Tiago Tezotto

Biochemical dissection of diageotropica and Never ripe tomato mutants to Cd-stressful conditions

In order to further address the modulation of signaling pathways of stress responses and their relation to hormones, we used the ethylene-insensitive Never ripe (Nr) and the auxin-insensitive diageotropica (dgt) tomato mutants. The two mutants and the control Micro-Tom (MT) cultivar were grown over a 40-day period in the presence of Cd (0.2 mM CdCl₂ and 1 mM CdCl₂). Lipid peroxidation, leaf chlorophyll, proline content, Cd content and antioxidant enzyme activities in roots, leaves and fruits were determined. The overall results indicated that the MT genotype had the most pronounced Cd damage effects while Nr and dgt genotypes might withstand or avoid stress imposed by Cd. This fact may be attributed, at least in part, to the fact that the known auxin-stimulated ethylene production is comprised in dgt plants. Conversely, the Nr genotype was more affected by the Cd imposed stress than dgt, which may be explained by the fact that Nr retains a partial sensitivity to ethylene. These results add further information that should help unraveling the relative importance of ethylene in regulating the cell responses to stressful conditions.

Opinion - Nickel and urease in plants: Still many knowledge gaps

We propose experimental strategies to expand our understanding of the role of Ni in plants, beyond the Ni-metallocenter of urease, still the only identified Ni-containing plant enzyme. While Ni has been considered an essential mineral for plants there is a clear lack of knowledge of its involvement in metabolic steps except the urease-catalyzed conversion of urea to ammonia and bicarbonate. We argue that urease (and hence, Ni) plays an important role in optimal N-use efficiency under various N regimes by recycling urea-N, which is generated endogenously exclusively from arginase action on arginine. We further suggest that urease and arginase may connect different metabolic compartments under stress situations, and therefore may be involved in stress tolerance. To determine possible non-urease roles of Ni we call for experimental manipulation of both Ni and N availability in urease-negative mutants. Plant ureases have been shown to have defense roles, distinct from their ureolytic activity, and we call for investigation of whether Ni helps maintain a urease conformation or stability for these non-ureolytic defense roles. The beneficial effects of Ni at upper concentration limits have not been fully examined. We posit a Ni strategy of plants whose growth/performance is stimulated by unusual amounts of soil Ni, for defense and/or for maximal N-use efficiency. While we know little about Ni and urease roles in N metabolism and defense, virtually nothing is known about Ni roles in plant-microbial consortia. And, much of what we know of Ni and urease is limited to only a few plants, e.g. soybean, potato and Arabidopsis, and we suggest studies vigorously extended to other plants.

Cadmium stress antioxidant responses and root-to-shoot communication in grafted tomato plants

Many aspects related to ROS modulation of signaling networks and biological processes that control stress responses still remain unanswered. For this purpose, the grafting technique may be a powerful tool to investigate stress signaling and specific responses between plant organs during stress. In order to gain new insights on the modulation of antioxidant stress responses mechanisms, gas-exchange measurements, lipid peroxidation, H2O2 content, proline, superoxide dismutasexa0(SOD), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX) and guaiacol peroxidase (GPOX) were analyzed in Micro-Tom grafted plants submitted to cadmium (Cd). The results observed revealed that higher amounts of Cd accumulated mainly in the roots and rootstocks when compared to leaves and scions. Macronutrients uptake (Ca, S, P and Mg) decreased in non-grafted plants, but differed among plant parts in all grafted plants. The results showed that thexa0accumulation of proline observed in scions of grafted plants could be associated to the lower MDA contents in the scions of grafted plants. In the presence of Cd, non-grafted plants displayed increased CAT, GR, GPOX and APX activities for both tissues, whilst grafted plants revealed distinct trends that clearly indicate signaling responses from the rootstocks, allowing sufficient time to activate defense mechanisms in shoot. The information available concerning plants subjected to grafting can provide a better understanding of the mechanisms of Cd detoxification involving root-to-shoot signaling, opening new possibilities on strategies which can be used to manipulate heavy metal tolerance, since antioxidant systems are directly involved in such mechanism.

Antioxidant and antiproliferative activities in different maturation stages of broccoli (Brassica oleracea Italica) biofortified with selenium.

