Tetrapyrrole Biosynthesis

The light-induced expression of Lhc proteins was found to coincide with the greening and maturation process of chloroplasts implying the action of a plastid signal. One potential signal could be attributed to the chlorophyll precursor Mg-Proto-Porphyrin-IX (Mg-Proto-IX). Feeding experiments with the iron chelator dipyridyl led to decreased Lhcb mRNA levels in Chlamydomonas reinhardtii (Johanningmeier and Howell 1984). The chelation of iron leads to an interruption of the heme feedback inhibition in the tetrapyrrole pathway which in turn causes accumulation of Mg-Proto-IX. This effect could be also observed in higher plants (Kittsteiner et al. 1991). Direct feeding of Mg-Proto-IX to Chlamydomonas cell cultures led to induction of nuclear heat-shock genes HSP70a/b/c (Kropat et al. 1997) supporting the notion that this chlorophyll precursor could mediate a retrograde signal which affects nuclear gene expression.

Mrna Biosynthesis Plants

Fig. 3 Plastid signals depending on tetrapyrrole and carotenoid biosynthesis. Plastid, cytosol and nucleus are depicted schematically. The photosynthetic apparatus is given schematically in a separate box. Genes are shown as white boxes. transcription start sites are indicated by a small arrow in front of the genes. Protein components are given as white ovals (for identity compare text). Tetrapyrrols are represented by grey squares. Repressive effects are given as a black line with a hammerhead. Influences of organellar processes on nuclear gene expression are indicated by thick black arrows, putative diffusion by dotted arrows. Transduction of these signals are not known and marked by question marks

Fig. 3 Plastid signals depending on tetrapyrrole and carotenoid biosynthesis. Plastid, cytosol and nucleus are depicted schematically. The photosynthetic apparatus is given schematically in a separate box. Genes are shown as white boxes. transcription start sites are indicated by a small arrow in front of the genes. Protein components are given as white ovals (for identity compare text). Tetrapyrrols are represented by grey squares. Repressive effects are given as a black line with a hammerhead. Influences of organellar processes on nuclear gene expression are indicated by thick black arrows, putative diffusion by dotted arrows. Transduction of these signals are not known and marked by question marks

Another line of evidence for involvement of chlorophyll precursors in retrograde signalling came from studies on carotenoid-deficient plants. Maize seedlings with defects in carotenoid synthesis exhibited a decreased accumulation of Lhcb mRNA while other nuclear-encoded transcripts for cytosolic enzymes were not impaired (Mayfield and Taylor 1984). Alternatively, disruption of carotenoid biosynthesis by blocking the phytoene desaturase (PDS) (catalysing an early enzymatic step in this pathway) with the herbicide norflurazon (NF) (Chamovitz et al. 1991) led to comparable effects as the genetic defects. The resulting carotenoid deficiency of plas-tids led to reduced photosynthetic efficiency followed by photo-oxidative damage of thylakoid membranes due to the loss of non-photochemical de-excitation mechanisms. This photo-oxidative stress within the plastid prevented conversion of proplastids into mature chloroplast and resulted in a decreased expression of nuclear Lhcb and RbcS genes (Oelmüller and Mohr 1986). Thus, it was concluded that intact plastids are required for expression of nuclear photosynthesis genes and that a "plastid factor" is required for a correct build-up of the photosynthetic machinery (Oelmüller 1989; Taylor 1989).

Using this NF-mediated repression as a tool a genetic approach was performed to get deeper insights into the nature of the plastid signal (Susek et al. 1993). First, a transgenic Arabidopsis line carrying a fusion construct consisting of the Lhcb1.2 (CAB3)-promoter (known to be down-regulated by NF treatment, see above) and reporter genes conferring hygromycin resistance and P-glucuronidase activity was created. Then the seed pool of this reporter line was mutagenised with ethyl methane sulphonate (EMS) and the resulting EMS mutant population was grown on plates with an NF-containing medium. By this means the seedling population was screened for individuals exhibiting a genetic defect which interrupted the down-regulation of the Lhcb expression under NF and, consequently, conferred hygromycin resistance. These mutants are regarded as defective in plastid signalling and, therefore, were named genomes uncoupled (gun) mutants. In the second screening step expression of the P-glucuronidase gene was tested and relative activity of the Lhcb1.2 promoter was estimated. All gun mutants exhibited Lhcb expression whereas in wild-type plants Lhcb transcription was almost abolished. In total six different gun mutant lines (gun1-gun6) were found in this screen (Susek et al. 1993). Since gunl is different from gun2-gun5 it is discussed in a separate section (Sect. 4).

