The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: II. THE EFFECTS OF 6-BENZY LADENINE AND GIBBERELLIC ACID UPON EMBRYONATED AND DETACHED COTYLEDONS

The presence of the embryo axis promotes starch hydrolysis incotyledons of Phaseolus vulgaris and, although reduced ratesof enzyme activity proceed in its absence, weight loss fromthe cotyledon is only significant in its presence. Applicationof gibberellic acid at various concentrations had no effectupon amylase or protease activity in either detached or embryonatedcotyledons: this was not the case for 6-benzyladenine whichpromoted both. In detached cotyledons amylolytic activity wasdirectly proportional to the concentration of applied 6-BA;concentrations below 10^–6 M were unable to completelysubstitute for the presence of the axis, while, above 10^–6M, hydrolytic activity higher than that of treated embryonatedcotyledons was observed. Increasing the concentration of 6-BAhad no effect, however, upon the amylolytic activity of embryonatedcotyledons. Detached cotyledons showed an increased chlorophyllcontent and 6-BA treatment further increased this; the proportionsof chlorophyll a and chlorophyll b remained unaltered. An inhibitor of amylase activity, destroyed by heating, is presentin embryonated cotyledons and can depress amylolysis in detachedcotyledons which appear to be inhibitor-free. The results intotal suggest that the developing axis may regulate reservehydrolysis in three distinct manners: firstly, by stimulatingthe synthesis of amylase via hormonal control, most probablya cytokinin; secondly, by checking the rate of amylolysis bymeans of an amylase inhibitor; and, thirdly, by being a sinkfor the products of reserve degradation.     

cytokinin-induced amylolytic activity in bean cotyledons: Identification of the regulated enzyme

Separation of an extract of cotyledons of Phaseolus vulgarison a column of Sephadex G-200 revealed three amylolytic fractions.The slower migrating fraction hydrolyzed/ß-Limit dextrinazure and was inhibited by EDTA. The activity of this fractionwas enhanced by the embryo axis and this effect could be fullyreplaced by kinetin or benzyladenine. These results suggestthat the bean embryo axis exerts a promotive influence on theactivity of a-amylase in the cotyledons and that this effectis mediated by cytokinins. The other two amylolytic fractions did not show a-amylase activity.No effect of the embryo axis or of cytokinins on their activitycould be noted. ¹Present address: The Thimann Laboratories, University of California,Santa Cruz, California, U.S.A. (Received June 27, 1979; )     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. II. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the radicle tips of germinatingPhaseolus vulgaris seeds revealed that cytokinins are transportedrapidly from the embryonic axis to the cotyledons, and are utilizedand metabolized extensively in these organs. The informationobtained on the transportation between the different parts ofthe embryo is consistent with the view that the mobilizationof food reserves from the cotyledons is controlled by cytokininswhich originate in the embryonic axis. Tentative identificationof the radioactive metabolites formed indicate that the appliedzeatin was altered by side-chain cleavage and by substitutionto the adenine ring. Phaseolus vulgaris, bean, germination, cytokinins, transport, radicle     

Changes in Abscisic Acid Content in Axis and Cotyledons of Developing Phaseolus vulgaris Embryos and their Physiological Consequences

Prevost, I. and Le Page–Degivry, M. Th. 1985. Changesin absicisic acid content in axis and cotyledons of developingPhaseolus vulgaris embryos and their physiological consequences.—J.exp. Bot. 36: 1900–1905.Changes in abscisic acid (ABA)content with time were measured in embryonic axes and in cotyledonsof Phaseolus vulgaris embryos using a radio–immunoassay.During embryogenesis, a similar pattern was observed in bothtissues: ABA increased to a maximum 29 d after an thesis, followedby a decrease as the seed matured. The level of ABA in the cotyledonswas always much higher than that in the axes. In in vitro cultures,the duration of the lag phase before germination of isolatedembryonic axes increased with ABA content. The presence of cotyledonsalways lengthened the lag phase; longer lag phases were associatedwith greater concentrations of ABA in the cotyledons. Moreoverthe presence of cotyledons stimulated the growth of seedlings. Key words: ABA distribution, embryo maturation, axis and embryo germinability     

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

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

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

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     

Vigna Radiata Seed Germination under Salinity

Salinity reduced mung bean (Vigna radiata Wilczek) radicle and root elongation, delayed and inhibited hypocotyl elongation and mobilization of reserves from the cotyledons to the embryo axis. Fresh and dry masses and water content of the embryo axes were reduced. Under salinity, a net leakage of K to the media increased with time and increasing NaCl concentrations. Sugars present in the cotyledons of seeds were of primary importance for growth of the embryo axis upto 18 h after sowing whereas breakdown of starch by amylase contributed later, the contribution being delayed and reduced with increasing NaCl concentration. Even when amylase activity in the cotyledons was progressively reduced with increasing NaCl concentration, the increasing contents of soluble sugars in the cotyledons indicated that sugars were not limiting for mung bean seedling growth under salinity.     

