Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Regulation by sucrose of storage compounds breakdown in germinating seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus albus L.) and Andean lupine (Lupinus mutabilis Sweet). II. Mobilization of storage lipid

Research of the regulatory function of sucrose in storage lipid breakdown was conducted on isolated embryo axes, excised cotyledons and whole seedlings of three lupine species grown in vitro on medium with 60 mM sucrose or without the sugar. Lack of sucrose in the medium caused significant increase in total lipid content in yellow, white and Andean lupine isolated embryo axes but in Andean lupine seedling cotyledons and excised cotyledons, lipid level was clearly lower in carbohydrates deficient conditions. Sucrose caused no significant effect on fatty acids spectra. The main fatty acid in yellow lupine seeds was linoleic acid, in white lupine oleic acid and in Andean lupine both oleic and linoleic acids. The main phospholipid in organs of three lupine species was phosphatidylcholine. In sugar-deficient conditions, content of phosphatidylcholine and some others phospholipids was decreased. The peculiar features of regulation by sugars of storage lipid breakdown in germinating lupine seeds and induction of autophagy in young carbohydrate starved embryo axes is discussed.     

Gibberellin-like activity in cotyledons and embryonic axes during pea seed germination

Endogenous gibberellin-like activity was determined in dry pea seeds (Pisum sativum cv. Bördi), in cotyledons and axes of germinating pea seeds and also in excised cotyledons and axes. During the first two days of pea seed germination, neither the embryonic axes nor the cotyledons show a mutual influence on gibberellin activity, but this appears after 72–96 h of germination. The gibberellin-like activity m cotyledons and axes of germinating seeds increased during the same period, but it decreased in isolated axes and excised cotyledons.     

The Biosynthesis of Cytokinins in Germinating Lupin Seeds

Imbibed intact seeds, and excised embryos and cotyledons ofyellow lupin (Lupinus luteus L. cv. Weiko III) have been incubatedwith [¹⁴C]-adenine to investigate cytokinin biosynthesis duringthe early stages of germination. Following incubation the tissueswere extracted and purified by solvent partition and chromatographyon cellulose phosphate, diethylaminoethyl cellulose and SephadexLH-20 columns. Using a variety of thin layer chromatographic,high performance liquid chromato-graphic and chemical procedures,incorporation of ¹⁴C into dihydrozeatin riboside and its nucleotidewas demonstrated in extracts of intact embryos, intact cotyledonsand excised embryos. However, radioactivity was not found associatedwith cytokinins in fractions derived from the isolated cotyledons.This is the first direct demonstration of cytokinin biosynthesisin germinating seeds and the results indicate that the capacityfor cytokinin biosynthesis is probably confined to the embryonicaxes. If this is so, the levels of [¹⁴CJ-dihydrozeatin ribosideassociated with intact embryo and intact cotyledon fractionsindicate that the synthesized cytokinin is transported to andaccumulates in the cotyledons. Key words: Lupinus luteus, cytokinin biosynthesis, seed germination     

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.     

Regulation by sucrose of storage compounds breakdown in germinating seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus albus L.) and Andean lupine (Lupinus mutabilis Sweet): I. Mobilization of storage protein

Research of the regulatory function of sucrose in storage protein breakdown was conducted on isolated embryo axes, excised cotyledons and whole seedlings of three lupine species grown in vitro on medium with 60 mM sucrose or without the sugar. Sucrose stimulated growth of yellow, white and Andean lupine isolated embryo axes and cotyledons but growth of seedlings was inhibited. Dry matter content was higher in sucrose-fed isolated organs and in seedling organs. Ultrastructure research revealed that lack of sucrose in the medium caused enhancement in storage protein breakdown. Protein deposits in cotyledons were smaller as well as soluble portion content in all studied organs was lower when there was no sucrose in the medium. In the same conditions, the activity of glutamate dehydrogenase was significantly higher. Increase in vacuolization of cells of white lupine root meristematic zone cells was observed and induction of autophagy in young carbohydrate-starved embryo axes is discussed.     

Lysyl tRNAs of lupinus luteus seeds

Lysine accepting transfer RNA of lupin seeds and lupin embryo axes can be fractionated into at least 5 species by reversed-phase chromatography (RPC-5). One main and two minor isoacceptors were observed in wheat and barley embryos. Changes in isoaccepting species of tRNA^1ys were followed in cotyledons of germinating lupin seedlings. Ribosome binding studies revealed that one of the main lupin tRNA^1ys species recognizes the AAG codon, the second AAA and the third one AAA and AAG.     

