Eduardo A. Pagano

Typical 2-Cys peroxiredoxins – modulation by covalent transformations and noncovalent interactions

2‐Cys peroxiredoxins are peroxidases devoid of prosthetic groups that mediate in the defence against oxidative stress and the peroxide activation of signaling pathways. This dual capacity relies on the high reactivity of the conserved peroxidatic and resolving cysteines, whose modification embraces not only the usual thiol–disulfide exchange but also higher oxidation states of the sulfur atom. These changes are part of a complex system wherein the cooperation with other post‐translational modifications – phosphorylation, acetylation – may function as major regulatory mechanisms of the quaternary structure. More importantly, modern proteomic approaches have identified the oxyacids at cysteine residues as novel protein targets for unsuspected post‐translational modifications, such as phosphorylation that yields the unusual sulfi(o)nic–phosphoryl anhydride. In this article, we review the biochemical attributes of 2‐Cys peroxiredoxins that, in combination with complementary studies of forward and reverse genetics, have generated stimulating molecular models to explain how this enzyme integrates into cell signaling in vivo.

Uptake of chromium by Salvinia minima: Effect on plant growth, leaf respiration and carbohydrate metabolism

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.

Chloroplast fructose-1,6-bisphosphatase: structure and function.

Redox regulation of photosynthetic enzymes has been a preferred research topic in recent years. In this area chloroplast fructose-1,6-bisphosphatase is probably the most extensively studied target enzyme of the CO2 assimilation pathway. This review analyzes the structure, biosynthesis, phylogeny, action mechanism, regulation and kinetics of fructose-1,6-bisphosphatase in the light of recent findings on structure–function relationship, and from a molecular biology viewpoint.

Seasonal variability of physiological and biochemical aspects of chromium accumulation in outdoor-grown Salvinia minima

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.

Grindelia chiloensis resin and biomass production in its native environment

Preliminary studies have shown that Grindelia chiloensis has higher resin contents than Grindelia camporum which has been previously evaluated as a new resin crop for arid environments. G. chiloensis may provide an additional source of germplasm variability from which to improve resin content and adaptation to cold, arid environments. A number of studies were conducted to evaluate several native populations of G. chiloensis growing in its native environment in southern Argentina. Three native stands of G. chiloensis from northeastern Patagonia were evaluated. All three sites are located around 42 S latitude, within 200 m from the seacoast, and receive an average precipitation below 160 mm/year. Native cover of G. chiloensis ranged between 8% and 11% and was not significantly different among populations. Average individual plant net primary productivity (NPP) was estimated with a single harvest at the end of the growing season (April 1995) and ranged between 92 g and 168 g dry weight (DW). No significant difference in NPP was found among populations. Plant density was significantly different among populations (P < 0.01) and ranged between 2.8 and 0.16 plants/m2. Crude resin (CR) content of the years biomass (stems, leaves, and flowers) was significantly different among populations (P < 0.01); CR content was 23.4%, 17.4%, and 16.7% for the three populations studied. Refined resin (RR, an estimate of resin acids content) was significantly different among populations and ranged between 16.3% and 11.5% for these samples. The ratio RRCR was around 69% and was not different among populations. Capitula and leaves did not differ in resin CR and RR contents, but these were significantly higher than those found in stems. This contrasts with what has been found in G. camporum in which capitula have significantly higher resin contents than leaves. This characteristic will influence the ideotype used in the selection of high productivity germplasm.

Detoxification of Cr(VI) in Salvinia minima is related to seasonal-induced changes of thiols, phenolics and antioxidative enzymes

In this study, protein- and non-protein-thiol-containing compounds (THCC), soluble phenolics (SP), proline (Pro), proteins and malondialdehyde (MDA) contents, and antioxidative enzyme activities were analyzed in floating and submerged leaves of Salvinia minima to establish their role against Cr-induced oxidative stress. We analyzed relationships among biochemical responses to different Cr(VI) concentrations to explore underlying mechanisms of Cr detoxification in plants growing under field conditions during summer and winter seasons. Significant increases in THCC were observed in submerged leaves from both seasons, while in floating leaves THCC increased only in summer being decreased in winter. Contrarily SP increased in floating leaves and decreased in submerged ones. MDA increased significantly in winter-leaves, but in summer-leaves remained unchanged. Antioxidative enzymes, i.e. guaiacol peroxidase (G-POD), superoxide dismutase (SOD) and catalase (CAT) showed different activity patterns. G-POD significantly increased in Cr-treated leaves from both seasons, while SOD increased in submerged leaves only, remaining practically unchanged in floating ones. CAT activity increased in floating leaves from both seasons, whereas in submerged ones was decreased or increased. Proteins increased in both leaf types during summer whereas decreased or remained unchanged in winter. Pro increased in winter-submerged leaves only. Results show that seasonal-induced changes occur in all measured parameters.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

BACKGROUND Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. RESULTS This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. CONCLUSION This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean.

