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)     

Contribution of the Pentose Phosphate Pathway and Glycolytic Pathway to Dormancy Breakage and Germination of Peanut (Arachis hypogaea L.) Seeds

Levels of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADPH oxidoreductase, E.C. 1.1.1.49 [EC] ) 6-phosphogluconate dehydrogenase(6-phospho-D-gluconate : NADP⁺ oxidoreduc tase, E.C. 1.1.1.44 [EC] )and aldolase (fructose 1, 6-diphosphate, D-glyceraldehyde, 3-phosphatelyase, E.C. 4.1.2.13 [EC] ) were assayed in the seeds of geneticallydormant and non-dormant pure lines of groundnut. In dormantlines cotyledons showed increased levels of activity of G-6-PDHand 6-PGDH during dry storage after-ripening. While the embryonicaxis did not exhibit detectable levels of enzyme activitiesimmediately after harvest, the activity started after a lapseof time during dry storage. When seeds of dormant lines wereincubated with kinetin (6-furfurylaminopurine) a distinct increasein the levels of both the enzymes was observed. The levels ofaldolase activity gradually decreased in the cotyledons andincreased in the embryonic axis of both control and kinetintreated seeds during the period of after-ripening. Comparedto control, kinetin treatment increased the aldolase activityin the embryonic axis and decreased it in the cotyledons. In non-dormant lines the activity of both the enzymes of PPpathway increased sharply both in the cotyledons and embryonicaxis while aldolase activity decreased in the cotyledons andincreased in the embryonic axis during germination i.e., from24 h to 96 h of germination. Abscisic acid caused inhibitionof enzyme activities to a large extent. Key words: PP pathway, dormancy breakage, germination, peanut     

Translatable mRNAs for Chloroplast-Targeted Proteins in Detached Radish Cotyledons during Senescence in Darkness

A protein-import system prepared with isolated chloroplastswas used to monitor changes in levels of mRNAs for chloroplast-targetedproteins during dark-induced leaf senescence. Biologically activechloroplasts were isolated from young (9-day-old) and aged (14-day-old)radish cotyledons. Poly(A)⁺-RNA was prepared from radish cotyledonsthat had been detached from seedlings and placed in darknessto accelerate senescence. The RNA was translated in a wheatgerm system, and the products were added to an import systemprepared with chloroplasts from young cotyledons. Electrophoreticanalysis of the imported proteins suggested that most chloroplast-targeted proteins decreased in abundance during dark treatmentof cotyledons. However, the relative abundance of 38 stromaland three thylakoid proteins increased transiently or continuouslyamong the products of translation of RNA isolated during thecourse of senescence. The efficiency of the uptake of precursorproteins by chloroplasts isolated from aged cotyledons was lowerthan that by chloroplasts from young tissue. The chloroplastsfrom aged cotyledons more efficiently imported at least onestromal protein and one thylakoid protein than chloroplastsfrom the young tissue. The relative abundance of these two proteinsincreased among the products of translation of RNA from senescingcotyledons when tested in the uptake system with chloroplastsfrom young cotyledons. These results suggest that some nucleargenes for chloroplast-targeted proteins are expressed in senescingcotyledons more efficiently than in young tissue, and that themachinery for import of proteins into chloroplasts changes duringaging of the tissue to allow more efficient import of certainproteins that may be responsible for the senescence of the chloroplasts. ¹Present address: Kihara Institute for Biological Research,Yokohama City University, Mutsukawa 3-122-20, Minami-ku, Yokohama,232 Japan     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

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     

Immunocytochemical Localization of Uricase in Peroxisomes of Soybean Cotyledons

Antibodies specific for nodule uricase were used for immunocytochemistryto demonstrate the presence of uricase in cotyledons of soybean(Glycine max) during germination and early seedling growth.The enzyme was localized exclusively in peroxisomes. ¹Permanent address: Department of Plant Cytology and Cytochemistry,University of Lodz, Lodz, Poland ²Current address: Department of Plant Science, University ofArizona, Tucson, AZ 85721, U.S.A.     