On the nature of the hormonal regulation of amylase activity in cotyledons of germinating peas

The effect of the embryo axis and various growth substanceson amylase activity in cotyledons of germinating peas was studied. The embryo axis brought about an elevation in the level of enzymeactivity. Gibberellic acid, kinetin, benzyladenine and zeatinriboside also promoted the activity, but none of these substancesbrought about a full replacement of the axis. Such a replacementwas caused by zeatin or by a simultaneous application of zeatinriboside and gibberellic acid. The results suggest that the regulative effect of the embryoaxis on amylase activity in cotyledons is brought about eitherby zeatin alone or a cooperative action of a cytokinin (or severalcytokinins) and a gibberellin (or several gibberellins). (Received November 30, 1974; )     

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

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

Studies into Causes of Non-germination of Aged Wheat Seeds

Excised embryos of aged non-germinating wheat seeds, when placedon sucrose/glucose germinated well and grew into normal plantlets,while on agar alone they remained ungerminated as in the intactseed. From the results of amylase assays in seeds of differentages it appears that failure of amylolytic activity in agedseeds could be a cause of unavailability of utilizable subtrateto the embryonic axis of aged seeds. This would bring abouta limitation in the capacity of germination and growth of theembryonic axis even before the embryo becomes non-viable. Wheat (Triticum aestimon L. cv. Sonalilca), embryo, germination, sucrose, amylase     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. III. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the cotyledons of germinatingbean seeds demonstrated that cytokinins are not readily exportedfrom the cotyledons to the embryonic axis during the early stagesof this process. In the cotyledons the applied zeatin is metabolizedextensively to metabolites which are polar and which occur atR_F 0·2–0·5 on paper chromatograms. Thesemetabolites are stable and are not readily exported from thecotyledons. In contrast the metabolites found at R_F 0–0·2are more readily exported. When exported to the radicles andplumules a large proportion of the translocated metaboliteswere converted to compounds which on paper co-chromatographedwith zeatin. This seems to suggest that the embryonic axis hasthe capacity to synthesize cytokinins and that some of the metabolitesformed during its catabolism can also be used for its synthesis. Phaseolus vulgaris, bean, germination, cytokinins, transport, cotyledons     

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; )     

Changes in the Content of Starch and Activities of some Enzymes of Carbohydrate Metabolism in Cotyledons of Germinating Seeds of Voandzeia subterranea

During the germination of the seeds of Voandzeia subterraneaL. Thouars, the content of starch in the cotyledons fell progressively.There was a substantial increase in amylase activity duringthe first 4 d. The activity of starch phosphorylase increasedafter 6 d when there was a rapid decline in amylase activity.The activities of hexokinase, glucose-6-phosphate dehydrogenase,and 6-phosphogluconate dehydrogenase increased, suggesting thepresence of an active phosphogluconate pathway in the cotyledons.     

Does osmotic regulation of amylase activity in bean cotyledons exist?

The hypothesis that the promotive effect of the embryo axis of the germinating bean seed on amylase activity in the cotyledons is mediated by an osmoregulative mechanism was examined. After 2 days of germination the action of the axis on amylolytic activity was already clearly revealed, whereas at the same time it did not have any influence on osmotic pressure in the cotyledons. When the axis was attached to one cotyledon during 4 days of incubation, osmotic pressure in the cotyledon was lower than its value in the cotyledons of the intact seedling, whereas amylolytic activity was similar in both treatments. It was concluded that the tested hypothesis is not valid in the case of the bean seedling. External osmotic agents brought about a decrease in the level of amylase in the cotyledons, but this does not prove that osmotic changes which are brought about by production of internal metabolites are involved in the regulation of amylase synthesis.     

