The objective of this work was to evaluate cowpea (Vigna unguiculata) cultivars regarding plant d... more The objective of this work was to evaluate cowpea (Vigna unguiculata) cultivars regarding plant development and the quantity and quality of soluble proteins in their grains, for breeding purposes. The experiment was conducted in a greenhouse, in a completely randomized experimental design, with the Paulistinha, BRS Novaera, Epace 10, and BR 17-Gurguéia cultivars. Leaf area, shoot fresh and dry matter, leaf protein content, number of nodules, and nodule and root dry matter were evaluated. In mature grains, soluble fractions and soluble amino acids were also quantified, and the electrophoretic analysis was performed with protein denaturation. There were no differences between cultivars for the plant development variables. However, protein quantity and quality in the grain differed between cultivars. 'BRS Novaera' and 'BR 17-Gurguéia' showed a higher soluble protein content in their grains. 'BRS Novaera' exhibited higher contents of two soluble sulfur amino acidsmethionine and cysteine-, not differing from 'BR 17-Gurguéia' regarding methionine content. Both cultivars presented protein band polymorphism, but BRS Novaera had an extra band for albumins. The BRS Novaera and BR 17-Gurguéia cultivars have the highest content of soluble proteins in their grains and the greatest protein polymorphism, which makes them suitable for improving the nutritional quality of cowpea.
We evaluated the mechanisms that control Cd accumulation and distribution, and the mechanisms tha... more We evaluated the mechanisms that control Cd accumulation and distribution, and the mechanisms that protect the photosynthetic apparatus of Brachiaria decumbens Stapf. cv. Basilisk and Panicum maximum Jacq. cv. Massai from Cd-induced oxidative stress, as well as the effects of simulated summer or winter conditions on these mechanisms. Both grasses were grown in unpolluted and Cdpolluted Oxisol (0.63 and 3.6 mg Cd kg-1 soil, respectively) at summer and winter conditions. Grasses grown in the Cd-polluted Oxisol presented higher Cd concentration in their tissues in the winter conditions, but the shoot biomass production of both grasses was not affected by the experimental conditions. Cadmium was more accumulated in the root apoplast than the root symplast, contributing to increase the diameter and cell layers of the cambial region of both grasses. Roots of B. decumbens were more susceptible to disturbed nutrients uptake and nitrogen metabolism than roots of P. maximum. Both grasses translocated high amounts of Cd to their shoots resulting in oxidative stress. Oxidative stress in the leaves of both grasses was higher in summer than winter, but only in P. maximum superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were increased.
Lysine biosynthesis has been extensively studied and the regu-was not. It would appear that the m... more Lysine biosynthesis has been extensively studied and the regu-was not. It would appear that the modulation by Ca 2 + and latory enzymes characterized in some of the most important ionic strength of LOR is a common feature among plant LOR enzymes. S-adenosylmethionine (SAM) did not produce any crop plants, however, much less is known about the lysine significant effect on either enzyme activity, indicating that it degradation pathway. Lysine 2-oxoglutarate reductase (LOR) only plays a role in the regulation of lysine biosynthesis. The and saccharopine dehydrogenase (SDH) have recently been effect of S-2-aminoethyl-L-cysteine (AEC) as both a substrate partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for further characterized these enzymes from rice endosperm in relation to Ca 2 + and ionic strength modulation. Optimum pH LOR, but was also able to inhibit LOR activity, possibly values of 7.0 and 8.0 were obtained for LOR and SDH, competing with lysine at the active site. The higher K m for respectively. The LOR domain of the polypeptide was modu-AEC compared to lysine may reflect a lower binding affinity for AEC. lated by Ca 2 + and ionic strength, whereas the SDH domain tion has been shown to be considerably elevated in the majority of plant tissues analysed, all of them have failed to exhibit over-accumulation of lysine in the seed (Azevedo et al. 1990, Diedrick et al. 1990, Heremans and Jacobs 1995). It has been suggested that in order to achieve lysine accumulation in seeds, the catabolism of lysine must be reduced (Brochetto-Braga et al. 1992, Azevedo and Lea 2000). In the opaque-2 mutant of maize, the activities of two enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8; also designated lysine h-ketoglutarate reductase [LKR]) and saccharopine dehydrogenase (SDH, EC 1.5.1.9), were shown to be strongly reduced, thus increasing the availability of lysine to be incorporated into storage proteins, or allowing more lysine to accumulate in the soluble form in the maize endosperm (Gaziola et al. 1999, Kemper et al. 1999). Falco
Theoretical and Experimental Plant Physiology, May 27, 2020
This work aimed to quantify the concentration of soluble amino acids, nitrogen (N) and Cd in the ... more This work aimed to quantify the concentration of soluble amino acids, nitrogen (N) and Cd in the developing seeds of maize plants that were grown in Cd-contaminated soil from seed sowing to the reproductive stages. The specific activities and feedback inhibition properties of lysine (Lys) metabolic enzymes were also determined in developing seeds. The potential maize yield was depressed by Cd exposure. Cadmium concentration in plant organs followed the decreasing order: roots [ stems = leaves [ developing seeds (37.04, 1.95, 1.46 and 0.22 mg kg-1 Cd, respectively). The relatively low Cd concentration in the remained developing seeds was a result from root-and earmediated reductions of the Cd translocation in plants. Plants under Cd exposure presented developing seeds with an increased N concentration (up to 7%) when compared to control plants. Furthermore, the level of soluble amino acids (particularly histidine, glycine, tyrosine, methionine, isoleucine and valine) was increased in the developing seeds of Cd-treated plants. In addition, changes in the feedback properties of dihydrodipicolinate synthase (DHDPS), an enzyme from lysine metabolism, were observed in developing seeds. In conclusion, the current study showed that maternal plant exposure to Cd can alter the concentration of soluble amino acids and the behavior of Lys metabolic enzymes in developing seeds. This study provided not only new information about the influence of the long-term Cd exposure on plants, but also data about protective plant strategies against Cd toxicity.
