GUN4 is required for efficient chlorophyll synthesis and photoautotrophic growth in Chlamydomonas reinhardtii

Mauro Ceol^1§, Cinzia Formighieri^2§, Manuela Mantelli², Jean-David Rochaix¹ and Roberto Bassi²

1Department of Molecular Biology and Department of Plant Biology, University of Geneva, 30 Quai Ernest-Ansermet, 1211 Geneva, Switerland

2Dipartimento Scientifico e Tecnologico, Universit di Verona, 15 Strada Le Grazie, 37234 Verona, Italy

§These authors contributed equally to this work.

In plants and algae tetrapyrrole synthesis occurs mainly in the chloroplast. This metabolic pathway leads to the synthesis of chlorophyll, heme, siroheme and phytochromobilin.

Contributions from many research groups led to the identification of all the enzymes involved in tetrapyrrole synthesis. At the same time the major regulatory mechanisms adopted by photosynthetic organisms to prevent photoxidative damage caused by the highly reactive intermediates produced in this pathway have been uncovered.

The precursor of all the tetrapyrroles, 5-aminolevulinic acid (ALA), is produced from glutamyl-tRNA by the subsequent action of two enzymes, glutamyl-glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase. ALA is then used to produce the cyclic tetrapyrrole protoporphyrin IX (ProtoIX), the last common precursor of chlorophyll, heme and phytochromobilin. ProtoIX can be used as a substrate by two enzymes, the iron chelatase (FeCh) and the magnesium chelatase (MgCh). The FeCh introduces a Fe²⁺ in the tetrapyrrolic ring to produce protoheme, the precursor of heme and phytochromobilin. In the other branch,

75 the MgCh leads to the production of Mg-protoporphyrin IX (MgProtoIX), the first specific precursor of chlorophyll (Masuda and Fujita, 2008; Tanaka and Tanaka, 2007).

MgCh is composed of three subunits, CHLI, CHLD and CHLH. CHLI (~45 kDa) is an AAA⁺ ATPase and contains a Mg²⁺ binding site (Jensen et al., 1998; Reid et al., 2003), CHLD (~83 kDa) is found stably associated with CHLI. Because CHLD contains an incomplete ATPase domain, it is not able to hydrolyze ATP (Jensen et al., 1998). The CHLH subunit is the larger of the three (~140 kDa) and can bind porphyrins (Karger et al., 2001). Thus it contains the active site for the chelation reaction. Recently another protein, GUN4, was reported to be associated with the MgCh, (Genome Uncoupled 4). This protein was identified in a genetic screen for mutants affected in chloroplast retrograde signaling (Larkin et al., 2003; Mochizuki et al., 2001;

Susek et al., 1993). GUN4 is a positive regulator of the activity of the MgCh and it has been shown to bind ProtoIX and Mg-Protoporphyrin IX (MgProtoIX). In vitro studies on the effect of GUN4 on MgCh activity showed that this protein leads to the full activation of the enzyme in the presence of physiological concentrations of Mg²⁺ and ProtoIX (Davison et al., 2005;

Verdecia et al., 2005).

The MgCh is the first enzyme committed specifically to chlorophyll synthesis and is subjected to various levels of regulation. Its activity is controlled by the redox state of the chloroplast.

The ATPase activity of the CHLI subunit is redox-dependent in vitro. Indeed it was shown to be inactive if fully oxidized by 50 µM CuCl2, oxidized CHLI re-activate after incubation with 5 mM DTT. Moreover reduction of a chloroplast extract was shown to stimulate the MgCh activity (Ikegami et al., 2007; Kobayashi et al., 2008). Other effectors that link the MgCh to the physiological state of the chloroplast are the concentration of stromal Mg²⁺ and the ATP/ADP ratio (Reid and Hunter, 2004). Interestingly, it was shown that upon illumination, the concentration of Mg²⁺ and the ATP/ADP ratio increase in the chloroplast (Ishijima et al., 2003;

Usuda, 1988). It is likely that the concurrent increase in these two parameters has a positive

effect on the MgCh activity, while the increase in the ATP/ADP ratio has a negative effect on the activity of the FeCh (Cornah et al., 2002).

