Effect of growth temperature on reserve mobilization and hydrolytic enzyme activities in Vigna mungo cotyledons

Seeds of Vigna mungo were allowed to germinate at 27, 18 and15°C, and time-course changes of hydrolytic enzyme activitiesand the mobilization rate of reserve components in cotyledonswere studied. The seeds germinated at 27 and 18°C grew normally,whereas the growth at 15°C was markedly retarded. In cotyledonsof seedlings grown at 27 and 18°C, amylolytic and proteolyticenzyme activities increased at early stages of growth and therates of starch and protein mobilization changed correspondingto the hydrolytic enzyme activities. At 15°C the enzymeactivities increased gradually during the experimental periodof 16 days, but the reserves in cotyledons remained almost unchangeduntil the end of the experimental period. Changes of zymogram patterns of amylolytic and proteolytic activitiesin cotyledons of seedlings grownat 27, 18 and 15°C wereexamined using polyacrylamide gel electrophoresis. The intensitiesof a main band of a-amylase and at least two bands of protease(gelatin-hydrolyzing activity) increased concurrently with invitro activities of amylolytic and proteolytic enzymes. At leastthree bands of starch phosphorylase were present in cotyledonsat early stages of germination and their intensities decreasedduring the growth of seedlings at 27, 18 and 15°C. (Received June 4, 1980; )     

Histochemical studies on mobilization of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark at 27 C, and time-sequence changes of mobilization of protein and starch reserves in cotyledons were observed by histochemical techniques. The distributions of amylase and protease activities in cotyledon sections were also examined during germination by use of the starch-polyacrylamide gel film and India ink-gelatin film methods, respectively. Amylolytic and proteolytic processes occurred more or less simultaneously during the germination. At the day 2 stage, low levels of hydrolytic enzyme activities were observed throughout cotyledon sections. At day 4, both amylase and protease activities appeared to increase in tissue areas farthest from vascular bundles, and the mobilization of starch and protein reserves also proceeded in these areas. At day 6, the reserves were found to remain only in the cells around vascular bundles. When cotyledons were detached from axis organs, allowed to imbibe water and incubated for 4 days at 27 C, the breakdown of reserves was markedly retarded and the patterns of enzyme localization in cotyledon sections appeared not as conspicuous as those in the sections from intact cotyledons. These histochemical results are discussed with reference to the previous results ofin vitro experiments.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Characteristics of hydrolase biosynthesis in Bacillus mesentericus grown on various media

The dynamics of the consumption of major carbon and nitrogen sources and the biosynthesis of hydrolytic enzymes were studied in Bacillus mesentericus grown on semisynthetic media. Conditions were chosen that provide the obtaining of the culture liquid with predominantly proteolytic or amylolytic activity. The replacement of maltose with native starch resulted in more intensive accumulation of the biomass and hydrolytic enzymes, and in more rapid (by 3-5 hr) transformation from the logarithmic to the stationary growth phase.     

The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: II. THE EFFECTS OF 6-BENZY LADENINE AND GIBBERELLIC ACID UPON EMBRYONATED AND DETACHED COTYLEDONS

The presence of the embryo axis promotes starch hydrolysis incotyledons of Phaseolus vulgaris and, although reduced ratesof enzyme activity proceed in its absence, weight loss fromthe cotyledon is only significant in its presence. Applicationof gibberellic acid at various concentrations had no effectupon amylase or protease activity in either detached or embryonatedcotyledons: this was not the case for 6-benzyladenine whichpromoted both. In detached cotyledons amylolytic activity wasdirectly proportional to the concentration of applied 6-BA;concentrations below 10^–6 M were unable to completelysubstitute for the presence of the axis, while, above 10^–6M, hydrolytic activity higher than that of treated embryonatedcotyledons was observed. Increasing the concentration of 6-BAhad no effect, however, upon the amylolytic activity of embryonatedcotyledons. Detached cotyledons showed an increased chlorophyllcontent and 6-BA treatment further increased this; the proportionsof chlorophyll a and chlorophyll b remained unaltered. An inhibitor of amylase activity, destroyed by heating, is presentin embryonated cotyledons and can depress amylolysis in detachedcotyledons which appear to be inhibitor-free. The results intotal suggest that the developing axis may regulate reservehydrolysis in three distinct manners: firstly, by stimulatingthe synthesis of amylase via hormonal control, most probablya cytokinin; secondly, by checking the rate of amylolysis bymeans of an amylase inhibitor; and, thirdly, by being a sinkfor the products of reserve degradation.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Impact of polychlorinated biphenyls on the activity of intestinal proteinases and carbohydrases in juvenile roach Rutilus rutilus (L.)

