Carlos G. Bartoli

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

Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min−1 mg−1 protein in the presence of 1 mm NaNO2. Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 ± 0.04 nmol min−1 mg−1 protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 ± 0.04 nmol min−1 mg−1 protein that was not affected either by omission of Ca2+ or by supplementation with Ca2+ and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors Nω-nitro-l-Arg methyl ester hydrochloride and Nω-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 μm S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 μm peroxynitrite (ONOO−) led to an increase in lipid-derived radicals (34%). The effect of ONOO− on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO− treatment significantly affected both O2 evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

Auxin signaling participates in the adaptative response against oxidative stress and salinity by interacting with redox metabolism in Arabidopsis

Auxin regulates gene expression through direct physical interaction with TIR1/AFB receptor proteins during different processes of growth and development in plants. Here we report the contribution of auxin signaling pathway to the adaptative response against abiotic stress in Arabidopsis. Phenotypic characterization of tir1/afb auxin receptor mutants indicates a differential participation of each member under abiotic stress. In particular, tir1 afb2 and tir1 afb3 mutants resulted more tolerant to oxidative stress. In addition, tir1 afb2 showed increased tolerance against salinity measured as chlorophyll content, germination rate and root elongation compared with wild-type plants. Furthermore, tir1 afb2 displayed a reduced accumulation of hydrogen peroxide and superoxide anion, as well as enhanced antioxidant enzymes activities under stress. A higher level of ascorbic acid was detected in tir1 afb2 compared with wild-type plants. Thus, adaptation to salinity in Arabidopsis may be mediated in part by an auxin/redox interaction.

Thylakoid-Bound Ascorbate Peroxidase Mutant Exhibits Impaired Electron Transport and Photosynthetic Activity

In chloroplasts, stromal and thylakoid-bound ascorbate peroxidases (tAPX) play a major role in the removal of H2O2 produced during photosynthesis. Here, we report that hexaploid wheat (Triticum aestivum) expresses three homeologous tAPX genes (TaAPX-6A, TaAPX-6B, and TaAPX-6D) mapping on group-6 chromosomes. The tAPX activity of a mutant line lacking TaAPX-6B was 40% lower than that of the wild type. When grown at high-light intensity photosystem II electron transfer, photosynthetic activity and biomass accumulation were significantly reduced in this mutant, suggesting that tAPX activity is essential for photosynthesis. Despite the reduced tAPX activity, mutant plants did not exhibit oxidative damage probably due to the reduced photochemical activity. This might be the result of a compensating mechanism to prevent oxidative damage having as a consequence a decrease in growth of the tAPX mutant plants.

Nitric oxide as a key component in hormone-regulated processes.

Nitric oxide (NO) is a small gaseous molecule, with a free radical nature that allows it to participate in a wide spectrum of biologically important reactions. NO is an endogenous product in plants, where different biosynthetic pathways have been proposed. First known in animals as a signaling molecule in cardiovascular and nervous systems, it has turned up to be an essential component for a wide variety of hormone-regulated processes in plants. Adaptation of plants to a changing environment involves a panoply of processes, which include the control of CO2 fixation and water loss through stomatal closure, rearrangements of root architecture as well as growth restriction. The regulation of these processes requires the concerted action of several phytohormones, as well as the participation of the ubiquitous molecule NO. This review analyzes the role of NO in relation to the signaling pathways involved in stomatal movement, plant growth and senescence, in the frame of its interaction with abscisic acid, auxins, gibberellins, and ethylene.

Antioxidant enzymes and lipid peroxidation during aging of Chrysanthemum morifolium RAM petals

Abstract To characterize the physiological status of petals over the senescence period, the rate of leakage of electrolytes and weight were measured in Chrysanthemum morifolium RAM petals. Both a significant increase in electrolyte leakage to the external medium and a concomitant decrease of flower weight were observed. Lipid peroxidation was evaluated by measurements of thiobarbituric acid reactive substance (TBARS) in Chrysanthemum morifolium RAM petals during senescence, that was arbitrarily divided into five stages. TBARS content was higher in petals from stage 5 (complete wilting) than in stage 1 (blooming). Non-significant differences were detected in ethylene production between petals classified in stages 1–3 (50 ± 4 pmol/g fresh weight/h). The activity of the enzymes involved in hydroperoxide metabolism was determined. The activities of superoxide dismutase (SOD), catalase, peroxidases and ascorbate peroxidase (AP) were measured. SOD activity showed a maximum value at stage 3 (245 ± 30 U/mg protein), followed by a decline. A progressive increase in peroxidase (5-fold increase) and AP (6-fold increase) activities was measured as a function of time. Petals in stage 1 showed lower catalase activity (20.8 ± 0.5 μmol/min/mg protein) than petals in stages 2–5 (43 ± 3 μmol/min/mg protein). The data presented here suggest that lipid peroxidation and membrane damage are involved in deterioration of Chrysanthemum morifolium RAM petals. The significant increase in the activity of SOD, catalase, peroxidases and AP in the initial stages of senescence, indicates that antioxidant defenses are triggered by coordinated mechanisms to control damage by aging in petals.

