The retrograde signaling protein GUN1 regulates tetrapyrrole biosynthesis Takayuki Shimizua, Sylwia M. Kacprzakb, Nobuyoshi Mochizukic, Akira Nagatanic, Satoru Watanabed, Tomohiro Shimadae,f, Kan Tanakae, Yuuki Hayashia, Munehito Araia, Dario Leisterg, Haruko Okamotob,h,1, Matthew J. Terryb,h, and Tatsuru Masudaa,2 aGraduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, 153-8902 Tokyo, Japan; bSchool of Biological Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom; cGraduate School of Science, Kyoto University, Sakyo-ku, 606-8502 Kyoto, Japan; dDepartment of Bioscience, Tokyo University of Agriculture, Setagaya-ku, 156-8502 Tokyo, Japan; eLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan; fSchool of Agriculture, Meiji University, Kawasaki-shi, 214-8571 Kanagawa, Japan; gPlant Molecular Biology, Faculty of Biology, Ludwig Maximilians Universität München, D-82152 Planegg-Martinsried, Germany; and hInstitute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom Edited by Krishna K. Niyogi, University of California, Berkeley, CA, and approved October 22, 2019 (received for review July 2, 2019) The biogenesis of the photosynthetic apparatus in developing photosynthesis-related genes (5–7). The genomes uncoupled seedlings requires the assembly of proteins encoded on both (gun) mutants that have a reduced ability to coordinate this nuclear and chloroplast genomes. To coordinate this process there nuclear response to chloroplast status were identified through needs to be communication between these organelles, but the the retention of LHCB1.2 expression after an inhibitory NF retrograde signals by which the chloroplast communicates with treatment (8) and have been the basis for retrograde signaling the nucleus at this time are still essentially unknown. The Arabi- research for the last 25 y. Of the original 5 gun mutants de- dopsis thaliana genomes uncoupled (gun) mutants, that show el- scribed, gun2 and gun3 lack a functional heme oxygenase 1 and evated nuclear gene expression after chloroplast damage, have phytochromobilin synthase (9), gun4 led to the identification of formed the basis of our understanding of retrograde signaling. the Mg-chelatase regulator GUN4 (10), and gun5 was mutated in Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis the gene encoding the H subunit of Mg-chelatase (SI Appendix, proteins and this has led to the development of a model for Fig. S1) (9). The GUN1 protein, which is a pentatricopeptide chloroplast-to-nucleus retrograde signaling in which ferrochelatase repeat (PPR) protein with a small MutS-related (SMR) domain, 1 (FC1)-dependent heme synthesis generates a positive signal pro- is also localized in the chloroplast (5). moting expression of photosynthesis-related genes. However, the In addition to these chloroplast-localized proteins, another molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying class of gun mutant has been identified which lacks nuclear- hypothesis for chloroplast-to-nucleus signaling. Here, we show that localized components of the signaling pathway. Screens for a GUN1 directly binds to heme and other porphyrins, reduces flux gun mutant phenotype identified multiple alleles of the blue light through the tetrapyrrole biosynthesis pathway to limit heme and photoreceptor cryptochrome 1 (11) and also suggested a role for protochlorophyllide synthesis, and can increase the chelatase activ- the red light photoreceptor phytochrome B and the transcription ity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabo- Significance lism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway. The signaling pathway between chloroplasts and the nucleus (retrograde signaling) is important for the correct development retrograde signaling | chloroplast | heme | tetrapyrrole | gun mutants of the photosynthetic apparatus of plant seedlings. The path- way is still not understood, but the majority of mutants with hloroplasts retain their own reduced genome that encodes altered signaling (gun mutants) implicate the tetrapyrrole Cfor protein subunits that are required for the functional as- molecule heme in this process. In this article, we have dem- sembly of all of the major photosynthetic complexes (1). How- onstrated that the major retrograde signaling protein GUN1 ever, the majority of photosynthetic proteins are encoded by the can bind tetrapyrroles and regulate the flow through the tet- nuclear genome and imported into the chloroplast. Thus, the rapyrrole biosynthesis pathway. The results support a role for efficient development of chloroplasts and the photosynthetic