Fluctuating temperatures terminate dormancy in Cynara cardunculus seeds by turning off ABA synthesis and reducing ABA signalling, but not stimulating GA synthesis or signalling

Fluctuating temperatures terminate seed dormancy in many species, including Cynara cardunculus (L.) var. sylvestris. Termination of physiological dormancy requires low ratios of abscisic acid (ABA)/gibberellins (GA). In a previous paper we have shown that physiological responses to fluctuating temperatures comprise a reduction of abscisic acid (ABA) content and sensitivity. However, a possible stimulation of GA synthesis was also suggested as part of the mechanism. That possible stimulation, as well as the identification of potential regulatory sites for ABA and GA metabolism and signalling involved in the termination of dormancy by fluctuating temperatures, are yet to be determined. In this study, we measured GA content and sensitivity in seeds incubated under constant and fluctuating temperatures. We also assessed the expression of several genes involved in ABA and GA metabolism and signalling. Our results show that fluctuating temperatures reduce ABA/GA ratios through a reduction in ABA accumulation during incubation but without altering GA synthesis as compared to that observed under constant temperatures. On the other hand, fluctuating temperatures did not increase sensitivity to GA. Fluctuating temperatures reduced the expression of CycaNCED and CycaABI5 (ABA synthesis and signalling genes) with a temporal pattern that coincides with the interruption of ABA accumulation that precedes germination of seeds incubated under fluctuating temperatures. However, fluctuating temperatures did not modify the expression of CycaCYP707A2 (ABA inactivation) as compared to that observed under constant temperatures. Consistent with our determinations of GA content and sensitivity, fluctuating temperatures did not modify the expression of GA synthesis (CycaGA3ox )a nd signalling genes (CycaRGL2 and CycaGAI) in relation to that observed at constant temperatures. These results show that fluctuating temperatures terminate dormancy in Cynara cardunculus seeds through an interruption in ABA accumulation and a reduction in ABA signalling exerted at the level of CycaNCED and CycaABI5 expression.

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

Abstractα-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA3-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA3-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.

Heme oxygenase up-regulation under ultraviolet-B radiation is not epigenetically restricted and involves specific stress-related transcriptions factors

Heme oxygenase-1 (HO-1) plays a protective role against oxidative stress in plants. The mechanisms regulating its expression, however, remain unclear. Here we studied the methylation state of a GC rich HO-1 promoter region and the expression of several stress-related transcription factors (TFs) in soybean plants subjected to ultraviolet-B (UV-B) radiation. Genomic DNA and total RNA were isolated from leaves of plants irradiated with 7.5 and 15 kJ m-2 UV-B. A 304 bp HO-1 promoter region was amplified by PCR from sodium bisulfite-treated DNA, cloned into pGEMT plasmid vector and evaluated by DNA sequencing. Bisulfite sequencing analysis showed similar HO-1 promoter methylation levels in control and UV-B-treated plants (C: 3.4±1.3%; 7.5: 2.6±0.5%; 15: 3.1±1.1%). Interestingly, HO-1 promoter was strongly unmethylated in control plants. Quantitative RT-PCR analysis of TFs showed that GmMYB177, GmMYBJ6, GmWRKY21, GmNAC11, GmNAC20 and GmGT2A but not GmWRK13 and GmDREB were induced by UV-B radiation. The expression of several TFs was also enhanced by hemin, a potent and specific HO inducer, inferring that they may mediate HO-1 up-regulation. These results suggest that soybean HO-1 gene expression is not epigenetically regulated. Moreover, the low level of HO-1 promoter methylation suggests that this antioxidant enzyme can rapidly respond to environmental stress. Finally, this study has identified some stress-related TFs involved in HO-1 up-regulation under UV-B radiation.