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     

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     

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; )     

A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris

Kinetin and the embryo axis acted similarly in bringing abouta promotion of amylase activity in cotyledons of Phaseolus vulgaris.No promotive effect of gibberellic acid or indole-3-acetic acidon amylase activity could be detected. It is suggested thatthe regulatory action of the embryo axis on starch degradationin the cotyledons of P. vulgaris is mediated by cytokinins. (Received May 4, 1970; )     

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     

Cytokinins in Root Nodules of Phaseolus mungo

Four cytokinins have been separated from extracts of root nodulesof Phaseolus mungo by thin-layer chromatography. Their activitywas determined on the basis of their ability to induce betacyaninsynthesis in cotyledons of Amaranthus caudatus. Zeatin and itsriboside showed greater activity than N⁶ (²-(isopentenyl)) aminopurine and its riboside in the bioassay. Phaseolus mungo, mung bean, cytokinins, isopentenyl, amino-purine, zeatin, betacyanin synthesis, Amaranthus caudatus     

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     

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.     

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     

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     

Sodium Recirculation and Loss from Phaseolus vulgaris L.

In a split-root experiment, ²²Na was supplied to Phaseolus vulgarisL. roots emerging from the stem, 2.5 cm above the main roots.Sodium exported from these upper roots was translocated a shortdistance upward in the stem and downward to the main roots.Most of the ²²Na arriving in the main roots was lost to themedium. Sodium loss from P. vulgaris roots into KCI or NaCl was similarand was not affected by oligomycin. The results confirm a previous hypothesis regarding the mechanismof sodium exclusion from the tops of sodium non-accumulatorplants. Phaseolus vulgaris L., bean, sodium transport     

Respiration Rate, Mitochondrial Activity and Mitochondrial Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

Parallel observations have been made on changes in respirationrate, mitochondrial activity, and the fine structure of mitochondria,in the cotyledons of Phaseolus vulgaris germinating at 25^? Cin the dark. While oxygen uptake of the intact cotyledons risesto a peak between the third and fifth days of germination, mitochondrialactivity falls from 36 hours onwards. In most of the storagecells, mitochondrial cristae have become swollen and the matrixhas darkened by the third day. The continulng rise in respirationrate beyond 36 hours could be accounted for by the increasein soluble oxidative activity, which occurs while mitochondrialactivity is falling. Alternatively, it could be mediated bymitochondria from the vascular bundle cells, where swellingand darkening are delayed till after the fifth day of germination,and where there is proliferation of cristae till the fourthday.     

The Messenger RNA Population in the Embryonic Axes of Phaseolus vulgaris During Development and Following Germination

Misra, S. and Bewley, J. D. 1985. The messenger RNA populationin the embryonic axes of Phaseolus vulgaris during developmentand following germination.—J. exp. Bot. 36: 1644–1652. Messenger RNAs were extracted from young, mid-maturation, late-maturation,mature-dry, and 20-h-germinated embryonic axes of Phaseolusvulgaris cv. Taylor's Horticultural. They were translated invitro in a rabbit reticulocyte lysate protein synthesizing system.Analysis of the translation products using two-dimensional polyacrylamidegel electrophoresis indicated that there were substantial changesin the messenger RNA populations of developing and germinatingaxes. The number of polypeptides synthesized increased sharplyat 20–22 d after pollination and then declined. Therewas a parallel increase and decrease in the Poly(A)⁺ contentof the seed axis. The analysis showed that certain messageswere present throughout development and were stored in maturedry seed. These messages were degraded upon subsequent rehydration.Some messages appeared during mid-maturation but declined duringlater stages of development and were absent from the matureseed. In the germinating seed a set of messages unique to germinationappeared. Key words: —Seed development, germination, mRNA, in vitro translation, Phaseolus vulgaris     

Diurnal Rb+ Transport from Roots to the Laminar Pulvinus in Phaseolus vulgaris L.

The primary leaves of kidney bean (Phaseolus vulgaris L.) openunder light and close in the dark by the deformation of thepulvinus resulting from diurnal distribution changes of K⁺,Cl^–, organic acid (or H⁺) and NO₃^–. When Rb⁺ was added as a tracer of K⁺ to the seedlings throughtheir roots, it was transported to the pulvinus cells duringthe light period but not during the dark period. Transpirationoccurred vigorously in the light but almost stopped in the dark.We concluded that Rb⁺ absorbed by the roots was carried to thepulvinus by the transpiration stream. Phaseolus vulgaris L., pulvinus, Rb⁺, diurnal transport transpiration stream     

Functional aspects of Abscisic Acid Metabolism in Cotyledons of Phaseolus vulgaris L. Seedlings

