Distribution of Physiological Activities in the Cotyledons of Germinating Seeds

From autoradiographical experiments on water-imbibing pea (Pisum sativum) or Phaseolus mungo cotyledons supplied with glucose-¹⁴C or iodoacetic acid-¹⁴C, it was shown that the compounds did not penetrate into the innermost part of the cotyledons but remained in the peripheral region during the early germination period. Changes in the rates of incorporation of ¹⁴C into alcohol from glucose-¹⁴C were examined during the early period of germination, and it was concluded that glucose supplied exo-genously was actively metabolized in the peripheral region. Endogenous alcohol formation was supposed to occur mainly in the peripheral region in the early phases of germination. On the basis of these results, the heterogeneity of the distribution of physiological activities in the cotyledon is discussed.     

Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: α-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.     

Activities of Hydrogen Peroxide-Scavenging Enzymes in Germinating Wheat Seeds

During imbibition and germination of wheat (Triticum aestivum)in the dark over 72 h, activities of the enzymes of the ascorbate(AsA)-dependent H₂O₂-scavenging pathway, AsA peroxidase, monodehydroascorbate(MDAsA) reductase, dehydroascorbate (DHAsA) reductase and glutathione(GSSG) reductase as well as superoxide dismutase (SOD), catalaseand guaiacol peroxidase were determined both in whole grainsand in isolated embryos and endosperm. With the exception of DHAsA reductase, activities of the otherenzymes assayed increased in germinating seeds, especially duringradicle emergence (between 24–48 h of imbibition). Theseincreases, particularly for AsA peroxidase, were much higherin the embryo than in the endosperm. Within 72 h of imbibition,activities per seed increased 116-fold for AsA peroxidase, 19-foldfor guaiacol peroxidase, 5-fold for catalase and only 1·4-foldfor SOD. In contrast to the decreases in DHAsA reductase, theother AsA recycling enzyme, MDAsA reductase, increased 5-foldwithin 72 h. The results indicate that, in wheat seeds, imbibition and germinationis associated with enhanced cellular capacity to detoxify H₂O₂.For this detoxification the operation of AsA peroxidase togetherwith the AsA-regenerating enzymes appears to be of particularimportance. Key words: Ascorbate peroxidase, germination, hydrogen peroxide detoxification, inhibition, wheat     

Starch Degradation Metabolism towards Sucrose Synthesis in Germinating Araucaria araucana Seeds

As starch is the main seed reserve material in both species of Araucaria of South America, A. araucana and A. angustifolia, it is important to understand starch breakdown in both embryo and megagametophyte tissues of Araucaria seeds. Sugar analysis by thin layer chromatography indicates that sucrose is the main sugar produced in both tissues. Enzyme reactions coupled to benzidine oxidation indicate that sucrose is the main sugar moved from the megagametophyte to the growing regions of the embryo via the cotyledons.

Phosphorylase was detected in both embryo and megagametophyte tissues by the formation of [³²P]glucose-1-P and by formation of [¹⁴C] amylopectin from [¹⁴C]glucose-1-P. The enzyme activity increases 5-fold in both embryo and gametophyte to a peak 18 hours after the start of imbibition. Debranching enzyme, α-glucosidase, and hexokinase are also present in both embryonic and megagametophytic tissues.

Branched glucan oligosaccharides accumulate during this time, reaching a maximum 40 hours after imbibition starts, and decline after germination occurs.

The pattern of activity of the enzymes studied in this work suggests that starch degradation is initiated by α-amylase and phosphorylase in the embryo and by phosphorylase mainly in the megagametophyte. Sucrose-P synthase seems to be the enzyme responsible for sucrose synthesis in both tissues.

     

Starch Deposition and Carbohydrase Activities in Developing and Germinating Soya Bean Seeds

Immature soya bean seeds accumulate starch as a transient reservematerial which is utilized later in development. Germinatingseeds also accumulate starch reserves, probably as a resultof gluconeogenesis from storage lipid. Developing beans showa rapid increase in ß-amylase activity which continuesinto early germination before declining. Distribution of ß-amylaseactivity is not consistent with its supposed role in starchdegradation. Soya bean seeds also contain -amylase and -glucosidaseactivities which could be responsible for starch mobilization. Glycine max (L.) Merr., soya bean, starch, carbohydrase, amylase, -glucosidase     

