COMMENTARY Surprising roles for bilins in a green alga Jean-David Rochaix1 Departments of Molecular Biology and Plant Biology, University of Geneva,1211 Geneva, Switzerland

It is well established that the origin of plastids whichservesaschromophoreofphyto- can be traced to an endosymbiotic event in chromes (Fig. 1). An intriguing feature of which a free-living photosynthetic prokaryote all sequenced chlorophyte genomes is that, invaded a eukaryotic cell more than 1 billion although they lack phytochromes, their years ago. Most from the intruder genomes encode two HMOXs, HMOX1 were gradually transferred to the host nu- andHMOX2,andPCYA.InPNAS,Duanmu cleus whereas a small number of these genes et al. (6) investigate the role of these genes in were maintained in the plastid and gave the green alga Chlamydomonas reinhardtii rise to the plastid genome with its associated and made unexpected findings. protein synthesizing system. The products of Duanmu et al. first show that HMOX1, many of the genes transferred to the nucleus HMOX2, and PCYA are catalytically active were then retargeted to the plastid to keep it and produce bilins in vitro (6). They also functional. Altogether, approximately 3,000 demonstrate in a very elegant way that these nuclear genes in plants and algae encode proteins are functional in vivo by expressing plastid proteins, whereas chloroplast ge- a cyanobacteriochrome in the chloroplast Fig. 1. Tetrapyrrole biosynthetic pathways. The nomes contain between 100 and 120 genes of C. reinhardtii,where,remarkably,the and chlorophyll biosynthetic pathways diverge at pro- (1). A major challenge for eukaryotic pho- photoreceptor is assembled with bound toporphyrin IX (ProtoIX). The negative feedback of heme tosynthetic organisms is to coordinate phytochromobilin chromophore. They fur- on GTR (Glu-tRNA reductase) is indicated. In C. rein- these two genetic systems during chloro- ther show that HMOX1 and PCYA are chlo- hardtii, conversion of protochlorophyllide (PChlide) to chlorophyllide (Chlide) can occur in a light-dependent plast development and to maintain plastids roplast whereas the animal-type (marked as “L”) and a light-independent manner (“D”). In functional under changing environmental HMOX2 is membrane-bound in the cytosol. the study of Duanmu et al. (6), the pathway is perturbed conditions. The situation is even more com- The existence of this HMOX associated with by introducing mammalian (BVR) plex with the existence of a third genetic cytosolic membranes in C. reinhardtii and for heme depletion. The activity of HMOX1 and PCYA system in plant and algal mitochondria, is demonstrated by introducing cyanobacteriochrome otherchlorophytesisrathersurprisingbe- (CBCR) reporter and by showing that it covalently binds which will not be discussed here. Although cause enzymes of this type are absent from PCB chromophore. The different steps affected in the gun many nuclear genes have been identified streptophyte algae and land plants. and hy mutants are indicated. ALA, δ-aminolevulinic acid; that are involved in chloroplast ex- To assess the role of HMOX1 and Chl, chlorophyll; CHL H/D/I, Mg-chelatase, subunit H/D/I; pression, the nucleus is also capable of sens- FC, Fe-chelatase; GSA, glutamate 1-semialdehyde; PCYA, HMOX2, the authors isolated mutants un- phytochromobilin synthase. ing the state of the chloroplast and to react able to produce either of these two to maintain chloroplast homeostasis. This enzymes. They show that, under conditions process is called retrograde signaling and of iron starvation, growth of the WT, but reinhardtii is able to produce chlorophyll in involves signals originating from the plastid not of the or hmox2 mutants, can be the dark, but at a lower level than in the light. and transmitted to the nucleus, where rescued by adding hemin to the growth These results point to an important role of they elicit a specific transcriptional response. medium, indicating that HMOX1 and bilins. Heme is known to exert a negative In recent years, several retrograde signaling feedback on chlorophyll synthesis, which HMOX2 are able to degrade exogenously fi pathways have been identified mostly supplied heme for iron acquisition. It is explains why chlorophyll de ciency occurs through genetic approaches (2, 3). Most likely that HMOX2 originates from the an- in land plants lacking HMOX. If this expla- of the mutants isolated, called gun mutants, cient eukaryotic host that was invaded dur- nation is also valid for the C. reinhardtii are affected in the tetrapyrrole biosynthetic ing endosymbiosis. This gene has been hmox1 mutant, Duanmu et al. reason that pathway (4, 5). In plants and algae, this maintained in chlorophyte but not in strep- the opposite effect would be expected if the pathway gives rise to heme and chloro- tophyte algae, perhaps because the former level of heme is decreased (6). This was phyll, with a branching point at the level more often experience iron limitation. achieved by expressing, in WT cells, a mam- malian biliverdin reductase, an that of protoporphyrin IX (Fig. 1). This com- A striking result of this study is that α pound is then converted to protoheme and photoautotrophic growth of C. reinhardtii converts biliverdin IX (BV) into bilirubin Mg-protoporphyrin IX by Fe- and Mg- cells is compromised in the absence of and phytocyanobilin (PCB) into phycocya- chelatase, respectively. In turn, heme acts HMOX1 whereas heterotrophic growth norubin, and is thereby expected to deplete as prosthetic group for a large number of in the dark is unaffected, and, moreover, hemoproteins, but a portion of heme is thethreefoldincreaseofchlorophyllthat Author contributions: J.-D.R. wrote the paper. converted by (HMOX) occurs upon a shift of WT cells from the The author declares no conflict of interest. to biliverdin IXa and by phytochromobilin dark to the light is not observed with the See companion article on page 3621. synthase (PCYA) to phytochromobilin, hmox1 mutant. In contrast to land plants, C. 1E-mail: [email protected].

