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Plant Signal Behav. 2010 February; 5(2): 196–199.
PMCID: PMC2884134

New insights into the retrograde signaling pathway between the plastids and the nucleus

Abstract

In plant cells, the genetic information required for biological activity is divided into three organelles—the nucleus, plastids and mitochondria. These organelles require tightly coordinated gene expression to accomplish the appropriate biological processes. Chloroplasts harness light energy and use it for carbon fixation in photosynthesis. However, majority of the proteins involved in photosynthesis is encoded by the nucleus genome. Thus, nuclearencoded photosynthesis-related proteins are targeted to plastids after their synthesis in the cytosol. Therefore, it is critical to regulate nuclear gene expression in response to the functional or metabolic state of the plastids; this process relies on signals from the plastids to the nucleus that are known as retrograde signals. Our genetic studies revealed that GENOMES UNCOUPLED 1 (GUN1) and Golden2-like1 (GLK1) mediate the retrograde signal that coordinates plastid protein import and nuclear gene expression. In this study, we propose a novel signaling pathway that regulates nuclear gene expression according to the rate of protein import into the plastid.

Key words: plastid protein import, plastid biogenesis, chloroplast, retrograde signaling, golden2-like

The endosymbiosis of free-living cyanobacteria into eukaryotic host cells created the primitive plastids about 1.2 billion years ago.1 This major advance involved the transfer of genetic information from the plastids to the host nucleus. At present, the plastid genome encodes fewer than 100 open reading frames (ORFs) in Arabidopsis thaliana,2,3 and most genes that were encoded by the cyanobacterium have been moved to the host nuclear genome. Therefore, to maintain the function of plastids, it is important to strictly control the expression of nuclear genes, which encode plastid-localized proteins. Consequently, a precise coordination system between the import of nuclearencoded plastid proteins and its expression in the nucleus has been established during evolution.

The Arabidopsis plastid protein import2 (ppi2) mutant lacks the major protein import receptor of plastids, atToc159.4 This mutant specifically fails to accumulate nuclear-encoded photosynthesis- related proteins. Interestingly, the reduction of protein accumulation into the plastid is not solely because of the lack of an import receptor but also because of the downregulation of relevant genes. Our quantitative real-time polymerase chain reactions (qRT-PCR) revealed that many of the genes that are downregulated in ppi2 encode photosynthesis-related proteins (Fig. 1A). Proteins required for photosynthesis are imported into the plastids after their synthesis on the cytoplasmic ribosome, and import of these proteins is mediated by the atToc159 protein import receptor.5 However, in the absence of atToc159, plastids shut down the expression of photosynthesis-related genes. These data clearly indicate that there is a retrograde signaling pathway that coordinates protein import with nuclear gene expression.

Figure 1
Gene expression profile of the ppi2 mutant. Quantitative analysis of gene expression in ppi2 by qRT-PCR. The mRNA levels were normalized to the levels of 18S rRNA. The expression level in wild-type was set to 1. Each bar, plotted here on a log scale, ...

When seedlings are treated with herbicides (e.g., norflurazon), plastid biogenesis is arrested and transcript levels of nuclear-encoded photosynthesis-related proteins are reduced.6 This observation raises the possibility that the phenomena observed in the ppi2 mutant and in the herbicide-treated seedling might be similar and that the retrograde signaling pathway from plastid-to-nucleus might be involved in the downregulation of nuclear genes in ppi2. Arabidopsis GUN loci are considered to be involved in the signal transduction pathway from the plastid to the nucleus because gun mutants (gun1 to gun5) accumulate nuclear photosynthesisrelated gene transcripts even under herbicide treatment.7 One of the GUN genes, GUN1 encodes a plastid-targeted pentatricopeptide repeat protein that has been proposed to transmit a retrograde signal under stressful conditions.8 An abscisic acid-insensitive mutant, abi4, also exhibits a gun phenotype, indicating that ABI4 regulates retrograde regulon in the nucleus. Therefore, GUN1 appears to repress the expression of nuclear genes through the activation of ABI4.

