correlates with cell deathprogression of the respective mutants (Pruzˇinska´ et al.2003; A. Pruzˇinska´ and S. Ho¨rtensteiner, unpublishedresults). Thus, in these mutants, the accumulation ofphotoreactive Chl catabolites can be suggested to causethe production of reactive oxygen species that in turninduce cell death (Mach et al. 2001). Surprisingly, othermutants have been described that do not develop anapparent cell death phenotype although they also accu-mulate pheide a due to reduced PaO activities (Vicentiniet al. 1995; Thomas et al. 1996). Besides pheide a, thesemutants also accumulate chlorophyllides, indicating thatthey have a genetic defect that is diﬀerent from acd1 orlls1. On the other hand, several lesion mimic mutantshave been identiﬁed that are aﬀected in genes of Chlbiosynthesis (Hu et al. 1998; Meskauskiene et al. 2001;Ishikawa et al. 2001).Altogether, it can be concluded that functional Chlmetabolism, i.e. biosynthesis, turnover and degradation,is important to prevent the accumulation of photody-namic intermediates. Furthermore, it becomes obviousthat Chl degradation via the PaO/RCCR pathway is avitally important process during plant senescence. Quitelikely it is also involved in cellular responses to a varietyof stresses that are linked to Chl breakdown, such as thehypersensitive reaction. The photodynamic properties ofChl enable the conversion of light energy to chemicalenergy during photosynthesis, but during senescence,photodynamism may turn into a threat. Thus, parallel tothe ‘‘invention’’ of Chl and the evolution of oxygenicphotosynthesis, plants evolved a mechanism for thedetoxiﬁcation of Chl. In unicellular photosynthesizers,such as Chlorella protothecoides, RCCR is absent and,consequently, RCC-like compounds are excreted intothe medium (Engel et al. 1991; Ho¨rtensteiner et al.2000a). However, the development of multicellularplants required, in addition to PaO, the appearance ofRCCR to enable the safe disposal of Chl catabolitesinside the vacuole.ReferencesCalibe A, Grimm R, Kaiser G, Lu¨beck J, Soll J, Heins L (1997)The chloroplastic protein import machinery contains a Rieske-type iron–sulfur cluster and a mononuclear iron-binding pro-tein. EMBO J 16:7342–7350Engel N, Jenny TA, Mooser V, Gossauer A (1991) Chlorophyllcatabolism in Chlorella protothecoides. 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The loss of green color during chlorophyll degradation—a prerequisite to prevent cell death?

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The loss of green color during chlorophyll degradation—a prerequisite to prevent cell death?

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