Marie Garmier

Leaf Mitochondria Modulate Whole Cell Redox Homeostasis, Set Antioxidant Capacity, and Determine Stress Resistance through Altered Signaling and Diurnal Regulation

To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H2O2 and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

Lack of Respiratory Chain Complex I Impairs Alternative Oxidase Engagement and Modulates Redox Signaling during Elicitor-Induced Cell Death in Tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I–deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin NEa induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.

Complex I Dysfunction Redirects Cellular and Mitochondrial Metabolism in Arabidopsis

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. However, the plant enzyme has structural and functional features that set it apart from its counterparts in other organisms, raising questions about the physiological significance of this complex in plants. We have developed an experimental model in which rotenone, a classic complex I inhibitor, has been applied to Arabidopsis (Arabidopsis thaliana) cell suspension cultures in order to dissect early metabolic adjustments involved in cell acclimation to mitochondrial dysfunction. Rotenone induced a transitory decrease in cellular respiration (0–4 h after treatment). Cell respiration then progressively recovered and reached a steady state at 10 to 12 h after treatment. Complex I inhibition by rotenone did not induce obvious oxidative stress or cell death but affected longer term cell growth. Integrated analyses of gene expression, the mitochondrial proteome, and changes in primary metabolism indicated that rotenone treatment caused changes in mitochondrial function via alterations in specific components. A physical disengagement of glycolytic activities associated with the mitochondrial outer membrane was observed, and the tricarboxylic acid cycle was altered. Amino acid and organic acid pools were also modified by rotenone treatment, with a marked early decrease of 2-oxoglutarate, aspartate, and glutamine pools. These data demonstrate that, in Arabidopsis cells, complex I inhibition by rotenone induces significant remodeling of metabolic pathways involving the mitochondria and other compartments and point to early metabolic changes in response to mitochondrial dysfunction.

Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences.

BackgroundMitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.ResultsIn this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence. Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.ConclusionsThese data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways.

Dissecting Phosphite-Induced Priming in Arabidopsis Infected with Hyaloperonospora arabidopsidis

Phosphite (Phi), a phloem-mobile oxyanion of phosphorous acid (H3PO3), protects plants against diseases caused by oomycetes. Its mode of action is unclear, as evidence indicates both direct antibiotic effects on pathogens as well as inhibition through enhanced plant defense responses, and its target(s) in the plants is unknown. Here, we demonstrate that the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) exhibits an unusual biphasic dose-dependent response to Phi after inoculation of Arabidopsis (Arabidopsis thaliana), with characteristics of indirect activity at low doses (10 mm or less) and direct inhibition at high doses (50 mm or greater). The effect of low doses of Phi on Hpa infection was nullified in salicylic acid (SA)-defective plants (sid2-1, NahG) and in a mutant impaired in SA signaling (npr1-1). Compromised jasmonate (jar1-1) and ethylene (ein2-1) signaling or abscisic acid (aba1-5) biosynthesis, reactive oxygen generation (atrbohD), or accumulation of the phytoalexins camalexin (pad3-1) and scopoletin (f6′h1-1) did not affect Phi activity. Low doses of Phi primed the accumulation of SA and Pathogenesis-Related protein1 transcripts and mobilized two essential components of basal resistance, Enhanced Disease Susceptibility1 and Phytoalexin Deficient4, following pathogen challenge. Compared with inoculated, Phi-untreated plants, the gene expression, accumulation, and phosphorylation of the mitogen-activated protein kinase MPK4, a negative regulator of SA-dependent defenses, were reduced in plants treated with low doses of Phi. We propose that Phi negatively regulates MPK4, thus priming SA-dependent defense responses following Hpa infection.

