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Abstract Background The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

Background: To investigate in the conventional techniques of the pedicle screws using triggered screw electromyography (t-EMG), considering different threshold cutoffs: 10, 15, 20 25 mA, for predicting pedicle screw positioning during surgery of the adolescent with idiopathic scoliosis (AIS)., Methods: Sixteen patients (4 males, 12 females, average age 16.6 years) were included, with an average curve magnitude of 50 degrees and placement of 226 pedicle screws. Each screw was classified as "at risk for nerve injury" (ARNI) or "no risk for nerve injury" (NRNI) using CT and the diagnostic accuracy of EMG considering different threshold cutoffs (10,15, 20 and 25 mA) in the axial and Sagittal planes for predicting screw positions ARNI was investigated., Results: The EMG exam accuracy, in the axial plane, 90.3% screws were considered NRNI. In the sagittal plane, 81% pedicle screws were considered NRNI. A 1-mA decrease in the EMG threshold was associated with a 12% increase in the odds of the screw position ARNI. In the axial and sagittal planes, the ORs were 1.09 and 1.12, respectively. At every threshold cutoff evaluated, the PPV of EMG for predicting screws ARNI was very low in the different threshold cutoff (10 and 15); the highest PPV was 18% with a threshold cutoff of 25 mA. The PPV was always slightly higher for predicting screws ARNI in the sagittal plane than in the axial plane. In contrast, there was a moderate to high NPV (78-93%) for every cutoff analyzed., Conclusions: EMG had a moderate to high accuracy for positive predicting value screws ARNI with increase threshold cutoffs of 20 and 25 mA. In addition, showed to be effective for minimizing false-negative screws ARNI in the different threshold cutoffs of the EMG in adolescent with idiopathic scoliosis (AIS).

We analyzed the effect of pH on Cr(III) accumulation, biomass production, and phenolic profile of Salvinia rotundifolia and Salvinia minima plants grown in the presence of increasing concentrations of CrCl 3 . Biomass accumulation, metal tolerance index, and photosynthetic pigment contents indicate that Salvinia rotundifolia seems to be more tolerant of Cr(III) than S. minima at different pHs. Increased metal accumulation by Salvinia species under increasing pH could be explained by changes of the protonation status of cell wall functional groups because both the highest and the lowest pH values used in the present study were outside of the levels at which Cr(III) species start to precipitate. The metal translocation factor indicates that in buffered conditions S. rotundifolia tend to retain more Cr(III) in lacinias than S. minima, probably through the involvement of insoluble phenolics. The results of the present study could be useful to the management of solution pH to maximize the removal of Cr(III) by aquatic plants. Environ Toxicol Chem 2019;38:167-176. © 2018 SETAC., (© 2018 SETAC.)

The effect of cadmium on roots of four citrus rootstocks was studied to assess the relationships between oxidative stress, carbohydrates, phenolics and antioxidant responses. Swingle citrumelo (SC), Rangpur lime (RL), Troyer citrange (TC) and Volkamer lemon (VL) genotypes were exposed to 0, 5 and 10µM Cd over 7 days, after which Cd accumulation was markedly higher in roots compared with stems and leaves. Malondialdehyde (MDA) and lipoxygenase (LOX) activity increased in Cd-treated SC and RL roots, suggesting that a lipid peroxidation is the main driver of plasma membrane damage. In contrast, in TC and VL genotypes, LOX-mediated lipid peroxidation does not appear to play a key role in Cd-induced lipid peroxidation, but H2O2 accumulation seems to be responsible of less plasma membrane damage. Catalase (CAT), superoxide dismutase (SOD) and guaiacol and syringaldazine peroxidases (G-POD and S-POD respectively) were differentially affected by Cd. Lipid profile and ATPase-dependant proton extrusion indicated higher disfunctionalities of root plasma membrane in SC and RL genotypes than in TC and VL genotypes. Differences in carbohydrates and phenolic compounds were also observed. Histochemical analysis of G-POD activity and lignin and suberin deposition revealed differences among genotypes. A model to explain the relationships among carbohydrates, soluble phenolics, lipid peroxidation and H2O2 accumulation in Cd-exposed roots was proposed.

