Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation Michael Moulin*, Alex C. McCormac†‡, Matthew J. Terry†, and Alison G. Smith*§ *Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom; and †School of Biological Sciences, University of Southampton, Boldrewood Campus, Southampton SO16 7PX, United Kingdom Edited by Diter von Wettstein, Washington State University, Pullman, WA, and approved August 8, 2008 (received for review April 2, 2008) Chloroplast biogenesis involves careful coordination of both plas- GTS HEMA GSA tid and nuclear gene expression, which is achieved in part by Glu ALA ALAD retrograde signaling from the chloroplast to the nucleus. This can FLU be demonstrated by the fact that the herbicide, Norflurazon (NF), PBGD which causes bleaching of chloroplasts, prevents the light induc- UROS UPM tion of photosynthesis-related genes in the nucleus. It has been UROD SIRB proposed that the tetrapyrrole pathway intermediate Mg-proto- CPO porphyrin IX acts as the signaling molecule in this pathway and Siroheme PPO accumulates in the chloroplasts and cytosol of the cell after NF treatment. Here we present data that demonstrate that this model Proto IX CHLD FC CHLH GUN4 is too simplistic. We have developed a sensitive liquid chromatog- CHLI HY2 HO raphy-mass spectrometry (LC/MS) method to measure tetrapyrrole PΦB Heme Mg-proto intermediates and have shown that no Mg-protoporphyrin IX, nor CHLM indeed any other chlorophyll-biosynthesis intermediate, can be Mg-proto ME detected in NF-treated plants under conditions in which nuclear CRD1 gene expression is repressed. Conversely when endogenous Mg- DVR protoporphyrin IX levels are artificially increased by supplemen- MV Pchlide DV Pchlide tation with the tetrapyrrole precursor, 5-aminolevulinic acid, the POR POR CAO DVR expression of nuclear-encoded photosynthetic genes is induced, Chlide b MV Chlide a DV Chlide not repressed. We also demonstrate that NF-treatment leads to a CHLG CHLG strong down-regulation of tetrapyrrole biosynthesis genes, con- Chlorophyll b Chlorophyll a sistent with the absence of an accumulation of tetrapyrrole inter- mediates. Finally, there is no correlation between nuclear-gene Fig. 1. The tetrapyrrole pathway in plants showing intermediates and genes expression and any of the chlorophyll biosynthetic intermediates analyzed in this study. Intermediates: Glu, glutamate; ALA, 5-aminolevulinic over a range of growth conditions and treatments. Instead, it is acid; Proto IX, protoporphyrin IX; Mg-proto, Mg-protoporphyrin; Mg-proto ME, Mg-protoporphyrin monomethyl ester; DV Pchlide, divinyl protochloro- possible that a perturbation of tetrapyrrole synthesis may lead to phyllide; MV Chlide, monovinyl chlorophyllide. Table S1 lists the enzymes that localized ROS production or an altered redox state of the plastid, correspond to the gene names. which could mediate retrograde signaling. he tetrapyrrole biosynthetic pathway leads to the synthesis of and hy2) are deficient in heme oxygenase and phytochromobilin Ta number of important products including the chlorophylls synthase respectively (8) (Fig. 1). Additional tetrapyrrole-related and hemes. The enzymatic steps of the pathway are well char- gun mutants have also been identified (10, 11). acterized (Fig. 1) (1–3), and genes for virtually all of the enzymes The observation that many gun mutants are impaired in have been identified in higher plants. The pathway is tightly tetrapyrrole biosynthesis led to the suggestion that one or more regulated to ensure a continuous cofactor supply to the cognate intermediates in the pathway might themselves act as signaling apoproteins whilst avoiding the phototoxic accumulation of molecules in plastid-nucleus signaling. Lesions in the pathway intermediates (3,4). This is exemplified by the coordination of that give rise to a gun phenotype would all be expected to be chlorophyll synthesis with the production of light-harvesting compromised in the accumulation of Mg-protoporphyrin IX chlorophyll proteins (LHCs) encoded by the nucleus, which is in (Mg-proto). Strand, et al. (11) reported that Mg-proto accumu- part mediated by retrograde signals from the chloroplast to the lated in wild-type (WT) plants after an NF treatment, but nucleus. These signals can be observed following treatment with accumulation was reduced in gun mutants. In addition, applica- the herbicide Norflurazon (NF), which causes photooxidative damage of chloroplasts in white light (WL) and leads to a dramatic reduction in the expression of Lhcb and other nuclear- Author contributions: M.M., A.C.M., M.J.T., and A.G.S. designed research; M.M. and A.C.M. encoded photosynthesis-related genes (5,6). A