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; )     

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.     

Synthesis and Turnover of {alpha}-Amylase in Cotyledons of Germinating Vigna mungo Seeds: Effects of Exogenously Applied End-Products and Plant Hormones

Pulse-chase experiments indicated that the higher levels ofa-amylase in detached and incubated cotyledons of Vigna mungothan those in cotyledons attached to the embryonic axis weredue to both faster synthesis and slower degradation of the enzymein the detached cotyledons than in the attached cotyledons.Levels of a-amylase in the cotyledons were examined in termsof possible effects of end-products and the effects of exogenouslyapplied plant hormones and growth regulators. Levels of a-amylaseactivity and content were reduced by high concentrations ofglucose and sucrose, and it is suggested that this effect wascaused mostly by osmotic stress and partly by end-product repression.The level of a-amylase was nearly twice that in controls after1 to 10µM GA₃ had been applied to the cotyledons. In addition,0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.S mM naphthaleneaceticacid also increased the level by 34% to 66% as compared to thecontrol. ABA and uniconazole both prevented the synthesis ofa-amylase. (Received July 4, 1994; Accepted November 14, 1994)     

Analysis of Lipids of Citrullus lanatus (cv. Sugar baby) During Seed Germination and Seedling Growth

Changes in the dry weight (dry wt.), total and neutral lipids,and fatty acid composition were determined in the cotyledonsand axis of Citrullus lanatus cv. Sugar baby seedlings duringtheir first 12 d growth in the dark, at 25 ? 1 ?C. The major stored reserves were mobilized between days 3 and8. The lipid concentration after day 4 decreased rapidly inthe cotyledons. On the first day the lipid concentration inthe axis was higher than in the cotyledons but decreased rapidlyand from day 5 up to the end of the experiments it remainedat very low values. In the cotyledons, the change in the fattyacid composition was relatively small compared to the significantchange observed in the axis. The neutral lipids of the cotyledonsdecreased sharply between days 2 and 6 while their fatty acidcomposition suffered no significant change. The axis, comparedto the cotyledons, contained a small but concentrated amountof neutral lipids, which decreased up to day 6. Thereafter,an increase was observed. The fatty acid composition of theaxis neutral lipids changed significantly, linoleic and oleicacid decreased while palmitic and linolenic acid increased. Key words: Citrullus lanatus, seed germination, seedling growth, lipids, fatty acids     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Genotypic Differences in the Temperature Responses of Tropical Crops: II. SEEDLING EMERGENCE AND LEAF GROWTH OF GROUNDNUT (ARACHIS HYPOGAEA L.) AND PEARL MILLET (PENNISETUM TYPHOIDES S. & H.)

Mohamed, H. A., Clark, J. A. and Ong, C. K. 1988. Genotypicdifferences in the temperature responses of tropical crops.II. Seedling emergence and leaf growth of groundnut (Arachishypogaea L.) and pearl millet (Pennisetum typhoides S. &H.).—J. exp. Bot. 39: 1129-1135. Measurements of seedling emergence and leaf growth of five milletand seven groundnut genotypes were made at soil temperaturesranging from 7 to 27?C. The rate of seedling emergence (R_e)varied greatly between millet genotypes but R_e was remarkablysimilar in groundnut genotypes. In pearl millet there is a strongcorrelation between the rate of germination and the rate ofleaf production, hourly leaf extension and seedling emergence.The results are discussed in terms of the thermal time requirementsof various processes. Key words: Temperature, emergence, groundnut, millet     

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     

Phosphoenolpyruvate Carboxylase and CarbonEconomy of Apple Seedlings

Seeds of apple cv. Golden Delicious were germinated and cultivatedin the greenhouse until the third leaf emerged. Respirationofgerminating seeds or photosynthesis of the first leaves wasmeasured by infra-red gas analysis and porometry, respectively.To study the role of phosphoenolpyruvate carboxylase (PEPC),the dominant carboxylase in the carbon economy, its CO₂ refixationpotentialwas related to the amount of CO₂ lost in respiration. With arange of 0.2 (dry seeds) to 18 (cotyledons) µmol CO₂ h^–1g^–1 PEPC activity resembled or exceeded the amount ofC0₂ lost in respiration before the third leaf developed. Itis concludedthat PEPC largely contributes to economize the carbonmetabolism of apple seedlings before they become photosyntheticallycompetent. Key words: Apple (Malus pumila Mill.) seedling, carbon economy, phosphoenolpyruvate carboxylase, photosynthesis, respiration     