In plants of Salvinia rotundifolia and Salvinia minima the effect of two Cr(VI) concentrations (5 and 20mgL(-1)) applied for 7days was assessed by measuring changes in biomass, photosynthetic pigments, Cr accumulation, malondialdehyde (MDA), membrane stability index (MSI), thiols (TT, NPT and PBT), and phenolics (SP and IP). Biomass in S. minima was decreased at highest Cr(VI) concentration, but there were no changes in S. rotundifolia. Metal accumulation was different in both species. S. minima accumulates more metal in fronds, but S. rotundifolia accumulates more metal in lacinias. Results also showed that S. minima translocates more Cr to fronds than S. rotundifolia, but at the whole plant level higher accumulation occurred in this last. Tolerance index (Ti) was higher in S. rotundifolia. Chl b and carotenoids were decreased only upon exposure to high Cr(VI) concentration in both species. Cr(VI) treatment did not enhance MDA accumulation. Cr exposure had no impact on MSI values when comparing with Cr-untreated values. Thiols in fronds and lacinias showed different distribution patterns between species. IP and NPT were higher in S. rotundifolia lacinias that accumulate more Cr than S. minima lacinias. Whilst SP and NPT were higher in S. minima fronds compared with S. rotundifolia ones. This may indicate that these species can cope with Cr(VI) toxicity, either through metal complexation and/or metal reduction or by the scavenging of ROS derived from Cr-induced oxidative stress. Based on Cr accumulation and biomass production, S. rotundifolia seems more suitable to remove Cr(VI) from polluted waters., (Copyright © 2016 Elsevier B.V. All rights reserved.)

UV-B radiation (UVBR) is a small fraction of the solar spectrum from 280 to 315nm. UVBR produces photomorphogenic acclimation responses in plants, modulating their cellular structure and physiology. Here, changes in the peel of harvested lemons after short time exposure to UVBR were analyzed and its potential effects against fungal infection were studied. In the flavedo, UVBR treatment induced variations in the respiratory profiles and increased the phenolic compound contents. Final products of the flavonoid pathway (flavones, flavonols and anthocyanins) increased more markedly than their precursors (flavanones and dihydroflavonols). The increased accumulation of soluble phenolics in the flavedo of treated lemons is associated with the high antioxidant activity found in the flavedo of these samples. Supporting the biochemical determinations, anatomical observations showed abundant intravacuolar deposits of phenolic compounds and an increase in the cell wall thickness in UVBR-treated samples. Metabolic and anatomical modifications associated to UVBR improved natural defenses against Penicillium digitatum, the causal agent of green mold disease. Our results suggest that mature postharvest lemons exposed to the artificial radiation showed phenotypic plasticity, allowing an acclimation response to UVBR which confers fruit resistance to pathogens. Thus, combination of UVBR with other treatments could represent an important improvement to control postharvest diseases on citrus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plants are continuously exposed to atmospheric particulate matter (dust), and their leaves are the main receptors of deposited dust. The objective of this study was to assess the effects of dust deposition on leaf gas exchange parameters of 17 native and non-native tree and shrub species growing in Gran San Miguel de Tucumán in northwestern Argentina. Maximum assimilation rate (), stomatal conductance (), transpiration rate (), internal CO concentration (), and instantaneous water-use efficiency (WUE) were measured in cleaned leaves (CL) and dusted leaves (DL) of different species on November 2010, July 2011, and September 2011. In almost all studied species, gas exchange parameters were significantly affected by dust deposition. Values for , , and of DL were significantly reduced in 11, 12, and 14 species compared with CL. Morphological leaf traits seem to be related to reduction. Indeed, L. and (Mart. ex DC.) Standl. species with pubescent leaves and thick ribs showed the highest reduction percentages. Contrarily, and WUE were increased in DL but were less responsive to dust deposition than other parameters. Increases of and WUE were significant in 5 and 11 species, respectively. Correlation analyses between /, /, and / pairs showed significant positive linear correlations in CL and DL of many studied species, including small and tall plants. These results suggest that leaf stomatal factors and shade-induced effect by accumulated dust are primarily responsible for the observed reductions in photosynthesis rate of DL., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

