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Tomato fruit ripening and postharvest display a complex net of metabolic pathways, which are controlled by different signaling molecules and hormones that coordinate this physiological process. The inhibition of the ethylene action with 1-methylcyclopropene (1-MCP) has proved to be an excellent tool for maintaining fruit quality during postharvest. Along with these findings, nitric oxide (NO) demonstrated different effects on several species in fruit postharvest. The aim of this work was to evaluate the joint effect of 1-MCP and a NO donor (S-nitrosoglutathione, GSNO) during tomato postharvest at 23 °C. Breaker tomatoes were harvested from a local grower and divided in the following treatments: untreated, 1 mM GSNO, 0.5 μL L−1 of 1-MCP, and the combination of 1 mM GSNO + 0.5 μL L−1 1-MCP. Fruit was stored at 23 °C and analyzed after 5 and 10 d. According to the results, the combination of 1-MCP and the NO donor delayed fruit softening at days 5 and 10, reduced the ethylene synthesis significantly at day 5 and elevated the respiration rate at 10 d, compared to the individual treatments of 1-MCP and GSNO alone. However, the solely application of 1-MCP, independently from the addition of GSNO, showed a strong effect in the rise of the total titratable acidity content, together with the citric acid content. 1-MCP also up-regulated the CAT, SOD and POD activities at 5 and 10 d after harvest. These findings may suggest that some fruit quality parameters in tomato postharvest are controlled by ethylene, while others need the coordination of nitric oxide as a signal molecule.

Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 μM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803., Instituto de Fisiología Vegetal

The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-l-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening., Instituto de Fisiología Vegetal

Results of studies on the response of plants to adverse growth conditions are used to understand restrictions to crop yield. However, this extrapolation requires certain experimental considerations that are rarely taken into account in the experimental designs. This chapter analyzes several hurdles and limitations of the experimental approaches commonly used to study plant stress tolerance. It is very common that basic studies under controlled (plants in pots growing in chambers such as phytotrons) or semi controlled (e.g., plants growing in a greenhouse) conditions attempt to extrapolate their results to crops grown in the field under more realistic conditions. Thus, the objective of this chapter is to analyze if this scaling up is possible, and in which situations. We analyze the scientific literature in this research field (mainly, but not restricted to, water stress), trying to elucidate the various limitations in this scaling up from molecular, physiological, biochemical, and agronomic studies.

Blueberries are a perishable fruit that loses its firmness and weight rapidly after harvest. High concentrations of ascorbic acid (AA) and phenolic compounds such as anthocyanins are important attributes contributing to the quality of the fruit. The aim of this work is to extend the postharvest life of three blueberries cultivars; ‘Mistý and ‘Blue Cuinex́, firm and soft cultivars respectively and ‘Blue Chiṕ with intermediate firmness. The fruit was treated separately or in combination with a nitric oxide donor, S-nitrosoglutathione (GSNO) and 1-methylcyclopropene (1-MCP) and stored at 4 °C for 14 d. The best treatment for ‘Blue Cuinex́ was the combination of 1- MCP and GSNO, while ‘Mistý softened slower when treated with 1-MCP, and ‘Blue Chiṕ was not affected by treatment. The 1-MCP+ GSNO treatment maintained higher concentrations of AA and GSH in ‘Blue Cuinex́. AA concentrations declined in ‘Mistý but did not respond to GSNO. The combination of 1-MCP and GSNO extended the postharvest life of ‘Blue Cuinex́ by improving the concentrations of AA and glutathione in the berries but not for ‘Mistý. These results demonstrate that blueberry cultivars respond differentially to 1-MCP and nitric oxide, and that the sequence of application could be used to slow fruit softening and maintain antioxidant properties., Instituto de Fisiología Vegetal

Plant domestication is a complex process in which natural and cultural factors play important roles delimiting evolutionary pathways of plants under cultivation. In order to deal with and understand the changes generated during this process, multi-disciplinary research is required, especially when a full picture of the domestication history of a taxon is to be assessed. We present here some advances in the study of Cucurbita maxima (squash) domestication from an integrated perspective, including experimental, morphometric and archaeobotanical approaches, which are discussed in the light of new data from physiological analyses. Modern material includes plants obtained from experimental fields, derived from crosses between domesticated (C. maxima ssp. maxima) and spontaneous/wild forms (C. maxima ssp. andreana), resulting in F1 and F2 generations. The archaeobotanical material includes remains recovered from sites in southern Peru and northwest Argentina ranging in date from 3,000 to 800 bp. Morphological and anatomical analyses were conducted on seeds, pericarps and peduncles (the stem of the flower or fruit) for reconstructing squash size and shape evolution under domestication. The results suggest the presence of hybrid forms, mainly from the earlier sites, but also from more recent ones. As expected, a linear evolutionary pathway was not found. Diversity and multiple crossings seem to have been a constant in squash cultivation over time, emphasising the role of gene flows between domestic and wild variants in the domestication process. Finally, we hypothesize the possible linkage between past gene flow and different dormancy patterns as part of management practices, allowing the maintenance of squash populations adapted to different environmental conditions., Instituto de Fisiología Vegetal, Laboratorio de Etnobotánica y Botánica Aplicada

Reduced GDP-l-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-l-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-l-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-l-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production., Instituto de Fisiología Vegetal