Another important regulatory mechanism of the MgCh is the distribution of this enzyme in the chloroplast. The enzyme that catalyzes the oxidation of protoporphyrinogen IX to ProtoIX, protoporphyrinogen IX oxidase (PPOX), is membrane-associated in the chloroplast (Che et al., 2000). Of the two enzymes that can use ProtoIX, the FeCh has been reported to be stably associated with the chloroplast membranes (Roper and Smith, 1997; Suzuki et al., 2002), whereas the localization of the MgCh depends on at least two factors. The CHLD and CHLH subunits are membrane associated at a concentration of 5 mM Mg²⁺ and are soluble at a concentration of 1 mM Mg²⁺ (Gibson et al., 1996; Luo et al., 1999; Nakayama et al., 1998), the CHLI subunit seems to be soluble regardless of the Mg²⁺ concentration (Nakayama et al., 1998). More recently Adhikari and colleagues reported an effect of the porphyrin concentration on the localization of GUN4 and of the CHLH subunit of the MgCh (Adhikari et al., 2009).

Using isolated pea chloroplasts, they showed that ALA feeding causes a redistribution of GUN4 and CHLH in the chloroplast. In unfed chloroplasts GUN4 localizes equally to the membrane and the soluble fraction. Upon feeding with ALA the membrane-associated fraction increased by 50%. A similar effect was observed for CHLH. In this case the membrane-associated fraction increased by 15% (Adhikari et al., 2009). ALA feeding is known to increase the endogenous level of porphyrins in the chloroplast. Indeed an increase of 20- to 30- fold of ProtoIX and MgProtoIX was reported (Adhikari et al., 2009). In this study it was proposed that GUN4 or a GUN4-porphyrin complex may stabilize the interaction of CHLH with the

77 membranes may help the formation of a complex with the MgMT, and interestingly the CHLH subunit of the MgCh has been reported to enhance the activity of the MgMT (Shepherd et al., 2005). The relocation of the MgCh to the membrane fraction has been suggested to be required to redirect the tetrapyrrole pathway towards chlorophyll synthesis (Tanaka and Tanaka, 2007).

Indeed, it is possible that the association of a larger fraction of the MgCh to the membrane, in the proximity of PPOX and the enhancing effect on the activity of MgMT lead to an increase in the rate of chlorophyll synthesis.

As mentioned previously GUN4 was identified in a screen for mutants affected in chloroplast retrograde signaling. The screen was based on the expression of a reporter gene under the control of the Lhcb1 promoter in Arabidopsis seedlings treated with norfluorazon (Susek et al., 1993). Five different gun mutants were identified and surprisingly four of them were affected in genes involved in the tetrapyrrole biosynthetic pathway. Two of the mutants were affected in the heme catabolic pathway, gun2 in the heme oxygenase and gun3 in the biliverdin reductase (Mochizuki et al., 2001; Susek et al., 1993). The gun5 mutant was affected in the CHLH subunit of the MgCh and the gun4 mutation was found to affect a protein that associates to MgCh to stimulate its enzymatic activity (Larkin et al., 2003; Mochizuki et al., 2001). It is interesting to note that the chloroplast-to-nucleus signaling pathway does not seem to require the CHLI subunit of MgCh, whereas a knock-out of the CHLD subunit behaves similarly to the gun5 mutant with respect of the derepression of the Lhcb1 promoter in the presence of norflurazon (Mochizuki et al., 2001; Strand et al., 2003).

The gun4 mutant of Arabidopsis shows an albino phenotype under standard laboratory conditions, but chlorophyll synthesis is not completely compromised as it can grow photoautotrophically in continuous dim light, where it develops pale green tissues and accumulates roughly 10% of the chlorophyll of the wt, 50% if fed with 5-aminolevulinic acid (Peter and Grimm, 2009). Interestingly, this effect is limited to continuous light conditions, as in dark-light cycles the plants did not accumulate detectable levels of chlorophyll (Peter and

Grimm, 2009) On sucrose supplemented media the mutant can develop leaves and flower-like structures (Larkin et al., 2003; Peter and Grimm, 2009). This result indicates that even if GUN4 is not essential for chlorophyll synthesis, it is nevertheless required in order to produce enough chlorophyll to sustain optimal plant growth and its depletion leads to increased photosensitivity (Larkin et al., 2003). A GUN4 knock-out in Synechocystis leads to a drastic decrease in chlorophyll content, abolishes photoautotrophic growth and causes accumulation of high levels of ProtoIX (Sobotka et al., 2008; Wilde et al., 2004). Interestingly, the GUN4 knock-out had decreased levels of the CHLH subunit of the MgCh and of the FeCh in Synechocystis, suggesting that GUN4 may have a role in the distribution of the porphyrins between the iron and the magnesium branch of the pathway (Sobotka et al., 2008).

Recently two groups independently published the crystal structure of GUN4 from cyanobacteria, Verdecia and colleagues from Synechocystis sp. PCC 6803 and Davidson and colleagues from Thermosynechococcus elongatus (Davison et al., 2005; Verdecia et al., 2005).

The region of the protein that binds the porphyrins is conserved in higher plants and it has been named the GUN4 domain. This domain is composed of 8 -helices ( 1 to 8) and 2 loops ( 2/ 3 and 6/ 7). The 8 helices form a “cupped hand” structure with a highly hydrophobic pocket that can bind porphyrins. The long 6/ 7 loop is itself hydrophobic and can probably move to close the hydrophobic pocket upon porphyrin binding (Davison et al., 2005; Verdecia et al., 2005).

In this study we report the identification and the characterization of a gun4 mutant in the green alga Chlamydomonas reinhardtii. We show that in this organism GUN4 is not essential for chlorophyll synthesis, but important to sustain chlorophyll accumulation. The mutant is light sensitive, but can still grow photoautotrophically in dim light, although at a slower rate compared to the wild type.

Results

Identification of the gun4 mutant

The gun4 mutant was isolated in a screen for pale green/low fluorescence after random insertional mutagenesis of a cw15 strain of Chlamydomonas reinhardtii with linearized pSL18 plasmid containing the paromomycin resistance cassette. We mapped the site of insertion by inverse PCR at position +589 in the second exon of the Gun4 gene (Figure 1A). The insertion caused deletions of 184 bp in the second exon and of 489 bp in the pSL18 plasmid (dashed line in Figure 1A). The knock-out was confirmed by RT-PCR with primers specific for the Gun4 CDS. No mRNA was amplified in the gun4 strain under these conditions (+RT in Figure 1B).

GUN4 is conserved among all the organisms that perform oxygenic photosynthesis. Alignment of the deduced amino acid sequence of Chlamydomonas with the GUN4 sequences of Arabidopsis thaliana, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus revealed a high sequence conservation in these species (Figure 1C).

Characterization and complementation of the gun4 mutant

Inactivation of GUN4 in Arabidopsis and Synechocystis causes light sensitivity and impairs photoautotrophic growth (Larkin et al., 2003; Sobotka et al., 2008). We performed a growth test on acetate-containing media (TAP) and minimal media (HSM) in different light conditions in order to analyze the light sensitivity and the ability of the gun4 mutant of Chlamydomonas to grow photoautotrophically. In TAP medium gun4 grows like the wild type in the dark or under dim light (TAP in Figure 2A). Under standard light conditions (60 µE) growth is very slow and the cells are pale green (TAP in Figure 2A). Under high light conditions the gun4 mutant bleaches and dies, whereas the wild type grows well (TAP in Figure 2A). In minimal media gun4 grows very poorly in dim light (HSM in Figure 2A). Under standard light conditions the mutant barely grows and is extremely pale compared to the wild type (HSM in Figure 2A).

Under high light gun4 does not survive (HSM in Figure 2A). The results in minimal media are similar to the phenotype observed for the Arabidopsis gun4 mutant which was able to grow to a certain extent in dim light (Larkin et al., 2003). On the other hand they differ from Synechocystis in which loss of GUN4 abolishes the ability of the organism to grow photoautotrophically (Sobotka et al., 2008).

The gun4 strain of C. reinhardtii was transformed with the wild type Gun4 gene tagged with HA under the control of the PsaD promoter. Two clones expressing different levels of the protein (clones 2.1 and 2.3 in Figure 2B) were subjected to the same light treatments, in order to confirm that the loss of the Gun4 gene is responsible for the observed phenotype. Under all

Arabidopsis and Synechocystis gun4 mutants accumulate very low levels of chlorophyll under any light conditions (Larkin et al., 2003; Sobotka et al., 2008). The gun4 mutant strain of Chlamydomonas is pale green, but it is not albino and the level of chlorophyll allows the cells to grow photoautotrophically, although at a considerably slower rate than the wild type. We quantified the chlorophyll content of dark adapted cultures grown in TAP media, a condition in which the mutant grows at a similar rate as the wild type. The chlorophyll content of the gun4 mutant under these condition was ~65% compared to the wild type, and the two complemented strains showed an intermediate phenotype, with clone 2.1 closer to the wild type value compared to clone 2.3 (Figure 3A).

Next we examined whether the C. reinhardtii mutant accumulates high levels of ProtoIX, as reported for the Synechocystis knock-out (Sobotka et al., 2008). We used dark-grown cultures

81 in order to avoid the inhibitory effects of light on the growth of the gun4 mutant. The gun4 strain accumulates ~30 times more ProtoIX than the wild type (Figure 3B). This difference is higher than in the case of Synechocystis. However, the analysis of Synechocystis was performed under dim light and standard light conditions, in which the strains were probably subjected to a certain degree of photoxidative damage (Sobotka et al., 2008), whereas in our case the cells were grown in the dark. The two strains complemented with the wild type copy of GUN4 did not accumulate ProtoIX (Figure 3B). Chlorophyll b is one of the chromophores for LHC proteins of the photosystems. It is interesting to note that in gun4 the chlorophyll a/chlorophyll b (Chl a/b) ratio is unaffected compared to the wt (Figure 3C).

Characterization of the photosynthetic parameters of the gun4 mutant through spectroscopic analysis.

Analysis of functional antenna size revealed a drastic reduction for both photosystems in the gun4 mutant with respect to wt in cells grown at 30 µE (Figure 4A and 4B). The functional photosynthetic antenna size parameter reflects the energy that can be actually harvested by the antenna of the individual photosystem and transferred to the reaction center.

PSII antenna size is measured based on the kinetics of chlorophyll fluorescence induction upon a flash of light on dark adapted cells in the presence of DCMU, which prevents the transfer of electrons to the plastoquinone pool. Saturation of the reaction center induces the dissipation of the excitation energy in the antenna as fluorescence. The fluorescence rises until it reaches the F_maxwith kinetics that depends on the antenna size. Thus, slower kinetics of fluorescence induction is indicative of a reduced antenna size. The actual relationship is the following: the size is inversely proportional to the time needed in order to reach the 2/3 of Fmax. The PSII antenna size of gun4 is approximately 40% of that of wt (Figure 4A). We can observe also a reduction in the PSI antenna size to 20% of the wt (Figure 4B). This parameter is inversely

proportional to the time needed to reach 2/3 of the maximum absorbance change at 705 nm, which reflects the photo-oxidation of P700, the reaction center of the PSI (Melis, 1989).

The concentration of PSI in the thylakoid membranes can be estimated spectrophotometrically from the amplitude of the light minus dark absorbance difference signal at 705 nm (P700) (Melis and Brown, 1980). The gun4 mutant possesses half of the functional PSI units compared to the wt (the data have been normalised to chlorophyll absorbance) (Figure 4C).

The density of functional photosystem units could be estimated by studying PSII and PSI contributions to the electrochromic effect on photosynthetic pigments. The electrochromic signal (ECS) is induced by an electrical field generated across the thylakoid membrane by the photochemical charge separation and its extent is linearly proportional to the photochemistry of the reaction center. Practically, ECS is detected as an absorption change at 520 nm. In the absence of the PSII inhibitors DCMU and hydroxylamine (HA), the ECS derives from PSII and PSI. In the presence of DCMU and HA the only contribution to the ECS comes from PSI. Thus PSII contribution is the difference between the signals measured in the absence and in the presence of DCMU and HA. DCMU inactivates the PSII electron acceptor side whereas HA destabilizes the manganese cluster responsible for oxygen evolution and slows the charge recombination between the electron donor and the electron acceptor sites of PSII, which would preclude correct estimation of the PSI/PSII ratio (Finazzi et al., 2006). Cells of wt and gun4 were grown in TAP at 30 µE, and dark adapted before the measurements. Samples with the same chlorophyll content were analyzed. The gun4 mutant had a low level of functional photosystem units compared to the wt (Figure 4D). Estimation of functional PSI units by ECS measurement is consistent with the result obtained by studying the P700 oxidation (Figure 4C).