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     

Structure and Expression of a-Amylase Gene from Vigna mungo

A single copy of the a-amylase gene, composed of three intronsand four exons, was found in Vigna mungo. Examination of levelsof a-amylase and its mRNA in detached cotyledons indicated thatattachment of the embryonic axis is not required for expressionof the gene in cotyledons of germinating seeds. (Received December 21, 1993; Accepted March 14, 1994)     

Development of {beta}-amylase activity and polymorphism in wheat seedling shoot tissues8

Increasing ß-amylase activity in wheat (Triticum aestlvum,var. Star) seedling shoot tissues was consistently accompaniedby the development of a characteristic polymorphism of the enzyme,as shown by electrophoresis employing amylopectin-containingpolyacrylamide gels. Very young shoot tissue contained one principalform of the enzyme (ß1), whereas two other major forms(ß2, ß3) appeared complementary to thisupon further growth. In vitro incubation experiments indicatedthat the polymorphism arose via a probably proteolytic conversionof ß1 into ß2 and ß3. The conversioninvolved neither an activation of ß-amylase nor asignificant modification of ß-amylase component plvalues. The electrophoretic ß-amylase patterns ofsubcellular leaf compartments suggested that ß1 issynthesized in the cytoplasm of leaf mesophyfi cells and thatthe other forms arise upon transfer of this ‘primary’form into the vacuole. The development of shoot ß-amylaseactivity did not require light, but appeared to be under thenegative control of the chloroplast and was stimulated by mineralnutrients. No clear relationship between ß-amylaseactivity and starch metabolism was evident, since the leaf activitywas largely absent from mesophyll protoplasts, could not beunequivocally demonstrated in the mesophyll chioroplasts, anddeveloped regardless of whether the tissues contained significantamounts of starch or not. Key words: Wheat, leaves, ß-amylase, polymorphism, compartmentation     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.     

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; )     

ON THE EFFECTS OF PLANT EXTRACTS ON THE {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM

Effects of ethanol extracts obtained from several plant materialson -amylase activity in rice endosperm were examined. Promotingeffect on the -amylase activity was estimated in terms of gibberellinA₃ equivalence using the embryoless endosperm. Inhibiting effectwas examined using intact seed. On chromatography of the extractfrom Prunus, the zones showing promotion of the -amylase activityalso exhibited the activity for the elongation of the leaf sheathof rice seedling. But this was not the case with the extractsfrom Pharbitis, Allium and Lupinus. (Received April 13, 1966; )     

The Effect of Temperature on Leaf Appearance in Rice

Temperature is the principal environmental determinant of cropleaf appearance. The objective of this study is to analyse whetherthere are different effects of day temperature (T_D) and nighttemperature (T_N) on main-stem leaf appearance in rice (OryzasativaL.). Plants of 12 rice cultivars were grown at five constant temperatures(22, 24, 26, 28 and 32 °C) and four diurnally fluctuatingtemperatures (T_D/T_N: 26 /22, 30 /22, 22 /26 and 22 /30 °C)with a constant photoperiod of 12hd^-1. The leaf appearance onthe main stem was measured. A constant change in leaf appearance rate was observed duringontogeny. The relation between the number of emerged leavesand days from seedling emergence was described by a power-lawequation with only one cultivar-specific parameter. Values forthis parameter were estimated for the five constant temperaturetreatments, and the relation between this parameter and temperaturewas quantified by a nonlinear model. Leaf appearance for thefour fluctuating temperature treatments could be accuratelypredicted on the basis of these relations in each cultivar.This indicated that there were no specific effects ofT_DandT_Nonleaf appearance in rice, in contrast with phenological developmentto flowering. The optimum temperature for leaf development wasfound to be substantially higher than for development to flowering. The final main-stem leaf number differed with diurnal temperatureconditions. When a diurnal temperature delayed flowering, itincreased the leaf number as well. This might explain whyT_DandT_Nhada different effect on development to flowering but not on leafdevelopment. Oryza sativa; rice; leaf appearance; leaf number; day and night temperature     

