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Senescence is regulated by a complex interplay of factors and regulatory circuits, which may be accelerated or delayed depending on the integrated signals. Using a forward genetic screen in Arabidopsis thaliana, we identified a mutant strongly delayed in its induction of senescence in response to prolonged darkness. This mutant, which corresponds to a novel loss-of-function allele of PIF5 (PHYTOCHROME-INTERACTING FACTOR 5), exhibits even slightly more extended survival of leaves in darkness than the previously reported pif5-3 TDNA knock-out line. In the present study, we additionally aimed at deciphering the metabolic and regulatory processes conferring this enhanced capacity for survival in pif5 mutants. We combined physiological, metabolomic and transcriptomic analyses, and discovered that the extended survival of mutant leaves in darkness was associated with reduced protein degradation, slight differences in amino acid catabolism related gene expression as well as strong reduction of amino acid transporter expression, which coincided with enhanced amino acid accumulation. Our findings suggest that enhanced survival in darkness could be mediated by moderate levels of protein degradation allowing build up and slow usage of amino acids as alternative respiratory substrates, while during irreversible senescence, strong degradative processes, together with enhanced amino acid transport either to the site of their metabolization inside the leaf, or to other organs in the plant, could promote the fast progression of senescence and antagonize survival. Comparative metabolomics and gene expression analyses suggested that the senescence regulatory network downstream of PIF5 organizes these irreversible stages of leaf senescence, promoting autophagy and amino acid export, possibly by direct binding of important senescence promoting factors like ORE1 to the promoters of some of the involved genes. The failure to induce these later stages may prolong the reversible phase of d

Photorespiration is a complex and tightly regulated process occurring in photosynthetic organisms. This process can alter the cellular redox balance, notably via the production and consumption of both reducing and oxidizing equivalents. Under certain circumstances, these equivalents, as well as reactive oxygen or nitrogen species, can become prominent in subcellular compartments involved in the photorespiratory process, eventually promoting oxidative post-translational modifications of proteins. Keeping these changes under tight control should therefore be of primary importance. In order to review the current state of knowledge about the redox control of photorespiration, we primarily performed a careful description of the known and potential redox-regulated or oxidation sensitive photorespiratory proteins, and examined in more details two interesting cases: the glycerate kinase and the glycine cleavage system. When possible, the potential impact and subsequent physiological regulations associated with these changes have been discussed. In a second part, we reviewed the extent to which photorespiration contributes to cellular redox homeostasis considering, in particular, the set of peripheral enzymes associated with the canonical photorespiratory pathway. Finally, some recent biotechnological strategies to circumvent photorespiration for future growth improvements are discussed in the light of these redox regulations.

Cysteines are important residues for protein structure, function, and regulation. Owing to their modified reactivity, some cysteines can undergo very diverse redox posttranslational modifications, including the reversible formation of disulfide bonds, a widespread protein regulatory process as well exemplified in plant chloroplasts for Calvin-Benson cycle enzymes. Both core- and peripheral-photorespiratory enzymes possess conserved cysteines, some of which have been identified as being subject to oxidative modifications. This is not surprising considering their presence in subcellular compartments where the production of reactive species can be important. However, in most cases, the types of modifications and their biochemical effect on protein activity have not been validated, meaning that the possible impact of these modifications in a complex physiological context, such as photorespiration, remains obscure. We here describe a detailed set of protocols for alkylation methods that have been used so far to (1) study the protein cysteine redox state either in vitro by submitting purified recombinant proteins to reducing/oxidation treatments or in vivo by western blots on protein extracts from plants subject to environmental constraints, and its dependency on the two major reducing systems in the cell, i.e., the thioredoxin and glutathione/glutaredoxin systems, and (2) determine two key redox parameters, i.e., the cysteine pKa and the redox midpoint potential.

Recycling of the 2-phosphoglycolate generated by the oxygenase reaction of Rubisco requires a complex and energy-consuming set of reactions collectively known as the photorespiratory cycle. Several approaches aimed at reducing the rates of photorespiratory energy or carbon loss have been proposed, based either on screening for natural variation or by means of genetic engineering. Recent work indicates that plant yield can be substantially improved by the alteration of photorespiratory fluxes or by engineering artificial bypasses to photorespiration. However, there is also evidence indicating that, under certain environmental and/or nutritional conditions, reduced photorespiratory capacity may be detrimental to plant performance. Here we summarize recent advances obtained in photorespiratory engineering and discuss prospects for these advances to be transferred to major crops to help address the globally increasing demand for food and biomass production.