l-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

Background: l-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the final step of the l-ascorbate biosynthesis pathway and at the same time is essential for complex I accumulation. Results: The active GLDH is localized within three different subcomplexes of complex I. Conclusion: Evidence is increasing that GLDH represents a complex I assembly factor. Significance: New insights into mitochondrial complex I assembly in Arabidopsis thaliana. l-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete “peripheral arm” of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the “membrane arm” of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves

The effects of red/far red (R/FR) ratios on leaf ascorbate (AA) and glutathione (GSH) accumulation were examined in common bean (Phaseolus vulgaris L.). Growth under low R/FR ratios resulted in a “shade” phenotype and much lower leaf AA and GSH contents than high (R/FR) ratios. Photosynthesis rates were unaffected by changes in the R/FR ratio but leaf respiration rates, pyridine nucleotide pools and antioxidant enzyme activities were decreased under the low R/FR regime. The GSH pool changed slowly in response to altered R/FR ratios but leaf ascorbate acclimated over a single photoperiod. We conclude that light quality signals, particularly R/FR ratios, are important regulators of antioxidant synthesis and accumulation. These acclimatory changes are an early response to changing light environment.

MiR393 regulation of auxin signaling and redox-related components during acclimation to salinity in Arabidopsis.

One of the most striking aspects of plant plasticity is the modulation of development in response to environmental changes. Plant growth and development largely depend on the phytohormone auxin that exerts its function through a partially redundant family of F-box receptors, the TIR1-AFBs. We have previously reported that the Arabidopsis double mutant tir1 afb2 is more tolerant to salt stress than wild-type plants and we hypothesized that down-regulation of auxin signaling might be part of Arabidopsis acclimation to salinity. In this work, we show that NaCl-mediated salt stress induces miR393 expression by enhancing the transcription of AtMIR393A and leads to a concomitant reduction in the levels of the TIR1 and AFB2 receptors. Consequently, NaCl triggers stabilization of Aux/IAA repressors leading to down-regulation of auxin signaling. Further, we report that miR393 is likely involved in repression of lateral root (LR) initiation, emergence and elongation during salinity, since the mir393ab mutant shows reduced inhibition of emergent and mature LR number and length upon NaCl-treatment. Additionally, mir393ab mutant plants have increased levels of reactive oxygen species (ROS) in LRs, and reduced ascorbate peroxidase (APX) enzymatic activity compared with wild-type plants during salinity. Thus, miR393 regulation of the TIR1 and AFB2 receptors could be a critical checkpoint between auxin signaling and specfic redox-associated components in order to coordinate tissue and time-specific growth responses and tolerance during acclimation to salinity in Arabidopsis.

Glutathione and ascorbic acid protect Arabidopsis plants against detrimental effects of iron deficiency

Iron is an essential micronutrient required for a wide variety of cellular functions in plant growth and development. Chlorosis is the first visible symptom in iron-deficient plants. Glutathione (GSH) and ascorbic acid (ASC) are multifunctional metabolites playing important roles in redox balancing. In this work, it was shown that GSH and ASC treatment prevented chlorosis and the accumulation of reactive oxygen species induced by iron deficiency in Arabidopsis leaves. In iron deficiency, GSH and ASC increased the activity of the heme protein ascorbate peroxidase at a similar level to that found in iron-sufficient seedlings. GSH was also able to preserve the levels of the iron-sulfur protein ferredoxin 2. GSH content decreased 25% in iron-deficient Arabidopsis seedlings, whereas the ASC levels were not affected. Taken together, these results showed that GSH and ASC supplementation protects Arabidopsis seedlings from iron deficiency, preserving cell redox homeostasis and improving internal iron availability.