In order to assess implications of Cd-induced oxidative stress in roots of the citrus rootstock Citrumelo, seedlings were hydroponically exposed to two relatively realistic Cd concentrations during 7 days. Our results showed that increasing Cd concentrations in external solution were associated with higher Cd accumulations in roots. At 5μM Cd the accumulation of Cd in roots was over 70-f higher than in aerial part (stem+leaves). Malondialdehyde (MDA), superoxide radical (O(2)(*-)), hydrogen peroxide (H(2)O(2)) and lipoxygenase activity (LOX) increased in Cd-exposed roots, suggesting a metal-induced oxidative stress. The Cd treatment enhanced the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and guaiacol-type peroxidase (G-POD), as well as the content of secondary metabolites i.e. soluble phenolics (SPs) and lignin. Histochemical analyses of roots showed that Cd, H(2)O(2), (O(2)(*-)), lignin and G-POD displayed a similar location pattern. Almost all analyzed parameters showed a similar dynamic tendency with increases under 5μM Cd followed by decreases under 10μM Cd, suggesting that a complex coordinated Cd-defensive mechanism is operating in Citrumelo roots exposed to environmental realistic Cd concentrations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Quinoa is a good source of protein and can be used as a nutritional ingredient in food products. This study analyses how much growing region and/or seasonal climate might affect grain yield and nutritional quality of quinoa seeds., Results: Seeds of ten quinoa cultivars from the Andean highlands (Bolivia/Argentina site) and Argentinean Northwest (Encalilla site) were analysed for seed yield, protein content and amino acid composition. Grain yields of five cultivars growing at Encalilla were higher, and four were lower, compared with data from the Bolivia/Argentina site. Protein contents ranged from 91.5 to 155.3 and from 96.2 to 154.6 g kg(-1) dry mass for Encalilla and Bolivia/Argentina seeds respectively, while essential amino acid concentrations ranged from 179.9 to 357.2 and from 233.7 to 374.5 g kg(-1) protein respectively. Significant positive correlations were found between the content of essential amino acids and protein percentage., Conclusion: It appears that there are clear variations in seed yield, total protein content and amino acid composition among cultivars from the two sites. Essential amino acid composition was more affected than grain yield and protein level. The study revealed that both environmental and climatic factors influence the nutritional composition of quinoa cultivars growing in different agroecological regions., (Copyright © 2011 Society of Chemical Industry.)