In general, the effects of Cadmium (Cd) on crop quality are evaluated solely by Cd accumulation i... more In general, the effects of Cadmium (Cd) on crop quality are evaluated solely by Cd accumulation in the edible plant parts; thus, the potential effects on several nutritional features are not often taken into account. This study evaluated Cd effects on the enzymatic activities of lysine (Lys) metabolism, storage protein and amino acid profile in maize. Stress parameters were also assessed. In vegetative organs, Cd was accumulated in the following decreasing order: roots > stems > leaves. Cadmium accumulation in grains decreased at grain maturity (from 0.25 to 0.07 µg g-1 DW). In leaves, neither hydrogen peroxide content nor superoxide dismutase, catalase, ascorbate peroxide and guaiacol peroxidase activities did not change in Cd-treated plants compared to control plants. Lipid peroxidation was not detected in immature grains and leaves of plants under Cd exposure, indicating that Cd accumulation mainly in the roots is a mechanism to avoid oxidative stress in aboveground parts of the plant. However, Lys metabolism in immature grains was modified, showing increases in the specific activities of aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) after Cd exposure. AK sensitivity to Lys feedback inhibition increased, but decreased in DHDPS in Cd-treated plants, suggesting differential regulation for these enzymes. In mature grains, the Lys content did not change, while the proline content increased by 54 % in Cd-treated plants. This is the first report on Cd effects on amino acid profile, storage protein contents and enzymes from Lys metabolism in grains of a cereal plant species.
The grain proteins of barley are deficient in lysine and threonine due to their low concentration... more The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise) and five antisense C-hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-hordein suppression did not only alter the metabolism of at least one aspartate-derived amino acid (threonine), but major changes were also detected in the metabolism of lysine and methionine. Modifications in the activities and regulation of aspartate kinase, dihydrodipicolinate synthase and homoserine dehydrogenase were observed in most transgenic lines. Furthermore the activities of lysine a-ketoglutarate reductase and saccharopine dehydrogenase were also altered, although the extent varied among the transgenic lines.
Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca 2+ content i... more Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca 2+ content in the distal fruit tissue. However, many other factors can also determine fruit susceptibility to BER. It is possible that during fruit growth, Ca 2+ imbalance can increase membrane leakiness, which may trigger the accumulation of reactive oxygen species, leading to cell death. Brassinosteroids are a class of plant hormones involved in stress defenses, specially increasing the activity of antioxidant enzymes and the accumulation of antioxidant compounds, such as ascorbic acid. The objective of this study was to understand the mechanisms by which 24-epibrassinolide (EBL) reduces fruit susceptibility to BER. Tomato plants 'BRS Montese' were cultivated in a greenhouse and were weekly sprayed with water (control) or EBL (0.01 µM) after full bloom. Plants and fruits were evaluated at 15 days after pollination (DAP). According to the results, EBL treatment inhibited BER development, increased fruit diameter, length, and fresh weight. EBL-treated fruit showed higher concentrations of soluble Ca 2+ and lower concentrations of cell wall-bound Ca 2+. EBL-treated fruit also had higher concentrations of ascorbic acid and lower concentrations of hydrogen peroxide, compared to water-treated fruit. EBL treatment increased the activity of the three main antioxidant enzymes known as ascorbate peroxidase, catalase, and superoxide dismutase. According to the results, EBL treatment maintained higher soluble Ca 2+ and antioxidant capacity, reducing fruit susceptibility to BER.