Ferroptosis is an oxidative iron-dependent form of cell death recently described in eukaryotic organisms like animals, plants and parasites. Here we report that a similar process takes place in the cyanobacterium Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis cells undergo a cell death pathway that can be suppressed by canonical ferroptosis inhibitors or by external addition of calcium, glutathione or ascorbic acid. Moreover, as described for eukaryotic cells ferroptosis, this pathway is characterized by an early depletion of antioxidants, and by lipid peroxidation. As in general prokaryotes membranes contain poorly oxidizable saturated or monounsaturated lipid molecules, it was thought that they were not susceptible to ferroptosis. Interestingly, cyanobacteria contain thylakoid membranes that are enriched in polyunsaturated-fatty-acid-containing phospholipids, which might explain their sensitivity to ferroptosis. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program., Instituto de Fisiología Vegetal

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program. Fil: Aguilera, Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentina Fil: Berdun, Federico Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentina Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Steelheart Molina, Maria Charlotte. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Alegre, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Bayir, Hülya. University of Pittsburgh; Estados Unidos Fil: Tyurina, Yulia Y.. University of Pittsburgh; Estados Unidos Fil: Kagan, Valerian E.. University of Pittsburgh; Estados Unidos Fil: Salerno, Graciela Lidia. Fundación para Investigaciones Biológicas Aplicadas; Argentina Fil: Pagnussat, Gabriela Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Martin, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentina

Attempts to improve the ascorbate (AsA) content of plants are still dealing with the limited understanding of why exists a wide variability of this powerful anti-oxidant molecule in different plant sources, species and environmental situations. In plant mitochondria, the last step of AsA synthesis is catalyzed by the enzyme L-galactone-1,4-lactone dehydrogenase (L-GalLDH). By using GalLDH-RNAi silencing plant lines, biochemical and proteomic approaches, we here discovered that, in addition to accumulate this antioxidant, mitochondria synthesize AsA to down-regulate the respiratory activity and the cellular energy provision. The work reveals that the AsA synthesis pathway within mitochondria is a branched electron transfer process that channels electrons towards the alternative oxidase, interfering with conventional electron transport. It was unexpectedly found that significant hydrogen peroxide is generated during AsA synthesis, which affects the AsA level. The induced AsA synthesis shows proteomic alterations of mitochondrial and extra-mitochondrial proteins related to oxidative and energetic metabolism. The most identified proteins were known components of plant responses to high light acclimation, programmed cell death, oxidative stress, senescence, cell expansion, iron and phosphorus starvation, different abiotic stress/pathogen attack responses and others. We propose that changing the electron flux associated with AsA synthesis might be part of a new mechanism by which the L-GalLDH enzyme would adapt plant mitochondria to fluctuating energy demands and redox status occurring under different physiological contexts., Instituto de Fisiología Vegetal

Nitric oxide (NO) and hydrogen peroxide (H2O2) have profound effects in growth and development processes in plant physiology. They act as signal molecules under normal conditions or biotic and abiotic stress situations. Fruit ripening is a highly coordinated process that needs the control of several biosynthetic pathways, including signal molecules such as NO and H2O2. On the other hand, enzymatic and nonenzymatic antioxidants act to maintain the equilibrium and to prevent oxidative stress. This forward look reviews the relationship between these two active species and their interplay with ethylene in climacteric and non-climacteric fruit ripening. Throughout we emphasize the influence of NO and H2O2 crosstalk coupled to the hormone control, chloroplast to chromoplast transition, and their implications in postharvest technology.

El Ca y el Mg intervienen en el crecimiento vegetal según su disponibilidad, y posiblemente su relación. Los suelos de la Región Pampeana se han considerado bien dotados de estos elementos, aunque diagnosticaron áreas con bajos contenidos. El objetivo del trabajo fue evaluar diferentes concentraciones de Ca en solución nutritiva sobre la producción de materia seca (MS), contenido de Ca y Mg en MS, y su exportación aérea en raigrás anual (Lolium multiflorum L.) y soja forrajera (Glycine max [L.] Merr). Adicionalmente, se evaluó el efecto de duplicar la concentración a igual relación Ca/Mg. Se realizó un ensayo con soluciones nutritivas de 1, 5, 9 y 12 mM Ca y 2 mM Mg. Se adicionó un tratamiento con 10 mM Ca y 4 mM Mg. Se realizaron tres cortes en raigrás y uno en soja. Se determinó que en raigrás los niveles de nutrición cálcica influyen sobre las concentraciones y exportación de Ca y Mg en MS, sin efectos sobre la producción de MS. La relación Ca/Mg ocasionalmente se relacionó con la concentración de Ca y Mg en MS y en la exportación de Ca y Mg. En soja, los niveles de nutrición cálcica influyen sobre la producción de MS, concentraciones de Ca en MS y en la exportación de Ca y Mg, mientras que no intervienen en la concentración de Mg en MS. En este cultivo, la relación Ca/Mg en solución ocasionalmente produjo efecto en la concentración de Ca en MS y en la exportación de Ca y Mg.