The differential spatial distribution of secondary metabolites in Arabidopsis leaves reacting hypersensitively to Pseudomonas syringae pv. tomato is dependent on the oxidative burst

Secondary metabolites (SMs) play key roles in pathogen responses, although knowledge of their precise functions is limited by insufficient characterization of their spatial response. The present study addressed this issue in Arabidopsis leaves by non-targeted and targeted metabolite profiling of Pseudomonas syringae pv. tomato (Pst-AvrRpm1) infected and adjacent uninfected leaf tissues. While overlap was observed between infected and uninfected areas, the non-targeted metabolite profiles of these regions differed quantitatively and clustering analysis underscores a differential distribution of SMs within distinct metabolic pathways. Targeted metabolite profiling revealed that infected tissues accumulate more salicylic acid and the characteristic phytoalexin of Arabidopsis, camalexin, than uninfected adjacent areas. On the contrary, the antioxidant coumarin derivative, scopoletin, was induced in infected tissues while its glucoside scopolin predominated in adjacent tissues. To elucidate the still unclear relationship between the accumulation of SMs and reactive oxygen species (ROS) accumulation and signalling, a catalase-deficient line (cat2) in which ROS signalling is up-regulated, was used. Metabolic analysis of cat2 suggests that some SMs have important interactions with ROS in redox homeostasis during the hypersensitive response to Pst-AvrRpm1. Overall, the study demonstrates that ROS availability influences both the amount and the pattern of infection-induced SM accumulation.

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

A mutation in the Arabidopsis mRNA polyadenylation factor suppresses the cell death associated with the immunity response mediated by salicylic acid and defective myoinositol biosynthesis. Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3′ end processing, through the regulation of SA production, is a key component of plant immune responses.

Light and Oxygen Are Not Required for Harpin-induced Cell Death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin NEa were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O2) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.

The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato

Secondary metabolism plant glycosyltransferases (UGTs) ensure conjugation of sugar moieties to secondary metabolites (SMs) and glycosylation contributes to the great diversity, reactivity and regulation of SMs. UGT73B3 and UGT73B5, two UGTs of Arabidopsis thaliana (Arabidopsis), are involved in the hypersensitive response (HR) to the avirulent bacteria Pseudomonas syringae pv. tomato (Pst-AvrRpm1), but their function in planta is unknown. Here, we report that ugt73b3, ugt73b5 and ugt73b3 ugt73b5 T-DNA insertion mutants exhibited an accumulation of reactive oxygen species (ROS), an enhanced cell death during the HR to Pst-AvrRpm1, whereas glutathione levels increased in the single mutants. In silico analyses indicate that UGT73B3 and UGT73B5 belong to the early salicylic acid (SA)-induced genes whose pathogen-induced expression is co-regulated with genes related to cellular redox homeostasis and general detoxification. Analyses of metabolic alterations in ugt mutants reveal modification of SA and scopoletin contents which correlate with redox perturbation, and indicate quantitative modifications in the pattern of tryptophan-derived SM accumulation after Pst-AvrRpm1 inoculation. Our data suggest that UGT73B3 and UGT73B5 participate in regulation of redox status and general detoxification of ROS-reactive SMs during the HR to Pst-AvrRpm1, and that decreased resistance to Pst-AvrRpm1 in ugt mutants is tightly linked to redox perturbation.

Changes in antioxidant expression and harpin-induced hypersensitive response in a Nicotiana sylvestris mitochondrial mutant

Abstract The present study was designed to investigate the role of plant mitochondria in redox sensing and hypersensitive response in a Nicotiana sylvestris mitochondrial mutant (CMSII mutant) that lacks a functional respiratory complex I. The abundance of compartment-specific transcripts encoding isoforms of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) was higher in the CMSII mutant. This suggests the presence of redox signalling, originating in the mitochondria, that affects the rest of the cell. In order to explore this further, the hypersensitive response induced in tobacco by harpin—a bacterial elicitor from Erwinia amylovora —was exploited as a model system for programmed cell death. Although the time course of harpin-induced necrosis was similar in the N. sylvestris wild-type and CMSII, several aspects of the hypersensitive response were found to be different in the mutant. For example, the accumulation of autofluorescent compounds, as observed under UV light, was lower in the mutant. In addition, presymptomatic transpiration was absent while cytosolic APX and PAL transcripts were enhanced. These results strongly suggest that mitochondrial functions (possibly mediated by redox changes) participate in the hypersensitive response to bacterial pathogens.