In this work, three different broccoli maturity stages subjected to biofortification with selenium were evaluated for antioxidant and antiproliferative activities. Antioxidant trials have shown that the maturation stages biofortified with selenium had significantly higher amounts of phenolic compounds and antioxidant activity, especially seedlings. Although non-polar extracts of all samples show antiproliferative activity, the extract of broccoli seedlings biofortified with selenium stood out, presenting cytocidal activity for a glioma line (U251, GI50 28.5 mg L(-1)).

Essentiality of nickel in plants: a role in plant stresses

The element Ni is considered an essential plant micronutrient because it acts as an activator of the enzyme urease. Recent studies have shown that Ni may activate an isoform of glyoxalase I, which performs an important step in the degradation of methylglyoxal (MG), a potent cytotoxic compound naturally produced by cellular metabolism. Reduced glutathione (GSH) is consumed and regenerated in the process of detoxification of MG, which is produced during stress (stress-induced production). We examine the role of Ni in the relationship between the MG cycle and GSH homeostasis and suggest that Ni may have a key participation in plant antioxidant metabolism, especially in stressful situations.

Simple procedure for nutrient analysis of coffee plant with energy dispersive X-ray fluorescence spectrometry (EDXRF)

Nutrient analysis is used to estimate nutrient content of crop plants to manage fertilizer application for sustained crop production. Direct solid analysis of agricultural and environmental samples by energy dispersive X-ray fluorescence spectrometry (EDXRF) was chosen as alternative technique to evaluate the simultaneous multielemental quantification of the most important essential elements in coffee (Coffea arabica L.) plants. Inductively coupled plasma atomic emission spectrometry and certified reference materials made from leaves were used to calibrate and check the trueness of EDXRF method for the determination of the concentration of several nutrients in coffee leaves and branches. Fluorescence spectrometry proved to be advantageous and presented low cost as loose powder samples could be used. Samples collected from a field experiment where coffee plants were treated with excess of Ni and Zn were used to verify the practical application of the method. Good relationships were achieved between certified values and data obtained by EDXRF, with recoveries ranging from 82 to 117 %.

Changes of Nutritional Status during a Phenological Cycle of Coffee under High Nitrogen Supply by Fertigation

High-technology coffee cultivation systems involving fertigation commonly apply high rates of nitrogen (N). However, there is little information on the plants uptake of N under these conditions. The objective of this study was to evaluate the changes in the nutritional status of coffee plants during a phenological cycle in response to N applied by fertigation. The study was conducted with 7-year-old trees of Coffea arabica L., under pivot irrigation and fertigation, with five N rates (0, 200, 400, 600, and 800 kg ha−1) applied on the plants. The changes in nutritional status were monitored during the phenological cycle, and the yield was measured at the end of the cycle. The N concentration increased with the N rates and varied with the phenological period, with the greatest concentration occurring during anthesis, the same as for the sulfur level. However, N rate did not affect the concentrations of other nutrients. The greatest concentrations of phosphorus, calcium, and magnesium were obtained in the final fructification phases and for potassium in the initial phases. Micronutrient concentrations were greater in the vegetative phases and anthesis but were reduced in the fruit- formation phase and increased again when the berries started to mature. The maximum yield, considering a relative production of 90%, was obtained with application of 415 kg ha−1 of N. Leaf concentrations of N were directly related to the N rates, showing that the nutritional status of coffee plants can be evaluated in the ripening phase to adjust the fertilization parameters for the next year.

INFLUENCE OF NITROGEN FERTILIZATION ON NICKEL ACCUMULATION AND CHEMICAL COMPOSITION OF COFFEE PLANTS DURING FRUIT DEVELOPMENT