This work focuses on the alterations in soybean root growth and activity during whole plant senescence and the contribution of roots to source-sink relations during plant development. The experiments were designed to analyze the activity of roots in relation to: a) whole plant senescence, b) total pod removal and c) root pruning (15, 25 and 50% of DW) during seed growth stages. Roots can grow until an advanced R5 stage and their specific activity decreases along the reproductive development but whole root activity declines from R6. However root respiration is maintained at a basal level until R8. Depodded plants showed a large increase of root dry matter (about 470%) and a large increase of AOX protein. Root pruning treatments showed a proportional increase of specific root respiration in 25 and 50% treatments but no differences of whole root respiration and dry matter partitioning at R7. These results indicate that roots are under the control of the requirements of above ground organs until final stages of seed growth but, after this, roots may survive independently for some time. This suggests that roots do not suffer a senescence-like process as leaves do. Also, plants have a high capacity to buffer changes in root biomass production and specific root activity under pod removal or partial root pruning., Instituto de Fisiología Vegetal

Ascorbic acid (AA) is a major redox buffer in plant cells. The role of ethylene in the redox signaling pathways that influence photosynthesis and growth was explored in two independent AA deficient Arabidopsis thaliana mutants (vtc2-1 and vtc2-4). Both mutants, which are defective in the AA biosynthesis gene GDP-L-galactose phosphorylase, produce higher amounts of ethylene than wt plants. In contrast to the wt, the inhibition of ethylene signaling increased leaf conductance, photosynthesis and dry weight in both vtc2 mutant lines. The AA-deficient mutants showed altered expression of genes encoding proteins involved in the synthesis/responses to phytohormones that control growth, particularly auxin, cytokinins, abscisic acid, brassinosterioids, ethylene and salicylic acid. These results demonstrate that AA deficiency modifies hormone signaling in plants, redox-ethylene interactions providing a regulatory node controlling shoot biomass accumulation., Instituto de Fisiología Vegetal, Centro de Investigación y Desarrollo en Criotecnología de Alimentos

This work presents the results of physiological studies developed to understand modifications linked to the reduction of seed dormancy provoked by domestication processes. The experiments performed compared wild and domesticated Cucurbita subspecies and their hybrids developed by reciprocal crossings. Seeds of two accessions of the wild subspecies presented dormancy, but it was largely reduced in seeds from the domesticated genotype, and partially reverted in hybrids, especially in those obtained when the domesticated genotype was used as the mother plant. In addition, naked embryos of all subspecies did not display dormancy when incubation was performed at 28°C, but embryo germination was progressively reduced only in the wild genotype under decreasing incubation temperatures (22 and 16°C). In the embryos, abscisic acid (ABA) concentrations were similar in both domesticated and wild subspecies, whereas in the seed coat, it was threefold higher in the wild subspecies. The naked embryos from the wild subspecies were far more responsive to ABA than those from the domesticated subspecies. These results indicate that dormancy in the wild subspecies is imposed by the seed coat tissues and that this effect is mediated by their high ABA content and the sensitivity of the embryos to ABA. These physiological aspects were apparently removed by domestication along with the temperature-dependent response for germination., Instituto de Fisiología Vegetal, Laboratorio de Etnobotánica y Botánica Aplicada, Facultad de Ciencias Naturales y Museo

Nitrogen (N) is an element with a high influence in seedling growth in the nursery. Nevertheless, absolute levels of nutrient concentration are irrelevant if the relationship among the major elements is not examined. In this work we studied the incidence of several N concentrations in two proportional relationships with potassium (K), keeping the relationship with phosphate (P) constant (i.e. 3:2:1 and 3:2:3 for N:P:K), during the rapid growing phase in the fertigation production of Austrocedrus chilensis seedlings. Seedling height, stem diameter at the collar level, total biomass, aboveground/underground biomass ratio (PSA/PSR), nutritional status and root growth potential (RGP) were measured at the end of the growing season. The two treatments combining lower concentration of N with a relationship 3:2:3 produced the highest effect on all morphological parameters assessed. The PSA/PSR was more equilibrated in the treatments with the relationship 3:2:3. The N concentration in the leaves was proportional to its concentration in the nutrient solution. The P content did not show differences among treatments, whereas the K level was higher for the treatment of lower N concentration with relationship 3:2:1. The RGP was low in all treatments. Our observations showed that the 3:2:3 relationship produced larger seedlings with a better balance of biomass partition, evidencing that the management of K in the nutrient solution has higher influence in the seedlings growth of Austrocedrus chilensis., El nitrógeno es uno de los nutrientes más influyentes sobre el crecimiento de los plantines en un vivero. Sin embargo, la concentración de cada nutriente carece de importancia si no se la analiza en relación al conjunto de ellos. En este trabajo sobre la producción bajo fertirriego de plantines de ciprés de la cordillera, se estudió la incidencia de distintos niveles de concentración de nitrógeno (50, 100, 150 y 200 mg L-1) en dos relaciones proporcionales con el potasio, manteniendo invariable la relación con el fósforo (i.e. 3:2:1 y 3:2:3 para N:P:K), durante la etapa de crecimiento rápido. Se evaluaron la altura, el diámetro al cuello del plantín, la biomasa total, la asignación entre la parte aérea y radical (PSA/PSR), el estado nutricional y el potencial de crecimiento radical (PCR). Los tratamientos con menor concentración de nitrógeno y de proporciones 3:2:3 produjeron el mayor incremento en los parámetros morfológicos analizados. La PSA/PSR fue más equilibrada en los tratamientos con relación 3:2:3. La concentración foliar de nitrógeno fue proporcional a su concentración en la solución nutritiva. La concentración foliar de fósforo no mostró diferencias, mientras que se observó una mayor concentración foliar de potasio en el tratamiento de menor concentración de nitrógeno y relación 3:2:1. El PCR mostró valores bajos en todos los tratamientos. Las observaciones indican que la relación 3:2:3 produjo plantines de mayor tamaño y con asignaciones de biomasa más balanceadas, evidenciando que el manejo del potasio tuvo gran influencia en el crecimiento del ciprés de la cordillera en vivero., Instituto de Fisiología Vegetal

This work studied modifications experienced by root mitochondria during whole plant senescence orunder light deprivation, using Arabidopsis thaliana plants with YFP tagged to mitochondria. During post-bolting development, root respiratory activity started to decline after aboveground organs (i.e., rosetteleaves) had senesced. This suggests that carbohydrate starvation may induce root senescence. Similarly, darkening the whole plant induced a decrease in respiration of roots. This was partially due to a decreasein the number of total mitochondria (YFP-labelled mitochondria) and most probably to a decrease inthe quantity of mitochondria with a developed inner membrane potential (ΔΨm, i.e., Mitotracker red-labelled mitochondria). Also, the lower amount of mitochondria with ΔΨm compared to YFP-labelled mitochondria at 10d of whole darkened plant, suggests the presence of mitochondria in a “standby state”. The experiments also suggest that small mitochondria made the main contribution to the respiratory activity that was lost during root senescence. Sugar supplementation partially restored the respiration of mitochondria after 10d of whole plant dark treatment. These results suggest that root senescence is triggered by carbohydrate starvation, with loss of ΔΨm mitochondria and changes in mitochondrial size distribution., Instituto de Fisiología Vegetal

The ability of plants to respond to a wide range of environmental stresses is highly flexible and finely balanced through the interaction of hormonal plant growth regulators and the redox signalling hub, which integrates information from the environment and cellular metabolism/physiology. Plant hormones produce reactive oxygen species (ROS) as second messengers in signalling cascades that convey information concerning changes in hormone concentrations and/or sensitivity to mediate a whole range of adaptive responses. Cellular redox buffering capacity that is determined largely by the abundance of ascorbate has a profound influence on the threshold at which hormone signalling is triggered and on the interactions between different hormones. Other antioxidants such as glutathione, glutaredoxins and thioredoxins are also central redox regulators of hormone signalling pathways. The complex network of cross-communication between oxidants and antioxidants in the redox signalling hub and the different hormone signalling pathways maximises productivity under stress-free situations and regulates plant growth, development, reproduction, programmed cell death and survival upon exposure to stress. This interactive network confers enormous regulatory potential because it allows plants to adapt to changing and often challenging conditions, while preventing boom or bust scenarios with regard to resources, ensuring that energy is produced and utilised in a safe and efficient manner Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Casalongue, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Simontacchi, Marcela Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Marquez Garcia, Belen. University of Leeds; Reino Unido Fil: Foyer, Christine H.. University of Leeds; Reino Unido

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., Instituto de Fisiología Vegetal

The aim of this work is to extend and improve the postharvest life of mature spinach leaves using clean technologies like the use of short pulses of light at low irradiance. After harvest spinach leaves were immediately sealed in polyethylene bags in the laboratory. These bags were placed in a dark chamber at 23 ◦C under continuous dark or with the application of light pulses (LP) consisting of 15 min each 2–6 h or 7 min each 2 h for 3 d. The chosen irradiance, 30 mol m−2 s−1 PPFD, corresponded to the light compensation point previously measured in spinach plants under greenhouse conditions. After the leaves were treated with LP for 3 d, all the samples were transferred to a chamber at 4 ◦C under continuous dark for another week. Senescence was triggered in leaves under continuous dark after 3 d of storage and delayed in those receiving LP. In addition ascorbic acid and glutathione contents were kept higher in LP-treated than in untreated spinach. These trends were conserved after storage under continuous dark and refrigeration for another week. When LP was applied in combination with 1-MCP the antioxidant capacity was further improved. These results demonstrate that short LP of low irradiance can be used to extend and to improve postharvest life of mature spinach leaves. Fil: Gergoff Grozeff, Gustavo Esteban. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Chaves, Alicia Raquel. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Bartoli, Carlos Guillermo. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Ascorbic acid (AA) is synthesized in plant mitochondria through the oxidation of L-galactono-1,4-lactone (L-GalL) and then distributed to different cell compartments. AA-deficient Arabidopsis thaliana mutants (vtc2) and exogenous applications of L-GalL were used to generate plants with different AA content in their leaves. This experimental approach allows determining specific AA-dependent effects on carbon metabolism. No differences in O2 uptake, malic and citric acid and NADH content suggest that AA synthesis or accumulation did not affect mitochondrial activity; however, L-GalL treatment increased CO2 assimilation and photosynthetic electron transport rate in vtc2 (but not wt) leaves demonstrating a stimulation of photosynthesis after L-GalL treatment. Increased CO2 assimilation correlated with increased leaf stomatal conductance observed in L-GalL-treated vtc2 plants Fil: Senn, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Gergoff Grozeff, Gustavo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Alegre, Matias Leonel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Barrile, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: de Tullio, M. C.. Universita Degli Studi Di Bari; Italia Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina

Light is one of the most important factors modulating processes and sequences in plants life, like fruit ripening and the concentrations of water and lipid soluble antioxidants. The aim of this work was to evaluate the most effective frequency of low irradiance light pulses (LP) during the night and to analyze its effect on plant and fruit growth, as well as on modifications of concentrations of soluble sugar, amino acids, antioxidants and organic acids. LP of 15 min each were applied over the plants in a temperature controlled greenhouse after fruit set till they turned to mature red, with a frequency of 2 and 4 h. LP induced no changes in the typical maturation indexes such as soluble solid, total acidity, pH or firmness; meanwhile there was an 18% increase in fruit yield when plants were exposed to 15 min LP every 2 h during the night. Furthermore, by analyzing the tomato cluster receiving this LP treatment separately, the biomass of the fruit was found to have increased by 28% compared with the same cluster in control plants. In coincidence with this, fruit treated with a frequency of 2 and 4 h LP showed an increase in lycopene concentration, concomitantly with a rise in the proportion of red mature fruit harvested from the whole plant. On the other hand, there was a drop in the concentration of soluble sugars and free amino acids, possibly conducing to a decrease in water soluble antioxidants (ascorbic acid and glutathione) and citric and malic acids concentration. Overall, these results showed that nocturnal LP treatments improved fruit yield in tomato plants with higher amounts of lycopene, which indicate earlier fruit ripening. Fil: Gergoff Grozeff, Gustavo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Senn, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Alegre, Matias Leonel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Chaves, Alicia Raquel. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina

Ascorbic acid (AA) is present in high concentration in plant tissues and participates of many vital processes. Its biosynthetic pathway in plants has been recently established. Nowadays, research is focused on understanding the regulation of this pathway. One of the aspects is to unravel the importance of each enzyme through the establishment of limiting reactions and specific controlling processes. Interestingly, the synthesis of AA presents a high interaction with several physiological processes. The photosynthetic activity directly affects AA synthesis, and the last reaction of its formation takes place in mitochondria depending on the mitochondrial electron transport chain. In addition, it is now known how some plant hormones regulate this pathway. The incident light constitutes a key environmental factor controlling the synthesis of AA in plants. Light regulates the levels of several enzymes, but its effect on AA biosynthesis is also mediated through modifications in the previously mentioned physiological processes. The knowledge about the regulation of AA synthesis will allow the development of manipulative strategies leading to effectively increase the concentration of AA in edible plant organs.

Senescence of detached spinach leaves either untreated or treated with 0.1 or 1.0 μL L −1 1-MCP has been investigated. 1-MCP treated leaves had higher chlorophyll content and photosystem II potential quantum yield (Fv/Fm) and lower solute leakage than untreated leaves after storage in darkness at 23 °C for 6 d, indicating a delay of senescence. Ethylene production was increased in spinach supplemented with 1-MCP after 3 d storage and then declined to the rates of untreated leaves. 1-MCP treated spinach had higher ascorbic acid and glutathione concentrations, and a low oxidised/reduced ratio for both antioxidants. Accumulations of ammonium and protein degradation were reduced by 1-MCP. The results presented here indicate that inhibition of ethylene sensitivity can be successfully used to extend the postharvest life of spinach leaves.

The aim of this work was to determine if changes in antioxidant contents are directly controlled by ethylene or are indirect effects associated with modulation of leaf senescence. Detached spinach (Spinacia oleracea L. cv Bison) leaves were treated with ethephon to induce ethylene production, placed in polyethylene bags and stored under darkness at 23 °C for 3 days. The ethephon treatment produced a rapid decrease on ascorbic acid (AA) content and an increase in its oxidised state before any leaf senescence parameters changed. Both AA synthesis and recovery from oxidised forms were affected by ethylene. In addition, leaves from Arabidopsis (Arabidopsis thaliana (L.) Heynh) ethylene insensitive mutants (ein2-1, ein3-1 and ein4) presented higher AA content than leaves from wt plants before dark storage. Furthermore, ein3-1 and ein4 but not ein2-1, showed slower leaf AA content declining than wt plants under dark-induced leaf senescence. The Arabidopsis mutant with constitutive triple response (ctr1-1) showed lower leaf AA content than wt plants but similar AA degradation rate with a higher oxidised state after dark storage for 3 days. These results demonstrate that ethylene is an important factor controlling AA content in mature leaves.

The fruit (pods) of Prosopis (Fabaceae) are frequently recovered from pre-Hispanic Argentinian archaeological sites, suggesting that this genus was of importance in ancient economies in this region. Yet it is only recently that archaeobotanists have begun to carry out systematic research into this genus. Therefore many questions remain to be addressed concerning the food value of Prosopis fruit, and its potential contribution. This paper examines starch from the pods of two species, Prosopis flexuosa and Prosopis chilensis, for the purposes of describing and classifying their morphological features and biometrical parameters. Pods of both species were gathered from two extremes (northern and southern) of the Hualfin Valley, Catamarca, Argentina. Starch abundance is estimated and compared with that of Zea mays, an economically important plant with high concentrations of starch. This paper reveals that Prosopis pods contain very low amounts of starch compared to starch-rich edible species. Despite this, the recovery of Prosopis starch grains can be successfully used to assess archaeological tool uses. Prosopis starch grains were found to be highly variable in shape. Grain size ranged between 10 and 20 μm. Granule irregularity and the high birefringence under polarized light are two of the most important diagnostic features. No significant statistical differences were found in the structure (morphology, size and hilum) of the starch of the same species from both localities. Finally, the implications for the role of this plant in past societies are discussed.

The effects of moderate heat shock (HS) treatments on the senescence of detached spinach leaves have been studied. At harvest, detached spinach leaves received moderate heat treatments (37, 40, 43 °C and room temperature) by immersion in water baths. The content of small HS proteins increased proportionally to the temperature applied, with undetectable levels in untreated leaves. A HS treatment at 40 °C delayed leaf senescence as indicated by higher chlorophyll content and potential quantum yield of PSII (Fv/Fm), and decreased solute leakage after storage for 7 d compared with untreated samples. A higher in vivo production of H 2 O 2 was observed as HS temperature increased. Oxidation of 2′,7′-dichlorodihydrofluorescein diacetate observed by in vivo confocal microscopy revealed that mitochondria were the main site of reactive oxygen species generation under either untreated or HS-treated leaves. Although high-temperature treatments did not prevent the loss of water soluble antioxidant concentrations, the ratio of reduced/oxidised forms of ascorbic acid was higher 3 d after HS treatment, compared with control leaves. The effect of HS treatments on physiologically based protective mechanisms that delay leaf senescence is discussed.

The NADH–ubiquinone oxidoreductase complex (complex I) (EC 1.6.5.3) is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animals and fungi (Opisthokonta), it contains an extra domain comprising trimers of putative γ–carbonic anhydrases, named the CA domain, which has been proposed to be essential for assembly of complex I. However, its physiological role in plants is not fully understood. Here, we report that Arabidopsis mutants defective in two CA subunits show an altered photorespiratory phenotype. Mutants grown in ambient air show growth retardation compared to wild–type plants, a feature that is reversed by cultivating plants in a high-CO2 atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contributes to sustaining efficient photosynthesis under ambient (photorespiratory) conditions. Fil: Soto, Débora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Cordoba, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Villarreal, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Schmitz, Jessica. Heinrich Heine Universitat; Alemania Fil: Maurino, Veronica G.. Heinrich Heine Universitat; Alemania Fil: Braun, Hans Peter. Leibniz Universitat Hannover; Alemania Fil: Pagnussat, Gabriela Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Zabaleta, Eduardo Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina

The aim of this study was to explore the role of the mitochondrial alternative oxidase (AOX) in the protection of photosynthesis during drought in wheat leaves. The relative water contents of water-replete and drought-exposed wheat plants were 97.2±0.3 and 75±2, respectively. Drought increased the amount of leaf AOX protein and also enhanced the rate of AOX-dependent O 2 uptake by the respiratory electron transport chain. The amount of the reduced, active form of the AOX protein was specifically increased by drought. The AOX inhibitor salicylhydroxamic acid (1 mM; SHAM) inhibited 70% of AOX activity in vivo in both water-replete and drought-exposed plants. Plants treated with SHAM were then exposed to low (100), high (350), or excess light (800 μmol photons m -2 s -1 ) for 90 min. SHAM did not modify chlorophyll a fluorescence quenching parameters in water-replete controls after any of these treatments. However, while the maximal quantum yield of photosystem II (PSII) electron transport (F v /F m ) was not affected by SHAM, the immediate quantum yield of PSII electron transport (Φ PSII ) and photochemical quenching (qP) were gradually reduced by increasing irradiance in SHAM-treated drought-exposed plants, the decrease being most pronounced at the highest irradiance. Non-photochemical quenching (NPO) reached near maximum levels in plants subjected to drought at high irradiance. However, a combination of drought and low light caused an intermediate increase in NPO, which attained higher values when AOX was inhibited. Taken together, these results show that up-regulation of the respiratory AOX pathway protects the photosynthetic electron transport chain from the harmful effects of excess light., Instituto de Fisiología Vegetal

Low temperatures severely limit photosynthesis and growth of chilling-sensitive species. The decrease in photosynthetic capacity may be partly due to chilling-associated oxidative damage to chloroplast components. Thus, we sought to determine the extent of lipid peroxidation and oxidative damage to thylakoid proteins in leaves of soybean ( Glycine max L. Merr.) exposed to chilling stress under light. The ratio of variable to maximum fluorescence ( F v / F m ) decreased in plants exposed for 24 h to 7 °C and 500 μmol m −2 s −1 of photosynthetic photon flux density (PPFD), but not in leaves chilled in darkness. The initial drop in F v / F m was exacerbated by treatment with the chloroplast protein synthesis inhibitor lincomycin, suggesting that concurrent repair ameliorated chilling-associated damage to photosystem II (PSII). The degree of oxidative damage to thylakoid proteins (i.e., carbonylation) and lipids (estimated as malondialdehyde content) did not change in response to chilling. Likewise, exogenous applications of the ascorbic acid precursor l -galactono-1,4-lactone (Gal) or of the α-tocopherol analog 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) did not prevent the chilling-induced decrease of F v / F m , although they were effective in protecting thylakoids from oxidative damage caused by paraquat. Our data suggest that chilling stress does not cause rampant oxidative damage to thylakoid proteins and lipids, and that other cell compartments may be more susceptible than thylakoids to oxidative damage associated with low temperature stress.

