Determination of Microgram Quantities of Stevioside from Leaves of Stevia rebaudiana Bert. by Two-Dimensional Thin Layer Chromatography

A method is described for the determination of stevioside fromleaves of Stevia rebaudiana Bert. Separation upon non-activatedsilica gel impregnated with boric acid in a two-dimensionalchromatographic system followed by clution and colorimetricassay is reported. Recuperation from chromoplates is over 95%.The observed ratio of the optical density of glucose: steviosidematches that expected theoretically so that glucose may be employedas a standard where pure stevioside is not available. Effects of the plant growth retardant, ancymidol, on the growthand morphology of the shoot system of cucumber (Cucumis sativusL. ) were investigated. Ancymidol inhibited stem elongation,reducing both number and length of internodes. Reduction inleaf area, attributable to a reduction in both cell size andnumber, was accompanied by an increase in chlorophyll per unitarea. The retardant decreased apical dominance and delayed anthesis.Gibberellic acid fully reversed ancymidol-induced inhibitionof stem elongation, internode length and production, and leafexpansion. GA_4/7 and ancymidol gave a synergistic promotionof stem elongation by increasing elongation of younger internodesand increasing internode production. Synergistic promotion ofpetiole elongation by this combination was also observed. Ancymidol,applied 7 d previously either to the shoot or root, severelyreduced the level of gibberellin-like activity in bleeding sapcollected from decapitated plants.     

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.     

Early Seedling Growth in Normal and Opaque-2 Zea mays L. cv. Maya: I. COMPOSITION AND MOBILIZATION OF THE ENDOSPERM PROTEIN RESERVE

Grain of op-2 Maya have a higher total protein content in bothendosperm and germ than normal maize. In large grain, differencesin total grain protein are due mainly to the endosperm whilein small grain, differences are due to the germ, particularlythe scutellum which is larger in op-2 kernels. Reserve proteincomposition was distinct: op-2 endosperms showed an increasein the proportions of albumins, globulins, and the glutelinG₃ fraction which was accompanied by a marked decrease in zeincontent in comparison with normal endosperms. During endosperm depletion there was a net increase in the salt-solublefraction prior to day 3 from imbibition; glutelins and zeinhowever decreased throughout, the former being degraded at initiallyfaster rates. In general, regardless of protein composition,the most abundant fraction was utilized most rapidly. Water uptake during imbibition was greater in op-2 grain, thoughno differences in axis growth in terms of dry weight or laminaarea were apparent at the end of reserve degradation. In bothnormal and op-2 seedlings, protein was mobilized both earlierand faster than endosperm dry matter and the shoot, principallythe first leaf lamina, received more reserve nitrogen than theroot.     

The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: I. CHANGES IN TOTAL AMYLOLYTIC AND PROTEOLYTIC ACTIVITY AND THE EFFECTS OF 6-BENZYLADENINE AND GIBBERELLIC ACID UPON WHOLE SEEDLINGS

Axis growth commenced only 20 h from imbibition in Phaseolusvulgaris when both uptake of water and oxygen were levellingoff. Cotyledonary dry material was exhausted by day 9, the greatestrates of transfer occurring from day 2. Utilization of reserveprotein proceeded relatively faster than dry matter and proportionatelymore was translocated to the shoot, particularly the leaf blades.Lamina protein and chlorophyll content showed a biphasic increasewith rapid synthesis prior to day 7 being followed by slowerrates up to maximum leaf area on day 17. The level of free aminoacids in the cotyledons fell continuously from day 3 and roseto peak levels in the axis between days 5 and 7. Soluble sugarsincreased throughout the period examined in the axis and accumulatedin the cotyledons prior to day 7. Exogenous application of GA₃ had marked morphological effectsupon the seedling though did not significantly alter the distributionof dry matter or protein reserves: 6-BA, in addition to distinctmorphological effects, delayed the mobilization of reservesto the axis. Total amylolytic activity in the cotyledon, optimal at pH 5.5,increased continually from day 3 to 9 whereupon activity abruptlydeclined. This increase was due to the appearance of -amylase;ß-amylase, while present, remained at a constant andcomparatively low level. Peak proteolytic activity occurredprior to that for amylases between days 5 and 7. 6-BA significantlyincreased both amylolytic and proteolytic activities in vitrothough did not alter the changes in levels of free amino acidsor sugars in the reserve tissue and axis. The discrepancy betweenin vivo and in vitro effects of 6-BA may be attributed to concomitanteffects upon seedling development whereby sink capacity is reduced.     

The Effect of Long and Short Day Length upon the Growth of Whole Plants and the Level of Soluble Proteins, Sugars, and Stevioside in Leaves of Stevia rebaudiana Bert.

Long-day conditions increase internode length, leaf area, anddry weight and reduce the interval between the appearance ofsuccessive leaf pairs in S. rebaudiana as compared with shortdays. Total soluble leaf sugars, protein, and stevioside contentare also augmented in both absolute and relative terms and thebiosynthesis of steviol, the aglucone present in stevioside,is increased by 45%. The interaction between dry matter, steviosidecontent, and day length is discussed and a theory for the biologicalrole of stevioside as a possible defence mechanism against insectherbivory is proposed: as such this is the first known caseof a tetracyclic diterpene glycoside assuming this function.     

