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. S2). The threshold of sensitivity elevated Mg-proto as a signaling molecule leading to the repres- is 200 fmol injected, although it is possible to detect the presence sion of nuclear genes, including that this intermediate is photo- of much lower levels from the diagnostic fragmentation pattern. toxic. Additionally, levels of Mg-proto and the repression of Table 1 (treatment 1) shows the level of tetrapyrrole inter- Lhcb expression are not always correlated. For example chlI mediates present in aerial tissues of 4-d-old dark-grown Arabi- mutants of Arabidopsis do not have a gun phenotype (8), even dopsis seedlings incubated in 1 mM ALA for 16 h, a treatment though they are compromised in Mg-proto production. More- that causes their accumulation. All of the chlorophyll branch over, in barley seedlings treated with NF, where Lhcb expression intermediates from Proto IX were detectable, including both is reduced, no porphyrins accumulated (14). One explanation for divinyl (DV)- and MV-protochlorophyllide (Pchlide), indicating these discrepancies is the difficulty in measuring tetrapyrrole that the order of enzymic steps is not fixed (18) (see Fig. 1). The intermediates quantitatively in plant material. To overcome this, presence of low levels of Chlide in this tissue was most likely we developed a novel liquid chromatography/mass spectrometry because the sampling and extraction was carried out under green (LC/MS) method that enables targeted metabolite profiling of safelight, which allowed some photoconversion, but the amount tetrapyrroles in seedlings. Each tetrapyrrole can be distinguished present was always less than 15% that in tissue harvested after by retention time and fragmentation signature, thus allowing unambiguous identification (17). Using this method, we have 2 min exposure to WL (data not shown). The same observation re-examined the proposed role of Mg-proto (and other chloro- has been previously observed but never quantified (e.g., ref. 19). phyll intermediates) as signaling molecules. Our results call into In gun5 seedlings grown under the same conditions, Proto IX was question the simple model of Mg-proto accumulation acting to elevated compared to WT, whereas the levels of Mg-porphyrins repress nuclear gene expression. were reduced, as expected for a Mg-chelatase mutant (8). In 4d-old etiolated seedlings grown in the absence of ALA (Table Results 1, treatment 2) neither Mg-proto nor its methylester were Measurement of Tetrapyrrole Intermediates by LC/MS in Arabidopsis detectable, but the other intermediates were all present, albeit at Seedlings. To establish the profile of the tetrapyrrole interme- lower levels than in the ALA-treated plants. Illumination of diates in WT seedlings after NF treatment, it was important to 3-d-old untreated seedlings with WL for 24h (treatment 3) led to

Moulin et al. PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 ͉ 15179 Downloaded by guest on September 23, 2021 seedlings (Table 1, treatment 6). In contrast, in dark-grown NF-treated seedlings the tetrapyrrole profile was similar to that in untreated seedlings, both with and without ALA feeding (Table 1, treatments 4 and 5), demonstrating that NF does not inhibit tetrapyrrole synthesis per se, but rather the absence of carotenoids causes photodestruction of the tetrapyrroles. The inability to measure tetrapyrroles in the tissue was not due to an artifact during extraction, because when plant samples were spiked with 50 pmol/gFW of Proto IX or Mg-proto standard (Fig. S3E), most of the standard was recovered. Nor was there a transient accumulation of porphyrins that had disappeared by the time of sampling: within 15 min of illumination of dark- grown seedlings, the tissues photobleached due to the absence of photoprotective carotenoids. At the same time Proto IX disap- peared, and we could not detect this intermediate, Mg-proto or Mg-protoME over the following 24 h by either MS or fluores- cence (data not shown). To ensure that growth conditions were not responsible for our inability to detect tetrapyrroles in NF-treated material, we analyzed material that had been grown under different conditions, including replicating those used by Strand et al. (11), namely growth in continuous light in NF for Fig. 2. Analysis of tetrapyrroles by LC/MS. WT Arabidopsis seedlings were 6 d. We also tested whether inclusion of sucrose in the medium ␮ grown for3dincomplete darkness with or without 5 M NF, followed by 24 h had any effect. In all cases there was no accumulation of in WL. Pigments were extracted from aerial tissue and analyzed by LC/MS as Mg-proto or any other tetrapyrrole intermediate (data not described in Experimental Procedures.