Development of Pyrimidine-metabolizing Enzymes in Cotyledons of Germinating Peas

Mechanisms controlling conversion of orotic acid-6-¹⁴C to uridine-5′-phosphate in cotyledons of germinating Alaska peas (Pisum sativum L.) were investigated. The content of 5-phosphoribosyl-1-pyrophosphate was very low in dry seeds, increased to a maximum after about 12 hours of imbibition, and then rapidly declined. Orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase activities more than doubled during the first 24 hours of germination and then also decreased. These results do not account for the continuous increases of orotate anabolism in such cotyledons as we observed previously. The initial increases in activities of these two enzymes were unaffected by cycloheximide, while the subsequent decreases were less rapid in the presence of this inhibitor. Activities of cotyledonary cytidine deaminase and uridine hydrolase also increased during imbibition, but the activity of only the latter showed a decrease after imbibition was completed. Cycloheximide inhibited the initial rapid increase in uridine hydrolase activity but had little effect on its subsequent decline. Cycloheximide had only slight inhibitory effects on the development of cytidine deaminase activity during the first 62 hours. The evidence suggests that uridine hydrolase might be synthesized de novo during the first few days of germination, but that the other three enzymes might not be.     

Acid Phosphatase in Cotyledons of Germinating Vigna mungo Seeds

A marked increase in acid phosphatase activity took place incotyledons of germinating Vigna mungo seeds. The attachmentof axis organs was not required for this development of enzymeactivity in cotyledons. DEAE-cellulose column chromatographyrevealed that the phosphatase is composed of at least threeforms. (Received August 19, 1981; Accepted October 30, 1981)     

Properties of Membrane-bound Protease in the Cotyledons of Germinating Pea Seeds

Teasterone 3-myristate as a new type of brassinosteroid derivative was found in lily anthers. Esterification only occurs with the hydroxyl group at C3. There is no endogenous esterified derivative of typhasterol, castasterone, or brassinolide in the lily tissues.     

Enzymic Degradation of Starch Granules in the Cotyledons of Germinating Peas

Starch, total alpha- and beta-amylase, and phosphorylase levels and the zymogram patterns of these 3 starch-degrading enzymes were determined in the cotyledons of smooth pea (Pisum sativum L.) during the first 15 days of germination. Starch is degraded slowly in the first 6 days; during this time, alpha-amylase is very low, beta-amylase is present at a constant level while phosphorylase gradually increases and reaches a peak on the fifth day. Beginning on the sixth day there is a more rapid degradation of starch which coincides with alpha-amylase production. One phosphorylase band and 2 beta-amylase bands are present in the zymogram of the imbibed cotyledon. An additional phosphorylase band and 1 alpha-amylase band appear during germination. Seeds imbibed in benzyladenine, chloramphenicol, and in cycloheximide show retarded growth and slower starch degradation and enzyme production than the controls. We conclude that alpha-amylase is the major enzyme involved in the initial degradation of starch into more soluble forms while phosphorylase and beta-amylase assist in the further conversion to free sugars.     

Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase,3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purifiedfrom glyoxysomes of germinating pumpkin cotyledons. Molecularweights of their subunits were as follows: malate synthase,60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase,72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoAdehydrogenase activities were exclusively localized in glyoxysomes,whereas malate dehydrogenase and citrate synthase activitieswere found in both glyoxysomes and mitochondria. Monospecificantibodies against malate dehydrogenase and citrate synthaseinhibited their activities present in glyoxysomes but in mitochondria.Immunocytochemical analysis using the protein A-gold techniquecombined with Lowicryl K4M embedding showed that the antigenicsites for these enzymes were found exclusively in glyoxysomes.These data indicates that malate dehydrogenase and citrate synthasepresent in glyoxysomes are immunologically different from thosein mitochondria, respectively. ¹ This is paper No. 9 in the series "Analytical Studies on MicrobodyTransition". ³ Present address: Meiji Institute of Health Science, Naruta,Odawara, Kanagawa 250, Japan. ⁵ Present address: Department of Biology, Faculty of Science,Kobe University, Rokkoudai, Nada, Kobe 657, Japan. (Received December 23, 1987; Accepted January 27, 1988)     

Synthesis of DNA and the Development of Amylase and Phosphatase Activities in Cotyledons of Germinating Seeds of Vaccaria pyramidata