tetrapyrroles in mediating retrograde signaling and open up apparatus requires coordination between the nuclear and chlo- the opportunity to develop a unifying hypothesis for this roplast genomes and this is achieved through reciprocal signaling pathway that takes account of all identified gun mutants. between the organelles. The nucleus controls the expression of many chloroplast proteins, including those responsible for reg- Author contributions: M.J.T. and T.M. designed research; T. Shimizu, S.M.K., N.M., T. Shimada, K.T., Y.H., M.A., and T.M. performed research; T. Shimizu, S.M.K., A.N., ulation of transcription of the chloroplast genome, via antero- S.W., T. Shimada, K.T., Y.H., M.A., D.L., H.O., M.J.T., and T.M. analyzed data; and N.M., grade signaling pathways (1). Information on chloroplast status M.J.T., and T.M. wrote the paper. can in turn regulate nuclear gene expression via retrograde sig- The authors declare no competing interest. naling pathways (2). These signaling pathways are termed bio- This article is a PNAS Direct Submission. genic retrograde signaling (3) to distinguish them from the This open access article is distributed under Creative Commons Attribution-NonCommercial- extensive role that signals from mature chloroplasts (termed NoDerivatives License 4.0 (CC BY-NC-ND). operational retrograde signaling) play in the response of plants Data deposition: All plasmid constructs are available from Addgene (accession IDs to the environment (2, 4). The expression of a large number of 136357–136363). nuclear genes encoding chloroplast proteins is dependent on the 1Present address: School of Life Sciences, University of Sussex, Brighton BN1 9QG, United presence of functional chloroplasts and mutations in genes af- Kingdom. fecting chloroplast function or treatments with inhibitors such as 2To whom correspondence may be addressed. Email: [email protected]. norflurazon (NF), an inhibitor of the carotenoid biosynthesis This article contains supporting information online at https://www.pnas.org/lookup/suppl/ enzyme phytoene desaturase, or the plastid translation inhibitor, doi:10.1073/pnas.1911251116/-/DCSupplemental. lincomycin (Lin), result in the strong down-regulation of many First published November 15, 2019. 24900–24906 | PNAS | December 3, 2019 | vol. 116 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1911251116 Downloaded by guest on September 24, 2021 factor ELONGATED HYPOCOTYL 5 (HY5) (11). There is a and support a role for tetrapyrroles as mediators of a single well-established link between light and retrograde signaling (12, biogenic chloroplast-to-nucleus retrograde signaling pathway. 13) and the involvement of these components reflects this. Other signaling components that have been proposed to have a role in Results biogenic retrograde signaling, such as PHD TRANSCRIPTION GUN1 Levels Affect Tetrapyrrole Metabolism. To explore the in- FACTOR WITH TRANSMEMBRANE DOMAINS 1 (PTM1) teraction of GUN1 with tetrapyrrole biosynthesis, we first tested and ABSCISIC ACID INSENSITIVE 4 (ABI4) have not stood whether GUN1 could alter the flow through the tetrapyrrole up to scrutiny as other groups have not been able to reproduce pathway (31). We performed these experiments using dark-grown the gun phenotype of the respective mutants (14, 15). However, seedlings as GUN1 is rapidly degraded in white light (26) and also overexpression of GOLDEN2-LIKE1, a regulator of chloroplast because we hoped that the absence of the significant changes in development (16), does cause a gun phenotype (13, 17). tetrapyrrole flow in white light would permit us to observe any The analysis of gun2-gun5 resulted initially in the hypothesis GUN1-dependent changes. Fig. 1A shows that feeding the pre- that Mg-protoporphyrin (MgProto) is a mobile retrograde signal cursor ALA to two gun1 mutant alleles in the dark resulted in between the chloroplast and the nucleus (18), but this hypothesis increased accumulation of protochlorophyllide (Pchlide) com- was not supported in further studies in which no correlation was pared to wild type (WT), while seedlings overexpressing GUN1 observed between MgProto levels and LHCB gene expression (24) had reduced accumulation of Pchlide (Fig. 1A). Small dif- (19–21). Instead, the identification of a dominant gun6 mutant ferences were also observed in the absence of added ALA (SI with increased ferrochelatase 1 (FC1) activity (22) led to the Appendix,Fig.S2B) consistent with previous observations of in- proposal that heme synthesized by FC1 is either the signal itself creases in Pchlide synthesis in a gun1 mutant