The mobilization of defence mechanisms in the early stages of pea seed germination against Ascochyta pisi

Ascochyta pisi is a necrotrophic pathogenic fungus, which mainly survives between seasons through infected seeds. Defence responses of pea embryo axes to A. pisi were investigated in the heterotrophic phase of seed germination and during the transition from the heterotrophic to the autotrophic phase. Germinated pea seeds, both non-inoculated and inoculated with A. pisi, were cultured in perlite for 96 h. Polarographic studies performed on intact embryo axes of germinating pea seeds infected with A. pisi showed a high respiratory intensity in time from 48 to 96 h after inoculation. Forty-eight-hour embryo axes of germinating pea seeds exhibited the highest respiration rate, which in infected axes was maintained at the following time points after inoculation. Moreover, at 72 and 96 h after inoculation, respiratory intensity was by 64% and 73% higher than in the control. Electron paramagnetic resonance analysis revealed a higher concentration of semiquinone free radicals with g values of g _||?=?2.0031?±?0.0004 and g _⊥?=?2.0048?±?0.0004 in infected axes than in the control. Generation of superoxide anion radical was also higher in infected axes than in the control but stronger at 72 and 96 h after inoculation. Starting from 72 h after infection, the level of Mn²⁺ ions in infected axes decreased in relation to the control. At the same time, the highest activity of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) was observed in 72-h infected axes. In turn, the activity of peroxidase (EC 1.11.1.7) up to 72 h after infection was lower than in the control. In 48-h infected embryo axes, a very high level of pterocarpan pisatin was observed. Infection of germinating pea seeds with A. pisi restricted mainly the growth of the epicotyl, but did not inhibit the increase in length and fresh weight of root embryo axes versus cultivation time. These results indicate that in pea during the stages of seed germination and early seedling growth, protective mechanisms are induced in embryo axes against A. pisi.     

Lupine embryo axes under salinity stress. I. Ultrastructural response

Embryonic root is the primary site of salinity perception in germinating seeds. To understand better the NaCl stress response of lupine embryo axes, ultrastructural approach combined with analysis of DNA degradation was used. In this study lupine embryo axes were cultured in vitro on the medium supplemented with two salt concentrations 250 and 500 mM to differ the reaction. To assess the rate of DNA damage, alkaline electrophoresis of isolated nuclei and DNA fragmentation analysis were performed. Results of these studies suggest programmed cell death induction under salinity stress. Moreover, ultrastructure observations revealed other characteristic features of programmed cell death like endoplasmic reticulum reorganization, increased level of vacuolization, chromatin condensation and starch grains degradation. Our comparative analysis of ultrastructure changes and DNA fragmentation speak in favour of programmed cell death in lupine (Lupinus luteus L. ‘Mister’) embryo axes treated for 12 h with 250 and 500 mM NaCl.     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.     

Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes

Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate.     

Comparative study of storage compound breakdown in germinating seeds of three lupine species

Storage lipid and protein breakdown in germinating seeds of yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupine (L. mutabilis Sweet) and regulatory function of sucrose were investigated. Less oil bodies were detected in organs of yellow lupine seeds, whereas the highest content of oil bodies was noticed in the Andean lupine seeds. Mature, air-dried yellow, white and Andean lupine seeds do not contain starch. Starch grains appear the earliest in white lupine seeds during imbibition. Sucrose deficiency in tissues enhances breakdown of storage lipid, protein and temporary starch in cotyledons. In sucrose starved embryo axes of all investigated lupine species, an increased level of vacuolization was noted. Interconnections between catabolism of storage protein and storage lipid in germinating lupine seeds were identified by applying ¹⁴C-acetate. To assess the importance of key processes in storage lipid breakdown NaF (inhibitor of glycolysis and gluconeogenesis), KCN, NaN₃ and SHAM (inhibitors of mitochondrial electron transport chain) and MSO (inhibitor of glutamine synthetase) were used. Radioactivity coming from ¹⁴C-acetate was released as ¹⁴CO₂ but mostly was incorporated into ethanol-soluble fraction of embryo axes and cotyledons. Respiratory inhibitors caused a significant decrease in ¹⁴CO₂ and ethanol fractions in all three lupine species studied. MSO stimulated release of ¹⁴CO₂ and radioactivity of ethanol fractions in yellow lupine organs fed with sucrose, but in Andean lupine MSO enhanced the production of ¹⁴CO₂ and radioactivity of ethanol fractions both in organs fed and not fed with sucrose. Different strategies of storage compound breakdown are proposed, depending on relative proportion in storage protein and lipid content in lupine seeds.     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Regulation of storage lipid metabolism in developing and germinating lupin (<Emphasis Type="Italic">Lupinus</Emphasis> spp.) seeds

