The ins and outs of peroxisomes: Co-ordination of membrane transport and peroxisomal metabolism

AbstractPeroxisomes perform a range of metabolic functions which require the movement of substrates, co-substrates, cofactors and metabolites across the peroxisomal membrane. In this review, we discuss the evidence for and against specific transport systems involved in peroxisomal metabolism and how these operate to co-ordinate biochemical reactions within the peroxisome with those in other compartments of the cell

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of L-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by electron transport through this complex, (b) the absolute requirement of the enzyme for oxidized cytochrome c (cyt c(ox)) as substrate, and (c) the coordinated response of respiration and AA synthesis to stress induced by hormone treatment.Instituto de Fisiología VegetalFacultad de Ciencias Agrarias y Forestale

Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.Instituto de Fisiología Vegeta

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of L-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by electron transport through this complex, (b) the absolute requirement of the enzyme for oxidized cytochrome c (cyt c(ox)) as substrate, and (c) the coordinated response of respiration and AA synthesis to stress induced by hormone treatment.Instituto de Fisiología VegetalFacultad de Ciencias Agrarias y Forestale

Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.Instituto de Fisiología Vegeta

Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress

Timely perception of adverse environmental changes is critical for survival. Dynamic changes in gases are important cues for plants to sense environmental perturbations, such as submergence. In Arabidopsis thaliana, changes in oxygen and nitric oxide (NO) control the stability of ERFVII transcription factors. ERFVII proteolysis is regulated by the N-degron pathway and mediates adaptation to flooding-induced hypoxia. However, how plants detect and transduce early submergence signals remains elusive. Here we show that plants can rapidly detect submergence through passive ethylene entrapment and use this signal to pre-adapt to impending hypoxia. Ethylene can enhance ERFVII stability prior to hypoxia by increasing the NO-scavenger PHYTOGLOBIN1. This ethylene-mediated NO depletion and consequent ERFVII accumulation pre-adapts plants to survive subsequent hypoxia. Our results reveal the biological link between three gaseous signals for the regulation of flooding survival and identifies key regulatory targets for early stress perception that could be pivotal for developing flood-tolerant crops

N-terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N-end rule pathway

The N-end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking.To investigate the impact of the Arg/N-end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT-TAILS) for relative quantification of N-terminal peptides in prt6, an Arabidopsis thaliana N-end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6).TMT-TAILS identified over 4000 unique N-terminal peptides representing c. 2000 protein groups. Forty-five protein groups exhibited significantly increased N-terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm a-cruciferin was delayed in prt6 seedlings. N-termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII-dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript. We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action

How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data

Peroxisomal ABC transporters: functions and mechanism

Peroxisomes are arguably the most biochemically versatile of all eukaryotic organelles. Their metabolic functions vary between different organisms, between different tissue types of the same organism, and even between different developmental stages or in response to changed environmental conditions. New functions for peroxisomes are still being discovered and their importance is underscored by the severe phenotypes that can arise as a result of peroxisome dysfunction. The β-oxidation pathway is central to peroxisomal metabolism, but the substrates processed are very diverse, reflecting the diversity of peroxisomes across species. Substrates for β-oxidation enter peroxisomes via ATP Binding Cassette (ABC) transporters of the ABCD subfamily and are activated by specific acyl CoA synthetases for further metabolism. Humans have three peroxisomal ABCD family members, which are half transporters that homodimerise and have distinct but partially overlapping substrate specificity; S. cerevisiae has two half transporters that heterodimerise and plants have a single peroxisomal ABC transporter that is a fused heterodimer and which appears to be the single entry point into peroxisomes for a very wide variety of β-oxidation substrates. Our studies suggest that the Arabidopsis peroxisomal ABC transporter AtABCD1 (COMATOSE/PXA1/PED3) accepts acyl CoA substrates, cleaves them before or during transport followed by reactivation by peroxisomal synthetases. We propose that this is a general mechanism to provide specificity to this class of transporters and by which amphipathic compounds are moved across peroxisome membranes