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     

Starch Synthesis During Dormancy Breakage in Oilseed of Corylus avellana

Dormancy in seed of Corylus avellana L. (hazel) is broken bya sustained period of cold stratification. During this timeboth cytological and metabolic changes occur. Starch was presentinitially at a low level but increased by 20% in the embryonicaxes of hazel seeds during stratification at 5 °C, whileit decreased rapidly and then remained constant in the embryonicaxes from seeds held at 20 °C. Histochemical study confirmedthis analytical result. A comparison of the developmental patternof starch level with bound and soluble ADP glucose-starch synthaseactivity at 5 and 20 °C showed that the accumulation ofstarch in the embryonic axes followed an enhanced activity ofthe granule-bound ADP glucose-starch synthase. Cold stratificationresulted in an increase in starch content, which was probablyas a result of gluconeogenesis from products of reserve lipidhydrolysis. Corylus avellana L., hazel, starch, ADP glucose-starch synthase, stratification     

Changes in some nitrogenous components during the germination of pea seeds

The changes in major nitrogenous components during the germination of pea seeds have been followed. During the period of rapid axis growth, 3 to 8 days following germination, the nitrogen content of the cotyledons declines rapidly with an accompanying increase of nitrogen in the developing axis. The accumulation of alcohol soluble nitrogen, primarily amino nitrogen, in the cotyledons and axis during germination indicates that the mobilization of nitrogen is facilitated by proteolysis and translocation of the products.     

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 I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Sugar and starch changes in pea cotyledons during germination

Changes in starch and sugar contents in the cotyledons during germination have been compared in a smooth (cv. Alaska) and a wrinkled (cv. Progress) cultivar of the garden pea ( Pisum sativum L.). In both cultivars there was an initial accumulation of sucrose due to the hydrolysis of sucrosyl oligosaccharides, but galactose did not accumulate in the cotyledons. Starch mobilization in the Progress pea was linear with time and started before the rise in α-amylase (EC 3.2.1.1) activity in the cotyledons; sucrose was synthesized in the cotyledons, and their excision from the axis resulted in an additional accumulation of this sugar. In the Alaska pea, the onset of starch hydrolysis coincided with the rise in α-amylase activity; no accumulation of sucrose was found in excised cotyledons, whilst the sucrose content decreased continuously in attached cotyledons.
The same sugars were found in the cotyledons of both cultivars, suggesting a common pathway for starch breakdown. Maltose, maltotriose and linear malto-dextrins were not present and only trace amounts of glucose were detected, suggesting a degradation of starch by phosphorylase after an initial attack by α-amylase. α-Amylase activity in the cotyledons was higher in the presence of the axis, but was influenced by the water content of the cotyledons. Transient changes in α-amylase activity correlated well with changes in the rate of starch hydrolysis, but after 2–3 days starch mobilization was reduced in excised cotyledons probably due to the resynthesis of starch.     

Hydrolytic enzyme activities and degradation of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark, and time-course changes in contents of protein fractions, starch, soluble α-amino nitrogen and reducing sugars and in hydrolytic enzyme activities in cotyledons were investigated. In cotyledons of germinated seeds, marked increases in proteolytic (caseolytic, globulytic and gelatin-hydrolyzing) activities and amylolytic activity occurred with concurrent mobilization of storage proteins and starch. Removal of axis organs from seeds at very early stages of germination caused the deteriorated breakdown of storage components and decreased development of proteolytic enzymes in the cotyledons, but this treatment did not significantly affect the appearance of amylolytic activity. The experimental results are discussed in comparison with the hydrolytic enzyme activities of germinating seeds of other leguminous species.     

