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.     

Implication of chlorophyll biosynthesis on chloroplast-to-nucleus retrograde signaling

The biogenesis and function of chloroplast are controlled both by anterograde mechanisms involving nuclear-encoded proteins targeted to chloroplast and by retrograde signals from plastid to nucleus contributing to regulation of nuclear gene expression. A number of experimental evidences support the implication of chlorophyll biosynthesis intermediates on the retrograde signaling, albeit an earlier-postulated direct link between accumulation of chlorophyll intermediates and changes in nuclear gene expression has recently been challenged. By characterization of Arabidopsis mutants lacking the chloroplast localized NADPH-thioredoxin reductase (NTRC) we have recently proposed that imbalanced activity of chlorophyll biosynthesis in developing cells modifies the chloroplast signals leading to alterations in nuclear gene expression. These signals appear to initiate from temporal perturbations in the flux through the pathway from protoporphyrin to protochlorophyllide rather than from the accumulation of a single intermediate of the tetrapyr-role pathway.Key words: chloroplast biogenesis, NADPH-thioredoxin reductase, porphyrins, ROS, signaling, tetrapyrrole, thioredoxinOrchestrated regulation of gene expression in the nucleus and plastids is crucial for the proper biogenesis of the organelle during the development and for the acclimation of plants to environmental cues. Multiple potential candidates for initiating plastidial signals have been recognized, including intermediates of the tetrapyrrole biosynthetic pathway, redox state of chloroplast electron transfer components and reactive oxygen species (ROS). These multiple signaling pathways are likely to interact with each others, resulting in a complex signaling network between plastid and nucleus (reviewed in ref. ¹).     

A new gun mutant of oilseed rape with a reduced porphyrin flux through Mg-chelatase

Plastid-to-nucleus retrograde signaling coordinates the expression of nuclear photosynthetic genes with the developmental and functional state of the plastid. These signals are essential not only for coordinating the expression of photosynthetic genes both in the plastome and nuclear genome, but also for plants to respond optimally to environmental stress. In the present study, we found that the expression of the nuclear genes that encode plastid and non-plastid photosynthesis-related proteins was still maintained or slightly higher in cr3529, a chlorophyll deficient mutant of oilseed rape that possesses an arrested development of chloroplasts, suggesting that the expression of photosynthesis-related nuclear genes was uncoupled from the normal dependence on the developmental state of the chloroplast in cr3529. When the development of the plastid in cr3529 and the wild type was completely inhibited by lincomycin, much higher expression of photosynthesis-related nuclear genes was observed in cr3529, suggesting that the genomes uncoupled (gun) phenotype of cr3529 is even more apparent than under normal growth conditions. Lincomycin treatment also derepressed the expression of plastid genes in cr3529. The determination of porphyrin flux through Mg-chelatase showed that the content of protoporphyrin IX and Mg-protoporphyrin decreased in cr3529. The obvious gun phenotype of cr3529 under normal growth conditions and the pattern of tetrapyrrole metabolism in cr3529 suggest that it is a new gun mutant that could be used to study the regulation of the expression of nuclear and plastid genes by plastid-to-nucleus retrograde signaling under more physiological conditions and the mechanism of plant stress responses mediated by plastid signals.     

PAPP5 Is Involved in the Tetrapyrrole Mediated Plastid Signalling during Chloroplast Development

The initiation of chloroplast development in the light is dependent on nuclear encoded components. The nuclear genes encoding key components in the photosynthetic machinery are regulated by signals originating in the plastids. These plastid signals play an essential role in the regulation of photosynthesis associated nuclear genes (PhANGs) when proplastids develop into chloroplasts. One of the plastid signals is linked to the tetrapyrrole biosynthesis and accumulation of the intermediates the Mg-ProtoIX and its methyl ester Mg-ProtoIX-ME. Phytochrome-Associated Protein Phosphatase 5 (PAPP5) was isolated in a previous study as a putative Mg-ProtoIX interacting protein. In order to elucidate if there is a biological link between PAPP5 and the tetrapyrrole mediated signal we generated double mutants between the Arabidopsis papp5 and the crd mutants. The crd mutant over-accumulates Mg-ProtoIX and Mg-ProtoIX-ME and the tetrapyrrole accumulation triggers retrograde signalling. The crd mutant exhibits repression of PhANG expression, altered chloroplast morphology and a pale phenotype. However, in the papp5crd double mutant, the crd phenotype is restored and papp5crd accumulated wild type levels of chlorophyll, developed proper chloroplasts and showed normal induction of PhANG expression in response to light. Tetrapyrrole feeding experiments showed that PAPP5 is required to respond correctly to accumulation of tetrapyrroles in the cell and that PAPP5 is most likely a component in the plastid signalling pathway down stream of the tetrapyrrole Mg-ProtoIX/Mg-ProtoIX-ME. Inhibition of phosphatase activity phenocopied the papp5crd phenotype in the crd single mutant demonstrating that PAPP5 phosphatase activity is essential to mediate the retrograde signal and to suppress PhANG expression in the crd mutant. Thus, our results suggest that PAPP5 receives an inbalance in the tetrapyrrole biosynthesis through the accumulation of Mg-ProtoIX and acts as a negative regulator of PhANG expression during chloroplast biogenesis and development.     

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.     

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.     