Ear photosynthesis may be an important source of C for grain growth in water-stressed plants of cereals. The main objectives of this work were to determine the stability of the photosynthetic apparatus and the photochemical efficiency of ears in plants subjected to post-anthesis drought. Plants of wheat (Triticum aestivum L. cv. Granero INTA) were grown in pots under a rain shelter and subjected to water stress (soil water potential around −0.6 to −0.8 MPa) starting 4 days after anthesis. Post-anthesis drought substantially accelerated the loss of chlorophyll, Rubisco and the light-harvesting complex of photosystem II (LHCII) in the flag leaf, but the degradation of these photosynthetic components was much less affected by water deficit in awns and ear bracts. Quantum yield of PSII (ΦPSII) decreased in leaves of water-stressed plants. In contrast, ear bracts had a higher ΦPSII than leaves, and ΦPSII of ear bracts did not decrease at all in response to drought. Removing the grains immediately before fluorescence measurements (less than 30 min) slightly reduced ΦPSII, indicating that CO2 supplied by grain respiration may contribute to the high photochemical efficiency of ears in droughted plants. However, other factors may be involved in maintaining high ΦPSII, since even in the absence of grains ΦPSII remained much higher in ear bracts than in the flag leaf. The relative stability of ear photosynthetic components and their relatively high photochemical efficiency may help to maintain ear photosynthesis during the grain filling period in droughted plants.

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 Vegetal, Facultad de Ciencias Agrarias y Forestales

Plant steroid hormones brassinosteroids (BRs) and the gaseous hormone ethylene (ET) alter the ascorbic acid–glutathione (AA–GSH) levels in tomato (Solanum lycopersicum L.) plants. The interaction of these hormones in regulating antioxidant metabolism is however unknown. The combined use of genetics (BR-mutants) and chemical application (BR/ET-related chemicals) shows that BRs and ET signalling pathways interact, to regulate leaf AA content and synthesis. BR-deficient (dx) leaves display low total AA but BR-accumulating (35S:D) leaves show normal total AA content. Leaves with either BR levels lower or higher than wild type plants showed a higher oxidised AA redox state. The activity of l-galactono-1,4-lactone dehydrogenase (l-GalLDH), the mitochondrial enzyme that catalyses the last step in AA synthesis is lower in dx and higher in 35S:D plants. BR-deficient mutants show higher ET production but it is restored to normal levels when BR content is increased in 35S:D plants. Suppression of ET signalling using 1-methylcyclopropene in dx and 35S:D plants restored leaf AA content and l-GalLDH activity, to the values observed in wild type. The suppression of ET action in dx and 35S:D leaves leads to the respective decreasing and increasing respiration, indicating an opposite response compared to AA synthesis. This inverse relationship is lacking in ET suppressed dx plants in response to external BRs. The modifications in the in vivo activity of l-GalLDH activity do not correlate with changes in the level of the enzyme. Taken together, these data suggest that ET suppresses and BRs promote AA synthesis and accumulation., Instituto de Fisiología Vegetal

This work studied the participation of ascorbic acid (AA) in the dormancy establishment of apical buds of poplar (Populus deltoides Bartram ex Marshall cv. Catfish 2) lateral branches. AA content was highest during the active growth period and decreased to the lowest content during bud dormancy. The accumulation of the oxidised form, dehydroascorbate, was similar in either growth or dormant bud stages. The supplementation of buds with AA at the end of the summer delayed bud set. Similarly, the supplementation of buds with gibberellic acid extended the growth period and kept a high AA content in the apoplast. These results suggest that a decrease in the accumulation of reduced AA might be needed for the dormancy establishment in apical buds of poplar lateral branches., Instituto de Fisiología Vegetal

Aging of petals was accompanied by a 9-fold increase in oxygen radical generation, and a 2-fold increase in the content of oxidized proteins. α-Tocopherol content increased at the onset of aging and decreased as aging progressed. Total thiol content followed the α-tocopherol pattern during aging. Petals' ascorbic acid content started to decline when the flowers were excised. Control petals were exposed to 1 mM paraquat during 24 h and neither the production of oxygen radicals nor the content of hydroperoxides were significantly affected by the treatment. Paraquat exposure did not significantly modified the activities of glutathione reductase and catalase, but superoxide dismutase activity was increased. α-Tocopherol content was not affected by paraquat, but total thiols content decreased by 50%. Flowers showing browning that involved 50–100% of the petal, were exposed to 1 mM paraquat over 24 h. The activities of superoxide dismutase, glutathione reductase, and catalase were not affected. Paraquat exposure led to a 3-fold increase in oxygen radical generation, and to a 59% increase in hydroperoxide content in aged petals. The data presented here suggest that oxidative stress and a concerted antioxidant response are associated with the initial stages of aging in Dendranthema morifolium petals.