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     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

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; )     

Aminoethoxyvinylglycine Effects on Late-Season Apple Fruit Maturation

Aminoethoxyvinylglycine (AVG) inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and thus blocks ethylene synthesis. Preharvest foliar application of AVG to apple (Malus domestica Borkh.) fruit retards several key events of maturation including climacteric ethylene production, starch conversion to sugars, fruit softening, and abscission zone development. Although the impact of AVG on apple fruit maturation is well known, the biochemical basis of these effects is not well understood. The effects of AVG application on Redchief Delicious apple fruit maturation were studied. AVG applied four weeks prior to harvest significantly reduced internal ethylene levels, amylose degradation, and accumulation of sucrose, glucose, and sorbitol. Because AVG application coincidentally inhibited starch degradation and the increase in internal ethylene, we investigated the enzymatic basis of starch mobilization in apple fruit. Amylase activity was somewhat reduced in AVG-treated fruit. Amylase activity was less in AVG-treated fruit during the early stages of starch mobilization. Starch phosphorylase activity increased dramatically during the later stages of starch mobilization, but was not affected by AVG treatment. Soluble starch synthase activity was also unaffected by AVG treatment and remained constant throughout the eight-week harvest period. Moreover, AVG did not affect the levels of amylopectin, fructose, malate, ascorbate, citrate, or anthocyanin. These results suggest that apple fruit ripening has both ethylene-dependent and -independent processes occurring simultaneously.     

Starch and triacylglycerol metabolism related to somatic embryogenesis in Papaver orientale tissue cultures

During somatic embryogenesis in Papaver orientale tissue cultures a permanent starch accumulation and a transient triacylglycerol accumulation were observed. The degradation of the lipids during plantlet development from embryoids was paralleled by an activity increase of the glyoxylate-cycle enzymes malate synthase (EC 4.1.3.2) and isocitrate lyase (EC 4.1.3.1). Fat accumulation and breakdown was interpreted as a reflection of seed formation and germination during normal development.     

Reserve Mobilization During Germination of Tagetes minuta L.

Levels of albumins, globulins and starch were measured in dryTagetes minuta L. achenes (seeds) germinating at 25 °C andin those thermo-inhibited at 35 °C. In addition, the compositionof amino acids, fatty acids and free sugars was determined. In germinating seeds, albumins increased, while globulins decreased.At 35 °C, only a delayed decrease in globulins occurred.However, in both, the amino acids serine and glutamate increased.Lipid composition remained constant except for a slight declinein linoleic acid in the germinating seeds. At 25 °C, starchlevels increased with time while at 35 °C, there was onlya transient increase. Sucrose was the major free sugar foundin the seeds at all times, but its levels declined in the germinatingseeds. This was accompanied by an increase in the proportionsof glucose and fructose. No such changes in sugar compositionwere observed in the thermo-inhibited seeds. Tagetes minuta L., seed germination, reserve mobilization, thermo-inhibition     

The Effect of Chilling and Moisture Status on the Germination, Desiccation Tolerance and Longevity ofAesculus hippocastanumL. Seed

Effects of 2 °C chilling on the threshold moisture contentsand water potentials for various physiological processes wereestimated forAesculus hippocastanumL. seed. Seed harvested atthe time of maximum seed fall exhibited a dual response to drying:partial drying from near 50% to 32–40% moisture contentprogressively increased germination percentage (at 16 °C)up to various peak values; further desiccation was detrimental,confirming that the seeds are ‘recalcitrant’. Themoisture content for optimum germination was increased by atleast 10% as the chilling period was raised from 0 to 9 weeks.A negative linear relationship was found between log₁₀mean timeto germinate and probit final germination, regardless of pre-treatment,indicating that partial desiccation and chilling are interchangeablein promoting germination of hydrated seed. For nearly fullyhydrated seeds, increasing the chilling period from 6 to 26weeks increased the viability-loss onset point for desiccationinjury from near 40% to about 48% moisture content without alteringthe drying rates of seed tissues. Extending moist chilling invarious seed lots from 0 to 26 weeks decreased subsequent longevityat 16 °C. For 26-week-chilled seeds longevity (the periodto lose one probit of germination) differed above and belowa threshold moisture content of 48%. It remained constant inthe moisture-content range 48–38%, but increased progressivelyas moisture content was raised above 48%. This threshold moisturecontent coincided with the value above which chilled seed pre-germinatedin storage. The results indicate that post-harvest desiccationand chilling alter the water relations of various physiologicalprocesses and a schematic summary is presented which relatesthe results to an axis water sorption isotherm.Copyright 1998Annals of Botany Company Aesculus hippocastanumL., horse chestnut, chilling, moisture content, water potential, desiccation tolerance, longevity, recalcitrant seed, embryo axis, maturation, germination.     