Seasonal variations in physiological and biochemical parameters of the aquatic fern Salvinia minima exposed to different Cr(VI) concentrations were studied. Growth, photosynthetic pigments, soluble carbohydrates, sucrose-related enzymes, lipid peroxidation, phenolics, and Cr accumulation in floating and submerged leaves were analyzed. Cr content was lower in winter than in summer, indicating that active metabolic events occurred in metal uptake. Leaf number and metal concentration factor were higher in summer than in winter. Relative growth rate (R(n)) indicated that growth was more affected by Cr in winter than in summer. Biochemical parameters showed great seasonal variations under increasing Cr. Hexose, starch, malondialdehyde and phenolic contents were greatest in winter, but R(n) and protein values were lowest. Sucrose content was highest in summer floating leaves. A great seasonal variability was observed in sucrose-related enzymes with the highest activities occurring in winter lipoxygenase was much higher in winter than in summer, indicating a strong lipid peroxidation. Results indicate that in Salvinia Cr causes seasonal perturbations in carbohydrate metabolism and oxidative stress by altering both sucrose-related enzymes and lipoxygenase activities. Variability in physiological and biochemical parameters seems to indicate that in outdoor conditions different mechanisms, in terms of Cr accumulation and tolerance, may occur in S. minima during summer and winter., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aim: To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2-min preincubation with 10 ppm NaClO followed by 2-min incubation with 6 mmol l(-1) CuSO(4) and 100 mmol l(-1) H(2)O(2) at 25°C., Methods and Results: After the application of lethal SOT or sublethal SOT (decreasing only the H(2)O(2) concentration), we analysed several conidia features such as germination, oxygen consumption, ultrastructure and integrity of the cellular wall and membrane. Also, we measured the production of reactive oxygen species (ROS) and the content of thiobarbituric acid-reactive species (TBARS). With the increase of H(2)O(2) concentration in the SOT, germination and oxygen consumption of conidia became inhibited, while the membrane permeability, ROS production and TBARS content of conidia increased. Several studies revealed ultrastructural disorganization in P. digitatum conidia after lethal SOT, showing severe cellular damage without apparent damage to the cell wall. In addition, mycelium of P. digitatum was more sensitive than conidia to the oxidative treatment, because growth ceased and permeability of the membranes increased after exposure of the mycelium to a SOT with only 50 mmol l(-1) H(2)O(2) compared to a SOT of 100 mmol l(-1) for these effects to occur on conidia., Conclusion: Our insights into cellular changes produced by the lethal SOT are consistent with the mode of action of the oxidant compounds, by producing both alteration of membrane integrity and intracellular damage., Significance and Impact of the Study: Our results allow the understanding of SOT effects on P. digitatum, which will be useful to develop a reliable treatment to control postharvest diseases in view of its future application in packing houses., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

Metabolic responses to chromium (Cr) exposure and metal uptake were investigated using Salvinia minima plants. Cr treatment reduced the dry weight of floating and submerged leaves, while photosynthetic pigments were not affected. Measurements of respiratory oxygen uptake with and without inhibitors (KCN and SHAM) demonstrated that total respiration, alternative oxidase capacity and residual respiration were higher in Cr-treated than in Cr-untreated leaves, but the highest values were observed in floating leaves. Cr affected the soluble sugar content. Sucrose concentration was, in general, higher in Cr-treated than in Cr-untreated leaves, while the glucose concentration showed an inverse pattern. Cr also affected soluble acid invertase activity, but affectation trend was different between both leaves. Highest values of invertase activity were observed in Cr-treated floating leaves. According to our data soluble acid invertase and sucrose seem to be related to alternative oxidase capacity and residual respiration in floating and submerged leaves exposed to Cr. Thereby, this study constitutes an important contribution to understand metabolic relationships between mitochondrial respiration, alternative respiratory pathway and soluble carbohydrates in plants exposed to heavy metals., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Morphological and physiological responses of seedlings to different solar UV-B irradiances were evaluated in two varieties of quinoa (Chenopodium quinoa Willd.), a crop species from Andean region of South America. Cristalina and Chucapaca varieties were grown at 1965m a.s.l in a glasshouse under natural light conditions for 18 days, and then transferred to outdoors under near-ambient (+UV-B) and strongly reduced (-UV-B) solar UV-B radiation. Exposition to -UV-B increased cotyledon area and seedling height in Cristalina variety whereas leaf number decreased compared to +UV-B. By contrast Chucapaca variety was not affected by UV-B treatments. Seedling fresh weight (FW), root length and leaf thickness did not show significant differences between +UV-B and -UV-B treatments. Mesophyll tissue was slightly affected by solar UV-B reduction. Chlorophyll content was differentially affected by UV-B treatments. Under +UV-B the highest value was observed in Cristalina variety, while in Chucapaca it was observed under -UV-B treatment. Chlorophyll content was slightly higher in leaves than in cotyledons, but there was no difference in the distribution pattern. Chlorophyll a/b ratio and carotenoid content did not show significant differences between UV-B treatments. Leaf UVB-absorbing compounds showed significant differences between UV-B treatments in Chucapaca only, while there were no significant differences in Cristalina variety. UVB-absorbing compounds of cotyledons did not show significant differences between +UV-B and -UV-B treatments. Sucrose, glucose and fructose showed different distribution patterns in cotyledons and leaves of the two varieties under near-ambient and strongly reduced UV-B. Results demonstrated that varieties of quinoa exhibit different morphological and physiological responses to changes in solar UV-B irradiance, but these responses cannot be used to predict the sensitivity to solar UV-B during a short-term exposition. Also, this study can be useful to learn about the plasticity of metabolic pathways involved in plant's tolerance to solar UV-B radiation.