(A) PDA trace across the complete spectrum (200–800 nm) for the two samples (offset for clarity). Note that shown). Finally, when using the slightly different HPLC condi- the scale for the 5 ␮M NF sample has been greatly expanded compared to the tions of Strand et al. (11) we were still unable to detect control (no NF). The regions corresponding to the elution range for the porphyrins or carotenoids by absorbance or in the mass spectrum majority of porphyrins and carotenoids are indicated. (B) Total ion current in NF-treated seedlings (Fig. S4). (TIC) chromatogram of the same traces sampled from m/z 200-1000. The inset shows an enlargement of the region corresponding to the elution times for Norflurazon Treatment Has a Dramatic Effect on Tetrapyrrole Biosyn- porphyrins. (C) Fluorescence spectra for the same traces at the excitation and thesis Genes. To characterize further the effect of NF treatment emission wavelengths specific for Mg-proto (410 nm and 595 nm respectively). on the tetrapyrrole pathway we analyzed the expression of Also shown is the emission spectrum of 1 pmol Mg-proto standard. tetrapyrrole biosynthesis-related genes using Affymetrix full genome microarrays through the GARNet/NASC Affymetrix facility (see Experimental Procedures). Seedlings were germi- a rapid photoconversion of Pchlide to Chlide, levels of which ␮ then increased steadily, mirroring the increase in chlorophyll nated on 5 M NF in the dark and then exposed to WL for 3 d. For WT seedlings (black bars in Fig. 3A), the majority of genes (data not shown). In illuminated gun5 seedlings there were Ͼ Ϫ detectable amounts of Proto IX, whereas in WT there was none. were repressed, with the strongest repression ( 2 fold; 50% in Fig. 3A) shown for HEMA1, encoding the rate-limiting enzyme glutamyl-tRNA reductase, and the genes associated with the Effect of NF Treatment on Tetrapyrrole Pathway Intermediates. The Mg-porphyrin branch of the pathway (Fig. 1, Table S1). The LC/MS method described above can accurately and reproducibly trunk pathway for tetrapyrrole synthesis was also downregulated measure Proto IX and Mg-proto in plant tissue corresponding to (Fig. 3A). These data indicate that tetrapyrrole synthesis is 20 pmol/gFW, much less than the 6 nmol/gFW reported by regulated by plastid signals during seedling development in the Strand, et al. (11) to accumulate after NF treatment of Arabi- light. To verify the reliability of the array data, we conducted dopsis seedlings. We therefore used this method to analyze the ␮ real-time RT-PCR on 12 tetrapyrrole biosynthesis genes (Fig. porphyrin content of seedlings grown in the presence of 5 M 3B). In general, there was good agreement between the microar- NF in the dark for 3 d, then illuminated with WL for 24 h, ray data and the real-time PCR results with only occasional conditions under which nuclear gene expression is repressed (see discrepancies (Fig. 3B). As the microarray experiment was later). Fig. 2A compares the photodiode array (PDA) trace after conducted with seedlings grown in the presence of sucrose we HPLC of extracts from treated and untreated tissue. Note that also analyzed these tetrapyrrole biosynthesis genes in the ab- the PDA scale for the NF-treated sample has been expanded sence of sucrose after 24 h. The comparison of the two growth greatly. As expected, carotenoids, which elute at about 25–35 conditions shows that with the exception of CHLI, which only min, were absent in the NF-treated tissue, since NF inhibits the responds to signals in the presence of sucrose and over the longer carotenoid biosynthesis enzyme phytoene desaturase. However, time period, the response to NF was strikingly similar (Fig. 3B). porphyrins were also severely reduced. In Fig. 2B the mass To determine the effect of known plastid signaling pathways, spectrum in the range m/z 200-1000 