As in cotyledons of Agrostemma githago, synthesis of DNA takesplace after germination in cotyledons of Vaccaria pyramidataand is followed by the formation of hydrolases, in particular,-amylase and acid phosphatase. If DNA synthesis is inhibitedby hydroxyurea, no, or only slight, enzyme activity develops.The possible role of this DNA synthesis is discussed. Key words: DNA synthesis, amylase activity, phosphatase activity, seed germination, cotyledons, Vaccaria pyramidata     

Regulation of De-N-Glycosylation Enzymes in Germinating Radish Seeds

The activities of the de-N-glycosylation enzymes endo-N-acetyl- [beta]-D-glucosaminidase (ENGase; EC 3.2.1.96) and peptide-N4- (N-acetyl-[beta]-D-glucosaminyl) asparagine amidase (PNGase; EC 3.5.1.52) were monitored during germination and postgerminative development in radish (Raphanus sativus L. cv Flamboyant). The ENGase activity was detected only during postgermination, whereas the PNGase activity was present at high levels in both stages. When germination was inhibited with abscisic acid or cycloheximide, PNGase activity was detected at a basic level and ENGase activity was not detected at all. PNGase is present as an active protein in dry seeds and is apparently synthesized during seed formation. Conversely, the absence of ENGase in dry seeds suggests that its activity is dependent on the protein synthesis that occurs during and after germination. Treatment with gibberellic acid confirmed the production of both de-N-glycosylation enzymes after germination, and demonstrated a temporal delay between the production of the two enzymes during this period. Our results suggest that the two de-N-glycosylation enzymes are differentially regulated during plant development.     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

Metabolic Changes in Axes of Germinating Vigna unguiculata Seeds as Related to Effects of Removal of Cotyledons

[¹⁴C]-Labeled amino acids and sucrose were fed to Vigna unguiculataseeds through cut-ends of cotyledons, and incorporations ofradioactivity into trichloroacetic acid- and 80% ethanol-insolublefractions of axes, respectively, were followed during 48 h ofthe post-imbibition development. The results of these studies,together with determinations of changes in dry weight and proteincontents after the onset of imbibition, indicated that the reservematerials stored in cotyledons were available for active growthof axes only after 12 h of post-imbibition. However, pulse-labelingexperiments, where [³H]-labeled leucine and uridine were feddirectly to axes attached to or detached from cotyledons, indicatedthat synthesis of protein and RNA in both axes was very pronouncedeven at earlier stages (2–8 h) of post-imbibition. Albuminand globulin proteins of axes disappeared most rapidly duringthe 6–12 h period of post-imbibition. Cycloheximide, -amanitinand cordycepin added to imbibing axes inhibited the degradationof major globulin proteins, whereas the inhibitors had littleeffect on the degradation of major albumin proteins. Both proteolyticand amylolytic activities were found to occur in embryonic axesof ‘dry’ seeds, and increased to higher levels asthe germination proceeded. Axes at early stages of germinationmay degrade the self-sustained reserve proteins and utilizethem for the synthesis of new proteins. (Received June 11, 1983; Accepted August 16, 1983)     

Salt Stress Induced Changes in Growth and Enzyme Activities in Germinating Phaseolus Mungo Seeds

NaCl salt stress induced changes in growth and enzyme activities in blackgram (Phaseolus mungo L.) seeds during germination were studied. A decrease in germination percentage, root length, shoot length, and fresh mass was noticed with an increase in NaCl concentration. With the increase in NaCl concentration and duration of stress proline content increased and catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities decreased.     

Effect of Water Stress on Germination and Reserve Carbohydrate Metabolism in Germinating Seeds of Ceratonia siliqua L.

Ceratonia siliqua L. seeds were germinated in water and undera range of PEG 4000-induced moisture stresses. Germination wasretarded as water stress was stronger. Axis growth, total dryweight decrease, and starch formation in the embryo were closelycorrelated to galactomannan depletion, the latter being inhibitedwith lower external water potential. Endosperm -galactosidasewas inhibited in PEG-germinated seeds, while embryo -galactosidaseactivity was not significantly affected. Soluble sugar contentwas higher in cotyledons of seeds geminated under stress conditions,mainly due to higher sucrose accumulation. The higher sugarcontent in the endosperm was mainly due to higher galactoseand mannose content. Water stress effected a delay in the raffinose-typeoligosaccharide depletion.