The activities of two aspartate kinase (EC 2.7.2.4) isoenzymes that have been partially purified ... more The activities of two aspartate kinase (EC 2.7.2.4) isoenzymes that have been partially purified from developing rice seeds, were studied in the presence of calcium, calmodulin inhibitors, S-adenosylmethionine, S-(2-aminoetyl)-L-cysteine, methionine, valine and increased salt concentrations. None of the compounds tested was able to produce any significant alteration in threonine-sensitive aspartate kinase activity. On the other hand, the activity of the lysine-sensitive aspartate kinase was slightly increased by calcium. The increase in activity was not observed when EGTA was added in combination with calcium. S-adenosylmethionine alone inhibited the activity by 12% and intensified the inhibition caused by lysine. S-(2-aminoethyl)-L-cysteine also inhibited the activity of aspartate kinase, but not to the same extent of lysine. Methionine and valine stimulated slight increases in activity, whereas KCl up to 500 mM did not cause any change in aspartate kinase activity. These results with rice aspartate kinase indicate that lysine-sensitive aspartate kinase is also synergistically inhibited by S-adenosylmethionine, as observed for other plants species. Although some increase in aspartate kinase activity was observed in the presence of calcium, the magnitude of the alterations was not sufficient to indicate a regulatory role of calcium on aspartate kinase.
Abiotic stresses negatively affect crop development and yield. However, the use of seaweed extrac... more Abiotic stresses negatively affect crop development and yield. However, the use of seaweed extracts can alleviate production losses through improvement of the antioxidant system and synthesis of compatible osmolytes. The aim of this study was to assess the effects of Ascophyllum nodosum extract on antioxidant response and yield of common bean plants (Phaseolus vulgaris) under drought stress. Therefore, two methods of application (foliar spray and soil irrigation) and two doses (5 and 10 mL L-1) were tested, whereas untreated plants were used as control. There were no significant differences in malondialdehyde and hydrogen peroxide contents among treatments, indicating that application of seaweed extract did not change the oxidative status in treated plants when compared to the control ones. On the other hand, the use of seaweed extract increased proline content even prior to drought stress (46.3 to 145.4% when compared to the control), and this increment remained until the 10 th day of stress (from 60.1 to 201.7% over the reference plants), supporting early evidences that A. nodosum extract is a plant elicitor. The yield parameters were not affected by the extract, probably due to the severe drought stress which plants were submitted. The results suggest that A. nodosum extract affects proline metabolism in Phaseolus vulgaris plants, making the synthesis of this osmolyte more responsive to drought stress because seaweed-treated plants presented a higher proline content than the control ones during the exposure to water deficit.
Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de ... more Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de postura e sua interferência na disponibilidade do ferro Protein concentration (phosvitin and lipovitelin) in egg yolks of Laying Hens (Gallus gallus) in different posture cycles and its interference in the iron availability
Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation ... more Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation of germination process, and both may result in viability loss. This study aimed to elucidate the biochemical basis of embryos survival of Araucaria angustifolia recalcitrant seeds during storage. After harvesting, seeds were stored at ambient conditions (without temperature and humidity control) and in a cold chamber (temperature of 10 ± 3 °C, and relative humidity of 45 ± 5 %). Moisture content, viability, H2O2 content, lipid peroxidation, protein content, and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), at 0, 15, 45 and 90 days of storage, were evaluated. Seed viability reduced about 40 % during the storage period accompanied by a reduction in soluble protein (about 64 % of reduction) in both storage conditions, and increased lipid peroxidation (about 115 % and 66 % for ambient and cold chamber conditions, respectively). H2O2 content used as a marker of oxidative stress was reduced during the period, possibly controlled by the action of CAT and APX, for which increased activities were observed. The results allowed the identification of seven SOD isoenzymes (one Mn-SOD, five Fe-SOD and one Cu/Zn-SOD), whose activities also increased in response to storage. Some biochemical damage resulting from storage was observed, but viability reduction was not due to failure of enzymatic protection mechanisms.
A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido a... more A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido aspártico, pela qual são também sintetizados os aminoácidos treonina, metionina e isoleucina. Além disso, a lisina é o principal aminoácido limitante em todos os cereais e por cerca de 30 anos a via do ácido aspártico tem sido estudada em plantas, com o intuito de desvendar e caracterizar os principais pontos chave na regulação das vias de biossíntese desses aminoácidos. Duas etapas distintas, uma primeira originada a partir do desenvolvimento da cultura de tecidos (anos 70-80) e a segunda a partir do desenvolvimento de técnicas para a transformação de plantas (anos 90), permitiram que mutantes bioquímicos e plantas trangênicas fossem produzidos com alterações específicas em passos metabólicos chave, levando à superprodução e acúmulo de treonina em vários tecidos das plantas. Entretanto, a acumulação de lisina em sementes não foi obtida. Tal fato, associado a estudos bioquímicos da via de degradação da lisina em cereais e em leguminosas, indicou que a manipulação da degradação seria tão ou mais importante que a manipulação da biossíntese de lisina para o acúmulo deste aminoácido em sementes dos cereais. Em milho, o uso e estudo de outros mutantes tais como o opaco-2 e variedades QPM (Quality Protein Maize) contribuíram significativamente para a compreensão dos eventos regulatórios. As estratégias para a obtenção de materiais ricos em lisina e sua relevância à manipulação de outros aminoácidos são revisados. Palavras-chave: lisina, nutrição, aminoácidos essenciais, cereais MANIPULATING CEREAL CROPS FOR HIGH LYSINE ACCUMULATION IN SEEDS ABSTRACT: The nutrition value of a protein is directly related to its amino acid composition. Some of these amino acids, termed essential amino acids, cannot be synthesized by humans and therefore must be supplied in the diet for adults and in particular for infants and children. Lysine is an essential amino acid synthesized via the aspartic acid metabolic pathway, in which threonine, methionine and isoleucine are also endproducts. Moreover, lysine is the first limiting amino acid in all cereal grains. For over 30 years, the aspartic acid metabolic pathway has been studied in higher plants with the aim of identifying and characterizing the key regulatory points controlling the biosynthetic pathway. Two clear distinct time periods, one begining with the development of tissue culture techniques (1970-80's) and the second with the development of plant transformation techniques (90's), has encouraged the production of biochemical mutants and transgenic plants with specific alterations in key enzymes of the pathway, leading to the overproduction and accumulation of threonine in all plant tissues. However, the accumulation of lysine in seeds has been particularly difficult to achieve. Such an observation, associated with the recent biochemical studies on lysine degradation in cereal and legume plant species, has indicated that the manipulation of lysine degradation is as important as the manipulation of lysine synthesis, if the goal of accumulating this amino acid in cereal seeds is to be achieved. In maize, the study and use of other mutants such as the opaque-2 and QPM (Quality Protein Maize) varieties, has contributed significantly to our understanding of the regulatory aspects of the aspartate pathway. The strategies of obtaining cereals rich in lysine and their relevance to the manipulation of other amino acids have been discussed.
