Wiktoria Ratajczak

Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.

A transfer of carbon atoms from fatty acids to sugars and amino acids in yellow lupine (Lupinus luteus L.) seedlings.

The metabolism of 14C-acetate was investigated during the in vitro germination of yellow lupine seeds. Carbon atoms (14C) from the C-2 position of acetate were incorporated mainly into amino acids: aspartate, glutamate, and glutamine and into sugars: glucose, sucrose, and fructose. In contrast to this, 14C from the C-1 position of acetate was released mainly as 14CO2. Incorporation of 1-14C and 2-14C from acetate into amino acids and sugars in seedling axes was more intense when sucrose was added to the medium. However, in cotyledons where lipids are converted to carbohydrates, this process was inhibited by exogenous sucrose. Since acetate is the product of fatty acid beta-oxidation, our results indicate that, at least in lupine, seed storage lipids can be converted not only to sucrose, but mainly to amino acids. Inhibitory effects of sucrose on the incorporation of 14C from acetate into amino acids and sugars in cotyledons of lupine seedlings may be explained as the effect of regulation of the glyoxylate cycle by sugars.

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.

The involvement of glutamate dehydrogenase in the adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos

Embryos of pea (Pisum sativum L. cv Sol) deprived of cotyledons were cultured for 3 days in medium with or without sucrose. Respiratory activity of embryos (intact) as well as the ability to oxidize glutamate by mitochondria isolated from embryos were studied. Respiration of intact embryos grown in sucrose supplemented medium was more intensive than in the starved ones. Transfer of the starved embryos to the sucrose-containing medium induced the increase in the intensity of O2 consumption. Mitochondria isolated from both starved and control embryos exhibited respiratory control. Mitochondria isolated from embryos cultured in the absence of sucrose showed higher (about 60 %) ability to oxidize glutamate and α-ketoglutarate than mitochondria from embryos grown in sucrose containing medium. The absence of sucrose in the medium led to a rapid increase in the specific activity of glutamate dehydrogenase (NADH-GDH and NAD-GDH) and it was accompanied by changes in izoenzymatic pattern of enzyme. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase may be responsible for the increase of glutamate oxidation by mitochondria of pea embryos. Electrophoretic separation of glutamate dehydrogenase isolated from embryos cultured in medium without sucrose showed the presence of ca. 17 isoenzymes while in non-starved embryos only 7 isoenzymes were identified. However, the addition of sucrose to starved embryos after 24 hours of cultivation led to a decrease in glutamate dehydrogenase activity (up to 40 %) but it did not cause the changes in isoenzymatic pattern. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase maybe responsible for the increase of glutamate oxidation by mitochondria of pea embryos. The posibility of glutamate dehydrogenase regulation by sucrose is discussed.

Sugars as a metabolic regulator of storage protein mobilization in germinating seeds of yellow lupine (Lupinus luteus L.)

The intensity of protein reserve activation in yellow lupine (Lupinus luteus L.) organs cultured in vitro in the presence of saccharose and without sugar in the medium was studied. Isolated embryo axes, excised cotyledons and seeds deprived of their testae were grown on Heller medium: a) with 60 mM saccharose (+S), b) without sugar (−S) and c) for 72 hours without saccharose + for 24 hours in the presence of saccharose (−S→+S). Using nitroanilide substrates, exo- and endopeptidase proteolytic activity was investigated in enzymatic extracts. Proteolytic activity was examined in organs isolated from swollen seeds and also in organs cultured for 24, 48, 72 and 96 hours. The proteolytic activity was higher in organs cultured on medium without saccharose. Stimulation of the proteolytic activity on the first day of culture was not significant, but intensified in the successive days of culture. The organ subcultured onto a medium with saccharose (−S→+S) caused a significant limitation of proteolytic activity, even to a markedly lower level in comparison to that activity level in the material continuously supplemented with saccharose. Observations of ultrastructure in Transmission Electron Microscopy revealed increased protein body degradation in the absence of saccharose in the medium and an increased degree of cell vacuolization, which may be indicative of intensifying autophagic processes under conditions of carbohydrate deficit.

Variability of globulin composition in cultivars and individually tested seeds of yellow lupin (Lupinus luteus L.)

A study on globulins, major storage proteins in yellow lupin seeds, called conglutins, was conducted using SDS polyacrylamide gel electrophoresis. In this paper, an extensive and not yet published list of yellow lupin conglutins is presented. The patterns of subunits of major conglutins in seeds of three yellow lupin cultivars were similar to each other, varying only in the level of some polypeptides. Investigations of seeds of cultivar Parys showed considerable quantitative differences in major subunits. Some minor subunits occurred only in some seeds and were absent in the others. Great differences were shown between single individuals in the amount of subunits of conglutin which is of the most nutritional value due to high content of methionine.

Metabolism of amino acids in germinating yellow lupin seeds. II. Pathway of conversion of aspartate to alanine during the imbibition

The incorporation of 14C-aspartate during the imbibition of yellow lupin seeds resulted in the production of 14C-alanine and 14CO2. On the basis of tracer and enzymatic assays, conducted in vitro on the extract obtained from lupin seeds, it is postulated that aspartate can be converted to oxaloacetate, then, by phosphoenolopyruvate and pyruvate to alanine. This pathway can be catalyzed by the following enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine aminotransferase.

Changes in the activity of phosphoenolpyruvate carboxylating enzymes in germinating yellow lupin seeds

The rate of phosphoenolpyruvate carboxylation by extracts from germinating lupin seeds was measured through the H14CO3 fixation. PEP carboxylation in seed axes increased during their imbibition, mainly as a result of the increase in the activity of PEP carboxylase [EC 4.1.1.31]. However, the activity of PEP carboxykinase [EC 4.1.1.38], present during the first 3 hours of imbibition, as well as the activity of PEP-carboxykinase [EC 4.1.1.49], after 24 hours of imbibition, have also been shown. Possible physiological role of the changes in the activity of PEP carboxylases during lupin seeds germination is discussed.