Leaf senescence - the coordinated, active process leading to the organized dismantling of cellular components to remobilize resources - is a fundamental aspect of plant life. Its tight regulation is essential for plant fitness and has crucial implications for the optimization of plant productivity and storage properties. Various investigations have shown light deprivation and light perception via phytochromes as key elements modulating senescence. However, the signalling pathways linking light deprivation and actual senescence processes have long remained obscure. Recent analyses have demonstrated that PHYTOCHROME-INTERACTING FACTORS (PIFs) are major transcription factors orchestrating dark-induced senescence (DIS) by targeting chloroplast maintenance, chlorophyll metabolism, hormone signalling and production, and the expression of senescence master regulators, uncovering potential molecular links to the energy deprivation signalling pathway. PIF-dependent feed-forward regulatory modules might be of critical importance for the highly complex and initially light-reversible DIS induction.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Recycling of the 2-phosphoglycolate generated by the oxygenase reaction of Rubisco requires a complex and energy-consuming set of reactions collectively known as the photorespiratory cycle. Several approaches aimed at reducing the rates of photorespiratory energy or carbon loss have been proposed, based either on screening for natural variation or by means of genetic engineering. Recent work indicates that plant yield can be substantially improved by the alteration of photorespiratory fluxes or by engineering artificial bypasses to photorespiration. However, there is also evidence indicating that, under certain environmental and/or nutritional conditions, reduced photorespiratory capacity may be detrimental to plant performance. Here we summarize recent advances obtained in photorespiratory engineering and discuss prospects for these advances to be transferred to major crops to help address the globally increasing demand for food and biomass production.

Being intimately intertwined with (C3) photosynthesis, photorespiration is an incredibly high flux-bearing pathway. Traditionally, the photorespiratory cycle was viewed as closed pathway to refill the Calvin-Benson cycle with organic carbon. However, given the network nature of metabolism, it hence follows that photorespiration will interact with many other pathways. In this article, we review current understanding of these interactions and attempt to define key priorities for future research, which will allow us greater fundamental comprehension of general metabolic and developmental consequences of perturbation of this crucial metabolic process.

Reduction of flux through photorespiration has been viewed as a major way to improve crop carbon fixation and yield since the energy-consuming reactions associated with this pathway were discovered. This view has been supported by the biomasses increases observed in model species that expressed artificial bypass reactions to photorespiration. Here, we present an overview about the major current attempts to reduce photorespiratory losses in crop species and provide suggestions for future research priorities.

Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.

Using a noninvasive, solid-state magic-angle spinning nuclearmagnetic resonance (MAS NMR) approach, we track ex vivo thebehavior of individual membrane components in isolated, activemitochondria (model system: potato tubers) during physiologicalprocesses. The individual phosphatidylcholine (PC), phosphatidylethanolamine(PE), and cardiolipin (CL) membrane constituents were identifiedas distinct lines by applying MAS 31P NMR on extracted lipidmembranes. However, the CL NMR signal appeared to be very broadin functional mitochondria, indicating a tight complex formationwith membrane protein. Calcium stress induced severe membranedegradation without recovery of a single CL NMR resonance. Thissuggests that calcium overload destroys the outer mitochondrialmembrane and does not modify strongly the CL protein complexesin the inner membrane; a conclusion confirmed by respiratorycontrols. Conversely, mitochondrial membrane disruption on timedegradation or mechanical stress generates clearly visible identicalCL NMR signals, similar to those observed in rehydrated lipidextracts. Similarly, noninvasive based NMR tracking of lipidsin response to diverse physiological stimuli can easily be usedfor other organelles and whole living cells. Sani, M.-A., Keech,O., Gardeström, P., Dufourc, E. J., Gröbner, G. Magic-anglephosphorus NMR of functional mitochondria: in situ monitoringof lipid response under apoptotic-like stress.

Mitochondria are a major site of reactive oxygen species production and controlling the peroxide levels in this compartment is essential. Peroxiredoxins (Prx) are heme-free peroxidases, which use reactive cysteines for their catalysis and reducing systems for their regeneration. One of the two Prxs present in poplar mitochondria, Prx IIF, expressed as a recombinant protein, was found to reduce a broad range of peroxides with electrons provided preferentially by glutaredoxin and to a lesser extent by glutathione, all the thioredoxins tested being inefficient. This protein is constitutively expressed because it is found in all tissues analyzed. Its expression is modified during a biotic interaction between poplar and the rust fungus Melampsora laricii populina. On the other hand, Prx IIF expression does not substantially vary under abiotic stress conditions. Nevertheless, water deficit or chilling and probably induced senescence, but not photooxidative conditions or heavy metal treatment, also led to a small increase in PrxIIF abundance in Arabidopsis thaliana plants., First published: 07 November 2006