Hordeins are the major storage proteins in barley grains and are responsible for their low nutrit... more Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.
Journal of Agricultural and Food Chemistry, Feb 12, 2005
Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabo... more Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabolism of lysine is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9). The final soluble lysine concentration in cereal seeds is controlled by both synthesis and catabolism rates. The production and characterization of high-lysine plants species depends on knowledge of the regulatory aspects of lysine metabolism and manipulation of the key enzymes. We have for the first time isolated, partially purified, and characterized LOR and SDH from developing sorghum seeds, which exhibited low levels of activity. LOR and SDH were only located in the endosperm and were very unstable during the isolation and purification procedures. LOR and SDH exhibited some distinct properties when compared to the enzymes isolated from other plant species, including a low salt concentration required to elute the enzymes during anion-exchange chromatography and the presence of multimeric forms with distinct molecular masses.
Tomato is an important crop worldwide. Cadmium (Cd) concentrations in fruits depend on tomato gen... more Tomato is an important crop worldwide. Cadmium (Cd) concentrations in fruits depend on tomato genotype. This work aimed to study the relation among Cd accumulation, tolerance mechanisms, and fruit features in two tomato cultivars with contrasting tolerance to Cd stress. Tolerant (Yoshimatsu) and sensitive (Tropic Two Orders) plants were grown in control and contaminated soils (0.04 and 3.77 mg/kg Cd, respectively) from the seedling stage to fruit production. Both cultivars were able to acclimatize to Cd exposure, probably through mechanisms associated with reductions in the magnesium status. Cadmium concentrations varied according to the following descending order: roots = leaf blades > (peduncle + sepals) > stem = fruits. However, the tolerant cultivar accumulated more Cd than did the sensitive one. Although Cd reached the fruits from the first to the fourth bunches, peduncle and sepals may act as a barrier to Cd entrance in tomato pulp and peel. The Cd-induced changes in the fruit mineral profile varied according to plant cultivar, organ, tomato tissue, and bunch position. Moreover, plant yield was not affected by the Cd stress, which was able to improve fruit size and weight in the tolerant cultivar. In conclusion, new insights about the Cd-induced effects on tomato development and fruit attributes were provided by growing plants in soil, which is the media generally used to cultivate this crop, rather than hydroponics. It was shown that tomato cultivars with contrasting tolerance to Cd toxicity can reach sexual maturity and produce fruits with no yield losses, despite impacts on development from long-term Cd exposure. This study also revealed the role of floral receptacle and its related structures in limiting, even partially, Cd translocation to the fruits. Furthermore, Yoshimatsu's capacity to produce bigger and heavier fruits, in plants under Cd exposure, may probably be associated with enhanced Cd accumulation.
Abstract Cadmium (Cd) is a heavy metal highly toxic to plants and human and animal health. The in... more Abstract Cadmium (Cd) is a heavy metal highly toxic to plants and human and animal health. The increased concentration of this metal in the environment in recent decades is the result of anthropogenic activities that include the production of mineral fertilizers, pesticides, intense mining, waste disposal, and foundries. Cd accumulation is known to induce oxidative stress in plant cells due to overproduction of reactive oxygen species, which may result in severe damage at the cellular level, ultimately impairing plant growth and productivity. In response to such stress, different molecules having enzymatic or nonenzymatic actions contribute to mitigate plant cellular damage and balance redox homeostasis in plant cells. This chapter aims to summarize current knowledge about the prospecting of important components of the molecular mechanisms associated with antioxidant machinery in plant response and tolerance to Cd toxicity. Insights into antioxidant response regulation, which includes hormones, signaling molecules, transcription factors, and microRNAs, are also presented.