The chronic effects that polychlorinated biphenyls (PCBs) have on the activities of proteinases and carbohydrates in intestinal mucosa and chyme in juvenile roach Rutilus rutilus (L.) have been studied for the first time. Upon consuming food with PCB 50.8 ng/g wet weight for 218 days, the exposition of fingerlings in aquariums with dirt bottoms (contents PCB 425.6 ng/g dry weight) reduces the proteolytic activity of mucosa to a greater degree than amylolytic activity; the activity of sucrase changes in different directions. In 2-year-old fish receiving PCB only with food (50.8 ng/g wet weight bottom), proteolytic and amylolytic activities of the mucosa are reduced 18 and 35% in comparison with the control (52 days); the sucrose activity is reduced 13% (52 days) and 22% (169 days). In most cases the relation of amylolytic activity to proteolytic activity in fish of the experimental groups is less than in the control. The enzyme activities in chyme change in different directions, depending on the terms and conditions of the experiment.     

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

Subcellular localization of the starch-degrading enzymes in Vicia faba leaves was achieved by an electrophoretic transfer method through a starch-containing gel (SCG) and enzyme activity measurements. Total amylolytic and phosphorolytic activities were found predominantly in the extrachloroplastic fraction, whereas the debranching enzymes showed homogenous distribution between stromal and extrachloroplastic fractions. Staining of end products in the SCG revealed two isoforms of [alpha]-amylase (EC 3.2.1.1) and very low [beta]-amylase activity (EC 3.2.1.2) in the chloroplast preparation, whereas [alpha]- and [beta]-amylase exhibited higher activities in the crude extract. However, it is unclear whether the low [alpha]- and [beta]-amylase activities associated with the chloroplast are contamination or activities that are integrally associated with the chloroplast. Study of the diurnal fluctuation of the starch content and of the amylase activities under a 9-h/15-h photoperiod showed a 2-fold increase of the total amylolytic activity in the chloroplasts concurrent with the starch degradation in the dark. No fluctuation was detectable for the extrachloroplastic enzymes. The possible roles and function of the chloroplastic and extrachloroplastic hydrolytic enzymes are discussed.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Control of the Breakdown of Food Reserves in Germinating Dicotyledonous Seeds -- a Reassessment

The correct interpretation of experimental data concerning theeffect of the embryonic axis upon food reserve mobilizationin dicotyledonous seeds depends on an awareness of the preciseincubation conditions used for treating the seeds. Under optimalincubation conditions excised cotyledons can develop hydrolyticactivities which are comparable to those produced in the intactsystem. A reasonable hypothesis to explain this suggests thatsuch hydrolytic enzyme activities automatically increase withincotyledons during germination and early seedling growth andare regulated by feed-back effects mediated by reserve breakdownproducts. If this conjecture is correct then the requirementfor an axis-derived hormonal influence on cotyledonary metabolismis not mandatory. Germination of seeds, hydrolytic activity, feed-back control, cytokinins     

Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

We previously reported on Vicia narbonensis seeds with largely decreased alpha- D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33-43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phospho enolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.     

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     

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5–3 M NaCl. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase