Several chloroplast retrograde signals have been identified so far: one dependent on plastid protein synthesis, a second on hydrogen peroxide, a third on singlet oxygen, a fourth on the redox state of the electron transport chain and a fifth on the tetrapyrrole biosynthetic pathway.
Figure 7: Model of the retrograde signaling pathways discussed below.
Plastid protein synthesis dependent signals
Plastid protein synthesis was proposed to affect nuclear gene expression based on studies on the barley mutant albostrians. This mutant is deficient in chloroplast ribosomes and therefore
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impaired in chloroplast protein synthesis (Bradbeer et al., 1979). In the pigment-deficient leaves, the mutant seedlings fail to express several nucleus-encoded chloroplast proteins that are normally induced by light, suggesting the absence of a signal required for correct light-induced gene expression in the ribosome-deficient cells (Hess et al., 1994).
Other evidences for the existence of a plastid protein synthesis-dependent retrograde signal derive from the use of transcriptional or translational inhibitors specific for the plastid ribosomes. Treatment of barley seedlings with the plastid RNA polymerase inhibitor tagetitoxin prevents the induction of nuclear genes that are expressed during early phases of development (Rapp and Mullet, 1991). Inhibition of chloroplast translation by treatment with chloramphenicol or lincomycin or mutations that affect essential components of the plastid translational machinery also have inhibitory effects on the expression of nuclear genes encoding chloroplast proteins (Sullivan and Gray, 1999). It is interesting to note that these treatments have an effect on gene expression only if applied within 3 days after germination, suggesting the involvement of a factor that is only present in the early phases of seedling development (Beck, 2005; Pogson et al., 2008).
Recently the mutant genome uncoupled 1 (gun1) has been reported to release the inhibitory effect of translational inhibitors on nuclear gene expression. In the same studies the signal mediated by GUN1 was shown to have inhibitory effects on cryptochrome CRY1 signaling, thus implicating an interaction between plastid protein synthesis and the light signaling pathways (Ruckle et al., 2007; Ruckle and Larkin, 2009). GUN1 is a member of the pentatricopeptide-repeat (PPR) protein family, it is nucleus-encoded and imported in the chloroplast. In addition to the PPR domain, GUN1 contains a small Muts-related (SMR) domain that can bind strongly to DNA. The role of this binding is still unknown (Koussevitzky et al., 2007). Downstream effectors of GUN1 are still unknown. Some evidence suggests that the transcription factor ABI4 is implicated in this pathway, but it is unlikely that this is the only nucleo-cytosolic factor involved in GUN1 signaling (Koussevitzky et al., 2007).
29 Reactive oxygen species dependent signaling
The chloroplast is one of the major sources of reactive oxygen species (ROS) in plants.
Chlorophyll molecules excited to the triplet state can transfer their energy to oxygen (3O2) giving rise to singlet oxygen (1O2). This event occurs mainly at the level of photosystem II if the electron transport chain does not work efficiently (Krieger-Liszkay, 2005). Photosystem I can directly transfer an electron to oxygen instead of NADP+ creating superoxide ions (O2
•-), that are converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) (Apel and Hirt, 2004).
Because of its short half life, singlet oxygen is probably exclusively localized in the chloroplast, its role was difficult to separate from the role of other ROS due to the difficulties in generating it specifically in plant cells. Recently the use of the Arabidopsis mutant flu led to great advances in the understanding of the singlet oxygen signaling pathway. FLU is a negative regulator of chlorophyll biosynthesis (see previous chapters on the regulation of chlorophyll synthesis) and its absence leads to the accumulation of protochlorophyllide, which generates singlet oxygen upon illumination (Meskauskiene et al., 2001). Illuminated flu plants arrest their development and eventually die. This event was thought to be caused by irreversible photoxidative damage, but the discovery of suppressors of the flu phenotype highlights the existence of a singlet oxygen induced programmed cell death. The executer1/flu double mutant grows like the wild type in conditions that are inhibitory for the flu plants. EXECUTER1 (EX1) has been shown to be an integrator of singlet oxygen signaling from the chloroplast. In its absence the plant no longer arrests its growth in response to protochlorophyllide-dependent generation of singlet oxygen (Wagner et al., 2004). In Arabidopsis there is also a homologue of EX1, named EXECUTER2 (EX2). In a triple mutant flu/ex1/ex2 induction of almost all the nuclear genes responsive to singlet oxygen is abolished, strengthening the idea that EX1, and
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EX2, are essential components of the singlet oxygen dependent retrograde signaling pathway (Lee et al., 2007).