The objective of this work was to evaluate cowpea (Vigna unguiculata) cultivars regarding plant d... more The objective of this work was to evaluate cowpea (Vigna unguiculata) cultivars regarding plant development and the quantity and quality of soluble proteins in their grains, for breeding purposes. The experiment was conducted in a greenhouse, in a completely randomized experimental design, with the Paulistinha, BRS Novaera, Epace 10, and BR 17-Gurguéia cultivars. Leaf area, shoot fresh and dry matter, leaf protein content, number of nodules, and nodule and root dry matter were evaluated. In mature grains, soluble fractions and soluble amino acids were also quantified, and the electrophoretic analysis was performed with protein denaturation. There were no differences between cultivars for the plant development variables. However, protein quantity and quality in the grain differed between cultivars. 'BRS Novaera' and 'BR 17-Gurguéia' showed a higher soluble protein content in their grains. 'BRS Novaera' exhibited higher contents of two soluble sulfur amino acidsmethionine and cysteine-, not differing from 'BR 17-Gurguéia' regarding methionine content. Both cultivars presented protein band polymorphism, but BRS Novaera had an extra band for albumins. The BRS Novaera and BR 17-Gurguéia cultivars have the highest content of soluble proteins in their grains and the greatest protein polymorphism, which makes them suitable for improving the nutritional quality of cowpea.
We evaluated the mechanisms that control Cd accumulation and distribution, and the mechanisms tha... more We evaluated the mechanisms that control Cd accumulation and distribution, and the mechanisms that protect the photosynthetic apparatus of Brachiaria decumbens Stapf. cv. Basilisk and Panicum maximum Jacq. cv. Massai from Cd-induced oxidative stress, as well as the effects of simulated summer or winter conditions on these mechanisms. Both grasses were grown in unpolluted and Cdpolluted Oxisol (0.63 and 3.6 mg Cd kg-1 soil, respectively) at summer and winter conditions. Grasses grown in the Cd-polluted Oxisol presented higher Cd concentration in their tissues in the winter conditions, but the shoot biomass production of both grasses was not affected by the experimental conditions. Cadmium was more accumulated in the root apoplast than the root symplast, contributing to increase the diameter and cell layers of the cambial region of both grasses. Roots of B. decumbens were more susceptible to disturbed nutrients uptake and nitrogen metabolism than roots of P. maximum. Both grasses translocated high amounts of Cd to their shoots resulting in oxidative stress. Oxidative stress in the leaves of both grasses was higher in summer than winter, but only in P. maximum superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were increased.
Lysine biosynthesis has been extensively studied and the regu-was not. It would appear that the m... more Lysine biosynthesis has been extensively studied and the regu-was not. It would appear that the modulation by Ca 2 + and latory enzymes characterized in some of the most important ionic strength of LOR is a common feature among plant LOR enzymes. S-adenosylmethionine (SAM) did not produce any crop plants, however, much less is known about the lysine significant effect on either enzyme activity, indicating that it degradation pathway. Lysine 2-oxoglutarate reductase (LOR) only plays a role in the regulation of lysine biosynthesis. The and saccharopine dehydrogenase (SDH) have recently been effect of S-2-aminoethyl-L-cysteine (AEC) as both a substrate partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for further characterized these enzymes from rice endosperm in relation to Ca 2 + and ionic strength modulation. Optimum pH LOR, but was also able to inhibit LOR activity, possibly values of 7.0 and 8.0 were obtained for LOR and SDH, competing with lysine at the active site. The higher K m for respectively. The LOR domain of the polypeptide was modu-AEC compared to lysine may reflect a lower binding affinity for AEC. lated by Ca 2 + and ionic strength, whereas the SDH domain tion has been shown to be considerably elevated in the majority of plant tissues analysed, all of them have failed to exhibit over-accumulation of lysine in the seed (Azevedo et al. 1990, Diedrick et al. 1990, Heremans and Jacobs 1995). It has been suggested that in order to achieve lysine accumulation in seeds, the catabolism of lysine must be reduced (Brochetto-Braga et al. 1992, Azevedo and Lea 2000). In the opaque-2 mutant of maize, the activities of two enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8; also designated lysine h-ketoglutarate reductase [LKR]) and saccharopine dehydrogenase (SDH, EC 1.5.1.9), were shown to be strongly reduced, thus increasing the availability of lysine to be incorporated into storage proteins, or allowing more lysine to accumulate in the soluble form in the maize endosperm (Gaziola et al. 1999, Kemper et al. 1999). Falco
Theoretical and Experimental Plant Physiology, May 27, 2020
This work aimed to quantify the concentration of soluble amino acids, nitrogen (N) and Cd in the ... more This work aimed to quantify the concentration of soluble amino acids, nitrogen (N) and Cd in the developing seeds of maize plants that were grown in Cd-contaminated soil from seed sowing to the reproductive stages. The specific activities and feedback inhibition properties of lysine (Lys) metabolic enzymes were also determined in developing seeds. The potential maize yield was depressed by Cd exposure. Cadmium concentration in plant organs followed the decreasing order: roots [ stems = leaves [ developing seeds (37.04, 1.95, 1.46 and 0.22 mg kg-1 Cd, respectively). The relatively low Cd concentration in the remained developing seeds was a result from root-and earmediated reductions of the Cd translocation in plants. Plants under Cd exposure presented developing seeds with an increased N concentration (up to 7%) when compared to control plants. Furthermore, the level of soluble amino acids (particularly histidine, glycine, tyrosine, methionine, isoleucine and valine) was increased in the developing seeds of Cd-treated plants. In addition, changes in the feedback properties of dihydrodipicolinate synthase (DHDPS), an enzyme from lysine metabolism, were observed in developing seeds. In conclusion, the current study showed that maternal plant exposure to Cd can alter the concentration of soluble amino acids and the behavior of Lys metabolic enzymes in developing seeds. This study provided not only new information about the influence of the long-term Cd exposure on plants, but also data about protective plant strategies against Cd toxicity.
