A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana

To identify potentially novel and essential components of plant membrane trafficking mechanisms we performed a GFP-based forward genetic screen for seedling-lethal biosynthetic membrane trafficking mutants in Arabidopsis thaliana. Amongst these mutants, four recessive alleles of GSH2, which encodes glutathione synthase (GSH2), were recovered. Each allele was characterized by loss of the typical polygonal endoplasmic reticulum (ER) network and the accumulation of swollen ER-derived bodies which accumulated a soluble secretory marker. Since GSH2 is responsible for converting γ-glutamylcysteine (γ-EC) to glutathione (GSH) in the glutathione biosynthesis pathway, gsh2 mutants exhibited γ-EC hyperaccumulation and GSH deficiency. Redox-sensitive GFP revealed that gsh2 seedlings maintained redox poise in the cytoplasm but were more sensitive to oxidative challenge. Genetic and pharmacological evidence indicated that γ-EC accumulation rather than GSH deficiency was responsible for the perturbation of ER morphology. Use of soluble and membrane-bound ER markers suggested that the swollen ER bodies were derived from ER fusiform bodies. Despite the gross perturbation of ER morphology, gsh2 seedlings did not suffer from constitutive oxidative ER stress or lack of an unfolded protein response, and homozygotes for the weakest allele could be propagated. The link between glutathione biosynthesis and ER morphology and function is discussed.     

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.     

Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, γ -glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine–synthase complex.     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.     

Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress

The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar ( Populus tremula × P. alba ) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.     

Specific delivery of AtBT1 to mitochondria complements the aberrant growth and sterility phenotype of homozygous Atbt1 Arabidopsis mutants

It has been shown that homozygous AtBT1::T-DNA Arabidopsis mutants display an aberrant growth and sterility phenotype, and that AtBT1 is a carrier that is exclusively localized to the inner plastidial envelope and is required for export of newly synthesized adenylates into the cytosol. However, a recent demonstration that AtBT1 is localized to both plastids and mitochondria suggested that plastidic AtBT1 is not necessary for normal growth and fertility of Arabidopsis. To test this hypothesis, we produced and characterized homozygous AtBT1::T-DNA mutants stably expressing either dually localized AtBT1 or AtBT1 specifically localized to the mitochondrial compartment. These analyses revealed that the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants was complemented when expressing both the dual-targeted AtBT1 and AtBT1 specifically delivered to mitochondria. These data confirm that (i) plastidic AtBT1 is not strictly required for normal growth and fertility of the plant, and (ii) specific delivery of AtBT1 to mitochondria is enough to complement the aberrant growth and sterility phenotype of homozygous AtBT1::T-DNA mutants. Furthermore, data presented here question the idea that the requirement for AtBT1 is due to its involvement in transport of newly synthesized adenylates from the plastid to the cytosol, and suggest that the protein may play as yet unidentified functions in plastids and mitochondria.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5′ phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using ³⁴SO₄^2– also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non‐stress and oxidative stress conditions. However, when germinating on sulfur‐deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.     

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae . The PAD2 gene encodes a γ-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1 . In contrast to the jasmonate-insensitive mutant coi1-1 , expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1 . Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.     

PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted alpha-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake-defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis.     

AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis

Iron-sulfur (Fe-S) clusters are vital prosthetic groups for Fe-S proteins involved in fundamental processes such as electron transfer, metabolism, sensing and signaling. In plants, sulfur (SUF) protein-mediated Fe-S cluster biogenesis involves iron acquisition and sulfur mobilization, processes suggested to be plastidic. Here we have shown that AtSufE in Arabidopsis rescues growth defects in SufE-deficient Escherichia coli. In contrast to other SUF proteins, AtSufE localizes to plastids and mitochondria interacting with the plastidic AtSufS and mitochondrial AtNifS1 cysteine desulfurases. AtSufE activates AtSufS and AtNifS1 cysteine desulfurization, and AtSufE activity restoration in either plastids or mitochondria is not sufficient to rescue embryo lethality in AtSufE loss-of-function mutants. AtSufE overexpression induces AtSufS and AtNifS1 expression, which in turn leads to elevated cysteine desulfurization activity, chlorosis and retarded development. Our data demonstrate that plastidic and mitochondrial Fe-S cluster biogenesis shares a common, essential component, and that AtSufE acts as an activator of plastidic and mitochondrial desulfurases in Arabidopsis.