Association of thymidine and uridine kinase activities with changes in nucleic acid levels during peanut fruit ontogeny

Various stages of pegs, cotyledons and embryonic axes from maturing peanut fruits were examined for their ability to phosphorylate thymidine and uridine. Highest specific activities during peg elongation were found just prior to increases in endosperm nuclei and embryo cell numbers. In the developing cotyledons and axes, the net kinase activities of crude extracts reached a maximum 1–2 weeks before maximal RNA and DNA contents were attained. An exception was the apparent lack of any relationship between uridine kinase activities and RNA levels in developing embryonic axes. The present results support the observation that peanut axes are devoid of thymidine and uridine kinases during the first 24 hr of germination, as fully developed fruits had very low specific activities for both of these phosphate transferases.     

Ascorbate and glutathione metabolism during development and desiccation of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The ascorbate-glutathione system was studied during development and maturation of beech (Fagus sylvatica L.) seeds, the classification of which in the orthodox category is controversial. This study revealed an increase in glutathione content after acquisition of desiccation tolerance, which was more intensive in embryonic axes than in cotyledons. During seed maturation, the redox status of glutathione markedly changed toward the more reducing state, especially in cotyledons. Ascorbic acid content decreased during maturation, mostly in cotyledons. Activities of the enzymes of the ascorbate-glutathione cycle—ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2)—were markedly higher in embryonic axes than in cotyledons throughout the study period. In the course of seed maturation, the activities of these enzymes decreased. Importance of the ascorbate-glutathione cycle in desiccation tolerance of beech seeds was discussed in relation to results for typical orthodox and recalcitrant seeds of other broadleaved species.     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Changes in Neutral Sugar Compositions of Cell Wall Polysaccharides during Redifferentiation of Cultured Carrot Cells and during Maturation of Soybean Seeds

Changes in the neutral sugar compositions of cell walls werestudied during regeneration of shoots and roots from culturedcarrot cells and during maturation of soybean seeds. There weremore arabinan and arabinose-rich acidic polysaccharides thangalactose-rich polysaccharides in the pectic fractions of thecell walls from cultured carrot cells and more galactan, arabinogalactanor both than the arabinose-rich polysaccharides in the samefractions from their mother tissue, i.e. root phloem tissue. The arabinose content of the cell walls decreased and the galactosecontent increased during root and shoot formation until galactoseexceeded arabinose in the cell walls of fully developed shootsand roots from cultured cells. The cell wall arabinose contentalso was higher than that of galactose in cotyledons and embryonicaxes of immature soybean seeds, and change in the neutral sugarcomposition of the cell wall during seed maturation was similarto that during the redifTerentiation of cultured carrot cells.During the very late stage of maturation, galactose in the cellwalls exceeded the content of arabinose. Results suggest that the redifferentiation of roots and shootsfrom cultured cells goes through a process of cell wall formationsimilar to that of embryogenesis or seed development in themother plants. Results also indicate that the predominant arabinanand arabinose-rich acidic polysaccharides have important functionsin cell walls during embryogenesis and in the eraly stages ofseed maturation and that galactan, arabinogalactan, or bothreplace these arabinose-rich polysaccharides after seed maturation. ²Present address: Department of Botany, the University of BritishColumbia, # 3529-6270 University Blvd.,Vancouver, B.C. V6T 2B1Canada (Received October 28, 1982; Accepted April 8, 1983)     

Increase of cytokinin activity in detached etiolated cotyledons of squash after illumination

Cytokinins nullified the lag period of the formation of chlorophyllin detached etiolated cotyledons of squash (Cucurbita moschataDuch. var. melonaeformis Makino cv. Tokyo). One hour after illumination,cytokinin activity in detached cotyledons rapidly increasedand maintained a certain level for another hour. ¹ Present address: Department of Enviromental Studies, Collegeof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan. (Received November 25, 1976; )     