To investigate whether known components of the plastid signaling pathway are working in the ppi2 mutant and to determine whether the effects of GUN proteins affect gene expressions, qRT-PCR and western blot analysis were performed.9 Surprisingly, accumulation of GUN4, a key regulator of Mg-proto IX synthesis, was decreased at both the mRNA and the protein levels. This suggests that the downregulation of photosynthesis-related genes is mediated in a GUN4-independent manner. Next, to assess the genetic relationship between the ppi2 mutation and the GUNABI4 pathway, we generated a abi4 ppi2 double mutant. If ABI4 exists downstream of the retrograde pathway in ppi2, abi4 ppi2 should restore gene expression. However, we did not observe restoration of gene expression in the double mutant compared with the ppi2 single mutant.9 These results suggest that the mechanism of gene repression in ppi2 is distinct from Mg-proto IX and the ABI4 pathway.

To identify the transcription factor that functions as a regulator of transcription in the retrograde signal, we performed large-scale transcriptome analysis using SuperSAGE.10 A member of golden2-like transcription factor, GLK1, was identified as a candidate signaling mediator because the transcription level of GLK1 was significantly repressed in ppi2. Several papers reported that the GLK transcription factors are required for chloroplast development in Arabidopsis, rice, maize and moss.1114 Waters et al. (2009) demonstrated that the GLK gene conducts the synchronized expression of photosynthesis- related genes independent of the phyB pathway.15 Furthermore, direct binding to the CCAATC motif that exists in photosynthesis-related GLK targets was confirmed by a chromatin immunoprecipitation assay.15 If GLK1 is a positive regulator of the plastid signaling pathway, GLK1 should alter photosynthetic gene expression in the ppi2 background. In fact, constitutive expression of GLK1 partially complemented repressed photosynthesisrelated gene expression.9 Moreover, we provided evidence that GUN1 functions up-stream of GLK1 and negatively regulates GLK1.

The plastid-encoded genes are transcribed by both a nuclear-encoded phage-type RNA polymerase (NEP) and a plastid-encoded cyanobacteriumtype RNA polymerase (PEP).16,17 PEP is composed of plastid-encoded core subunits and nuclear-encoded sigma factors (SIGs) that provide promoter specificity to PEP. Plastid-encoded photosynthesis genes are transcribed exclusively by PEP.18,19 Interestingly, expression of a certain class of SIG gene is decreased in ppi2 (Fig. 2A). Moreover, SuperSAGE analysis revealed that SIG6 was also downregulated in ppi2.9 According to previous literature, SIG2 and SIG6 are thought to have a specific function in the chloroplast biogenesis of cotyledons.20,21 The CCAATC/GATTGG motif recognized by GLK exists within the first 1,000 bp upstream of the downregulated SIG genes (Fig. 2B). In accordance with these findings, downregulation of plastid-encoded photosynthesis-related genes such as large subunit of Rubisco (LSU) may be caused by a decrease of SIG expression, which is caused by GLK1 repression.

Figure 2
SIGs expression is affected by retrograde signal. (A) Accumulation of SIG1, SIG2 and SIG3 transcripts in ppi2. The qRT-PCR and quantification were performed according to Figure 1. (B) Position of GLK1 recognition motif (CCAATC) in SIGs promoter. The black ...

A chlorophyll intermediate, Mg-protoporphyrin IX (Mg-proto IX), was identified as a signaling molecule of the retrograde signal pathway because it was highly accumulated in seedlings treated with norflurazon.22 Moreover, Mg-proto IX accumulated both in the chloroplast and in the cytosol during stress conditions.23 Taken together, these results suggest that Mg-proto IX is a negative signaling molecule from plastids, which shuts off photosynthetic gene expression in the nucleus. In contrast, recent papers showed that there is no interrelation between the degree of gene expression in the nucleus and the accumulation of Mg-proto IX.24,25 Similarly, our data showed that the steadystate level of Mg-proto IX was not involved in repression of nuclear photosynthesis-related genes in ppi2. On the other hand, Mg-proto IX feeding experiments proposed a strict dependence of nuclear DNA replication on the replication of organelle DNA, which can be bypassed by the addition of a tetrapyrrole intermediate in Cyanidioschyzon merolae and tobacco BY-2 cells.26 The precise role of chlorophyll intermediates remains obscure.

The data reviewed here have placed emphasis on the function of GLK1 involved in retrograde signaling. However, some important questions still remain unclear. For example, how does GLK1 receive a signal(s) from defective plastids? What are the signaling mediators in the cytosol? It will be interesting to identify retrograde signaling mutants and use chemical genetics approaches for elucidating the complicated pathway between the plastids and the nucleus.

Footnotes

References

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