GUN control in retrograde signaling: How GENOMES UNCOUPLED proteins adjust nuclear gene expression to plastid biogenesis

Communication between cellular compartments is vital for development and environmental adaptation. Signals emanating from organelles, so-called retrograde signals, coordinate nuclear gene expression with the developmental stage and/or the functional status of the organelle. Plastids (best known in their green photosynthesizing differentiated form, the chloroplasts) are the primary energy-producing compartment of plant cells, and the site for the biosynthesis of many metabolites, including fatty acids, amino acids, nucleotides, isoprenoids, tetrapyrroles, vitamins, and phytohormone precursors. Signals derived from plastids regulate the accumulation of a large set of nucleus-encoded proteins, many of which localize to plastids. A set of mutants defective in retrograde signaling (genomes uncoupled, or gun) was isolated over 25 years ago. While most GUN genes act in tetrapyrrole biosynthesis, resolving the molecular function of GUN1, the proposed integrator of multiple retrograde signals, has turned out to be particularly challenging. Based on its amino acid sequence, GUN1 was initially predicted to be a plastid-localized nucleic acid-binding protein. Only recently, mechanistic information on the function of GUN1 has been obtained, pointing to a role in plastid protein homeostasis. This review article summarizes our current understanding of GUN-related retrograde signaling and provides a critical appraisal of the various proposed roles for GUNs and their respective pathways.

This review summarizes new insights in GUN-mediated retrograde signaling, and highlights outstanding questions and challenges that should be addressed in future research.     

Plant signaling systems. Plastid-generated signals and their role in nuclear gene expression

Current concepts are outlined regarding the chloroplast effects on expression of the nuclear genes encoding plastid proteins. The major types of plastid-generated signals are considered. The signal molecules are shown to include the reactive oxygen species, the redox state of the components of photosynthetic electron transport, in particular plastoquinones, the redox-active molecules of plastid stroma, such as thioredoxin and glutathione, and also the intermediates of tetrapyrrole biosynthesis (Mg-protoporphyrin IX and its monomethyl ester). The sophisticated regulatory network is emphasized as a channel matching up the expression of nuclear and plastid genes. The plastid-generated signals help plants adapt to the changing and frequently adverse environmental conditions.     

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll(ides) by spectrofluorometry

General equations which permit the determination of the amounts of any two closely related fluorescent compounds which can be distinguished by 77 degrees K but not by 293 degrees K spectrofluorometry have been described. This was achieved in the presence or absence of a third interfering compound, without prior separation of the fluorescent species. The adaptation of the generalized equations to the determination of the amounts of monovinyl (MV) and divinyl (DV) Mg-protoporphyrins or of MV and DV protochlorophyll(ides) in the presence or absence of Mg-Protos [Mg-protoporphyrin IX (Mg-Proto), Mg-Proto monoester, Mg-Proto diester or a mixture of those three tetrapyrroles] interference, was then demonstrated over a wide range of MV/DV tetrapyrrole proportions. These equations are likely to be very useful for the study of the intermediary metabolism of the monovinyl and divinyl chlorophyll biosynthetic routes in plants.     

Oxygen and reactive oxygen species-dependent regulation of plant growth and development

Oxygen and reactive oxygen species (ROS) have been co-opted during evolution into the regulation of plant growth, development, and differentiation. ROS and oxidative signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant development from seed and bud dormancy, liberation of meristematic cells from the quiescent state, root and shoot growth, and architecture, to flowering and seed production. Moreover, the phytochrome and phytohormone-dependent transmissions of ROS waves are central to the systemic whole plant signaling pathways that integrate root and shoot growth. The sensing of oxygen availability through the PROTEOLYSIS 6 (PRT6) N-degron pathway functions alongside ROS production and signaling but how these pathways interact in developing organs remains poorly understood. Considerable progress has been made in our understanding of the nature of hydrogen peroxide sensors and the role of thiol-dependent signaling networks in the transmission of ROS signals. Reduction/oxidation (redox) changes in the glutathione (GSH) pool, glutaredoxins (GRXs), and thioredoxins (TRXs) are important in the control of growth mediated by phytohormone pathways. Although, it is clear that the redox states of proteins involved in plant growth and development are controlled by the NAD(P)H thioredoxin reductase (NTR)/TRX and reduced GSH/GRX systems of the cytosol, chloroplasts, mitochondria, and nucleus, we have only scratched the surface of this multilayered control and how redox-regulated processes interact with other cell signaling systems.

Oxygen and reactive oxygen species regulate plant growth, development, and differentiation through multiple interlinked signaling pathways.

Advances

• Developmentally regulated hypoxia and reactive oxygen species (ROS) production are key features of the stem cell niches, providing information about stem cell position, the environment, and metabolic state.
• Protein cysteine oxidation is central to oxygen and ROS signaling. However, S-nitrosylation, S-glutathionylation, S-sulfhydration, and S-sulfenylation modifications can occur on the same cysteine. The influence of each modification on stability, localization, and function remains unknown.
• Numerous intersecting ROS signaling pathways are probable and likely depend on the site of ROS production and the nature of the oxidized receptor protein. ROS sensors such as the hydrogen peroxide (H₂O₂)-INDUCED Ca²⁺ INCREASES 1 (HPCA1) leucine rich receptor kinase translate redox signals into protein modifications to regulate signaling cascades. H₂O₂ perception/transduction is dependent on thiol-dependent mechanisms policed by the ferredoxin/thioredoxin (TRX), NAD(P)H TRX reductase C (NTRC), reduced glutathione (GSH), and glutaredoxin (GRX) systems.
• ROS waves transmit redox signals from cell to cell in the apoplast, and probably through plasmodesmata. Long-distance transport of H₂O₂ and other ROS, therefore, appears to be unnecessary. Similarly, contact sites between organelles allow ROS transfer.
• Convergence points for oxygen and ROS signaling occur on proteins such as ROH OF PLANT 2 (ROP2) GTPase,RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and TRX-h to regulate meristematic activity via TARGET OF RAPAMYCIN (TOR) kinase activity.

     

Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia

Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell. Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production and by regulating mitochondria-dependent apoptosis pathways.