During the early stages of germination and vegetative development,cotyledons of intact bean (Phaseolus vulgaris L.) seedlingsshowed active ABA catabolism causing a low endogenous ABA content.At the end of the substrate mobilizing phase, when the cotyledonsbecame senescent, a drastic increase of the endogenous ABA contentlinked with a decrease of the ABA catabolic activity was observed.This indicates that a close connection exists between senescenceand endogenous ABA content and metabolism in bean cotyledons. Removal of the apical growth region induced in the cotyledonsactivation of the ABA catabolism and the endogenous ABA concentrationdecreased below the detection limit (0.1 ng/g fr wt) at theonset of the outgrowth of the axillary buds. From then, apicaldominance was restored and the cotyledons returned to the senescentstate, which was correlated with a drastic increase of theirendogenous ABA content. ¹ Bevoegd verklaard navorser N. F. W. O. ² Wetenschappelijk medewerker F. K. F. O. (Received November 25, 1980; Accepted February 13, 1981)     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium in the Elongating Stem ofPhaseolus vulgarisL. A Physiological Assessment

Plasmolytic disruption of plasmodesmata interconnecting metaphloemsieve element-companion cell complexes with small and largephloem parenchyma cells in the elongating region of internode2 ofPhaseolus vulgarisL. seedlings did not affect accumulationof phloem-imported¹⁴C-photosynthates and⁸⁶rubidium. The membrane-impermeantdye, 5(6) carboxyfluorescein, loaded into leaf phloem as themembrane-permeant diacetate ester, was found not to move radiallyout of the importing sieve elements in the internode elongationregion. In contrast, the apoplasmic tracer, Calcuofluor White,rapidly moved laterally throughout all tissues of the elongationzone. Hexoses, sucrose and potassium were identified as themain osmotica in internode apoplasmic sap. Label asymmetry in[¹⁴C](fructosyl)sucrose was retained on accumulation by excisedstem segments. Uptake of [¹⁴C]sucrose and⁸⁶rubidium by stemsegments exhibited saturation kinetics. Sucrose uptake was inhibitedby the slowly penetrating sulphydryl reagent, para-chloromercuribenzenesulphonicacid.In vitrorates of sucrose uptake, at apoplasmic concentrations,corresponded to its predictedin vivorate of delivery to thestem ground tissues from mature sieve elements when respiratorylosses were assumed to be confined to the stem phloem. For potassium,the total delivery rate could be accounted for by itsin vitrorateof uptake. Overall, it was concluded that radial transport,in the elongation zone of internode 2 ofPhaseolus vulgarisL.seedlings, follows an apoplasmic route from mature sieve elementsto stem ground tissues.Copyright 1998 Annals of Botany Company PhaseoluLes vulgaris, apoplasm, elongating stem, French bean, photosynthates, potassium, radial transfer, symplasm.     

Effects of Nutrient Supply on Pre-emergence Growth and Nutrient Absorption in Wheat (Triticum aestivumL.) and Sugarbeet (Beta vulgarisL.)

Nutrient absorption in wheat and sugarbeet was studied duringpre-emergence growth by adding 0, 7, 10.5 or 14 mol m^-3nitrogen(N) to the growth medium. Seedling growth and carbon, N and¹⁵Ncontents of the seedling parts were measured. Differences betweenthe natural abundance of¹⁵N in seeds and in nutrient solutionwere used to determine the proportion of N in the organs originatingfrom seed reserves and from absorption. Absorption began laterfor wheat than for sugarbeet and had less effect on seedlinggrowth. The absorbed N was found mainly in roots. Compared towheat, sugarbeet seedling N content was greatly altered andthe hypocotyl showed increased elongation when nutrients wereadded. Most of the absorbed N was found in the radicle and hypocotylwith less in the cotyledons. Sugarbeet seedling emergence andearly growth could be decreased by adverse conditions occurringafter sowing by affecting mineral availability in the soil orthrough altered root absorption.Copyright 1998 Annals of BotanyCompany Triticum aestivumL., wheat,Beta vulgarisL., emergence, natural isotopic composition, seedling, seed reserves     

Response of Cultured Embryos of Phaseolus vulgaris and Hordeum vulgare to Farnesol

Excised embryos of Phaseolus vulgaris incubated in a mediumcontaining 10 mg dm^–3 farnesol showed enhanced root growthwhereas the leaves remained rudimentary At lower concentrationsof exogenous farnesol normal leaf development occurred and rootgrowth was comparable to untreated cultures. Enhanced root growthalso occurred when excised embryos of Hordeum vulgare were treatedwith farnesol but only at 10 mg dm^–3 and this treatmentdid not prevent leaf growth X-ray micro-probe analysis of leavesrevealed an increased phosphorus content in P vulgaris and adecreased sulphur content in H vulgare in comparison to untreatedplants. Hordeum vulgare L., barley, Phaseolus vulgaris, bean, embryo culture, farnesol, X-ray microprobe analysis, root growth     

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

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