Estudio de los diferentes aspectos agronómicos y fisiológicos del zinc en plantas hortícolasFitoextracción y biofortificación

Resumen

Universidad de Granada Facultad de Ciencias Departamento de Fisiología Vegetal ESTUDIO DE DIFERENTES ASPECTOS AGRONÓMICOS Y FISIOLÓGIOS DEL ZINC EN PLANTAS HORTÍCOLAS: FITOEXTRACCIÓN Y BIOFORTIFICACIÓN Yurena Barrameda Medina RESUMEN TESIS DOCTORAL (TESEO) Granada, Junio 2016 Zn has been recognized as an essential element for growth, development and differentiation of all types of life, including microorganisms, plants and animals. This is involved in numerous physiological processes as are the activation of enzymes, protein synthesis, metabolism of carbohydrates, lipids, nucleic and auxins acids, structural and functional integrity of biomembranes, gene expression and regulation and the reproductive development (formation pollen). However, this can become toxic beyond a threshold range of soil from 100 to 400 mg kg-1. Under these conditions, the Zn excess accumulates in plant tissues causing physiological alterations, growth inhibition and even reaching cause death reaching of them. Therefore, plants have developed a series of physiological and molecular to deal with different types of stress mechanisms as well as the toxic effects of metals or metalloids, maintaining metabolic homeostasis and deadening the generation of reactive oxygen species (ROS). Furthermore, approximately 30% of the cultivated land in the world is deficient in Znso producing an alteration in various physiological processes, causing rapid growth inhibition, development and final yield of the plant. Consequently, most cultivated crops contain low concentrations of Zn which can lead to increased Zn malnutrition in humans. Recently, the Biofortification has been proposed as a way to improve the nutritional quality of food plant, and is defined as the process of increasing the bioavailable concentration of essential elements such as Zn, in the edible parts of crops, through agricultural practices or genetic selection. Therefore, the fundamental objectives of this thesis were as follows: 1. Carrying out a study of the different strategies in plants Lactuca sativa and Brassica oleracea to the toxicity of Zn (0.5 mM), such as carboxylates metabolism and oxidative metabolism and glutathione as well as osmoprotectans compounds in order to define key physiological processes to select and / or generate resistant Zn toxicity of plants and horticultural plants using these programs decontamination (phytoextraction) in areas contaminated by this trace element. 2. Considering that the two horticultural plants used in this research are widely used in biofortification programs with trace elements, the aim of the second part of this thesis will be conducting a program of agronomic biofortification with Zn consisting of a supraoptimal application this element (10-100 uM) in order to analyze: the accumulation of Zn in the edible part of the plant and check the response of certain parameters of nutrirtional quality and NO3- assimilation in L. sativa and the synthesis and accumulation of bioactive compounds in B. oleracea. - Comparative study of the toxic effect of Zn in Lactuca sativa and Brassica oleracea plants: I. Growth, distribution, and accumulation of Zn, and mebolism of carboxylates. Zinc (Zn) is an essential micronutrient for plants, animals, and microorganisms. However, in environmental situations of heavy-metal soil pollution, Zn constitutes a major problem for worldwide agricultural production. Organic anions are compounds that have a special structure which proves indispensable to tolerate excess Zn. The aim of the present work was to determine whether carboxylate metabolism is a key physiological process to select and/or generate plants tolerance to Zn toxicity (0.5 mM). For this, we make a comparative analysis of the toxic effect of Zn between two horticultural plants of great agricultural interest, i.e. Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco. The tolerance under Zn-toxicity conditions was greater in B. oleracea than in L. sativa despite to show a higher Zn concentration in shoot. Regarding organic anions metabolism, in leaves of L. sativa the enzyme malate dehydrogenase (MDH) notably increased its activity under Zn-toxicity conditions and both under control conditions as well as in Zn toxicity the main organic anion was malate. With respect to B. oleracea under Zn-toxicity conditions the MDH activity declined with respect to control increasing malate levels in leaves, also under control as well as toxicity conditions the citrate synthase (CS) activity was high and the predominant organic anion was citrate. These results suggest that both in programmes of phytoextraction as well as in biofortification with Zn that are based on the fertilization enriched with this element, B. oleracea is more effective than L. sativa and that the organic anion citrate could be determinant in the tolerance and greater concentration of this element in leaves. - Role of GSH homeostasis under Zn toxicity in plants with different Zn tolerance. Tripepthide glutathione (GSH) is a pivotal molecule in tolerance to heavy metals, including Zinc (Zn). The aim of our work is to examine the role of GSH metabolism in two different horticultural plants under Zn toxicity in order to select and/or generate plants tolerant to Zn toxicity. We show a comparative analysis of the toxic effect of 0.5 mM Zn between Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco. In L. sativa the accumulation of Zn resulted in an increase in reactive oxygen species (ROS), while enzymes of GSH metabolism and the activities of the antioxidant enzymes were negatively affected. On the contrary, B. oleracea showed the existence of a