The PAP/SAL1 retrograde signaling pathway is involved in iron homeostasis

Plant Molecular Biology - There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde...     

A nucleotide metabolite controls stress-responsive gene expression and plant development

Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1) protein is a negative regulator of stress and abscisic acid (ABA) signaling and exhibits both an inositol polyphosphatase and a 3',5'-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3'-phosphoadenosine-5'-phosphate (PAP), yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt toxicity target in Arabidopsis.     

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 mitochondrial half-size ABC transporter is involved in cadmium tolerance in Chlamydomonas reinhardtii

Five cadmium-sensitive insertional mutants, all affected at the CDS1 (' cadmium-sensitive 1 ') locus, have been previously isolated in the unicellular green alga Chlamydomonas reinhardtii . We here describe the cloning of the Cds1 gene (8314 bp with 26 introns) and the corresponding cDNA. The Cds1 gene, strongly induced by cadmium, encodes a putative protein (CrCds1) of 1062 amino acid residues that belongs to the ATM/HMT subfamily of half-size ABC transporters. This subfamily includes both vacuolar HMT-type proteins transporting phytochelatin-cadmium complexes from the cytoplasm to the vacuole and mitochondrial ATM-type proteins involved in the maturation of cytosolic Fe/S proteins. Unlike the Δsphmt1 cadmium-sensitive mutant of Schizosaccharomyces pombe that lacks a vacuolar HMT-type transporter, the cds1 mutant accumulates a high amount of phytochelatin–cadmium complexes. By epitope tagging, the CrCds1 protein was localized in the mitochondria. Even though mitochondria of cds1 do not accumulate important amounts of 'free' iron, the mutant cells are hypersensitive to high iron concentrations . Our data show for the first time that a mitochondrial ATM-like transporter plays a major role in tolerance to cadmium.     

ROS and redox signalling in the response of plants to abiotic stress

The redox state of the chloroplast and mitochondria, the two main powerhouses of photosynthesizing eukaryotes, is maintained by a delicate balance between energy production and consumption, and affected by the need to avoid increased production of reactive oxygen species (ROS). These demands are especially critical during exposure to extreme environmental conditions, such as high light (HL) intensity, heat, drought or a combination of different environmental stresses. Under these conditions, ROS and redox cues, generated in the chloroplast and mitochondria, are essential for maintaining normal energy and metabolic fluxes, optimizing different cell functions, activating acclimation responses through retrograde signalling, and controlling whole-plant systemic signalling pathways. Regulation of the multiple redox and ROS signals in plants requires a high degree of coordination and balance between signalling and metabolic pathways in different cellular compartments. In this review, we provide an update on ROS and redox signalling in the context of abiotic stress responses, while addressing their role in retrograde regulation, systemic acquired acclimation and cellular coordination in plants.     

The Yeast Translational Allosuppressor, Sal6: A New Member of the Pp1-like Phosphatase Family with a Long Serine-Rich N-Terminal Extension

The allosuppressor mutation, sal6-1, enhances the efficiency of all tested translational suppressors, including codon-specific tRNA suppressors as well as codon-nonspecific omnipotent suppressors. The SAL6 gene has now been cloned by complementation of the increased suppression efficiency and cold sensitivity caused by sal6-1 in the presence of the omnipotent suppressor sup45. Physical analysis maps SAL6 to chromosome XVI between TPK2 and spt14. The SAL6 gene encodes a very basic 549-amino acid protein whose C-terminal catalytic region of 265 residues is 63% identical to serine/threonine PP1 phosphatases, and 66% identical to yeast PPZ1 and PPZ2 phosphatases. The unusual 235 residue N-terminal extension found in SAL6, like those in the PPZ proteins, is serine-rich. The sal6-1 mutation is a frameshift at amino acid position 271 which destroys the presumed phosphatase catalytic domain of the protein. Disruptions of the entire SAL6 gene are viable, cause a slight growth defect on glycerol medium, and produce allosuppressor phenotypes in suppressor strain backgrounds. The role of the serine-rich N terminus is unclear, since sal6 phenotypes are fully complemented by a SAL6 allele that contains an in-frame deletion of most of this region. High copy number plasmids containing wild-type SAL6 cause antisuppressor phenotypes in suppressor strains. These results suggest that the accuracy of protein synthesis is affected by the levels of phosphorylation of the target(s) of SAL6.     

An Arabidopsis cotyledon-specific albino locus: a possible role in 16S rRNA maturation

We report here the isolation and characterization of a cotyledon-specific albino locus of Arabidopsis, WHITE COTYLEDONS (WCO). This recessive mutation in the WCO locus, located on the top of Chromosome 1, results in albino cotyledons but green true leaves. An accumulation profile of chlorophylls and ultrastructure of chloroplasts indicate that WCO is necessary for development of functional chloroplasts in cotyledons but is dispensable in true leaves. This was further supported by the fact that the mutants request feeding of sucrose for their survival at the early seedling stage where true leaves have not emerged, but the mutants which have developed true leaves are able to grow autotrophically without sucrose supplementation. The wco mutants accumulate low levels of chloroplast mRNA encoding photosynthesis-related proteins and have a specific defect in 16S rRNA maturation in a cotyledon-specific manner. Although wco mutants exhibited abnormal chloroplasts and chloroplast gene expression in cotyledons, nuclear genes for photosynthetic components are expressed at similar levels to those found in wild-type siblings. This lack of suppression of the nuclear genes is not due to a defect in the signaling of the so-called "plastid factor" to the nucleus since normal suppression of the nuclear genes was observed in response to the photo-oxidative damage due to norflurazon application.     

In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast

The photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. To ensure that the photosynthetic complexes are assembled stoichiometrically and to enable their rapid reorganization in response to a changing environment, the plastids emit signals that regulate nuclear gene expression to match the status of the plastids. One of the plastid signals, the chlorophyll intermediate Mg-ProtoporphyrinIX (Mg-ProtoIX) accumulates under stress conditions and acts as a negative regulator of photosynthetic gene expression. By taking advantage of the photoreactive property of tetrapyrroles, Mg-ProtoIX could be visualized in the cells using confocal laser scanning spectroscopy. Our results demonstrate that Mg-ProtoIX accumulated both in the chloroplast and in the cytosol during stress conditions. Thus, the signaling metabolite is exported from the chloroplast, transmitting the plastid signal to the cytosol. Our results from the Mg-ProtoIX over- and underaccumulating mutants copper response defect and genome uncoupled5, respectively, demonstrate that the expression of both nuclear- and plastid-encoded photosynthesis genes is regulated by the accumulation of Mg-ProtoIX. Thus, stress-induced accumulation of the signaling metabolite Mg-ProtoIX coordinates nuclear and plastidic photosynthetic gene expression.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.