is shown for the two samples. the impact of NF treatment in the gun1,5 double mutant was Again there is nothing in the region corresponding to porphyrins examined (8, 20). Overall on NF gene expression was always (18–26 min) (Fig. 2B and Inset). Specific query of the mass greater in gun1,5 than WT, but the increase varied (Fig. 3A). spectrum for individual tetrapyrroles found no Mg-proto or Interestingly, the genes that showed the strongest response to Proto IX present. Although small peaks at the corresponding gun1,5 are those known to be important regulatory points in the elution times were seen, these were below threshold and did not pathway. For example, the regulator GUN4 was strongly inhib- give the correct fragmentation pattern (Fig. S3 D and G). ited following NF treatment, but was expressed in gun1,5 on NF Similarly, Fig. 2C shows the fluorescence trace from the HPLC, at a similar level to WT in the absence of NF. The HEMA1 and with the excitation and emission wavelengths for Mg-proto; CHLH genes showed a similar response (Fig. 3). It should also there is virtually no signal in the NF-treated tissue. In fact, the be noted that in some cases expression of genes was greater in only tetrapyrroles that could be measured with confidence were gun1,5 compared to WT when both genotypes were grown in the DV-Pchlide and MV-Chlide, at 2–5% of those seen in untreated absence of NF. This has been seen before for HEMA1 (21), but

15180 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803054105 Moulin et al. Downloaded by guest on September 23, 2021 expression of other chloroplast tRNA synthetase genes was also maintained following NF treatment (data not shown). Three other major tetrapyrrole biosynthesis genes failed to show downregulation after NF treatment. HEMA2 and FC1 were both moderately up-regulated, as has been observed previously (22, 23). These two genes have been shown to be important for non-photosynthetic heme synthesis. Similarly expression of PPO2, encoding the mitochondrial isoform of protoporphyrino- gen IX oxidase, was also increased by NF. It is possible that the strong expression of HEMA2, PPO2, and FC1 is to ensure supply of cofactor for hemoproteins that may be required in response to the severe oxidative stress induced by NF treatment (23). In summary, although steady-state transcript levels may not be a direct representation of the levels of tetrapyrrole enzyme activity, these data are consistent with the observation that NF-treated seedlings have a severely reduced capacity to syn- thesize intermediates such as Mg-proto in the light.

Is There a Relationship Between Tetrapyrrole Intermediates and Nuclear Gene Expression? To investigate further the relationship between tetrapyrroles and nuclear gene expression we used ALA feeding in the dark to increase all of the chlorophyll biosynthesis intermediates including Mg-proto (Table 1). Analysis by RT- PCR of the expression of Lhcb1.1 and FC2 (Fig. 3) (22), showed that in both WT and gun5, there is an increase in steady state transcript levels in ALA-fed tissue rather than Mg-proto re- pressing gene expression (Fig. S5). We extended this analysis to a total of 16 different conditions with seedlings grown in either complete darkness, or light/dark cycles, and treated with NF, ALA, or both, and investigated if there was any correlation, either negative or positive, between these intermediates and the repression of nuclear gene expres- sion. The expression of Lhcb1.1 was determined by RT-PCR (Fig. S6), and levels of all seven chlorophyll biosynthetic inter- mediates were determined by LC/MS (Fig. S7). Fig. 4 shows a graphical representation of relative levels of Lhcb1.1 transcript under the different conditions compared with levels of each of the intermediates, expressed as a percentage of the values for seedlings grown in the dark without treatments. There is clearly no relationship between Mg-proto and Lhcb1.1 transcript abun- dance. Although in condition 16 there is no measurable Mg- proto and the Lhcb1.1 transcript is highest, under conditions 1–3 both transcript and metabolite levels are low. Similarly, in Fig. 3. The effect of NF treatment on expression of genes encoding the condition 10, when there is an extremely high level of Mg-proto, PLANT BIOLOGY tetrapyrrole