Oxidizing compounds such as sodium hypochlorite (NaCIO) and hydrogen peroxide (H2O2) are widely used in food sanitization because of their antimicrobial effects. We applied these compounds and metals to analyze their antifungal activity against Penicillium digitatum, the causal agent of citrus green mold. The MICs were 300 ppm for NaClO and 300 mM for H2O2 when these compounds were individually applied for 2 min to conidia suspensions. To minimize the concentration of these compounds, we developed and standardized a sequential treatment for conidia that resulted in loss of viability on growth plates and loss of infectivity on lemons. The in vitro treatment consists of preincubation with 10 ppm of NaClO followed by incubation with 100 mM H2O2 and 6 mM CuSO4 (cupric sulfate). The combination of NaClO and H2O2 in the presence of CuSO4 produces a synergistic effect (fractional inhibitory concentration index of 0.36). The sequential treatment applied in situ on lemon peel 24 h after the fruit was inoculated with conidia produced a significant delay in the fungal infection. The in vitro treatment was effective on both imazalil-sensitive and imazalil-resistant strains of P. digitatum and Geotrichum candidum, the causal agent of citrus sour rot. However, this treatment inhibited 90% of mycelial growth for Penicillium italicum (citrus blue mold). These results indicate that sequential treatment may be useful for postharvest control of citrus fruit diseases.

Plants are autotrophic and photosynthetic organisms that both produce and consume sugars. Soluble sugars are highly sensitive to environmental stresses, which act on the supply of carbohydrates from source organs to sink ones. Sucrose and hexoses both play dual functions in gene regulation as exemplified by the upregulation of growth-related genes and downregulation of stress-related genes. Although coordinately regulated by sugars, these growth- and stress-related genes are upregulated or downregulated through HXK-dependent and/or HXK-independent pathways. Sucrose-non-fermenting-1- (SNF1-) related protein pathway, analogue to the protein kinase (SNF-) yeast-signalling pathway, seems also involved in sugar sensing and transduction in plants. However, even if plants share with yeast some elements involved in sugar sensing, several aspects of sugar perception are likely to be peculiar to higher plants. In this paper, we have reviewed recent evidences how plants sense and respond to environmental factors through sugar-sensing mechanisms. However, we think that forward and reverse genetic analysis in combination with expression profiling must be continued to uncover many signalling components, and a full biochemical characterization of the signalling complexes will be required to determine specificity and cross-talk in abiotic stress signalling pathways.

The effect of low temperature on growth, sucrose-starch partitioning and related enzymes in salt-stressed and salt-acclimated cotyledons of quinoa (Chenopodium quinoa Willd.) was studied. The growth of cotyledons and growing axes in seedlings grown at 25/20 degrees C (light/dark) and shifted to 5/5 degrees C was lower than in those only growing at 25/20 degrees C (unstressed). However, there were no significant differences between low-temperature control and salt-treated seedlings. The higher activities of sucrose phosphate synthase (SPS, EC 2.4.1.14) and soluble acid invertase (acid INV, EC 3.2.1.25) were observed in salt-stressed cotyledons; however, the highest acid INV activity was observed in unstressed cotyledons. ADP-glucose pyrophosphorylase (ADP-GPPase, EC 2.7.7.27) was higher in unstressed cotyledons than in stressed ones. However, between 0 and 4days the highest value was observed in salt-stressed cotyledons. The lowest value of ADP-GPPase was observed in salt-acclimated cotyledons. Low temperature also affected sucrose synthase (SuSy, EC 2.4.1.13) activity in salt-treated cotyledons. Sucrose and glucose were higher in salt-stressed cotyledons, but fructose was essentially higher in low-temperature control. Starch was higher in low-temperature control; however, the highest content was observed at 0day in salt-acclimated cotyledons. Results demonstrated that low temperature induces different responses on sucrose-starch partitioning in salt-stressed and salt-acclimated cotyledons. Data also suggest that in salt-treated cotyledons source-sink relations (SSR) are changed in order to supply soluble sugars and proline for the osmotic adjustment. Relationships between starch formation and SuSy activity are also discussed.