RNA synthesis in germinating red bean seeds

Nucleic acids from ³²P-labelled germinating red bean seeds wereinvestigated by means of MAK column chromatography. 1) In cotyledons,synthesis of D-RNA occurred in the early stages of germination,3 to 24 hr after the onset of imbibition. ³²P was also incorporatedinto rRNA continuously at rather a moderate rate. DNA-RNA hybridizationexperiments revealed that the proportion of heterogeneous RNA(D-RNA) to rRNA decreased gradually. Nucleotide analysis suggestedthat tRNA was synthesized de novo, and that its CCA-end exchangewas remarkable at early stages of germination. 2) In embryos,however, the incorporation of ³²P into rRNA was very much greaterthan into D-RNA, and the exchange reaction at CCA-end of tRNAwas not detected. The role of D-RNA, found in cotyledons inthe initial stages of germination, was discussed. ¹Present address: Research Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya,Japan. (Received May 10, 1972; )     

The Role of ABA in the Control of Apple Seed Dormancy Re-appraised by Combined Gas Chromatography-Mass Spectrometry

Measured by GC—MS²—SIR³, endogenous ABA⁴ in embryonicaxes of seeds of Malus pumila L. cv. Golden Delicious decreased8-fold and cotyledon ABA by only 60%, during 10–50 d ofstratification at 5 ?C, after ABA leaching during an initial24 h soaking. During stratification, the percentage germinationof embryos transferred to 17?C showed a significant linear dependenceon log_e of ABA levels in the axes at transfer. Between 50 and70 d, ABA levels increased markedly in axes and testa both ofstratified seeds and seeds allowed to re-dry at 17 ?C afterinitial soaking. The ability of fully stratified axes with elevatedendogenous ABA to germinate indicated that stratification haddecreased their ABA sensitivity. Changes in cotyledon ABA couldnot account for the promotory effect of cotyledons on germinationduring the first 30 d of stratification. Loss of testa inhibitionof germination during stratification was not linked with changesin testa ABA. Stratification markedly increased the sequestrationin the axes of exogenous ABA supplied via the cotyledons. Changesboth in axis ABA levels and sensitivity were thus correlatedwith dormancy release, but subject to modifying control by thecotyledons and testa not involving ABA. Rehydration of driedseeds affected axis ABA later during storage via mechanismsunconnected with dormancy. Key words: ABA, seed dormancy, stratification     

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.     

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)     

Glutamate synthase in developing pea cotyledons: Occurrence of NADH-dependent and ferredoxin-dependent enzymes

NADH-dependent and ferredoxin-dependent glutamate synthasesfrom developing pea cotyledons were separated by gel filtrationon a Sephadex G-200 column. The substrate requirements, molecularweights and effect of some inhibitors on both glutamate synthaseenzymes were investigated. ¹ Department of Agricultural Chemistry, Kyoto University, Kyoto606, Japan. ² To whom inquiries should be addressed. (Received August 9, 1979; )     

Stability of Polysome-Associated mRNA in Potato Tuber Cells during Aging of Tissue Discs

The stability of polysome-associated mRNA in potato tuber discsin the early stage of aging was examined by pulse-chase labelingexperiments and the change in the translational capacity ofthe RNA was studied using a wheat germ translation system. Theincorporation of pulse-fed ³H-uridine into polysomal RNA wasnot arrested immediately after the addition of actinomycin Dto the tissue, but increased by 25% during 4 hr of chasing.The radioactivity in the polysomal RNA then decreased by only30% of the value at the 4th hr during the next 9 hr in the presenceof actinomycin D. The remaining radioactivity in the polysomalRNA was stable at least for 18 hr. The proportion of radioactivityin polyadenylated RNA to that in non-polyadenylated RNA didnot vary appreciably during the chasing period. Non-polyadenylatedRNA of high molecular weight degraded faster than that of lowmolecular weight, but polyadenylated RNA did not show such size-selectivedegradation. The translational capacity of the polysomal RNAalso decreased by about 23% within 9 hr during the period ofinhibited RNA synthesis. In vivo experiments of ¹⁴C-leucineincorporation into proteins in the absence of RNA synthesissuggested that stable polysome-associated mRNA was actuallyfunctioning in the cells. SDS-polyacrylamide gel electrophoresisof the in vitro translation products indicated that mRNA codingfor polypeptides with relatively high molecular weights turnedover slightly faster than those for low molecular weight polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received May 12, 1982; Accepted August 26, 1982)     