Hydrogen peroxide is produced by the photosynthetic apparatus under different stress conditions and changes in the expression of nucleus-encoded stress proteins or light-induced proteins have been linked to hydrogen peroxide (Slesak et al., 2007; Vandenabeele et al., 2003;
Vandenabeele et al., 2004). Studies with reporter genes have shown that application of exogenous hydrogen peroxide stimulates the expression of high light-inducible genes like ascorbate peroxidases APX1 and APX2, the zinc finger transcription factors ZAT10 and ZAT12 and the early light inducible protein ELIP2 (Davletova et al., 2005; Karpinski et al., 1999; Rossel et al., 2007). Recently an interaction between the singlet oxygen and the hydrogen peroxide signaling pathways has been proposed, in particular it has been shown with transcriptomic analysis that hydrogen peroxide has an antagonistic effect to singlet oxygen on nuclear gene expression (Laloi et al., 2007).
The interaction between retrograde signaling pathways increases the complexity of the chloroplast-to-nucleus communication and may explain the adaptability of the plant cell to many different environmental conditions.
Signaling dependent on the redox state of the photosynthetic electron transport chain
The redox state of the electron transport chain, and in particular of the plastoquinone pool is an indicator of the efficiency of photosynthesis. If the photosynthetic reactions are not working properly, modifications in the expression of nuclear genes may be necessary to adjust them. The photosynthetic electron transport chain was therefore a likely candidate as the origin of retrograde signals. Even if gene regulation linked to the redox state of the plastoquinone pool has been found, evidence for it seems to be largely dependent on the plant species, the experimental conditions and the developmental stage. Moreover the distinction between redox signals and other signaling pathways has proven very difficult (Pogson et al., 2008). In the
31 unicellular alga Dunaliella tertiolecta, it was shown that the expression of the LHCB gene is altered in response to reduction or oxidation of the plastoquinone pool (Durnford and Falkowski, 1997). In plants the regulation of some genes has been linked to the redox state of the plastoquinone pool. Indeed in Arabidopsis 54 genes have been shown to be strictly regulated by the redox state of the plastoquinone pool, whereas many more seemed to be regulated together with other chloroplast signals (Fey et al., 2005b). In Chlamydomonas the redox-dependent retrograde signal has been partially characterized using strains carrying mutations in the cytochrome b6f acceptor site Q0, or lacking the cytochrome b6f complex. These strains were unable to induce the genes of the tetrapyrrole biosynthetic pathway upon illumination (Shao et al., 2006). Interestingly this inhibitory effect is specific for the cytochrome b6f complex and independent from the redox state of the plastoquinone pool as treatment of the cells with DCMU (an inhibitor of electron transfer from PSII to the plastoquinone) or DBMIB (an inhibitor of electron transfer from plastoquinone to the cytochrome b6f) have no effect on the light induction of the genes analyzed (Shao et al., 2006).
Recently the STN7 kinase has been linked to long term adaptation and redox dependent retrograde signaling. This kinase is necessary for the state transition process, a short term adaptation to changing light conditions (Bellafiore et al., 2005). In the stn7 plants the regulation of a set of nuclear genes is perturbed and the plants show reduced growth if the plants are subjected repeatedly to light of different wavelengths. This has led to the hypothesis of a dual role for the STN7 kinase in short and long term adaptation (Bonardi et al., 2005; Rochaix, 2007; Wagner et al., 2008).
Tetrapyrrole biosynthetic pathway dependent retrograde signaling
The possible involvement of chlorophyll precursors in the regulation of nucleus-encoded chloroplast proteins was suggested by studies on dipyridyl- treated cells (dipyridyl inhibits the FeCh and the Mg-protoporphyrin IX monomethyl ester cyclase) and on protoporphyrin IX
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accumulating mutants of Chlamydomonas reinhardtii. In these conditions the light-dependent accumulation of the LHCB transcripts was prevented, this effect was correlated with the overaccumulation of cyclic tetrapyrroles, as the treatment with dyoxoheptanoic acid, an inhibitor of earlier steps in the tetrapyrrole pathway, had no effect on LHCB mRNA accumulation (Johanningmeier, 1988; Johanningmeier and Howell, 1984). Another proof of the role of chlorophyll precursors in the retrograde signaling in Chlamydomonas came from the studies on chloroplast HSP70. It was shown that the induction of this gene by light was impaired in the MgCh mutant brs1, but not in a mutant affected in an enzyme that acts in a subsequent step of the chlorophyll biosynthetic pathway. Moreover feeding of Mg-protoporphyrin IX and Mg-Mg-protoporphyrin IX methylester, but not of Mg-protoporphyrin IX in the dark was sufficient to induce the expression of HSP70 in the dark (Kropat et al., 1997, 2000). In the same studies a correlation was observed between the induction of the HSP70 gene and a transient increase in the cellular concentration of chlorophyll precursors upon illumination, supporting a model in which the accumulation of cyclic tetrapyrroles has an important role in triggering a signal to modulate nuclear gene expression (Kropat et al., 2000). Why protoporphyrin IX does not seem to have a role in retrograde signaling is more difficult to explain. In fact feeding the cells with this molecule causes an increase in the cellular level of Mg-protoporphyrin and Mg-protoporphyrin methyl ester, an event that, according to the model proposed, should lead to an induction of the transcription of the HSP70 gene which is however not observed. The explanation given by the authors is that feeding of Mg-protoporphyrin IX increases the cellular concentration of this tetrapyrrole in every compartment of the cell including the cytosol and the nucleus, whereas enzymatic conversion of protoporphyrin IX to Mg-protoporphyrin IX leads to an increase of the latter only in the chloroplast, and another factor, probably light, is required for the export of the Mg-protoporphyrin IX to the cytosol (Beck, 2005). Convincing evidence of the export of Mg-protoporphyrin IX from the chloroplast is still missing.
