Increases in activities of membrane-bound hydrolases in pea cotyledons during germination

Membrane-bound proteinase and acid phosphatase activities, butnot cytosol proteinase activity, in pea cotyledons increasedafter lag phases during germination. The activity hydrolyzingN--benzoyl-D,L-arginine P-nitroanilide in the membrane fractionincreased rapidly in the imbibition stage. Whether the increasesare due to de novo synthesis of the enzyme proteins was studied. ¹ Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received May 28, 1973; )     

Einfluss von hydroxyharnstoff auf die DNA-synthese und keimung der samen vonAgrostemma githago sowieVaccaria pyramidata

Application of hydroxyurea to intact seeds ofAgrostemma githago as well asVaccaria pyramidata causes a strong inhibition of radicular³H-thymidine incorporation into DNA, but germination rate is only insignificantly reduced. The seedling development, however, is strongly affected. Consequently, DNA synthesis is not a prerequisite for the radicle protrusion.     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

Cell Cycle Activation During Imbibition and Visible Germination in Embryos and Megagametophytes of Jack Pine (Pinus banksiana Lamb.)

Flow cytometric determination of cell cycle activation duringimbibition and visible germination in five families of jackpine (Pinus banksiana Lamb.) embryos and megagametophytes revealedthat in seeds that had undergone no imbibition the majorityof cells were in the 2C state. As the imbibition period increased,less of the nuclei were blocked in the G0/G1 state and morebecome active in the cell cycle. The augmentation in the nucleiactive in the 2C–4C cycle as well as those with DNA levelshigher than the 4C state occured gradually and preceeded radicleemergence. In megagametophyte tissue examined at various stagesof imbibition, cell cycle activity became apparent rapidly followingimbibition. In nuclei of green and white embryos examined separatelythe ²frequency distributions were significantly different forall three families after 144h. As imbibition period increased,fewer nuclei from the green embryos were blocked in the 2C state,and more became active in the 2C–4C cell cycle. This wasnot the case for white embryos where no significant linear relationwas noted. Cell cycle activity in the hypocotyl+cotyledons regionand the emerging radicle were examined separately. Functionalrelations found in the hypocotyl+cotyledons region were notevident in the radicle. As visible germination proceeded, cellcycle activity in the hypocotyl + cotyledons region for thisperiod of germination showed a reversal of the activity notedduring imbibition: fewer nuclei were active and once again ahigher proportion were found in the 2C state. cell cycle; C levels; DNA content; flow cytometry; germination; imbibition; jack pine; megagametophyte; Pinus banksiana Lamb     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Relation between DNA synthesis and germination of vaccaria Pyramidata seeds

Dormant seeds of Vaccaria pyramidata are characterized by a high level of [¹⁴C]Ieucine incorporation into proteins, a high level of [³H]uracil incorporation into RNA and an undiminished synthesis of poly(A)⁺-RNA; however, DNA synthesis is very much reduced. Germination of dormant Vaccaria seeds can be induced either partially by cytokinins or more completely by cytokinins and ethylene. Cytokinins cause a drastically increased [³H]thymidine incorporation in cotyledons of imbibing seeds. This effect is enhanced by ethylene. The incorporation of [³H]thymidine in radicles is also stimulated, but to a lesser extent. Cytokinin-dependent stimulation of [³H]thymidine incorporation can be referred to as an activation of nuclear DNA synthesis.     

Metabolic activities in germinated ancient lotus seeds

Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi) wereunearthed in a western suburb of Beijing in 1984, and determinedto be 58070-years-old by radiocarbon dating. Pretreatment withconcentrated sulphuric acid for 6 h promoted 75% of the seedstested to germinate. Over 3 weeks of incubation after the additionof water, storage starch, total soluble sugar and globulin inthe cotyledons decreased whereas albumin and soluble -aminonitrogen increased. These changes in cotyledonary componentswere similar to the changes seen in four present-day Indianlotus varieties, although the content of reducing sugar in Taizilotus was significantly lower. The polypeptide compositionsin extracts from the cotyledons and embryonic axis of Taiziseeds, as analysed by SDS-PAGE, were similar to those of present-dayJiangxi seeds. Taizi lotus also showed the ability to incorporateradioactive leucine into the trichloroacetic acid (TCA)-insolublefraction of a cotyledonary extract during the 24 h post-imbibitionperiod. The incorporation was inhibited when seeds of Taizilotus were allowed to rehydrate for 24 h in the presence of-amanitin or cycloheximide. Key words: Ancient lotus, Nelumbo nucifera, protein synthesis, seed germination, seed viability     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

{beta}-Amylase Activity as an Index for Germination Potential in Rice

Seeds of different vigour also differ in their germination ability.In rice (Oryza sativa), this difference was correlated withthe level of incorporation of ³⁵S-methionine into 25-60% ammoniumsulphate precipitable material that was rich in amylase proteins.This protein fraction, from dry seeds, contained no -amylaseactivity. In contrast, ß-amylase activity was presentin all seed stocks capable of 99% germination, although thelevel was lower in seeds that grew slowly when germinated. Inlow viability low vigour stock (i.e. extensively deterioratedseeds) ß-amylase activity was absent. Alpha-amylaseactivity in all stocks was detected only after 24 h from thestart of imbibition. These results indicate that ß-amylaseactivity is reliable indicator of the germination ability ofrice seed stocks and of their vigour during germination.Copyright1995, 1999 Academic Press Rice (Oryza sativa L.,), germination, ß-amylase, -amylase, seed vigour     