Changes in Cell Fine Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

When germination begins, the storage cells of Phaseolus vulgariscotyledons are packed with starch grains and protein bodies.Digestion of these reserves starts in cells furthest away fromthe vascular bundles and is practically completed in eight daysat 25° C. After the reserves are hydrolysed, the storagecells die. The changes in fine structure during the processof digestion and protoplasmic breakdown are described. Vascularbundle and epidermal cells survive till the cotyledons absciss,but in these tissues also profound changes occur in cellularorganization. The observations on fine structure are discussedwith reference to the metabolic activities of the cotyledons.     

Water Status Changes During Development in Relation to the Germination and Desiccation Tolerance of Aesculus hippocastanum L. Seeds

Seed growth characteristics of Aesculus hippocastanum were examinedin detail during development from about 70 to 140 d after anthesis(DAA), mainly in 1988 and 1989. Mean fresh and dry weights increasedfor both the axis and the whole seed up to the time of peakseed fall at 135 DAA with no cessation before fruit abscission.Water per seed increased up to 100 DAA, after which no furtherincrease occurred; moisture content declined for the embryonicaxis and whole seed respectively from above 75 and 65% at 95DAA to 65 and 50% at 130 DAA. At fruit shedding in 1990 waterpotential values of -1·2, -2·6 and -1·1MPa were observed for the testa, cotyledon and axis tissuesrespectively; relevant sorption isotherms are presented. Decreases in seed moisture content during development were accompaniedby increases in desiccation tolerance and in germinability,both reaching their maximum at the time of peak seed fall. Atmaturity, only about 10% viability was retained on drying seedto 20% moisture content; it is confirmed that the seeds are'recalcitrant'. The exact relationship between moisture contentand germination during development was dependent on the deptof dormancy, as judged by the period of chilling required; eachduration of chilling at 2°C within the range 3-12 weeksyields a curve of sigmoid shape. No germination occurred at26°C without chilling, but nearly full germination can beobserved for samples collected at 6 weeks before maximum seedfall with 12 weeks chilling. The rate of moisture loss duringdesiccation at 15°C and 15% rh becomes reduced during development.The ontogeny of these 'recalcitrant' seeds is compared withthat of 'orthodox' seeded species and the implication of sigmoid-shapedcurves for the relationship between seed moisture content andgermination are considered.Copyright 1993, 1999 Academic Press Aesculus hippocastanum L., horse chestnut, seed development, water status, germination, desiccation intolerance, desiccation rate     

Evidence for the involvement of cytokinins in the regulation of proteolytic activity in cotyledons of germinating beans

The proteolytic activity of an extract of cotyledons of Phaseolusvulgaris increased during the first 7 days of germination, bothwhen casein was the substrate and when the endogenous proteinserved as such. The increase was partially dependent on thepresence of the embryo axis. The effect of the axis was replaceableby kinetin or zeatin, but not by gibberellic acid or IAA. During the period of 3–6 days after the beginning of incubation,the level of amino acids in cotyledons attached to the embryoaxis was lower than in detached ones. No such difference couldbe detected in the first 2 days of incubation. On the otherhand, the influence of the axis on protease activity alreadywas clearly detectable on the second day of germination. Applicationof casein hydrolysate to the seeds brought about an increasein the concentration of amino acids in the cotyledons, but nosimultaneous decrease in the protease level could be detected.We concluded that these results do not confirm the hypothesisthat the influence of the axis on proteolytic activity is dueto its function as a sink for amino acids produced in the cotyledons. (Received August 9, 1979; )     

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.