Senescence of chrysanthemum (Chrysanthemum morifolium RAM cv. Unsei) petals was associated with a marked deterioration of the plasma membrane and loss of water. The rates of ethylene synthesis declined after full bloom, and became non-detectable by the time the first symptoms of senescence were apparent. The drop in ethylene production correlated with a reciprocal increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content. At full bloom, pre-treatment of petals with 1 mM ACC increased their rates of ethylene production several fold, but the capacity to convert exogenous ACC into ethylene decline thereafter. Inhibitors of ethylene synthesis (i.e. aminoxyacetic acid) or ethylene action (i.e. silver thiosulfate) had no effect on senescence, implying that the minute amounts of ethylene synthesized by petals are below the threshold to cause any acceleration of this process. However, petal senescence was significantly accelerated by a 24 h treatment with 3 ppm of ethylene at full bloom, or by continuous treatment with ethephon and ACC at concentrations that produce a significant increase of ethylene synthesis. Thus, our results show that petal senescence in some varieties of chrysanthemum is sensitive to exogenous ethylene, and that the extended vase life that is characteristic of these flowers may be due to their low rates of ethylene synthesis.

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., Instituto de Fisiología Vegetal

The plant hormones brassinosteroids (BR) and ethylene (ET) act together to regulate plant metabolism. We used BR and 1-methylcyclopropene (1-MCP, an ET action inhibitor) to elucidate the interactions between both hormones for the regulation of mitochondrial respiratory pathways in papaya fruit. The exogenous application of the 24-epibrassinolide (epiBR) enhanced the alternative oxidase (AOX) capacity. While treatment with Brz2001 (Brz is a specific inhibitor of the BR synthesis) also enhanced AOX capacity, these effects lacked in fruit treated simultaneously with epiBR and Brz. Changing the BR level had no effect on ET emission rate in the first 24 h, but a reduction in ET emission was observed in Brz-treated fruit on the fifth day. Together with Brz, epiBR increased the ET production on the fifth day, following the day in which the treatment was carried out. When the ET sensitivity of fruit was inhibited by the application of 1-MCP, the effects of epiBR and Brz were opposite to those obtained without 1-MCP. AOX capacity was slightly inhibited by epiBR in fruit pre-treated with 1-MCP. Data suggest that BR and ET act antagonistically, therefore regulating, directly or indirectly, AOX capacity during papaya fruit ripening., Instituto de Fisiología Vegetal

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) 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., Instituto de Fisiología Vegetal

The population of the epiphytic weeds Tillandsia recurvata and Tillandsia aeranthos on forestry and ornamental trees in different areas of Argentina has increased, causing deleterious effects on their hosts. As a result, an experiment was performed to control these epiphytes with different herbicides. During October 1988/June 1989, in the La Plata woods (34°55′S) the following treatments were applied to ten different host species which were actively growing: Atrazine 10 g 1 −1 ; Dichlobenil 5 g 1 −1 ; Simazine 5 g 1 −1 of formulated product and their combination. All the selected herbicides, alone or in combination, controlled the epiphytes without causing phytotoxicity to the host plant, owing to the different absorption systems of the epiphytes and the hosts. This is a new approach to control these epiphytic weeds and an important tool for preserving natural forests which are very susceptible to the invasion of epiphytes.

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. Fil: Iglesias, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Terrile, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: D'ippólito, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Casalongué, Claudia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Plant mitochondria include gamma-type carbonic anhydrases (γCAs) of unknown function. In Arabidopsis, the γCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2–3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H2O2 dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility., Instituto de Fisiología Vegetal

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.

The effects of growth irradiance and respiration on ascorbic acid (AA) synthesis and accumulation were studied in the leaves of wild-type and transformed Arabidopsis thaliana with modified amounts of the mitochondrial alternative oxidase (AOX) protein. Plants were grown under low (LL; 50 μmol photons m-2 s-1), intermediate (IL; 100 μmol photons m-2 s-1), or high (HL; 250 μmol photons m-2 s-1) light. Increasing growth irradiance progressively elevated leaf AA content and hence the values of dark-induced disappearance of leaf AA, which were 11, 55, and 89 nmol AA lost g-1 fresh weight h-1, from LL-, IL-, and HL-grown leaves, respectively. When HL leaves were supplied with L-galactone-1,4-lactone (L-GalL; the precursor of AA), they accumulated twice as much AA and had double the maximal L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activities of LL leaves. Growth under HL enhanced dehydroascorbate reductase and monodehydroascorbate reductase activities. Leaf respiration rates were highest in the HL leaves, which also had higher amounts of cytochrome c and cytochrome c oxidase (CCO) activities, as well as enhanced capacity of the AOX and CCO electron transport pathways. Leaves of the AOX-overexpressing lines accumulated more AA than wild-type or antisense leaves, particularly at HL. Intact mitochondria from AOX-overexpressing lines had higher AA synthesis capacities than those from the wild-type or antisense lines even though they had similar L-GalLDH activities. AOX antisense lines had more cytochrome c protein than wild-type or AOX-overexpressing lines. It is concluded that regardless of limitations on L-GalL synthesis by regulation of early steps in the AA synthesis pathway, the regulation of L-GalLDH activity via the interaction of light and respiratory controls is a crucial determinant of the overall ability of leaves to produce and accumulate AA., Facultad de Ciencias Naturales y Museo, Instituto de Fisiología Vegetal, Facultad de Ciencias Agrarias y Forestales

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 NaNO 2. 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., Instituto de Fisiología Vegetal

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 Vegetal

Fil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico La Plata. Instituto de Fisiologia Vegetal; Argentina

Ascorbic acid is synthesized from galactono-γ-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-γ-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV., Instituto de Fisiología Vegetal