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.

     

Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3.1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination.     

Protein Bodies of Germinating Seeds of Vicia faba: CHANGES IN FINE STRUCTURE AND BIOCHEMISTRY

Changes in fine structure and starch, total nitrogen and solublesugar contents were followed during the first 3 weeks of germinationin Vicia faba cotyledons. Following hydration the reserves were mobilized and loss ofprotein from the cotyledons began after 4 days; concomitantfine structural changes included the swelling and coalescenceof protein bodies, the disappearance of their contents, andthe differentiation of the provascular tissue in the cotyledonwithin which ‘transfer cells’ developed. There wassome development of rough endoplasmic reticulum during the earlypart of germination. A pattern of protein-body degradation appeared in the cotyledonsduring germination, those cells nearest to the vascular bundlesand to the epidermis being the first to lose their reserves.After 3 weeks' growth the parenchyma cells of the cotyledonwere very vacuolate and typical of senescent tissue, while thevascular bundle cells still retained their contents and remainedactive-looking.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Sucrose Compartmentation in the Palisade Parenchyma of Vicia faba L

Intracellular sucrose compartmentation in the palisade parenchyma of Vicia faba L. leaflets was investigated by comparing the specific radioactivity of photosynthetically labeled [¹⁴C]sucrose in samples enriched in vacuole to that in samples enriched in cytoplasm. Brief centrifugation of leaflet punches was used to sediment most of the palisade parenchyma cytoplasm in the adaxial ends of the cells. The punches were quick-frozen, freeze-substituted, and embedded in methacrylate. Samples enriched in cytoplasm or in vacuoles were obtained from paradermal sections. After pulse-labeling, the sucrose specific radioactivity in vacuole-enriched samples was fairly constant. Sucrose specific radioactivity in cytoplasm-enriched samples was about 2.5 times that in vacuole-enriched samples initially and declined thereafter. Earlier interpretation of intracellular sucrose compartmentation (Plant Physiol 1975 55: 704-711) had predicted larger specific activity differences (up to 20 times) between the cytoplasm and vacuole. The difference between the actual and predicted behavior is ascribed to the observed extent of cross-contamination in samples and, more importantly, to the confinement of sucrose to extrachloroplastic regions of the cytoplasm.     

Protein Bodies of Developing Seeds of Vicia faba

Changes in fine structure and starch, nitrogen, and solublesugar content were followed through to maturation in developingcotyledons of Vicia faba. Various ultrastructural changes wereobserved in the developing cotyledons, notably an increase inthe number of membrane-bound ribosomes which corresponded withthe onset of storage protein deposition. The build-up of storageprotein was shown to occur in the cytoplasm within membrane-boundvacuoles which subsequently became the protein bodies of themature seed, retaining the original tonoplast as the boundingmembrane of the protein body. Nuclei became lobed during thelater phases of maturation; phytoferritin was observed in plastidsof mature seeds. The deposition of reserves in the cotyledonswas complete by 85–90 days after flowering, followingwhich water was lost until the seed became hard and ‘ripe’by no days after flowering.     

Release of Sucrose from Vicia faba L. Leaf Discs

The release of sucrose from leaf discs of Vicia faba L. to a bathing medium was studied for evidence of a relationship between this release and mesophyll export of photosynthate in vivo. Sucrose was released specifically over hexoses and represented over 85% of total photosynthate released. The sucrose appeared to be derived from the mesophyll tissue directly and release did not require concurrent photosynthesis. The data indicated two separate channels for sucrose release. The first was sensitive to inhibition by 1 millimolar p-chloromercuribenzenesulfonic acid and the second was promoted by lowering the Ca²⁺ concentration below 0.1 millimolar. Flow through both channels was about equal when tissue that had been actively photosynthesizing for several hours was used. The rate of release was not dependent on the extracellular pH, but was inhibited by 10 micromolar carbonylcyanide p-trifluromethoxyphenylhydrazone. Lowering the Ca²⁺ concentration below 0.1 millimolar or raising the K⁺ concentration above 100 millimolar stimulated sucrose release. The stimulation by high K⁺ was not reversed by adding Ca²⁺. The data supported the postulate that Ca²⁺ removal or K⁺ addition changed the permeability of the mesophyll plasma membrane to sucrose.     

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     

The Effect of Temperature on Development and Dry-matter Accumulation of Vicia faba Seeds

Vicia faba plants were grown at 16 °C and the temperatureraised to 21 or to 26 °C, 51 d post anthesis (p.a.). Temperatureincrease accelerated pod and seed development, stimulated dry-matteraccumulation, starch and protein synthesis. However, the durationof dry-matter accumulation of seeds was shortened, resultingin a decrease of final seed weight. The effect of temperature on storage capacity (determined bycell number and cell size) was also investigated. Formationof new cells in the layer under the epidermis continued fora much longer period than previously has been described in theliterature. At all stages of development cotyledons containedyoung and small cells with small nuclei at the outside and theybecame larger and older towards the centre of the cotyledon.Cells at the centre were only able to divide at an early stageof seed development up to day 59 p.a. Thereafter cell divisionoccurred mainly in the first cell layer under the epidermisup to 63–67 d p.a. Nuclear counts of macerated cotyledoncells did not show an effect of temperature on the number ofcells. It has, however, been questioned if this method was suitableto measure accurately the number of cells at the last stagesof seed development. The rate of cell expansion was stimulated by higher temperaturesbut the duration of expansion was shortened and the final sizeof cells was not affected by the different temperature treatments.Senescence of the pod wall was accelerated at higher temperaturesand it is possible that transport of sucrose from the pod wallto the seeds terminated earlier than at lower temperatures.This may have resulted in an inhibition of cell formation atthe periphery of the cotyledons and in an inhibition of starchand protein synthesis. Vicia faba, protein, starch, cell size, cell number, temperature     

Acyl Lipid Metabolism in Germinating Hazel Seeds (Corylus avellana L.)