In general, the effects of Cadmium (Cd) on crop quality are evaluated solely by Cd accumulation i... more In general, the effects of Cadmium (Cd) on crop quality are evaluated solely by Cd accumulation in the edible plant parts; thus, the potential effects on several nutritional features are not often taken into account. This study evaluated Cd effects on the enzymatic activities of lysine (Lys) metabolism, storage protein and amino acid profile in maize. Stress parameters were also assessed. In vegetative organs, Cd was accumulated in the following decreasing order: roots > stems > leaves. Cadmium accumulation in grains decreased at grain maturity (from 0.25 to 0.07 µg g-1 DW). In leaves, neither hydrogen peroxide content nor superoxide dismutase, catalase, ascorbate peroxide and guaiacol peroxidase activities did not change in Cd-treated plants compared to control plants. Lipid peroxidation was not detected in immature grains and leaves of plants under Cd exposure, indicating that Cd accumulation mainly in the roots is a mechanism to avoid oxidative stress in aboveground parts of the plant. However, Lys metabolism in immature grains was modified, showing increases in the specific activities of aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) after Cd exposure. AK sensitivity to Lys feedback inhibition increased, but decreased in DHDPS in Cd-treated plants, suggesting differential regulation for these enzymes. In mature grains, the Lys content did not change, while the proline content increased by 54 % in Cd-treated plants. This is the first report on Cd effects on amino acid profile, storage protein contents and enzymes from Lys metabolism in grains of a cereal plant species.
The grain proteins of barley are deficient in lysine and threonine due to their low concentration... more The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise) and five antisense C-hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-hordein suppression did not only alter the metabolism of at least one aspartate-derived amino acid (threonine), but major changes were also detected in the metabolism of lysine and methionine. Modifications in the activities and regulation of aspartate kinase, dihydrodipicolinate synthase and homoserine dehydrogenase were observed in most transgenic lines. Furthermore the activities of lysine a-ketoglutarate reductase and saccharopine dehydrogenase were also altered, although the extent varied among the transgenic lines.
Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca 2+ content i... more Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca 2+ content in the distal fruit tissue. However, many other factors can also determine fruit susceptibility to BER. It is possible that during fruit growth, Ca 2+ imbalance can increase membrane leakiness, which may trigger the accumulation of reactive oxygen species, leading to cell death. Brassinosteroids are a class of plant hormones involved in stress defenses, specially increasing the activity of antioxidant enzymes and the accumulation of antioxidant compounds, such as ascorbic acid. The objective of this study was to understand the mechanisms by which 24-epibrassinolide (EBL) reduces fruit susceptibility to BER. Tomato plants 'BRS Montese' were cultivated in a greenhouse and were weekly sprayed with water (control) or EBL (0.01 µM) after full bloom. Plants and fruits were evaluated at 15 days after pollination (DAP). According to the results, EBL treatment inhibited BER development, increased fruit diameter, length, and fresh weight. EBL-treated fruit showed higher concentrations of soluble Ca 2+ and lower concentrations of cell wall-bound Ca 2+. EBL-treated fruit also had higher concentrations of ascorbic acid and lower concentrations of hydrogen peroxide, compared to water-treated fruit. EBL treatment increased the activity of the three main antioxidant enzymes known as ascorbate peroxidase, catalase, and superoxide dismutase. According to the results, EBL treatment maintained higher soluble Ca 2+ and antioxidant capacity, reducing fruit susceptibility to BER.