screen for Ara- performed research; M.M. contributed new reagents/analytic tools; M.M., A.C.M., M.J.T., bidopsis mutants defective in the response to NF revealed the and A.G.S. analyzed data; and M.M., M.J.T., and A.G.S. wrote the paper. involvement of the tetrapyrrole pathway. In the original screen The authors declare no conflict of interest. a total of five nonallelic gun (genomes uncoupled) mutants were This article is a PNAS Direct Submission. identified, in which Lhcb1.2 was up-regulated in the light in the ‡Present address: Mambo-Tox Ltd, Southampton University Science Park, Southampton, presence of NF. The gun1 mutant lacks a chloroplast-localized SO16 7NP, United Kingdom. pentatricopeptide repeat protein (7), whereas the other four §To whom correspondence should be addressed. E-mail: [email protected]. mutants are defective in the tetrapyrrole pathway: gun5 has a This article contains supporting information online at www.pnas.org/cgi/content/full/ mutation in CHLH, a subunit of Mg-chelatase (8), gun4 lacks a 0803054105/DCSupplemental. regulator of Mg-chelatase (9), and gun2 and gun3 (allelic to hy1 © 2008 by The National Academy of Sciences of the USA 15178–15183 ͉ PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803054105 Downloaded by guest on September 23, 2021 Table 1. Estimation of tetrapyrroles in aerial tissue of Arabidopsis WT and gun5 seedlings determined by LC/MS pmol⅐gϪ1 FW Treatment Proto IX Mg-proto Mg-proto ME DV-Pchilde MV-Pchlide DV-Chlide MV-Childe 1 ϩ ALA Dk WT 165 Ϯ 74 47 Ϯ 22 280 Ϯ 181 2450 Ϯ 1394 2116 Ϯ 1362 200 Ϯ 35 287 Ϯ 124 gun5 313 Ϯ 115 21 Ϯ 597Ϯ 45 1996 Ϯ 1272 1170 Ϯ 845 318 Ϯ 82 294 Ϯ 141 2Dk WT 32 Ϯ 9 ND ND 510 Ϯ 218 1172 Ϯ 600 79 Ϯ 14 172 Ϯ 45 gun5 39 Ϯ 11 ND ND 354 Ϯ 121 151 Ϯ 72 109 Ϯ 15 99 Ϯ 12 324hWL WT ND ND ND 137 Ϯ 49 358 Ϯ 175 2098 Ϯ 1155 3421 Ϯ 878 gun5 Present ND ND 51 Ϯ 16 201 Ϯ 114 1436 Ϯ 812 654 Ϯ 105 4 ϩ ALA ϩ NF Dk WT 101 Ϯ 53 47 Ϯ 22 108 Ϯ 54 2355 Ϯ 1318 1390 Ϯ 889 58 Ϯ 25 243 Ϯ 131 gun5 410 Present 71 2614 1565 136 401 5 ϩ NF Dk WT Present ND ND 481 Ϯ 217 689 Ϯ 357 219 Ϯ 56 250 Ϯ 73 gun5 Present ND ND 536 Ϯ 335 226 Ϯ 144 79 Ϯ 10 184 Ϯ 110 6 ϩ NF 24 h WL WT ND ND ND 7 Ϯ 15Ϯ 113Ϯ 573Ϯ 36 gun5 ND ND ND 14 Ϯ 85Ϯ 125Ϯ 911Ϯ 5 Seedlings were grown for4dinthedark (Dk) or3dinthedark then 24 h in light (WL). For treatments 4–6, seedlings were grown with 5 ␮M NF. ALA (1 mM) was applied to the seedlings 16 h before harvest for treatments 1 and 4. Values are the mean of at least four independent experiments Ϯ standard deviation, except for gun5 under treatment 4. The limit of sensitivity for accurate quantification is equivalent to 20 pmol⅐gϪ1 FW. Present, compound identified unambiguously by the mass of the major ion and its fragmentation pattern but below the quantification threshold; ND, correct mass/fragmentation pattern not detected. tion of exogenous Mg-proto to protoplasts inhibited Lhcb1.2 use a method that could identify the compounds unambiguously, expression, whereas porphobilinogen, protoporphyrin IX (Proto and that was both reproducible and sensitive. The analysis of IX) and heme had no effect. These results supported previous tetrapyrroles has conventionally been carried out by HPLC observations that manipulation of Mg-proto levels using inhib- separation followed by either spectrophotometric or fluorimet- itors affected expression of photosynthesis-related genes (12, ric detection, but the work-ups necessary for this analysis can 13). Similarly, barley Mg-chelatase mutants xantha-f, -g and -h introduce errors in quantification, and often the different inter- expressed Lhcb genes in the presence of NF in the light, whereas mediates coelute so that they cannot easily be distinguished from the barley xantha-l mutant, defective in a later enzyme, Mg-proto one another (18). We have developed a method for detection by monomethylester cyclase (CRD1) (Fig. 1), did not (14). The tandem MS with an ion-trap instrument, which generates unique chlm mutant of Arabidopsis, lacking Mg-proto methyltrans- fragmentation patterns that are diagnostic for the individual ferase, accumulated high levels of Mg-proto and also showed severe repression of Lhcb expression in the light (15). Most molecules, so permitting unequivocal identification (17). All of recently a report by Ankele, et al. (16) described in vivo the intermediates from Proto IX to monovinyl chlorophyllide visualization of Mg-proto and its export from chloroplasts to the (MV-Chlide) can be detected [supporting information (SI) Fig. cytosol following NF treatment. S1], and quantified by reference to standard curves of known PLANT BIOLOGY However, there are difficulties with a model that proposes amounts of the compound (Fig.