The phenotype of the gun mutants varies from pale yellowish to undistinguisha-ble from wild-type. The mutants gun2-gun5 were mapped to the tetrapyrrole synthesis pathway (Fig. 2) (Surpin et al. 2002) and demonstrated reduced accumulation of Mg-Proto-IX under NF treatment which causes down-regulation of Lhcb expression in wild-type (Strand et al. 2003). The gun2 and gun3 mutant alleles were identified to encode the haem oxygenase and phytochromobiline synthase, respectively. The genetic lesions cause an overproduction of haem which activates a feedback loop that inhibits the trnE-reductase (HEMA), the first step of tetrapyrrole biosynthesis. This prevents accumulation of Mg-Proto-IX. Both mutants are allelic with hyl and hy2 (hypocotyl) mutants found in a screen for photomorphogenesis mutant which is consistent with the function of tetrapyrroles as chromophores of phyto-chromes (Mochizuki et al. 2001) . The mutants gun4 and gun5 were found to be directly involved in chelation of magnesium into protoporphyrin IX, the step which generates Mg-Proto-IX. gun4 was found to encode an activator of the Mg-chelatase and gun5 was affected in CHL-H, a subunit of Mg-chelatase (Fig. 3). GUN4 is a small soluble protein 22-kDa in size that can bind either the substrate proto-IX or the product of the chelation reaction, Mg-Proto-IX. The binding constant of GUN4 and Mg-Proto-IX was found to be lower than that of GUN4 and Proto-IX, however, only the latter couple is able to activate Mg-chelatase. By this means GUN4 could avoid accumulation of phototoxic Mg-Proto-IX and could control the chlorophyll biosynthesis pathway (Mochizuki et al. 2001; Larkin et al. 2003; Strand 2004).

In cyanobacteria GUN4 was shown to modulate enzyme activities of the Mg-chelatase and ferrochelatase that produces haem (Wilde et al. 2004). Thus, GUN4 may function as a global controller of the haem and chlorophyll branches. Since haem or its precursor Proto-IX is exported to mitochondria a control step at this point tetrapyrrole synthesis appears to be ideal for regulation and signalling.

Recently, the presence of Mg-Proto-IX in the cytosol could be visualised by confocal laser scanning technology. The actual low amount of Mg-Proto-IX in the plant cell was increased by circumventing the HEMA feedback inhibition by direct ALA feeding of NF treated seedlings (Ankele et al. 2007). This favours the model that Mg-Proto-IX is directly transported into the cytosol (Strand 2004) over the model which involves a Mg-Proto-IX sensing protein (like GUN4) and a subsequent cytosolic signal transduction cascade (Larkin et al. 2003) . How this can be reconciled with the high photo-toxicity of Mg-Proto-IX still has to be resolved.

The gun5 mutant was found to possess a mutated allele of CHL-H which provides Mg-Proto-IX for the [CHL-LCHL-D]^ complex in which the Mg insertion into Proto-IX occurs (Willows and Hansson 2003). Mutations in any of the Mg-chelatase subunits resulted in decreased Chl level. Interestingly, mutations in the CHL-I do not result in a gun phenotype although these mutants produce even less amounts of Mg-Proto-IX than gun5 (Mochizuki et al. 2001). This is consistent with the phenotype of a number of other mutants with defects in tetrapyrrole biosynthesis [ch42 (chlorata), cs, crd1 (copper response defect) (now called chl27)], which all exhibit no gun phenotype (Koncz et al. 1990; Tottey et al. 2003). ch42 and cs accumulate less Mg-Proto-IX than wild-type, however, it has not been investigated if this occurs also upon NF treatment. In contrast, the crd mutant accumulates more Mg-Proto-IX compared to wildtype. Especially the observations with the ch42 and cs mutants suggest that Mg-Proto-IX levels do not exclusively account for the tetrapyrrole-mediated signal. This is supported by a recent study on Chlamydomonas mutants with defects in the Mg-chelatase. These mutants exhibit reduced levels of Mg-tetrapyrroles but increased levels of soluble haem. It was shown that haem can mimic the activating role of Mg-Proto-IX on the induction of HSP70A promoter and other Mg-Proto-IX inducible genes. It was concluded that both tetrapyrroles can act as retrograde signals and that the respective signalling pathways converge at the same cis-elements (von Gromoff et al. 2008).

Further support for the idea that a developmental signal descends from the CHL-H (GUN5) subunit came from expression analyses of the nuclear transcripts of AtSig1-6 genes. They encode sigma factors that are crucial for promoter recognition of the plastid encoded RNA-polymerase (compare Rolland et al. 2008). These factors were found to be repressed after NF-treatment in wild-type but not in the gun5 background. A similar de-repression in the gun5 mutant was also found for plastid transcripts depending on PEP activity like psbA, psaA, psaC whereas genes transcribed by the nucleus-encoded RNA-polymerase were not affected. This suggests that GUN5 might act via regulation of nuclear-encoded components of the plastid gene expression machinery in early plastid development (Ankele et al. 2007).

A recent publication reported that CHL-H might be a plastid localised ABA receptor (Fig. 3) (Shen et al. 2006) . The authors found that the Arabidopsis cch (constitutive chlorina) mutant was deficient in ABA-related responses. The genetic lesion in this mutant was found to be a stronger allele of gun5. Therefore, the cch mutant displays a "gun phenotype". It could be further shown that direct ABA feeding to wild-type plants led to an increase in Mg-Proto-IX levels but to decreased Chl levels. This suggests that a component downstream of the Mg-chelatase plays an additional role in the tetrapyrrole and ABA crosstalk. Whether or not ABA deficiency caused by NF treatment is related to the putative ABA receptor function of the Chl-H subunit has to be studied in the future.

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