The activities of two aspartate kinase (EC 2.7.2.4) isoenzymes that have been partially purified ... more The activities of two aspartate kinase (EC 2.7.2.4) isoenzymes that have been partially purified from developing rice seeds, were studied in the presence of calcium, calmodulin inhibitors, S-adenosylmethionine, S-(2-aminoetyl)-L-cysteine, methionine, valine and increased salt concentrations. None of the compounds tested was able to produce any significant alteration in threonine-sensitive aspartate kinase activity. On the other hand, the activity of the lysine-sensitive aspartate kinase was slightly increased by calcium. The increase in activity was not observed when EGTA was added in combination with calcium. S-adenosylmethionine alone inhibited the activity by 12% and intensified the inhibition caused by lysine. S-(2-aminoethyl)-L-cysteine also inhibited the activity of aspartate kinase, but not to the same extent of lysine. Methionine and valine stimulated slight increases in activity, whereas KCl up to 500 mM did not cause any change in aspartate kinase activity. These results with rice aspartate kinase indicate that lysine-sensitive aspartate kinase is also synergistically inhibited by S-adenosylmethionine, as observed for other plants species. Although some increase in aspartate kinase activity was observed in the presence of calcium, the magnitude of the alterations was not sufficient to indicate a regulatory role of calcium on aspartate kinase.
Abiotic stresses negatively affect crop development and yield. However, the use of seaweed extrac... more Abiotic stresses negatively affect crop development and yield. However, the use of seaweed extracts can alleviate production losses through improvement of the antioxidant system and synthesis of compatible osmolytes. The aim of this study was to assess the effects of Ascophyllum nodosum extract on antioxidant response and yield of common bean plants (Phaseolus vulgaris) under drought stress. Therefore, two methods of application (foliar spray and soil irrigation) and two doses (5 and 10 mL L-1) were tested, whereas untreated plants were used as control. There were no significant differences in malondialdehyde and hydrogen peroxide contents among treatments, indicating that application of seaweed extract did not change the oxidative status in treated plants when compared to the control ones. On the other hand, the use of seaweed extract increased proline content even prior to drought stress (46.3 to 145.4% when compared to the control), and this increment remained until the 10 th day of stress (from 60.1 to 201.7% over the reference plants), supporting early evidences that A. nodosum extract is a plant elicitor. The yield parameters were not affected by the extract, probably due to the severe drought stress which plants were submitted. The results suggest that A. nodosum extract affects proline metabolism in Phaseolus vulgaris plants, making the synthesis of this osmolyte more responsive to drought stress because seaweed-treated plants presented a higher proline content than the control ones during the exposure to water deficit.
Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de ... more Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de postura e sua interferência na disponibilidade do ferro Protein concentration (phosvitin and lipovitelin) in egg yolks of Laying Hens (Gallus gallus) in different posture cycles and its interference in the iron availability
Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation ... more Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation of germination process, and both may result in viability loss. This study aimed to elucidate the biochemical basis of embryos survival of Araucaria angustifolia recalcitrant seeds during storage. After harvesting, seeds were stored at ambient conditions (without temperature and humidity control) and in a cold chamber (temperature of 10 ± 3 °C, and relative humidity of 45 ± 5 %). Moisture content, viability, H2O2 content, lipid peroxidation, protein content, and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), at 0, 15, 45 and 90 days of storage, were evaluated. Seed viability reduced about 40 % during the storage period accompanied by a reduction in soluble protein (about 64 % of reduction) in both storage conditions, and increased lipid peroxidation (about 115 % and 66 % for ambient and cold chamber conditions, respectively). H2O2 content used as a marker of oxidative stress was reduced during the period, possibly controlled by the action of CAT and APX, for which increased activities were observed. The results allowed the identification of seven SOD isoenzymes (one Mn-SOD, five Fe-SOD and one Cu/Zn-SOD), whose activities also increased in response to storage. Some biochemical damage resulting from storage was observed, but viability reduction was not due to failure of enzymatic protection mechanisms.
A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido a... more A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido aspártico, pela qual são também sintetizados os aminoácidos treonina, metionina e isoleucina. Além disso, a lisina é o principal aminoácido limitante em todos os cereais e por cerca de 30 anos a via do ácido aspártico tem sido estudada em plantas, com o intuito de desvendar e caracterizar os principais pontos chave na regulação das vias de biossíntese desses aminoácidos. Duas etapas distintas, uma primeira originada a partir do desenvolvimento da cultura de tecidos (anos 70-80) e a segunda a partir do desenvolvimento de técnicas para a transformação de plantas (anos 90), permitiram que mutantes bioquímicos e plantas trangênicas fossem produzidos com alterações específicas em passos metabólicos chave, levando à superprodução e acúmulo de treonina em vários tecidos das plantas. Entretanto, a acumulação de lisina em sementes não foi obtida. Tal fato, associado a estudos bioquímicos da via de degradação da lisina em cereais e em leguminosas, indicou que a manipulação da degradação seria tão ou mais importante que a manipulação da biossíntese de lisina para o acúmulo deste aminoácido em sementes dos cereais. Em milho, o uso e estudo de outros mutantes tais como o opaco-2 e variedades QPM (Quality Protein Maize) contribuíram significativamente para a compreensão dos eventos regulatórios. As estratégias para a obtenção de materiais ricos em lisina e sua relevância à manipulação de outros aminoácidos são revisados. Palavras-chave: lisina, nutrição, aminoácidos essenciais, cereais MANIPULATING CEREAL CROPS FOR HIGH LYSINE ACCUMULATION IN SEEDS ABSTRACT: The nutrition value of a protein is directly related to its amino acid composition. Some of these amino acids, termed essential amino acids, cannot be synthesized by humans and therefore must be supplied in the diet for adults and in particular for infants and children. Lysine is an essential amino acid synthesized via the aspartic acid metabolic pathway, in which threonine, methionine and isoleucine are also endproducts. Moreover, lysine is the first limiting amino acid in all cereal grains. For over 30 years, the aspartic acid metabolic pathway has been studied in higher plants with the aim of identifying and characterizing the key regulatory points controlling the biosynthetic pathway. Two clear distinct time periods, one begining with the development of tissue culture techniques (1970-80's) and the second with the development of plant transformation techniques (90's), has encouraged the production of biochemical mutants and transgenic plants with specific alterations in key enzymes of the pathway, leading to the overproduction and accumulation of threonine in all plant tissues. However, the accumulation of lysine in seeds has been particularly difficult to achieve. Such an observation, associated with the recent biochemical studies on lysine degradation in cereal and legume plant species, has indicated that the manipulation of lysine degradation is as important as the manipulation of lysine synthesis, if the goal of accumulating this amino acid in cereal seeds is to be achieved. In maize, the study and use of other mutants such as the opaque-2 and QPM (Quality Protein Maize) varieties, has contributed significantly to our understanding of the regulatory aspects of the aspartate pathway. The strategies of obtaining cereals rich in lysine and their relevance to the manipulation of other amino acids have been discussed.