Nutritional and physiological significance of micronutrients in coffee plants, especially with regard to nickel (Ni) is still unknown. The dynamics of nitrogen (N), phosphorus (P), potassium (K) and Ni accumulation in coffee fruits, as well as their relationships with total soluble protein, amino acids, reducing sugars, and starch content during coffee fruit development (green, ripe, and dry fruits), were investigated. Coffee trees received three N fertilizer rates (0, 150, and 300 kg of N ha−1) as ammonium sulfate split into three applications per year. Nitrogen fertilization increased reducing sugars and starch concentrations in ripe fruits. In contrast, green fruits showed the highest amino acid and Ni concentrations. Fruit Ni concentration decreased in both green and ripe fruits as N rates increased; thus, indicating the possibility of either a N-associated dilution effect on Ni concentration or that Ni uptake by roots and/or transport to developing fruit was limiting. Plant nutritional status and fruit development stage influenced the coffee grain chemical composition. Furthermore, the variation in reducing sugars and starch content was more closely linked to the stage of fruit development than to N supply. A supposed relationship among the decreased of caffeine, starch, amino acids, and proteins with Ni content during green fruit development suggests a fundamental role for Ni in coffee fruit ripening. The interaction between N and Ni metabolism during fruit ripening might influence the chemical parameters involved in the coffee grain quality. This is the first report documenting changes in Ni concentrations of coffee fruit as a function of N fertilization rates and the development stage, but further research is needed to better understand the significance of N-Ni interaction in developing coffee fruit.

Deletion of the single UreG urease activation gene in soybean NIL lines: characterization and pleiotropic effects

AbstractnThe soybean eu3-a mutant (formerly, eu3-e1) lacks all ureolytic activity. Eu3 encodes urease accessory (Ni insertion) protein, UreG. Eu3 (Glycine max v1.1 Glyma08g08970.1) is the only UreG-encoding gene in the soybean genome. Here we show that the eu3-a lesion is a 2.4xa0kb deletion, beginning 50xa0bp upstream of the transcription start, and covering 94xa0% of the deduced amino acid sequence of UreG, explaining the urease activity-null phenotype of eu3-a. We established near-isogenic lines (NILs), eu3-a versus Eu3, and confirmed a striking accumulation of endogenous urea in eu3-a and none in Eu3, and the greater resistance of eu3-a leaf discs to external urea. However, incubation of discs in light alleviated urea sensitivity in Eu3, apparently by providing C skeletons for urea-released NH3. Hence, urea, endogenous and exogenous, is apparently less toxic than urea-derived NH3. eu3-a and Eu3 plants, however, did not differ in seed Ni content, nor in pool sizes of metabolites that generate urea via arginine. The NILs differed in patterns of nodulation by Bradyrhizobium japonicum and by Bradyrhizobium elkanii. Ureases have been reported to be fungitoxic. However, eu3-a and Eu3 NIL seedlings were equally sensitive to the pathogenic Basidiomycete fungus, Rhizoctonia solani. Since eu3-a accumulates at least some apo-urease, we examined a specific anti-fungal role of the seed urease in seedlings of a second NIL pair, differing in the presence (Eu1) and absence (eu1-a) of the seed urease. Again, this second NIL pair exhibited equal sensitivity to R. solani. To our knowledge, we report only the second test of fungal resistance influenced by urease levels in intact plants.

Expression of metallothionein genes in coffee leaves in response to the absence or excess of Cu and Zn

Copper (Cu) and zinc (Zn) are plant nutrients; however, intracellular concentration must be kept within the sufficiency limits in order to adequately maintain metabolic functions. Like other organisms, plants have metallothioneins (MTs), which are cysteine-rich peptides that have been associated with metal homeostasis and detoxification because of their capacity to bind divalent metals. Nonetheless, the physiological roles of MTs are not completely understood and much is still unknown concerning their characterization in many higher plant species. In this study, MT expression was analyzed in leaves of Coffea arabica plants exposed to the absence or high levels of Cu and Zn in a nutrient solution, aiming to investigate the role of MTs in the maintenance of Cu and Zn homeostasis and in detoxification of excessive levels of these nutrients. At the same time, the studied MT sequences underwent phylogenetic analysis, showing that in C. arabica three of the studied MTs showed characteristics of three of the four different groups of plant MTs. It was observed that CaMT4 and CaMT15 belonged to group I but also presented characteristics of group II. CaMT3 showed protein characteristics of MT group II, and CaMT8 was positioned in type III MT clade. The expression levels of genes CaMT3, CaMT4, CaMT8, and CaMT15 were evaluated in leaves. The results indicated that MT type I, II, and III studied in C. arabica leaves are probably involved in Cu homeostasis and detoxification. However, further studies are needed to identify other MTs in coffeexa0plants and specifically those involved in response to Zn, since the evaluation here showed a low response to excessive concentrations of this metal.