Photoperiodic sensitivity of the cotyledon and first true leaf of Xanthium

Three days after germination of Xanthium seeds, the seedlingswith only the first pair of opposite leaves attained full photoperiodicsensitivity and flowered in response to a 16-hr dark periodtreatment. The cotyledons of three- to six-day-old seedlingswere completely insensitive to inductive dark treatment. Six-day-oldseedlings could not be induced to flower with a 9-hr dark period.But with a dark period of 10 hr or longer, floral inductionoccurred and the optimum dark period was 15 hr. Developmentof the flowering apices started within 2 days after the inductivedark treatment and was completed by 9 to 11 days. ¹This work was supported by the United States Atomic EnergyCommission Contract AT (11-1)-1338. ²Present address: Plant Physiology Section, Crops Research Institute,P.O. Box 3785, Kumasi, Ghana, West Africa. (Received March 28, 1974; )     

Cell elongation in the soybean root: The influence of inhibitors of RNA and protein biosynthesis

A possible requirement for RNA and protein synthesis duringcell elongation of intact seedling tissue was studied usingthe soybean seedling foot with the elongating zone being delineatedby India ink marks at 2 and 7 mm back of the root tip. In contrastto most excised plant tissues, there was marked net synthesisof RNA and protein during cell elongation of the intact root.AD and CH were potent inhibitors of cell elongation in the soybeanroot. CH essentially eliminated protein synthesis, whether measuredby net accumulation of protein or by ¹⁴C-leiicine incorporation,while completely inhibiting cell elongation after a short lag.AD, on the other hand, only partially inhibited protein synthesiswhile causing almost total inhibition of cell elongation aftera lag. The capacity of the tissue to synthesize protein in thepresence of AD was correlated with the maintenance of functionalpolyribosomes, thus suggestive that m-RNA associated with theregulation of cell elongation is more unstable (i.e., a shortermean life) than total root m-RNA. FU did not inhibit cell elongation,protein synthesis or the level of functional polyribosomes.The requirement for RNA synthesis during cell elongation ofthe seedling root, as in excised plant tissues, appears to berestricted to the AMPrich species of RNA presumed to be m-RNA. ¹This research was supported by NIH grant GM 10157. ²Purdue University AES paper No. 3359. ³Present address: Dept. of Botany, National Taiwan University,Taipei, Taiwan.     

Application of 31P NMR to Monitor Phosphorus Compounds and their Changes During Germination of Legume Seeds

Extracts of embryonic axes and cotyledons of 19 species of legumeswere analysed by ³¹P nuclear magnetic resonance; the effectof different extraction procedures on the solubilization ofthe several phosphocompounds was investigated. The contentsof phytic acid, inorganic phosphate, phosphomonoesters and phosphodiesterswere determined. The relative contents of these phosphocompoundswere very different when embryonic axes and cotyledons werecompared, and were greatly influenced by the taxonomic affiliationof the corresponding species. Phytic acid and inorganic phosphatewere by far the major phosphocompounds found in cotyledon extracts;an unusual phosphodiester component was detected only in embryonicaxes and was particularly abundant in species belonging to theLotoideae sub-family. The changes in the amount of the phosphodiestercompound were followed during germination and the results suggestthat it is not a phosphorus reserve. Furthermore, this componentwas preferentially present in the roots of the new plants. Itwas partially hydrolysed by RNAse and to a smaller extent byproteases, but not affected by DNAse. The hypothesis that thisunusual component might be a ribonucleoprotein is discussed. Key words: ³¹P NMR, legumes, seed components, phosphorus compounds, solubilization