A decreasing percentage of radioactivity from [U-¹⁴C]-oleateand [U-¹⁴C]-palmitate was recovered in the lipid fractions ofgerminating hazel cotyledons. The pattern of incorporation ofthe acids into the cotyledon glycerides was consistent withtheir degree of saturation. The relative incorporation intothe cotyledon phospholipids changed during germination. Radioactivityfrom both acids was recovered in increasingly unsaturated fattyacids in the cotyledon hpids. Diversion of both [U-¹⁴C]-fatty acids into acyl lipid synthesisoccurred in the germinating embryonic axes. Both were increasinglyrecovered in mixed glyceride groups which contained diglyceridesand highly unsaturated triglycerides. The two acids gave differentpatterns of incorporation in the axis phospholipids. Desaturationof the acids occurred to a lesser extent than in the germinatingcotyledons.     

Identification and Metabolism of 1-(Malonylamino)cyclopropane-1-carboxylic Acid in Germinating Peanut Seeds

Peanut seeds (Arachis hypogea L. Yue-you 551) contain 50 to 100 nanomoles per gram conjugated 1-aminocyclopropanecarboxylic acid (ACC). Based on paper chromatography, paper electrophoresis, and gas chromatography-mass spectrometry, it was verified that the major ACC conjugate was N-malonyl-ACC (MACC). Germinating peanut seeds converted [2-¹⁴C]ACC to ethylene 70 times more efficiently than N-malonyl-[2-¹⁴C]ACC; when ACC was administered, most of it was metabolized to MACC. Germinating peanut seeds produced ethylene and converted l-[3,4-¹⁴C]methionine to ethylene; this ethylene biosynthesis was inhibited by aminoethoxyvinylglycine. These data indicate that MACC occurs in peanut seeds but does not serve as the source of ethylene during germination; ethylene is, however, synthesized from methionine via ACC.     

Nucleic Acids of Developing Seeds of Vicia faba L.

Changes have been followed in the base composition of RNA andthe amounts of RNA and DNA during the development of Vicia fabaseeds. Both acids were synthesized most rapidly during days36–46 of the 72-day developmental period. Changes foundwere related to the development of the embryo and to the synthesisof storage protein.     

Nucleic Acid Metabolism of Vicia faba during Germination and Growth

During the growth of Vicia faba seedlings in the absence of an external nitrogen supply, the cotyledons decreased rapidly in dry weight and nucleic acid content. In the developing shoot the dry weight increased rapidly for four weeks and then very slowly over the next two weeks growth; the nucleic acid content of the shoot increased to a maximum after 4 weeks growth and decreased in amount during the next 2 weeks. On the other hand the roots increased in both dry weight and nucleic acid content throughout the growth period, although they only accounted for a small proportion of the total dry weight and nucleic acid content of the plant. These changes during germination and growth are discussed in relation to those occurring during these developmental stages in other plants.     

在非光合条件下外源蔗糖能促进蚕豆的气孔开放

蚕豆植株经暗饥饿处理40h后,取其下表皮,再用超声波“原位分离”下表皮上的保卫细胞对,然后在无菌、非光合条件下,用外源蔗糖处理蚕豆下表皮上的保卫细胞对,考察其对气孔开放的效应。结果发现,在1d的无菌培养过程中,蔗糖显著促进了气孔的开放。100mmol/L的蔗糖在10mmol/L的MES-NaOH/KOH(pH6.1)缓冲液中,开度分别增加2.0/2.6μm;在1μmol/L的气孔开放促进剂fusicoccin(FC)的存在下,100mmol/L的蔗糖在MES-NaOH/KOH(pH6.1)缓冲液中分别增加开度5.0/5.5μm。不同浓度（5～200mmol/L）的蔗糖处理结果表明气孔开度的增加与蔗糖浓度呈一定的正相关,浓度为100mmol/L的蔗糖处理表现出最大促进作用。同时还初步观察到,蔗糖可维持保卫细胞存活率和叶绿体的完整性。     

Anaerobic Metabolism in Germinating Seeds of Echinochloa crus-galli (Barnyard Grass) : METABOLITE AND ENZYME STUDIES

Echinochloa crus-galli, a problem weed in rice fields, has the rare ability to germinate and to grow in a totally oxygen-free environment. After 7 days growth in the light or dark under N₂, E. crus-galli var. oryzicola produces a 2- to 3-centimeter nonpigmented shoot.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

The Composition of the Broad Bean Seed (Vicia faba)

The composition of the seed of the Broad Bean (Vicia faba) hasbeen investigated and the results of alternative analyticalmethods compared. The seeds used in the present work were higherin nitrogen and phosphorus and lower in structural carbohydrate,magnesium and calcium than the seeds used in previous Germananalytical work. Such differences are attributed to differenceof variety and of conditions of maturation of the seed.     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.