Changes in the Levels of Total Soluble Proteins and Sugars During Leaf Ontogeny in Stevia rebaudiana Bert.

Leaf d. wt and the levels of soluble sugars and proteins showa two-phase development during leaf growth in Stevia rebaudiana.The initial large increases in leaf size are due mainly to waterintake up to an area of around 9–10 cm². Increases inabsolute protein content were initially slow though in the secondphase increased it rapidly with dry matter and soluble sugarcontent. In relative terms, however, the concentration of free sugarsdeclined throughout leaf growth. The data indicate that leafprotein synthesis is most probably dependent upon a carbon supplyfrom in situ photo synthesis which only becomes significantat 80 per cent of full leaf area. Stevia rebaudiana Bert., leaf ontogeny, protein content, sugar content     

Activities of Transaminases, Amylases and Proteases during Endosperm Degradation in Normal and Opaque-2 Zea mays L. cv. Maya

Transaminase, amylase and protease activities were comparedin seedlings of normal and Opaque-2 (o2) maize. Transaminaseactivity, greater in normal maize, was highest in the scutellumfrom which it decreased rapidly in activity from day 2 afterimbibition; only low activity was observed in endosperm andaxis tissue. Amylolytic activity, optimal around pH 5, was greater in normalmaize at all stages of endosperm degradation. Activity whichwas low on day 2, rose to a peak on day 6 and declined afterwards.The level of free sugars in the endosperm of normal was higherthan in o2 maize, and in both varieties was highly correlatedwith amylolysis. Protease activity, optimal at pH 3.6, was also greater in normalendosperms and increased up to day 6 and activity was maintainedat this level until around day 14. Although the activity ofall three enzyme systems examined was greater in normal maizethere were no apparent differences in the overall growth ofnormal and o2 seedlings during this period. Zea mays L, maize, corn, endosperm, enzyme activity, transaminase, amylase, protease     

The Effect of Time of Nitrate Application Upon the Growth of Barley Cultivars of Differing Endogenous Nitrogen Levels: Longer Term Experiments

The effect of time of nitrate application was examined in cultivarswith either a high or low endogenous nitrogen content in thegrain. In low-nitrogen cultivars delay in the application ofnitrate beyond day two from planting leads to considerable reductionsin total dry matter, particularly of the shoot. Maximum leaflength of all the main-stem leaves was reduced and their dateof emergence retarded by several days. There was no effect uponthe final length of tiller leaves, though tiller emergence wasappreciably retarded. These effects were absent or less markedin cultivars with a high endogenous nitrogen content or wherelow-nitrogen cultivars were supplied with nitrate during germination.The results in total indicate that inadequate nitrogen supplyto the seedling axis produces irreversible detrimental effectsupon plant growth which may ultimately reduce yeild. Hordeum vulgare L. barley, growth nitrogen content     

The Effects of Grain Nitrogen and Applied Nitrate on Growth, Photosynthesis and Protein Content of the First Leaf of Barley Cultivars

Five cultivars of barley with widely differing grain nitrogencontents were compared. In the absence of exogenous nitratesupply plants grown from high nitrogen grain showed a more rapidleaf emergence, greater leaf size, especially of the first leaf,higher photosynthetic rate and greater total souble proteinand Fraction 1 protein content of the first leaf, than plantsgrown from low nitrogen grain. However, early supply of nitrateto plants grown from low nitrogen grain enabled these to performas well as those from grain with a high nitrogen content. Regressionanalysis showed that Fraction 1 content of the first leaf isclosely correlated with grain nitrogen which exerts a progressivelygreater effect on content of this protein as application ofexogenous nitrate is delayed. The more rapid photosyntheticrate of plants grown with high nitrogen, and the consequentgreater rate of dry matter accumulation, is attributable mainlyto effects of nitrogen availability on leaf area and much lessto effects on leaf protein.     

The Utilization of Endosperm Reserves During Early Growth of Barley Cultivars and the Effect of Time of Application of Nitrogen

The cultivars Akka and Hiproly with high grain nitrogen andJulia and Foma with low grain nitrogen and Proctor containinghigh or low nitrogen were compared. The high nitrogen forms germinated more rapidly and by day 6had a higher axis dry weight than the low nitrogen types. Byday 6 dry weight of tops and roots was similar in the high nitrogencultivars but that of the tops was less in the low nitrogenforms. Analyses of the partition of nitrogen from endospermto axes confirmed that for all types this was maximal after48 h from planting and occurred at a similar relative rate sothat 50 per cent of the endosperm nitrogen was translocatedby 84–96 h from planting. Loss of endosperm dry weightwas much slower and here high nitrogen forms translocated drymatter at a significantly faster relative rate than the lownitrogen types. Nitrate supplied at planting or on day 2 increased the rateof endosperm depletion in the low nitrogen types and resultedin their achieving similar axis dry weights to high nitrogenforms which were largely unaffected by nitrate supply. Delayin nitrate application had progressively less effect on endospermbreakdown. There was a greater uptake of nitrate by the lownitrogen forms and rate of uptake was rapid. Uptake of nitrateby high nitrogen types was initially rapid but was followedby a much slower phase. The results indicate that endogenous and exogenous sources ofnitrogen are equally suitable for seedling growth. The significanceof grain nitrogen content in relation to endosperm breakdownis discussed as is the strategy of partition of endosperm reservesbetween root and shoot.