Some Effects of Potassium Deficiency on Seedling Development

Seedlings with leaf-like photosynthetic cotyledons such as Cucumissativus, Sinapis alba and Trifolium pratense have higher growthrates and a greater requirement for an external supply of potassiumthan hypogeal species (Pisum sativum, Vicia faba) or epigealspecies with very fleshy cotyledons (Phaseolus vulgaris, Lupinusalbus, Lupinus angustifolius). The initial stages of seedlingdevelopment in the latter two groups involve mainly transferof reserves from cotyledons to axis and a relatively small increasein total seedling dry weight takes place during the first 2to 3 weeks. Potassium may be drawn either from the supply already presentin the cotyledons or from an external source. Species with expandingphotosynthetic cotyledons utilize the reserve potassium duringcotyledon development and it is not transported to the epicotyl.Thus, in these species an external supply of potassium is essentialfor full development of the photosynthetic system and the roots.In species whose cotyledons are not major photosynthetic organsthe reserve potassium is transported from the cotyledons tothe developing axis. In these species culture in a nutrientsolution deficient in potassium or in distilled water increasesroot dry weight but reduces plumule weight (although to a lesserextent than in species with leaf-like cotyledons). In general,the response of seedlings to culture in a nutrient solutiondeficient in potassium was very similar to culture in distilledwater only. Thus, although all the species studied had similar levels ofpotassium in the seed (µg potassium/mg dry weight) itssite of utilization differs. It appears that seedlings withleaf-like cotyledons are more susceptible during early seedlinggrowth to deficiencies in external factors, such as potassium,than are species with fleshy cotyledons.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Growth Analysis of Soybean Seedlings During the Lifespan of the Cotyledons

Patterns of seedling growth of Glycine max in light and darknesswere compared during the period from germination to cotyledonabscission. Fitted growth curves and the derived functions,relative growth rate, unit leaf rate, leaf area ratio and specificleaf area, were used to assess the relative importance in seedlinggrowth of cotyledon storage reserves, cotyledon photosynthesisand leaf photosynthesis. The cotyledons are of an intermediatetype with a predominant storage and a minimal photosyntheticfunction. Cotyledon reserves support seedling growth until theprimary leaves expand, after which growth depends on leaf photosynthesis. Glycine max (L.) Merr., soybean, cotyledons, growth analysis, seedling development     

The Photosynthetic Net Carbon Dioxide Exchange Potential in Conventional and 'Leafless' Phenotypes of Pisum sativum L. in Relation to Foliage Area, Dry Matter Production and Seed Yield

The effect of the ‘leafless’ mutation (in whichtendrils replace leaflets and the stipules are reduced to avestigial form) upon foliage area, photosynthetic net CO₂ uptakepotential, dry matter production and seed yield in Pisum sativumwas studied by comparing two near-isogenic lines of genotypeafafstst and ++++. The mutation is of potential agronomic valuein that it offers improved lodging resistance, crop drying andharvester throughput. In the conventional phenotype the total foliage area of themain axis attained a plateau (456 cm²) at day 56 from seedlingemergence, whereas corresponding values for the ‘leafless’mutant showed a total area of 208 cm² at day 68 with no indicationof a plateau. The agronomic consequence of this is discussed.During the vegetative phase of the plant the maximum CO₂ uptakepotential in the fully expanded conventional leaf was 8·5mg CO₂ leaf^–1 h^–1 and in the ‘leafless’mutant this value was 7·0 mg CO₂ leaf^–1 h^–1.For most ‘leaves’ of the latter phenotype this valuewas between 30 and 60 per cent less than for their conventionalcounterpart. There was a consistently higher photosyntheticpotential per unit area in tendrils of the ‘leafless’mutant than in leaflets of the conventional phenotype. The respectivemean specific values for the two phenotypes were 53 and 37 mgCO₂ dm^–2 h^–1. The problem of obtaining a meaningfulsurface area value for tendrils is discussed and the cylindricalnature of tendrils is taken into account. The ‘leafless’ mutant consistently accumulated 50per cent less dry matter than did conventional plants in theperiod from seedling emergence to anthesis and yield of maturedry seed per plant showed a reduction of 50 per cent both inseed number and total seed weight. The implications for future breeding and selection programmesaimed at haulm reduction are discussed in relation to evaluatingthe ability of the background genotype to produce adequate tendrilsin the presence of afafstst. Triticum aestivum, wheat, callus culture, organogenesis     

Lipoxygenase, Hydroperoxide Lyase and Volatile C6-Aldehyde Formation from C18-Fatty Acids during Development of Phaseolus vulgaris L.

Kidney bean plants (Phaseolus vulgaris) were found to have thecapability to produce C₆-aldehydes (hexanal and hexenals) fromlinoleic and linolenic acids. The various organs tested hadlipoxygenase and hydroperoxide lyase activities responsiblefor the C₆-aldehyde formation. Young leaves showed relativelyhigh activities for C₆-aldehyde formation. However, the activitiesof the leaves decreased gradually with leaf expansion. Seedlingsand seeds containing cotyledons showed low activities for C₆-aldehydeformation, because of the occurrence of an inhibitory factorin the cotyledons. The substrate specificity of the enzymeswas essentially the same among the various developmental stagesof leaves tested. (Received February 5, 1982; Accepted March 19, 1982)