Considering the reduced antenna size of both photosystems (Figure 4A and 4B), we would have expected a higher photosystem units-chlorophyll ratio in gun4 compared to the wt. Surprisingly gun4 showed a lower amount of functional photosystem units compared to the wt (Figure 4D).

Moreover, gun4 displayed a PSI / PSII activity ratio higher than the wt (Figure 4E). We

83 conclude that a large fraction of photosystem units does not contribute to the photochemistry in the mutant. In particular, the fraction of nonfunctional PSII complexes is greater than the fraction of nonfunctional PSI complexes.

If the activity of PSI is higher than the activity of PSII, the cytochrome f of the cytochrome b6f complex should be in an oxidized state. The rate of the cytochrome f reduction / oxidation is an appropriate parameter to test the connectivity between the cytochrome b6f complex and the photosystems, because it depends on the concentration of reduced plastoquinone (PQH2) and oxidized plastocyanin (PC) (Finazzi et al., 2006; Finazzi et al., 2002). Illumination with actinic light results in the oxidation of cytochrome f, which rapidly reaches a plateau. After turning off the light the cytochrome f reduction can be measured. Cytochrome f redox changes can be calculated as the difference between the absorption at 554 nm and a baseline drawn between 545 and 573 nm (Finazzi et al., 1997) and corrected for the contribution of the electrochromic signal (5% of the signal observed at 520 nm) (Finazzi et al., 1999). We measured the cytochrome f redox changes in dark adapted or pre-illuminated (400 µE for 20 minutes) wt and gun4 cells. Cytochrome f redox state is unaffected by light treatment in wt cells (Figure 5A), as previously reported (Finazzi et al., 2006). On the contrary the cytochrome f is more oxidized in gun4 upon illumination compared to the wt, and also compared to the redox state measured in dark adapted gun4 cells (Figure 5B). These data are compatible with a higher activity of PSI than PSII in the mutant.

Photosynthetic electron transport can be estimated also by measuring the kinetics of P700 oxidation. The kinetics of P700 photo-oxidation in vivo upon a saturating actinic light can reflect either linear electron transport between the photosystems (LEF) or cyclic electron transport around PSI (CEF). More specifically a slow P700 oxidation indicates the occurrence of CEF, whereas a fast P700 oxidation reflects the establishment of LEF (DalCorso et al., 2008). DCMU inhibits electron transfer from PSII to the plastoquinone pool and can be used to discriminate between LEF and CEF. DCMU causes an increase in the P700 oxidation only if

the LEF is the major electron transfer pathway active during the measurement. P700 oxidation was investigated in wt and gun4 cells pre-illuminated for 20 minutes at a light intensity of 400 µE.

DCMU caused an increase in the oxidation of P700 in the wt, indicating that LEF is the main electron transfer pathway active in this strain (Figure 5C). On the contrary, P700 oxidation was insensitive to DCMU in gun4 (Figure 5D). This result may suggest that CEF is the main electron transfer pathway in the mutant. In agreement with this hypothesis, comparison of P700 oxidation in wt and gun4 cells in the absence of DCMU revealed a more reduced state of P700 in the mutant (Figure 5E). It is important to note that in DCMU-treated cells, the kinetics of P700 photo-oxidation depends on the number of electrons trapped between the two photosystems (Melis, 1982) Thus the extent of the DCMU effect depends also on the pre-existing redox state of the PQ pool. If the PQ pool is more oxidized in gun4 compared to the wt, because of a higher ratio of functional PSI / PSII, P700 oxidation would be less sensitive to the DCMU treatment. Moreover, the establishment of CEF would lead to a faster reduction of P700

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