The main storage compound in lupin seeds is protein, whose content can reach up to 45–50 % of dry matter. However, seeds of some lupin species can also contain quite a large amount of storage lipid. The range of lipid content in lupin seeds is from about 6 to about 20 % of dry matter. Storage lipid in developing seeds is synthesized mainly from sugars delivered by mother plants. During seed germination, one of the main end-products of storage lipid breakdown is also sugars. Thus, the sugar level in tissues is considered an important regulatory agent, during both lipid accumulation and lipid breakdown. Generally, in developing legume seeds, there is a strong negative relation between accumulation of storage protein and storage lipid. Results obtained in developing lupin cotyledons cultured in vitro pointed to the possibility of a positive relation between protein and lipid accumulation. Such a positive effect could be caused by nitrate. During lupin seed germination and seedling development, the utilization of storage lipid is enhanced under sugar deficiency conditions in tissues and is controlled at the gene expression level. However, under sugar starvation conditions, autophagy is significantly enhanced, and it can cause disturbances in storage lipid breakdown. The hypothesis of pexophagy, i.e., autophagic degradation of peroxisomes under sugar starvation conditions during lupin seed germination, has been taken into consideration. The flow of lipid-derived carbon skeletons to amino acids was discovered in germinating lupin seeds, and this process is clearly more intense in sucrose-fed embryo axes. At least four alternative or mutually complementary pathways of carbon flow from storage lipid to amino acids in germinating lupin seeds are postulated. The different strategies of storage compound breakdown during lupin seed germination are also discussed.     

Redistribution of Nutrient Elements from Cotyledons of Two Species of Annual Legumes During Germination and Seedling Growth

The distribution of dry matter and various mineral nutrientsbetween testa and embryo of seeds of Lupinus albus and L. angustifoliusis described It was found that lupin seeds at either end ofa pod contained less dry matter and minerals than seeds in themiddle of the fruit. The transport of dry matter, N, P, K, S,Ca, Mg, Na, Fe, Zn, Mn and Cu from cotyledons of parent seedsof both species to the seedling axis was measured from germinationto the time of cotyledon death. N, P, K and S were retrievedfrom cotyledons with over 90 per cent efficiency, dry matter,Mg, Na, Fe, Zn, Mn and with 59–90 per cent efficiency,and Ca with 26–31 per cent efficiency. There was littlechange in the efficiency of nutrient retrieval from cotyledonswhen seedlings were grown in different culture media. Both speciesshowed a linear relationship between the loss of each elementand dry matter throughout the experiment, and a similar proportioningbetween root and shoot of the amount of a specific nutrientmobilized from cotyledons of parent seeds. Lupinus albus L., Lupinus angustifolius L., lupin, transport, of dry matter and mineral nutrients     

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60 mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP⁺-dependent (EC 1.1.1.42) and NAD⁺-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.     

The influence of the embryonic axis and cytokinins on reserve mobilization in germinating lupin seeds

The influence of the embryonic axis and cytokinins (CKs) onreserve mobilization has been examined in yellow lupin (Lupinusluteus L. cv. JSG 6167) seed during germination and during earlygrowth for up to 9 d in the dark. The study included determinationof starch, soluble sugars, proteins, and amino acid content.Amylolytic and proteolytic enzyme activity was also measuredin untreated cotyledons with intact embryo (attached) or detachedcotyledons (embryo removed), and in detached cotyledons followingtreatment with CKs namely, dihydrozeatin, (diH)Z, and 6-benzylaminopurine,BAP. Generally, the detached cotyledons showed reduced mobilizationand decreased enzymatic activity in comparison to attached cotyledons,indicating the importance of the embryonic axis in this process.However, a rise in protease activity and free amino acid contentwas detected in 9-d-old detached cotyledons suggesting thatthe end products do not necessarily inhibit enzyme activity.While (diH)Z was partially effective in inducing reserve mobilizationand enzymatic activity in detached cotyledons, the effect ofBAP was more pronounced and appeared to replace the embryonicaxis. The embryonic axis of this species has recently been shownto synthesize CKs which are transported to the cotyledons, arehighly stabe and induce cotyledon expansion and chlorophyllsynthesis. The results of the present investigation and previousstudies from this laboratory collectively indicate that theregulation of reserve mobilization in yellow lupin seeds appearsto be mediated, at least in part, by a stimulus, probably aCK, emanating from the embryonic axis. Key words: Lupinus luteus, cytokinins, benzylaminopurine, dihydrozeatin, embryonic axis, lupin seeds, reserve mobilization     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

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.