pathway. (A) Data from full genome microarray analysis of WT there is still easily detectable Lhcb1.1 transcript. Moreover, none (dark bars) and gun1,5 double mutant (light bars) seedlings grown Ϯ 5 ␮MNF in the presence of 1.5% (wt/vol) supplementary sucrose. Following germina- of the other intermediates showed a convincing correlation with tion in D for 3 d, seedlings were allowed to de-etiolate under WL for 3 d. Data Lhcb1.1 expression. shown are the mean and range of two independent experiments. (B) Relative expression levels of 12 tetrapyrrole synthesis genes (see label above each Discussion panel) measured in WT and gun1,5 mutant seedlings grown Ϯ 5 ␮M NF. For Mg-Proto Does Not Accumulate after NF Treatment. Several studies each gene, top panels show data from seedlings grown identically to those in have identified Mg-proto as having the potential to be involved A, and bottom panels are for seedlings grown without sucrose for3dDand in chloroplast-to-nucleus communication, and this has become 1 d WL. Transcript levels were measured using real-time RT-PCR and corrected the dominant hypothesis (24). A central feature of this premise for equal cDNA template according to expression of 40S. Values shown are the was measurements in NF-treated seedlings, which demonstrated mean Ϯ SE from at least three independent experiments. A dark circle indi- cates the corresponding value in the microarray experiment shown in A. that Mg-proto levels increased to 6,380 pmol/gFW after NF treatment compared to 414 pmol/gFW in green WT seedlings, whereas other intermediates did not accumulate (11). Mg-proto has been reported to be visualized in vivo in NF-treated seed- is also apparent here for FC2 (encoding ferrochelatase-2), lings, but only after subsequence ALA feeding (16). CHLH, CHLD, and GUN4 (the first two in the absence of Reliable measurement of Mg-proto levels in plant cells is sucrose only), further supporting a regulatory role for plastid critical to the verification of this hypothesis and to this end, we signals in restricting tetrapyrrole synthesis during normal seed- developed an LC-MS approach that is equally sensitive, but more ling development in the light. reliable compared to conventional methods, in that compounds Not all genes were strongly down-regulated after NF treat- are identified unambiguously. Using this method the estimation ment. The GluTS gene showed a significant up-regulation. The of Mg-proto levels in dark-grown WT seedlings fed ALA (43 product of this enzyme, glutamyl-tRNAGlu, is also used for pmol/gFW) is in general agreement with data for light-grown chloroplast protein synthesis, suggesting that its response is tobacco leaves kept in darkness (40–50 pmol/gFW Mg-proto) driven by the need to synthesize new chloroplast proteins. The (25), and etiolated barley leaves fed ALA (250 pmol/gFW for

Moulin et al. PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 ͉ 15181 Downloaded by guest on September 23, 2021 more moderate response (29). It is known that the HEMA1 gene is under direct photoreceptor control while a more weakly light-regulated gene, GSA1, is not, and it was hypothesized previously (30) that the moderate light regulation seen for many tetrapyrrole genes may be mediated through plastid signaling from the developing chloroplasts. The observation here that most genes respond quite strongly to the loss of chloroplast integrity would be consistent with this hypothesis. One significant observation from the microarray data was that GluTS, HEMA2, PPO2, and FC1, genes encoding isoforms that have previously been implicated in having a major role in heme synthesis (22, 31) under stress conditions (23), were all induced following NF treatment. The UPM1 gene also showed strong induction under these conditions. This suggests a strong require- ment for siroheme in responding to stress. However, as the substrate for the enzyme encoded by UPM1, uroporphyrinogen III, is the first photosensitive tetrapyrrole, it is also possible that strong induction of UPM1 may have an immediate role in mitigating further photo-oxidative damage. One final possibility is that induction of siroheme may be part of the signaling cascade leading to sustained down regulation of nuclear gene expression. These hypotheses clearly require further testing.