detoxification mechanism of these ROS. Moreover, while in L. sativa increased the oxidized GSH (GSSG) and phytochelatins (PCs) concentration with the reduction of leaves biomass, in B. oleracea the higher concentration of reduced GSH and its use in the detoxification of ROS seems to be a major mechanism to provide tolerance to Zn toxicity without reducing leaf biomass. Our results suggested that under Zn toxicity, B. oleracea is more efficient and tolerant than L. sativa through the detoxification of lipid peroxidation products due to the reduced GSH. - Role of some nitrogenous compounds protectors in the resistance to zinc toxicity in Lactuca satica cv. Phillipus and Brassica oleracea cv. Bronco Zinc (Zn) pollution in the soil represents a major problem for crop production worldwide. In the present work, two horticultural plants exhibiting different tolerance to Zn, Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco, were exposed to Zn to evaluate the contribution of compatible osmolytes such as proline (Pro), glycine betaine (GB) and ɣ-aminobutyric acid (GABA) in the mechanism (s) of tolerance to Zn stress. This study confirms the higher susceptibility of L. sativa to Zn stress: lettuce plants experienced a strong reduction in biomass, while the levels of Pro and GB increased. These results suggest that in L. sativa, the increase of Pro and GB does not represent a mechanism of resistance to toxicity, but it is likely a symptom of Zn stress. Conversely, in B. oleracea, a slight decrease in Pro levels, mainly catalysed by degradation through proline dehydrogenase, was observed; a similar behaviour affected GB levels. On the other hand, GABA synthesis was slightly, but significantly, increased. The presence of high levels of GABA in Zn-stressed B. oleracea would suggest that reactive oxygen species detoxification could be essential to improve the resistance to toxicity under metal stress conditions. The data obtained in this fourth chapter of Doctoral Thesis show as an increase in Zn induced a differential response in plants L. sativa and B. oleracea. Our results suggest that in both programs phytoextraction as biofortification programme with Zn, L. sativa is less effective than B. oleracea: In L. sativa an increase of the MDH and CS activities and high levels of malate not seem to promote tolerance in this species. Likewise, the activities of enzymes and antioxidant compounds not appear to be a powerful mechanism involved in the mechanisms of tolerance. In addition, in this species the accumulation of PCs seems to be rather a survival mechanism that tolerance. Finally, the accumulation of compounds such as Pro or GB, seems to be a symptom of toxicity than an induction Zn promoted to provide tolerance to Zn in this species. Based on our results we can define L. sativa as species susceptible to the toxicity of Zn due to an accumulation of Zn in leaves resulted in a reduction in the biomass of this organ. In B. oleracea a decrease of the MDH activity as well as high activity of the enzyme CS was accompanied by a high concentration of organic anion citrate, which could be decisive in tolerance and greater accumulation of Zn the leaves of B. oleracea. Furthermore, accumulation of reduced GSH and its use in the detoxification of ROS, through the induction of enzymes, such as APX, GST, and GPX, and the detoxification of toxic ompounds, such as methylglyoxal, by increasing activities Gly I and Gly II, appear to be a key mechanism to provide tolerance to Zn toxicity in this species. However, Zn stress in B. oleracea produced an increase in the Pro degradation and an increase in the GABA levels. These two processes resulted in improved of Zn tolerance in B. oleracea and suggest that the ROS detoxifying under Zn stress may be essential to provide tolerance to metals. Based on these results, we can define B. oleracea as a species less sensitive than L. sativa to Zn toxicity, since an increase of Zn did not affect negatively the biomass of the aerial part due to the chelation and transport of this trace element to the aerial part, as well as a powerful detoxification of ROS by increased production of antioxidant compounds. In short, we can conclude that when carrying out a program of phytoextraction of Zn, B. oleracea be more efficient than L. sativa, accumulating a greater amount of this element in the aerial part without diminishing its biomass. Furthermore, these data reveal that both metabolism carboxylates, glutathione or osmoprotectants accumulation compounds such as GABA, are key processes in the tolerance in non-hyperaccumulator plants, as B. oleracea. - Zinc biofortification, antioxidant properties and ionomic profile in two leafy vegetables: Lactuca sativa and Brassica oleracea Zn deficiency is considered as a major risk factor for human health. Here, the effect of Zn biofortification a complementary solution for mineral malnutrition, were examined in two leafy vegetables, Lactuca sativa and Brassica oleracea in order to select Zn-efficient plants. Zn supply did not affected the biomass, but the concentration of malondialdehyde increased in both plants. Zn-use efficiency increased with respect to Zn concentration, total Zn accumulation (TZnA), and Zn utilization efficiency (ZnUtE). Nevertheless, Zn-use efficiency in L. sativa was lower than in B. oleracea. L. sativa showed lower levels of phenolic compounds, and reduced ascorbate, reduced glutathione and γ-aminobutyric acid than in B. oleracea. Furthermore, both plants had higher concentrations of Ca, Mg, Fe, and Mn, especially upon 20 µM Zn in L. sativa and 10-20 µM in B. oleracea. Our results indicate that the application of 20 µM Zn in L. sativa and 20-80 µM Zn in B. oleracea promoted the Zn accumulation and improved the leaf essential-nutrient