Communication between organelles and the nucleus is essential for fitness and survival.Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signaling pathways that collectively regulate the nuclear transcriptome. Here we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild type and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by over-accumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype.Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.

Tetrapyrroles are macrocyclic molecules with various structural variants and multiple functions in Prokaryotes and Eukaryotes. Present knowledge about the metabolism of tetrapyrroles reflects the complex evolution of the pathway in different kingdoms of organisms, the complexity of structural and enzymatic variations of enzymatic steps, as well as a wide range of regulatory mechanisms, which ensure adequate synthesis of tetrapyrrole end-products at any time of development and environmental condition. This review intends to highlight new findings of research on tetrapyrrole biosynthesis in plants and algae. In the course of the heme and chlorophyll synthesis in these photosynthetic organisms, glutamate, one of the central and abundant metabolites, is converted into highly photoreactive tetrapyrrole intermediates. Thereby, several mechanisms of posttranslational control are thought to be essential for a tight regulation of each enzymatic step. Finally, we wish to discuss the potential role of tetrapyrroles in retrograde signaling and point out perspectives of the formation of macromolecular protein complexes in tetrapyrrole biosynthesis as an efficient mechanism to ensure a fine-tuned metabolic flow in the pathway. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steadystate levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.During the last decades, almost all enzymes of tetrapyrrole biosynthesis and their complex network of transcriptional regulation have been comprehensively studied (Tanaka et al., 2011). These studies revealed a complex control of the expression of genes encoding enzymes in the light-regulated chlorophyll (Chl)-synthesizing branch of tetrapyrrole metabolism. In brief, 5-aminolevulinic acid (ALA) is synthesized in a transfer RNA (tRNA) GLU -mediated pathway, and eight molecules of ALA are ultimately converted in a series of enzymatic steps to protoporphyrin IX. The polymeric magnesium (Mg) chelatase complex consisting of the three different subunits CHLH, CHLI, and CHLD directs protoporphyrin IX into the Mg branch of tetrapyrrole biosynthesis. Methylation of magnesium protoporphyrin (MgP) by MgP methyltransferase (CHLM) at the C13 of pyrrole ring C initiates the formation of the typical fifth ring. The product of this step, magnesium protoporphyrin monomethylester (MgPMME), is then converted to divinyl protochlorophyllide (PChlide) by an oxidative cyclase complex. NADPH:protochlorophyllide oxidoreductase (POR) synthesizes chlorophyllide (Chlide). PChlide and Chlide are most likely the main substrates of a divinyl reductase that reduces the C7-C8 double bond, forming a monovinyl product. The two final steps of Chl a and b synthesis are likely ...

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNAGlu by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.

SUMMARYThe GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light-or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O 2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to 1 O 2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.

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Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed PI .I Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome TI of Sacchuromyces cerevisiae. The human protein migrates on a polyacrylainide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but inRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differeniiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength given rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and plO5Mcm.

Chlorophyll is indispensable for life on Earth. Dynamic control of chlorophyll level, determined by the relative rates of chlorophyll anabolism and catabolism, ensures optimal photosynthesis and plant fitness. How plants post-translationally coordinate these two antagonistic pathways during their lifespan remains enigmatic. Here, we show that two Arabidopsis paralogs of BALANCE of CHLOROPHYLL METABOLISM (BCM) act as functionally conserved scaffold proteins to regulate the trade-off between chlorophyll synthesis and breakdown. During early leaf development, BCM1 interacts with GENOMES UNCOUPLED 4 to stimulate Mg-chelatase activity, thus optimizing chlorophyll synthesis. Meanwhile, BCM1's interaction with Mgdechelatase promotes degradation of the latter, thereby preventing chlorophyll degradation. At the onset of leaf senescence, BCM2 is up-regulated relative to BCM1, and plays a conserved role in attenuating chlorophyll degradation. These results support a model in which post-translational regulators promote chlorophyll homeostasis by adjusting the balance between chlorophyll biosynthesis and breakdown during leaf development.