Do Tetrapyrroles Have a Role in Plastid Signaling? The conclusion drawn from our data is that NF-induced downregulation of Fig. 4. Lack of correlation between tetrapyrrole intermediates and Lhcb expression. WT Arabidopsis seedlings were grown in a total of 16 different nuclear gene expression can occur without any accumulation of conditions, chosen to vary the level of tetrapyrrole intermediates and/or Mg-proto, and there is no correlation between levels of chloro- expression of Lhcb1.1. In stage 1 (I), seedlings were grown for4dincomplete phyll biosynthetic intermediates and Lhcb expression. A simple darkness (D) or in 3 d in the dark followed by 1 d in white (100 ␮mol⅐mϪ2⅐sϪ1) signaling mechanism in which the accumulation of a tetrapyrrole light (L) in the absence (Ϫ) or presence (ϩ)of5␮M NF. In stage 2 (II), seedlings intermediate inhibits nuclear gene expression is thus highly were subsequently treated for a further 16 h in complete darkness (D) or in unlikely. Indeed, time course studies showed that any effect of white light (L) in the absence (Ϫ) or presence (ϩ) of 1 mM ALA. Lhcb1.1 Mg-proto would be extremely short-lived and, from the data expression was monitored by RT-PCR and quantified relative to ACTIN (Fig. S6) presented here, light-dependent. This is not consistent with the with the values of the dark-grown untreated sample (condition 7) set as 100%. Tetrapyrrole intermediates were measured by LC/MS (Fig. S7). The values are proposal of a sustained accumulation of Mg-proto, whether in shown with increasing relative expression of Lhcb1.1. Data shown are mean the chloroplast or following transport to the cytosol (16). ϮSE (n Ն 2). Although the data presented here do not suggest an alternative model, we do note that one short-lived, light-dependent process might be the generation of ROS signals (32). It is conceivable Mg-proto and Mg-proto ME combined) (26). In contrast, we that signaling is via a specific Mg-proto-derived ROS, as under found that Mg-proto failed to accumulate after NF treatment, optimal conditions no Mg-proto appears to accumulate. How- and was well below the 20 pmol/gFW threshold for unambiguous ever, Mg-proto would be expected to generate singlet oxygen in identification and quantitation. The most likely explanation for the presence of light (32), and analysis of specific ROS marker the discrepancy between our results and those of Strand, et al. genes suggests that NF treatment generates a much more (11) is that the HPLC conditions used by the latter are not complex ROS signal during chloroplast destruction (A.C.M. and optimal for resolution of pigment molecules, so that the fluo- M.J.T., unpublished results). The effects of NF treatment on rescence they detected was due to contaminating substances. nuclear gene expression would therefore be unlikely to correlate Significantly, Mochizuki and colleagues have carried out a with an individual tetrapyrrole intermediate, but such a scenario simultaneous re-evaluation of the Mg-proto hypothesis using a may explain why signaling has been suggested to coincide with molecular genetic approach, and have similarly concluded that Mg-proto under some experimental conditions. Strand, et al. Mg-proto cannot itself be acting as a signaling intermediate (27). (11) reported that superoxide generation was not reduced in gun mutants compared to WT. Although details of the conditions The Chlorophyll Synthesis Pathway Is Strongly Downregulated after were not provided, this would suggest that this ROS species at NF Treatment. To understand why no accumulation of tetrapyr- least is not a major component of any plastid signal profile. There roles was observed following NF treatment, we examined tetra- is evidence for regulation of nuclear gene expression by both 1 pyrrole biosynthesis gene expression under these conditions. All H2O2 (33) and O2 (34) and a role for these ROS, perhaps in genes of the chlorophyll biosynthesis pathway were strongly combination, needs to be explored further. Alternatively, regu- inhibited by NF treatment, although the most severe effects were lation of nuclear gene expression in response to the redox state seen for those encoding the enzymes of the Mg-porphyrin of plastids is well established (35), and NF treatment and branch. This is consistent with the need to eliminate the synthesis photo-oxidative destruction of tetrapyrroles may impact on this of potentially dangerous Mg-porphyrins while retaining some pathway. capacity for heme synthesis to respond to the