quality for human health. Nevertheless, in B. oleracea higher Zn concentration, TZnA, phenolics, antioxidants and nutrients concentrations were observed. B. oleracea is emerging as a better choice and more efficient candidate in Zn-biofortification programs. The best approach to increase human Zn levels is to encourage greater consumption of both green leafy vegetables, especially B. oleracea. - Zn-biofortification enhanced nitrogen metabolism and photorespiration process in a green leafy vegetable: Lactuca sativa L. Excessive rates of N fertilizers may result in elevated concentrations of nitrate (NO3-) and high NO3- concentrations are accumulated in the edible parts of leafy vegetables, as lettuce. The main objective of this work was to determine whether the NO3- accumulation and the nitrogen use efficiency (NUE) was affected by the application of different dosages of Zn, to ascertain the influence of this trace element in a biofortification programme in Lactuca sativa cv. Phillipus plants. For that, we analyzed the effect of Zn on NO3- assimilation, photorespiration, and the final products of those processes. Zn-doses in a range of 80-100 µM produced an increase on Zn concentration, provoking a decrease of NO3- since an increase of this element were sufficient to promote the nitrogen assimilation by an increase of the nitrate reductase (NR), glutamine synthetase (GS), aspartate aminotransferase (AAT), and the photorespiration processes. As result, we observed an increase on reduced N, total N concentration (TNC) and N utilization efficiency (NUtE). Consequently, under 80 µM-Zn the amino acid concentration increased significantly, include the concentration of amino acid essential for human health, ie; Leu, Ile and Thr. Adequate Zn fertilization is an important critical player in lettuce, making it possible to increase lettuce productivity and quality, especially with 80 µM Zn, and could decrease the level of Zn deficiency such as the toxic level of NO3- in human health. - Improved of amino acid profile and phytochemical content in Brassica oleracea cv. Bronco under a Zn-biofortification programme. Millions of hectares of cropland are affected by Zinc (Zn) deficiency and approximately one third of the human population suffers from an inadequate intake of Zn. The main aim of the current study was to determine the potential effect of a Zn-biofortification programme on the Zn concentration, the amino acid profile and the phytochemicals content in a green leafy vegetable, such as Brassica oleracea cv. Bronco. Our results indicate that supplementation of 80-100 µM Zn is optimal for maintaining the normal growth of plants and to promote the Zn concentration in the edible part of B. oleracea. Moreover, an increase of the Zn doses can induce an accumulation for the total amino acid concentration in the same range, such as of the glucosinolates (GSLs) levels (aliphatic, indolic and total GSLs), and the phenolic concentration (sinapic acid derivatives, flavonols and total phenols). On the basic of our results, an intake of B. oleracea grown under 80-100 µM Zn may increase the intake of this micronutrient and other elements beneficial for the human health. In general terms we can define the doses used in this study as optimal or supra-optimal for both plants, since a increase in the application of Zn produced no deleterious effects on the edible part of both plants maintain its quality in terms of antioxidants as well the balance of nutrients, especially with the dose of 20 µM in L. sativa and 20-80 µM in B. oleracea. However, overall we can define B. oleracea as the most efficient species in this type of program. These data are consistent with data obtained in the first part of this thesis, where we found that this species was able to accumulate more Zn under toxicity conditions than L. sativa. In addition due to an increase in the need to increase production in recent years, it has been fertilized excess nitrogen may be toxic to the human being by its accumulation in form of NO3- in the edible part of plants. In this sense, and knowing that L. sativa is hyperaccumulator of this toxic compound, in this study it was examined whether proper fertilization with Zn could promote efficiency in the use of N. Our data reveal that L. sativa is able to respond efficiently in a biofortification program with Zn, increasing the concentration of this micronutrient in the aerial part promoting the nitrogen and photorespiration metabolisms, as well as increased accumulation of amino acids essential for human health, especially supplemented plants 80 µM of Zn. In addition Zn may interact synergistically or antagonistically with other macro and micronutrients. In this sense, and knowing that B. oleracea is an important source of bioactive compounds, was carried out a study on the effect of Zn the amino acid profile and metabolism of glucosinolates and phenolics due to the beneficial properties that these compounds may offer human health. Based on our results, the Zn can clearly promote the synthesis of primary compounds such as amino acids, and secondary such as glucosinolates and phenolic compounds, especially when Zn dose applied in this species is in a range of 80-100 µM. In short we can conclude that consumption of both horticultural species cultivated under a Zn biofortification program could promote increased intake of this micronutrient as well as other elements beneficial to human health. However from the point of view of quality, a consumption of B. oleracea could provide a greater amount of beneficial elements. While a crop of L. sativa under these conditions could result in a decrease in nitrogen fertilization maintaining its production and increase the synthesis of essential amino acids to humans.