33 The characterization of the FLP proteins in Chlamydomonas has shown that there are probably two retrograde signals dependent on the tetrapyrrole pathway. In fact the two FLP isoforms are encoded by a single gene, whose messenger is alternatively spliced, the production of the shorter or of the longer form is under control of signals from the chloroplast and from the light perception (Falciatore et al., 2005).
The retrograde signal dependent on the tetrapyrrole pathway has been investigated also in higher plants. In the early ‘90s a genetic screen allowed for the isolation of 5 genome uncoupled mutants (GUN). The approach was based on the repression of the expression of photosynthetic genes in seedling treated with Norfluorazon (NF), a herbicide that blocks the synthesis of carotenoids. The gun mutants were expressing a reporter gene under the control of the Lhcb promoter even if treated with NF (Susek et al., 1993). Four of the gun mutants characterized present lesions in genes involved in the tetrapyrrole biosynthetic pathway. Two of them, gun2 and gun3, encode the enzymes heme oxygenase and phytochromobilin synthase that are necessary to produce phytochromobilin from heme. In their absence the cells accumulate heme, which inhibits the GluTR, the first enzyme of the tetrapyrrole pathway (see previous chapter on the regulation of chlorophyll biosynthesis) (Pontoppidan and Kannangara, 1994;
Vothknecht et al., 1998). The gun5 mutant is affected in the gene for the H subunit of the MgCh and gun4 had a mutation in a protein that binds to the MgCh to increase the kinetics of the reaction (Larkin et al., 2003; Mochizuki et al., 2001). The common phenotype of these four mutants is a decrease in the concentration of the chlorophyll precursors below the branching point of the tetrapyrrole pathway (Mochizuki et al., 2001; Strand et al., 2003). It has been proposed that accumulation of Mg-protoporphyrin IX may cause the repression of nuclear genes in the dark or during NF treatment in the light in seedlings (Strand et al., 2003). Evidence of the presence of Mg-protoporphyrin IX in the cytosol of NF treated wild type seedling (Ankele et al., 2007) led to the hypothesis that in certain conditions Mg-Protoporphyrin IX can
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exit the chloroplast to activate nuclear factors that are responsible of the repression of a set of nucleus-encoded chloroplastic proteins (Nott et al., 2006; Strand, 2004; Strand et al., 2003).
The characterization of an Arabidopsis line knocked-out for the Mg-protoporphyrin methytransferase seemed to confirm this model (Pontier et al., 2007). Recently, more precise analysis of the chlorophyll precursor concentration in seedlings treated with NF challenge the role of Mg-protoporphyrin IX. Two groups independently report that in NF treated seedlings exposed to light, the Mg-protoporphyrin IX concentration was lower than in untreated samples, and that accumulation of this tetrapyrrole is not sufficient to repress the LHCB mRNA accumulation in gun mutants (Mochizuki et al., 2008; Moulin et al., 2008). Another discrepancy between the experimental data and the role proposed for the Mg-protoporphyrin IX in retrograde signaling is the fact that mutants affected in the CHLI subunit of MgCh do not seem to have a gun phenotype, even if the enzyme activity is impaired like for the mutants of the other two subunits (Mochizuki et al., 2001).
In conclusion it is now clear that the tetrapyrrole biosynthetic pathway is a source of retrograde signals. One of the main open questions is the identification of the actual signal. The accumulation of Mg-protoporphyrin IX by itself does not seem to be sufficient.
Another evidence against the model proposed is the high toxicity of the tetrapyrroles. In fact accumulation of these compounds may lead to a rapid photo-oxidation of essential cellular components that may cause cell death. It is possible that one of the enzymes of the pathway is by itself capable of triggering a signal to repress nuclear transcription. This role has been proposed for the CHLH subunit of the MgCh, for the GUN4 protein and for the Mg-protoporphyrin IX methyltransferase (Larkin et al., 2003; Nott et al., 2006; Pontier et al., 2007).
Interestingly it has been recently shown that porphyrins promote the association of the MgCh and the GUN4 protein with the chloroplastic membrane, a mechanism that may favor the Mg branch versus the Fe branch of the tetrapyrrole pathway as the protoporphyrinogen IX oxidase,
35 the enzyme that produces the protoporphyrin IX, is membrane associated (Adhikari et al., 2009). The association of the complex with the membrane may also be important to transmit a signal to the cytosol.
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