Senescence is an active process allowing the reallocation of valuable nutrients from the senescing organ towards storage and/or growing tissues. Using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs), we investigated the fate of mitochondria and chloroplasts during dark-induced leaf senescence. Combining in vivo visualization of fates of the two organelles by three-dimensional reconstructions of abaxial parts of leaves with functional measurements of photosynthesis and respiration, we showed that the two experimental systems displayed major differences during 6 d of dark treatment. In whole DPs, organelles were largely retained in both epidermal and mesophyll cells. However, while the photosynthetic capacity was maintained, the capacity of mitochondrial respiration decreased. In contrast, IDLs showed a rapid decline in photosynthetic capacity while maintaining a high capacity for mitochondrial respiration throughout the treatment. In addition, we noticed an unequal degradation of organelles in the different cell types of the senescing leaf. From these data, we suggest that metabolism in leaves of the whole DPs enters a ‘stand-by mode’ to preserve the photosynthetic machinery for as long as possible. However, in IDLs, mitochondria actively provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. Finally, the heterogeneity of the degradation processes involved during senescence is discussed with regard to the fate of mitochondria and chloroplasts in the different cell types.

När ett blad gulnar genomgår det både morfologiska och metaboliska förändringar. Denna process benämns senescence och en förbättrad förståelse av dess mekanismer är viktiga både ur ett grundvetenskapligt perspektiv och för potentiella bioteknologiska applikationer. Denna avhandling rapporterar om flera viktiga aspekter relaterade till de cellulära och metaboliska mekanismer som sker under senescencen med tonvikt på mitokondriernas bidrag till denna process. I ett första steg utvecklade vi metoder för att isolera antingen mycket funktionella mitokondrier eller mycket rena mitokondrier från blad av Arabidopsis thaliana. Dessa metoder användes sedan till för att studera mitokondriella bidrag till cellens redox balans och att uppskatta mitokondriernas kapacitet under senescence-processen. Framför allt jämfördes induktionen av senescencen berodende på olika mörkerbehandlingar av Arabidopsis. Jämförelse mellan individuellt mörklagda blad med hela mörklagda växter visade en betydande skillnad i metabolisk strategi mellan de två mörkerbehandlingarna. Genom att integrera data från mätningar av fotosyntes, respiration och konfokal mikroskopi med transcriptomics- och metabolomics-profiler föreslår vi att metabolismen hos blad från helt mörklagda växter antar ett ”stand-by läge” för att kunna bibehålla fotosynteskapaciteten så länge som möjligt. I kontrast till detta visar mitokondrier från individuellt mörklagda blad en hög aktivitet och kan därmed producera energi och kolskelett för degraderingen av cellkomponenter, vilket möjliggör återvinning av näringsämnen. Vi har även studerat dynamiken av det mikrotubulibaserade cytoskelettet under mörkerindicerad senescence. Mitokondriernas rörlighet påverkades av en tidig nedbrytning av mikrotubuli hos individuellt mörklagda blad men inte hos blad där hela växten mörkerbehandlats. Dessutom verkade ett flertal mikrotubuliassocierade proteiner (MAP’s) att vara involverade i buntningen av mikrotubuli runt kloroplasterna. Sammanfattningsv, When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process. As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomics and metabolomics profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamic of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the

Glutaredoxins (Grxs) are small ubiquitous proteins of the thioredoxin (Trx) family, which catalyze dithiol–disulfide exchange reactions or reduce protein-mixed glutathione disulfides. In plants, several Trx-interacting proteins have been isolated from different compartments, whereas very few Grx-interacting proteins are known. We describe here the determination of Grx target proteins using a mutated poplar Grx, various tissular and subcellular plant extracts, and liquid chromatography coupled to tandem mass spectrometry detection. We have identified 94 putative targets, involved in many processes, including oxidative stress response [peroxiredoxins (Prxs), ascorbate peroxidase, catalase], nitrogen, sulfur, and carbon metabolisms (methionine synthase, alanine aminotransferase, phosphoglycerate kinase), translation (elongation factors E and Tu), or protein folding (heat shock protein 70). Some of these proteins were previously found to interact with Trx or to be glutathiolated in other organisms, but others could be more specific partners of Grx. To substantiate further these data, Grx was shown to support catalysis of the stroma β-type carbonic anhydrase and Prx IIF of Arabidopsis thaliana, but not of poplar 2-Cys Prx. Overall, these data suggest that the interaction could occur randomly either with exposed cysteinyl disulfide bonds formed within or between target proteins or with mixed disulfides between a protein thiol and glutathione.