3218–3219 | PNAS | February 26, 2013 | vol. 110 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1300399110 Downloaded by guest on September 28, 2021 the heme pool (Fig. 1). However, in this accompanied by a sudden increase in ox- coordination of with COMMENTARY case, contrary to the heme feedback hy- ygen evolution and, as Duanmu et al. chloroplast development (5). pothesis, the chlorophyll level decreased suggest,bythereleaseofhemefrom The results of this work raise a number of during a dark-to-light shift, raising the damaged hemoproteins (6). In contrast, questions. Do chlorophyte species lacking possibility that BV or PCB is responsible in the light, the plastid-derived bilins ap- phytochromes possess an alternative regula- for the light-dependent accumulation of pear to have an inhibitory action on nu- tory bilin-based system for regulating chlo- chlorophyll. This proposal is in agreement clear genes involved in photosynthesis. rophyll synthesis in the light? If so, how does with feeding experiments with biliverdin in Altogether, these considerations raise it work? Are there unknown PCB-based which the WT chlorophyll level in hmox1 the possibility that bilins are part of a ret- photoreceptors in these algae? Was the bilin was restored even though photoautotro- rograde signaling pathway that evolved in biosynthetic pathway uniquely maintained phic growth was only partially rescued. C. reinhardtii and more generally in for ensuring smooth daily transitions from However, how the bilins influence chloro- chlorophytes for the detoxification of re- dark to light with minimal photodamage? phyll accumulation is not yet clear. active oxygen species generated during How widely distributed is this bilin-medi- To further examine the role of bilins in the the transition from dark to light. Heme ated retrograde signaling pathway among regulation of chlorophyll synthesis during a has been proposed previously as positive oxygenic photosynthetic organisms with or dark/light transition, Duanmu et al. (6) per- retrograde signal for the regulation of nu- without phytochromes? Clearly, future re- form a global comparative transcriptomic clear gene expression in C. reinhardtii (7) search in this area is likely to provide new analysis with WT and hmox1 cells in the and recently also in Arabidopsis for the answers to these intriguing questions. presence or absence of biliverdin. Several im- portant results emerged from this study. First, a small set of 76 nuclear genes was identified 1 Martin W, et al. (2002) Evolutionary analysis of Arabidopsis, 5 Woodson JD, Perez-Ruiz JM, Chory J (2011) Heme synthesis by cyanobacterial, and chloroplast genomes reveals plastid phylogeny plastid ferrochelatase I regulates nuclear gene expression in plants. that are up-regulated by bilins in a light-in- and thousands of cyanobacterial genes in the nucleus. Proc Natl Curr Biol 21(10):897–903. dependent way. These genes include several Acad Sci USA 99(19):12246–12251. 6 Duanmu D, et al. (2013) Retrograde bilin signaling enables 2 Nott A, Jung HS, Koussevitzky S, Chory J (2006) Plastid-to-nucleus Chlamydomonas greening and phototrophic survival. Proc Natl Acad oxygen-dependent redox enzymes (mono- – retrograde signaling. Annu Rev Plant Biol 57:739 759. Sci USA 110:3621–3626. 3 and dioxygenases, proteins containing re- Pogson BJ, Woo NS, Förster B, Small ID (2008) Plastid signalling to 7 von Gromoff ED, Alawady A, Meinecke L, Grimm B, Beck CF – dox cofactors and enzymes associated with the nucleus and beyond. Trends Plant Sci 13(11):602 609. (2008) Heme, a plastid-derived regulator of nuclear gene expression 4 Susek RE, Ausubel FM, Chory J (1993) Signal transduction mutants in Chlamydomonas. Plant Cell 20(3):552–567. oxidative amino acid metabolism). A shift of Arabidopsis uncouple nuclear CAB and RBCS gene expression from of an algal culture from dark to light is chloroplast development. Cell 74(5):787–799.

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