Effects of solar and supplemental UV-B radiation on UV-B-absorbing compounds and malondialdehyde (MDA) accumulations in the peel of lemons collected in summer and winter were analyzed. UV-B-absorbing compounds were higher in flavedo than in albedo tissue in both seasons; however, the highest values were observed in summer. These compounds were also higher in outer than in inner flavedo surface. Lemons were categorized as sun-, semisun- and shaded-lemon according to localization inside the tree canopy. Depending on-tree localization UV-B-absorbing compounds were higher in flavedo of sun-lemon than in semisun- and shaded-lemon. Supplementary UV-B radiation (22 kJ m(-2) day(-1) UV-BBE) induced UV-B-absorbing compound synthesis in on-tree and postharvest lemons. Two minutes of supplemental UV-B irradiation in summer lemons produced a strong increment (300%) of UV-B-absorbing compound content, whereas in winter lemons a slight increase (30%) was observed only after 3 min of irradiation. By contrast, UV-B-absorbing compound accumulation was not observed in albedo. MDA accumulation showed approximately a similar trend of UV-B-absorbing compounds. According to our results, solar UV-B was not required for UV-B-absorbing compound accumulation in lemon peel. Relationships between UV-B-absorbing compounds, MDA, reactive oxygen species and pathogen protection are also discussed.

Plants of Citrus aurantifolia grown in a greenhouse without solar UV radiation (UVR) were transferred outdoors to evaluate the effect of solar UV-B radiation (UVBR, 280-315 nm) in prior-developed leaves, constituted by apical bud and those fully expanded before being taken outdoors, and post-developed leaves, formed by those expanded outdoors. Results demonstrated that over a 40 d outdoor period leaf chlorophyll content and distribution pattern were different with and without solar UVBR. Chlorophyll a, chlorophyll b and total chlorophyll contents in both treatments were higher in prior-developed leaves than in post-developed ones. However, highest values were observed in prior-developed leaves under solar UVBR, whereas in post-developed leaves an opposite trend was observed. Carotenoids content in prior-developed leaves was higher with solar UVBR, whereas in post-developed leaves there were no significant differences in both with and without solar UVBR. In addition, prior-developed leaves under solar UVBR accumulated flavonoids, but not anthocyanins. Growth parameters (e.g. DW, DW/FW ratio, LMA, plant height, length and width of foliar lamina) did not show significant differences between plants grown with and without solar UVBR. Thus, our results demonstrated that C. aurantifolia leaves exhibited a different sensibility to solar UVBR according to development stage in relation to photosynthetic pigments and UV-B absorbing compounds production. In addition, the solar UVBR was not necessary as inductor of photosynthetic protection mechanisms in a short-time growth period. On the other hand, our results also demonstrated that solar UVBR acted as an effective feeding deterrent against citrus leafminer.