stress conditions Experimental Procedures imposed. There has always been a close link between the response to light and plastid signals (28). It is therefore inter- Plant Material and Growth Conditions. The Arabidopsis thaliana L. gun5 mutant and the gun1,5 double mutant both in the Columbia (Col-0) WT esting that nearly all of the tetrapyrrole genes responded strongly background have been described previously (8, 20). For pigment analysis seeds to NF while a previous study using a miniarray system showed were surface-sterilized and plated onto medium containing 0.8% agar and that only a small cohort of genes (HEMA1, CHLH, CRD1, and 1/2 ϫ MS salts Ϯ 5 ␮M NF. Plates were placed at 4 °C in darkness for 2 d followed CAO) responded strongly to light with most genes showing a by a 30 min light irradiation to synchronize germination. Seeds were germi-

15182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803054105 Moulin et al. Downloaded by guest on September 23, 2021 nated in darkness at 22 °C for 3 d, followed by 24 h illumination in 100 energy level, 70%). Data were analyzed using Xcalibur (Thermo Fisher Scien- ␮mol.mϪ2.sϪ1 WL. Alternatively, to reproduce the conditions of Strand, et al. tific). Quantification was carried out using the linear range of a standard curve (11), seedlings were placed in continuous WL for 6 d. For ALA treatment, after constructed with known amounts of porphyrins (Fig. S2). 3 d in complete darkness 1 mM ALA was added and seedlings were incubated in darkness for 16 h before harvesting. For microarray and real-time RT-PCR Gene Expression Analysis. For microarray analysis total RNA extraction was experiments, growth conditions (including growth medium, light sources and carried out as previously described (35) but with the addition of a further treatments) were exactly as described previously (21). purification step using the Qiagen RNeasy kit (Qiagen) according to manu- facturer’s instructions. Microarrays were carried out using the Affymetrix Liquid Chromatography/Mass Spectrometry (LC/MS) Analysis. For the extraction ATH1 Arabidopsis Genome Array (22K) by the GARNet/NASC facility. Further of pigments, seedlings were ground in liquid N2,20␮l(1␮M) deuteropor- details of the conditions used are available at http://affymetrix.arabidopsis. phyrin internal standard was added and the powder homogenized in info/narrays/experimentpage.pl?experimentidϭ51. Analysis of the normal- methanol:0.1M NH4OH (90:10, vol/vol). The extract was centrifuged at ized data were conducted using Microsoft Excel and Silicon Genetics Gene- 10,000 ϫ g for 10 min, the supernatant kept, and the pellet re-homogenized Spring®7 (Agilent Technologies) software packages. For real-time RT-PCR with acetone:0.1M NH4OH (80:20, vol/vol). The supernatants were combined analysis, total RNA extraction was also carried out as previously described (36) and the procedure repeated twice. The pooled supernatants were dried under but with an additional step for removal of polysaccharides as given in SI Text. N2 flow, resuspended in 100 ␮l acetone: 0.1M NH4OH (80:20, vol/vol) and then The gene-specific primer pairs used are shown in Table S2. centrifuged at 10,000 ϫ g for 10 min. The supernatant was collected and kept in darkness at 4 °C until analysis. ACKNOWLEDGMENTS. The gun mutants were gifts from Enrique Lo´pez-Juez LC/MS analysis was carried out essentially as described (17). The extract was (Royal Holloway College, London, United Kingdom) and Joanne Chory (Salk separated on a Finnigan Surveyor HPLC (conditions detailed in SI Text). Elution Institute for Biological Studies, La Jolla, CA). We thank Nobuyoshi Mochizuki (Kyoto University, Kyota, Japan) for providing information before publica- was followed by an online formic acid treatment (4 ␮l/min) and then MS with DECA tion. We also thank John Gray (University of Cambridge, Cambridge, U.K.) for a Finnigan LCQ XP mass spectrometer with an ESI source (Thermo Fisher the gift of the Norflurazon, and GARNet (NASC, Nottingham, U.K.) for pro- Scientific). Detection was in positive ion mode using SIM-MS (Single Ion viding the microarray service. This work was supported by the Biotechnology ϩ Monitoring-MS) of [MϩH ] ions with the m/z values given in Fig. S1 (MS-MS and Biological Sciences Research Council [Grant Number 51/P17214], Gatsby parameters: capillary temperature, 300 °C; capillary voltage, 3.0 kV; collision Charitable Foundation, and EU Framework Program 5.

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