Seedling Growth in Phaseolus vulgaris L. The Early Growth of Cultivars Selected for Seedling Cold Tolerance

Silbernagel has described a test for selecting cultivars ofPhaseolus vulgaris L. which exhibit rapid seedling emergenceat low temperatures, and using this test has identified threecold-tolerant cultivars. We have compared the growth of thesecultivars with that of three control cultivars. Both at 25/20°C and at 20/15 °C day/night temperatures, Silbernagel'scultivars emerged more rapidly than the controls. This was dueto more rapid hypocotyl elongation, not to earlier germination.Rapid hypocotyl elongation was associated with high relativegrowth rates of the seedling axis and rapid loss in weight ofthe cotyledons. After emergence, cotyledons of all cultivarscontinued to lose weight at a constant exponential rate. Relativegrowth rates of the axes were not constant but declined withtime. There was no evidence that genotypic differences in growthrates before emergence were reflected in growth rates afteremergence. A simple quantitative analysis suggested that thedecline in axis relative growth rate after emergence was dueto a declining contribution from cotyledonary reserves. There were significant differences between cultivars in theinitial weight of the seedling axis. Axis weight (A) was notlinearly proportional to seed weight (S), but the curvilinearallometric relationship A = 0.0773 S^0.697 satisfactorily accountedfor most of the variation in initial axis weight between cultivars.In all cultivars, axis weights at emergence were smaller inthe cool regime than in the warm, because low temperatures depressedaxis relative growth rates relative to the rate of emergence.The biggest difference between the Q₁₀ of relative growth rate,and of emergence rate, and hence the biggest effect of low temperatureon axis weight at emergence, occurred in the cold-susceptiblecultivar Seafarer. However, genotypic cold tolerance duringthe period when growth is dependent on reserves did not appearto guarantee cold tolerance during the main period of growth. Phaseolus vulgaris, L., bean, seedling growth, temperature, cold tolerance     

Strcture of the Mature Embryo of Theobroma cacao L.

The mature embryo of Theobroma cacao L. consists of an embryonicaxis and two cotyledons. The cotyledonary tissue comprises thebulk of the embryo and contains mature and maturing vasculartissue surrounded by a bundle sheath. The cells of the groundtissue are filled with starch grains, anthocyanin pigments,or mucilage. The embryonic axis consists of epicotyl, hypocotyl,and root meristem. Maturation of the vascular tissue has begunin the procambium, where xylem maturation is ahead of phloemmaturation. Mucilage cells are found throughout the tissue of the axis.Most of the cells of the embryonic tissue contain starch grains.     

Effect of iso-osmotic levels of salts and PEG-6000 on enzymes in germinating pea seeds

Effects of iso-osmotic levels of salts (NaCl, CaCl₂, Na₂SO₄) and PEG-6000 on the activity of hydrolytic and nitrogen assimilatory enzymes in pea embryo axis and coty ledon were studied. The activity of nitrate reductase and nitrite reductase in embryo axis and cotyledon and the activity of protease and α-amylase in cotyledon decreased with decreasing medium osmotic potential as compared to control at all the stages of seedling growth. The activity of protease and amylase increases with increasing levels of stress in embryo axis. Sodium chloride induced, stress had more deleterious effects on the activity of nitrate reductase, nitrite reductase and αamylase followed by other salts and PEG-6000. On the other hand, CaCl₂ induced salt stress was more depressive for protease activity. The maximum increase in the activity of protease and amylase was observed in embryo axis at higher concentration of salts and PEG-6000.     

Host‐mediated induction of α‐amylases by larvae of the Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is irreversible and observed from the initiation of the feeding period

Larvae of Zabrotes subfasciatus secrete α‐amylases that are insensitive to the α‐amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non α‐amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible α‐amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible α‐amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10‐day‐old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible α‐amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible α‐amylases remained up‐regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible α‐amylase isoforms. Incubations of brush border membrane vesicles with the α‐amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes. © 2010 Wiley Periodicals, Inc.     

胚轴对萌发豌豆子叶中淀粉酶活性表达的影响

萌发豌豆的上、下肢轴均能诱导子叶中淀粉酶活性,外源GA和6—BA具有类似胚轴的作用。离体子叶的淀粉酶凝胶电泳只有一条活性极低的酶带,连生子叶中有两条酶带,其中由胚轴诱导新出现了一条活性很高的同工酶带,它的活性受亚胺环己酮的强烈抑制,而受放线菌素D影响不大。推测豌豆子叶中存在淀粉酶的长寿命mRN—A,胚轴和外源激素的作用在于促进mRNA的翻译。