Cadmium (Cd) uptake effects on sucrose content, invertase activities, and plasma membrane functionality were investigated in Rangpur lime roots ( CITRUS LIMONIA L. Osbeck). Cadmium accumulation was significant in roots but not in shoots and leaves. Cadmium produced significant reduction in roots DW and increment in WC. Leaves and shoots did not show significant differences on both parameters. Sucrose content was higher in control roots than in Cd-exposed ones. Apoplastic sucrose content was much higher in Cd-exposed roots than in control ones. Cd-exposed roots showed a significant decrease in both cell wall-bound and cytoplasmic (neutral) invertase activities; while the vacuolar isoform did not show any change. Alterations in lipid composition and membrane fluidity of Cd-exposed roots were also observed. In Cd-exposed roots phospholipid and glycolipid contents decreased about 50 %, while sterols content was reduced about 22 %. Proton extrusion was inhibited by Cd. Lipid peroxidation and proton extrusion inhibition were also detected by histochemical analysis. This work's findings demonstrate that Cd affects sucrose partitioning and invertase activities in apoplastic and symplastic regions in Rangpur lime roots as well as the plasma membrane functionality and H (+)-ATPase activity.

Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.

UV-B radiation (280-320 nm) is harmful to living organisms and has detrimental effects on plant growth, development and physiology. In this work we examined some mechanisms involved in plant responses to UV-B radiation. Seedlings of quinoa (Chenopodium quinoa Willd.) were exposed to variable numbers of UV-B radiation doses, and the effect on cotyledons was studied. We analyzed (1) cotyledons anatomy and chloroplasts ultrastructure; (2) peroxidase activity involved in the lignification processes; and (3) content of photosynthetic pigments, phenolic compounds and carbohydrates. Exposure to two UV-B doses induced an increase in the wall thickness of epidermal cells, which was associated with lignin deposition and higher activity of the peroxidase. The chloroplast ultrastructure showed an appearance typical of plants under shade conditions, likely in response to reduced light penetration into the mesophyll cells due to the screening effect of epidermal lignin deposition. Exposure to UV-B radiation also led to (1) enhancement in the level of phenolics, which may serve a protective function; (2) strong increase in the fructose content, a fact that might be related to higher requirement of erythrose-4P as a substrate for the synthesis of lignin and phenolics; and (3) reduction in the chlorophyll concentration, evidencing alteration in the photosynthetic system. We propose that the observed lignin deposition in epidermal tissues of quinoa is a resistance mechanism against UV-B radiation, which allows growing of this species in Andean highlands.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in lambda gt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3'-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39,253 daltons (Da), showing 82-88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb) KpnI-SacI fragment of the lambda gt11 derivative was subcloned between the KpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates of Escherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, the E. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5.(ABSTRACT TRUNCATED AT 250 WORDS)

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (Mr) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an Mr of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at - 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP(+)-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa Mr = 6324 M(-1) · cm(-1)). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg(2+) or Mn(2+), and was free of interfering enzyme activities. Ca(2+) was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg(2+) or Mn(2+) was used as cofactor. In the presence of 5 millimolar Mg(2+), Ca(2+) appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca(2+). However, with Mn(2+) (60 micromolar) as cofactor, Ca(2+) exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca(2+) behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg(2+) or Mn(2+) as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg(2+) as cofactor, but this effect was low with Mn(2+). From these results we suggest that Ca(2+) plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.

The invertase of Ricinus communis complexes with proteins, polyvinylpyrrolidone, polyethylene glycol, heparin and dextran sulfate. This association produces an increase of invertase activity. The minimal concentration of activator giving the maximal activation was attained at the same molarity for a given amount of enzyme for all macromolecules studied. These conditions are used for the molecular weight determination of the activating substance. The method may be used for the molecular weight determination of polymeric substances with a molecular weight in the range from 5000 to 1000,000 Da.

Quantitative determinations of total and soluble proteins, total and free sugars, starch, total lipids, tanins, ash (Ca, Na, K, Fe, and P), and caloric value were carried out on quinoa flour. Results show that the amount of soluble proteins was higher than the standard value for wheat and maize and was very close to that of barley's. The yield of free sugars like glucose (4.55%), fructose (2.41%) and sucrose (2.39%) were also of importance. Iron and calcium levels were higher than the reported values for maize and barley. The same occurred for the caloric value (435.5 Kcal/100 g). The content of saponins was also examined since its effect on red blood cells of group A and O has been related as a potential problem of the Andes population. From the chemical analysis a more complete view about quinoa as human food was presented.