Hordeins are the major storage proteins in barley grains and are responsible for their low nutrit... more Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.
Journal of Agricultural and Food Chemistry, Feb 12, 2005
Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabo... more Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabolism of lysine is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9). The final soluble lysine concentration in cereal seeds is controlled by both synthesis and catabolism rates. The production and characterization of high-lysine plants species depends on knowledge of the regulatory aspects of lysine metabolism and manipulation of the key enzymes. We have for the first time isolated, partially purified, and characterized LOR and SDH from developing sorghum seeds, which exhibited low levels of activity. LOR and SDH were only located in the endosperm and were very unstable during the isolation and purification procedures. LOR and SDH exhibited some distinct properties when compared to the enzymes isolated from other plant species, including a low salt concentration required to elute the enzymes during anion-exchange chromatography and the presence of multimeric forms with distinct molecular masses.
Tomato is an important crop worldwide. Cadmium (Cd) concentrations in fruits depend on tomato gen... more Tomato is an important crop worldwide. Cadmium (Cd) concentrations in fruits depend on tomato genotype. This work aimed to study the relation among Cd accumulation, tolerance mechanisms, and fruit features in two tomato cultivars with contrasting tolerance to Cd stress. Tolerant (Yoshimatsu) and sensitive (Tropic Two Orders) plants were grown in control and contaminated soils (0.04 and 3.77 mg/kg Cd, respectively) from the seedling stage to fruit production. Both cultivars were able to acclimatize to Cd exposure, probably through mechanisms associated with reductions in the magnesium status. Cadmium concentrations varied according to the following descending order: roots = leaf blades > (peduncle + sepals) > stem = fruits. However, the tolerant cultivar accumulated more Cd than did the sensitive one. Although Cd reached the fruits from the first to the fourth bunches, peduncle and sepals may act as a barrier to Cd entrance in tomato pulp and peel. The Cd-induced changes in the fruit mineral profile varied according to plant cultivar, organ, tomato tissue, and bunch position. Moreover, plant yield was not affected by the Cd stress, which was able to improve fruit size and weight in the tolerant cultivar. In conclusion, new insights about the Cd-induced effects on tomato development and fruit attributes were provided by growing plants in soil, which is the media generally used to cultivate this crop, rather than hydroponics. It was shown that tomato cultivars with contrasting tolerance to Cd toxicity can reach sexual maturity and produce fruits with no yield losses, despite impacts on development from long-term Cd exposure. This study also revealed the role of floral receptacle and its related structures in limiting, even partially, Cd translocation to the fruits. Furthermore, Yoshimatsu's capacity to produce bigger and heavier fruits, in plants under Cd exposure, may probably be associated with enhanced Cd accumulation.
Abstract Cadmium (Cd) is a heavy metal highly toxic to plants and human and animal health. The in... more Abstract Cadmium (Cd) is a heavy metal highly toxic to plants and human and animal health. The increased concentration of this metal in the environment in recent decades is the result of anthropogenic activities that include the production of mineral fertilizers, pesticides, intense mining, waste disposal, and foundries. Cd accumulation is known to induce oxidative stress in plant cells due to overproduction of reactive oxygen species, which may result in severe damage at the cellular level, ultimately impairing plant growth and productivity. In response to such stress, different molecules having enzymatic or nonenzymatic actions contribute to mitigate plant cellular damage and balance redox homeostasis in plant cells. This chapter aims to summarize current knowledge about the prospecting of important components of the molecular mechanisms associated with antioxidant machinery in plant response and tolerance to Cd toxicity. Insights into antioxidant response regulation, which includes hormones, signaling molecules, transcription factors, and microRNAs, are also presented.
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