Structure of the Mg-chelatase cofactor GUN4 reveals a novel hand-shaped fold for porphyrin binding

In plants, the accumulation of the chlorophyll precursor Mg-protoporphyrin IX (Mg-Proto) in the plastid regulates the expression of a number of nuclear genes with functions related to photosynthesis. Analysis of the plastid-to-nucleus signaling activity of Mg-Proto in Arabidopsis thaliana led to the discovery of GUN4, a novel porphyrin-binding protein that also dramatically enhances the activity of Mg-chelatase, the enzyme that synthesizes Mg-Proto. GUN4 may also play a role in both photoprotection and the cellular shuttling of tetrapyrroles. Here we report a 1.78-Å resolution crystal structure of Synechocystis GUN4, in which the porphyrin-binding domain adopts a unique three dimensional fold with a “cupped hand” shape. Biophysical and biochemical analyses revealed the specific site of interaction between GUN4 and Mg-Proto and the energetic determinants for the GUN4 • Mg-Proto interaction. Our data support a novel protective function for GUN4 in tetrapyrrole trafficking. The combined structural and energetic analyses presented herein form the physical-chemical basis for understanding GUN4 biological activity, including its role in the stimulation of Mg-chelatase activity, as well as in Mg-Proto retrograde signaling.     

Transient accumulation of Mg-protoporphyrin IX regulates expression of PhANGs - New evidence for the signaling role of tetrapyrroles in mature Arabidopsis plants

Genetic and physiological studies have revealed evidence for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis associated nuclear genes (PhANGs). It has been proposed that the tetrapyrrole pathway intermediate Mg-protoporphyrin IX (Mg-proto IX) acts as the signaling molecule in the pathways and accumulates in the chloroplasts and cytosol of the cell after treatment with the herbicide Norflurazon (NF). However, the role of Mg-Proto IX in plastid signaling has been challenged by two recent reports. In this paper, new evidence is presented supporting Mg-Proto IX as a plastid-signaling molecule in mature Arabidopsis seedlings. Fluorescence HPLC and confocal microscope observation verified that a short-term (<96 h) NF treatment resulted in a large accumulation of Mg-Proto IX accompanying with Lhcb repression, whereas the long-term NF treatments caused marked changes of tetrapyrrole pools, while Lhcb expression was continuously repressed. These results may explain the discrepancies among different reports. Reactive oxygen species (ROS) eliminator treatments only partly reversed the NF-induced repression of Lhcb. Therefore, the NF generates both ROS signals and Mg-Proto IX signals. Furthermore, our data suggested that plastid signal transduction through plastid GUN1 protein is independent of tetrapyrrole export from the plastid.     

GUN4-porphyrin complexes bind the ChlH/GUN5 subunit of Mg-Chelatase and promote chlorophyll biosynthesis in Arabidopsis

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O₂. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex.     

C-terminal residues of oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis

Oryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg(2+) and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension.     

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.     

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.     

Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Intermediates in the formation of the chlorophyll isocyclic ring

Cell-free, organelle-free synthesis of Mg-2,4-divinylpheoporphyrin a₅ (MgDVP) from Mg-protoporphyrin IX monomethyl ester (Mg-Proto Me) has been described (Wong and Castelfranco 1984 Plant Physiol 75: 658-661). This system consists of plastid membrane and stromal fractions and requires O₂, NAD(P)H and S-adenosylmethionine (SAM). The synthetic 6-methyl-β-ketopropionate analog of Mg-Proto Me was converted to MgDVP by the same catalytic system in the presence of O₂ and NADPH. SAM was not required. A compound (X) displaying the kinetic behavior of an intermediate was isolated from reaction mixtures with Mg-Proto Me as the substrate, but not with the 6-methyl-β-ketopropionate analog as the substrate. X was identified as the 6-methyl-β-hydroxypropionate analog of Mg-Proto Me by conversion to the dimethyl ester with CH₂N₂ and comparison with authentic 6-β-hydroxydimethyl ester. X was converted to MgDVP by the same catalytic system in the presence of O₂ and NADPH. We conclude that the conversion of Mg-Proto Me to MgDVP proceeds through the 6-β-hydroxy and the 6-β-ketopropionate esters in agreement with earlier suggestions.