Pigment synthesis in Cucurbita moschata cotyledons as influenced by CPTA and several inhibitors

2-(4-Chlorophenylthio) triethylamine hydrochloride (CPTA) inducedthe accumulation of a number of carotenes in pumpkin (Cucurbitamoschata) cotyledons, a tissue which does not normally accumulatethese pigments. Lycopene accumulated concomitantly with a decreasein -carotene and ß-carotene. CPTA appeared to inhibitcyclases involved in the synthesis of -carotene and ß-caroteneand to stimulate enzymes involved in lycopene synthesis. Cycloheximidereduced this CPTA induced enzyme synthesis while diphenylaminereduced CPTA induced carotene accumulation. Actinomycin D alonereduced accumulation of carotenes, but it did not affect CPTAinduced carotene accumulation. Rather lutein, violoxanthin andneoxanthin decreased and -carotene and ß-caroteneaccumulated. ¹Present address: Morioka Branch, Vegetable and Ornamental CropsResearch Station, Ministry of Agriculture and Forestry, Shimokuriyagawa,Morioka, Japan. (Received August 12, 1974; )     

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)     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Penicillin Induction of Gibberellin and {alpha}-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of -amylase in embryoless riceendosperm and enhances the gibberellin-induced response. Penicillininduction of -amylase can be prevented by inhibitors of nucleicacid and protein synthesis, CCC and 2,4-DNP. A characteristic gibberellin-like activity in the extracts frompenicillin-treated endosperms becomes detectable after 12 hfrom the addition of penicillin. This gibberellin-like activityis located on paper chromatograms at the R_F typical for GA₃and its formation is blocked by CCC, an inhibitor of GA biogenesis.Glucose has no effect on the biosynthesis of either gibberellinor -amylase induced by penicillin. The time-course study of the levels of different constituentsshows that penicillin probably induces RNA and DNA synthesisin the first place, which results in gibberellin biosynthesis,which in turn stimulates the synthesis of -amylase. The possiblemode of action of penicillin in higher plants is discussed.     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )     

Composition, Degradation and Utilization of Endosperm During Germination in the Oil Palm (Elaeis guineensis Jacq.)

The insoluble carbohydrate and lipid fractions, and -D-galactosidase,ß-D-mannosidase and isocitrate lyase activities werestudied in the various tissues of oil palm (Elaeis guineensisJacq.) kernels prior to and during germination. In ungerminatedkernels insoluble carbohydrate and lipid constituted 36 and47% of endosperm dry weight respectively. During germinationthe thick endosperm cell walls became markedly thinner, concurrentwith a significant decrease in the percentage of insoluble carbohydrateand an increase in -galactosidase and ß-mannosidaseactivity in both degraded and residual endosperm. The proportionof lipid in degraded endosperm also increased significantly.The insoluble carbohydrate appears to be a galactomannan locatedin the secondary walls of the endosperm. No galactomannan wasdetected in oil palm embryos or haustoria. Isocitrate lyasewas present in, and confined to, tissues of the haustorium ofgerminating kernels. The enzyme was not active in endospermat any stage of germination, nor was it active in embryos beforeor at the end of imbibition. The results suggest that galactomannan is the second largestcomponent of oil palm endosperm and that it is utilized morerapidly than lipid during the early stages of germination. Thefact that isocitrate lyase activity is confined to the haustoriumsuggests that in Elaeis gluconeogenesis, the conversion of triglycerideto carbohydrate, takes place entirely within the cotyledon ofthe seed. Elaeis guineensis, galactomannan, galactosidase, germination, isocitrate lyase, mannosidase, oil palm     

Synchronous Alterations of RNA Synthesis and DNA-dependent RNA Polymerase Activities during Development of Dictyostelium discoideum

The relation of RNA synthesis and RNA polymerase activitiesduring the development of Dictyostelium discoideum was investigated.Sucrose gradient analysis of the pulse-labeled RNA in intactcells indicated that the ratio of the synthesis rate of rRNAto that of messenger-like RNA (mRNA$heterogeneous nuclear RNA)was highest in the vegetative stage and relatively low in theearly phase of spore germination and in the morphogenetic stage.The levels of the activities of RNA polymerases I and II, whichwere determined in three different ways (distinction between-amanitin-resistant and -sensitive activities in sonicated nuclearor whole cell extracts, separation of D-I and D-II by DEAE-Sephadexchromatography, and separation of P-I and P-II plus P-III byphosphocellulose chromatography), changed during development.The modulation of the enzyme activities was roughly parallelto the alteration in the relative synthesis rates of these twoRNA species. The results suggested that the changes in the ratesof RNA synthesis during development of D. discoideum are mostlydue to the modulation of RNA polymerase activity per se. ¹ Present address: Department of Immunology and Virology, SaitamaCancer Center Research Institute, Ina-machi, Saitama 362, Japan. (Received August 22, 1981; Accepted December 10, 1981)     

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