Uptake of benzyladenine by excised watermelon cotyledons

The uptake of 8-[¹⁴C]N⁶-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.     

Effects of benzyladenine and abscisic acid on the disassembly process of photosystems in anArabidopsis delayed-senescence mutant,ore9

Using wild-type (WT) leaves and those from anore9 delayed-senescenceArabidopsis mutant, we investigated the delaying and accelerating effects of benzyladenine (BA) and abscisic acid (ABA), respectively, on the degradation process of the photosynthetic apparatus during dark-induced senescence (DIS). In the mutant, delays were seen for both the breakdown of chlorophyll (Chl) and the decrease in photochemical efficiency of photosystem II (Fv/Fm). Moreover, each step was prolonged in the disassembly process of the Chl-protein complexes. In the presence of BA, Chl degradation was retarded to a similar extent for both the mutant and the WT, but the decrease in Fv/Fm was not. However, in the presence of ABA, the two processes were accelerated in both genotypes. Therefore, although theore9 mutation causes this functional delayed-senescence, it may not be related to the non-functional delay that happens afterwards. In contrast, BA seems to affect both processes.     

Starch content and distribution in excised watermelon cotyledons treated with benzyladenine

We have examined the effect of benzyladenine (BA) on amyloplast number and distribution in semithin cross-sections of excised watermelon ( Citrullus vulgaris Schrad ., cv. Fairfax) cotyledons grown in the dark. The sections were stained with Lugol solution and observed with an immersion objective. In control cotyledons, amyloplasts were always more abundant and contain more starch grains than in BA-treated cotyledons. The higher starch content was correlated with a higher ratio between starch synthetase (EC 2.4.1.21) and amylase (EC 3.2.1.1) activities in the controls. In the central zone of the mesophyll, the amyloplasts contained more starch grains than in the abaxial and adaxial (palisade) zones. The average number of amyloplasts per cell was different in the three zones, and followed a different time course in control and treated cotyledons. BA decreased this number, particularly in the adaxial zones. Our data seem to indicate a different function of starch in the central and in adaxial zone of the mesophyll. In the central zone starch is probably used as a source of carbohydrates for export to the embryo axis, while in the adaxial zone, where the transformation from amyloplast to etioplast is particularly pronounced, starch may be a reserve for organelle differentiation. BA stimulates the utilization of starch for both functions.     

Synthesis of DNA in excised watermelon cotyledons grown in water and benzyladenine

Excised watermelon cotyledons were grown in water and benzyladenine, which greatly promotes growth, breakdown of reserves and development of organelles. In order to investigate the involvement of DNA synthesis in these benzyladenine-induced effects, [³H]thymidine was applied continuously (for 3 d) or administered briefly (5 h) to excised cotyledons at various stages of development. Autoradiographic analysis of squashed and sectioned cotyledons showed that both the cytoplasm (mainly in the region of the plastids) and most of the nuclei were labelled. Both types of labelling were promoted by benzyladenine treatment. The highest percentage of labelled nuclei was found in the early stages of growth (first day after excision of cotyledons), long before the burst of enzymatic activities involved in the germination processes. The possible meaning of the increase of nuclear DNA, apart from the normal replicative synthesis preceding cell division, is discussed.Abbreviations BA N⁶-benzyladenine - DNase deoxyribonuclease - EtBr ethidium bromide - FUdR fluorodeoxyuridine - [³H]T [methyl-³H]thymidine     

Effect of benzyladenine on the polypeptide pattern of plastids in excised watermelon cotyledons

We have shown in a previous paper that plastids of watermelon ( Citrullus vulgaris Schrad., cv. Fairfax) cotyledons differentiate into amyloplasts when the cotyledons are grown in water and into prochloroplasts when they are grown in benzyladenine (BA) solution. In the present work we have tested whether this large difference in development of the plastids is accompanied by equally conspicuous changes in their polypeptide pattern. Cotyledons were grown for 4 days in the dark either on distilled water or on 10⁻⁵ M BA. Alternatively they were transfered to 10⁻⁵ M BA after 4 days of growth in water.
Plastids of control cotyledons had a rather simple polypeptide pattern. The only prominent protein bands were the two subunits of ribulose bisphosphate carboxylase (EC 4.1.1.39). Contamination with storage protein was present. Plastids from BA-treated cotyledons had a much more complex polypeptide pattern. No storage protein contamination was observed. Polypeptide bands present only as traces in the control and having molecular weights of 32, 44, 48 and 49 kDa increased in intensity after an exposure of only 6 h to BA. The 32-kDa band seemed to be most dependent on the presence of the hormone. Contrary to what was observed in plastids, BA seemed to have no influence on the polypeptide pattern of mitochondria.     

Changes in abscisic acid levels during dormancy release in freesia corms

A high level of free-abscisic acid (ABA) was detected when corms were still in deep dormancy. The level of free-ABA decreased as the corm dormancy disappeared and increased temporarily after complete release from dormancy. A gradual slight increase of bound-ABA was observed during dormancy release.Treatment of dormant corms with benzyladenine (BA) increased sprouting but the sprouts did not show normal growth. Ethylene treatment induced complete sprouting and subsequent normal growth. Changes in ABA levels and ethylene production are discussed in relation to dormancy release in freesia corms.     

Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons

Excised watermelon (Citrullus vulgaris Schrad.) cotyledons were grown in the dark in the presence of 0.1 mM benzyladenine (BA). Under these conditions reserve breakdown and organelle differentiation progress very slowly. Treatment with BA accelerates, breakdown of reserves and stimulates development of organelles. Electron micrographs of cells from treated cotyledons show a larger number of plastids with a more developed inner membrane system. The levels of plastid pigments and enzymes are increased while starch content is reduced. Glyoxysomal enzyme levels are increased by BA during the first three days of development and their decline is accelerated thereafter. Also the activity of hydroxypyruvate reductase (EC 1.1.1.81.), a peroxisomal enzyme, is increased, but this increase is not followed by a decay phase. In water controls, hydroxypyruvate reductase bands together with glyoxysomal enzymes after equilibrium centrifugation in a sucrose gradient. In treated cotyledons the equilibrium position of glyoxysomal enzymes is uchanged while that of hydroxypyruvate reductase is shifted to a lower density.Abbreviations BA benzyladenine - RuDP ribulose-1,5-diphosphate - HPR hydroxypyruvate reductase     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.     

Current advances in abscisic acid action and signalling

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.     

Early changes in morphology and polypeptide pattern of plastids from watermelon cotyledons induced by benzyladenine or light are very similar

Watermelon ( Citrullus vulgaris Schrad. cv. Fairfax) cotyledons were excised from the embryo and grown in the dark for 4 days. They were then transferred to 10 μm benzyladenine (BA) solution or illuminated with white light. We have compared changes in ultrastructure of the plastids and of their polypeptide pattern induced by the two treatments.
At the end of the 4-day-period in the dark the plastids differentiated to amyloplasts and had few polypeptides: only the two subunits of ribulose bisphosphate carboxylase (RuBP) were clearly observed. Both light and BA induced starch depletion and gratia formation after 12–24 h. BA was less efficient than light in inducing thylakoid formation and more efficient in inducing starch depletion. After 6 h both factors induced the appearance of the same new polypeptides in the 28–53 kDa range. Most prominent among them is a 32 kDa band. Light is much more effective in inducing the formation of a 29 kDa band than is BA. In mature chloroplasts this band stains very strongly, while the 32 kDa band disappears. We suggest that the 29 kDa polypeptide is the light harvesting complex (LHC), since a purified LHC preparation from cotyledons grown either on water in light or on BA in the dark migrates on the polyacrylamide gel as a single 29 kDa band.     

Free and bound abscisic acid in relation to water potential in aging cotyledons of Lupinus albus

To evaluate the capacity for biosynthesis of abscisic acid (ABA) in the cotyledons of developing plants of Lupinus albus L. (= L. termis Forssk.) cv. Giza 1, and the physiological role which the compound may perform during senescence, the levels of free and bound forms of ABA have been estimated in conjunction with the natural changes in the water potential of the tissues during a period of 18 days after sowing. In the cotyledons of the dry seeds, the bound form of ABA is about three times as abundant as the free form. Peaks of the free ABA occur on days 3 and 8, when the water potential reaches minimum values of –1060 and –950 kPa respectively. Since the concentration of the bound ABA does not drop during days 1–8, it is suggested that the peaks of free ABA are due to synthesis in the expanding cotyledons in response to the two water potential minima. During the post expansion period (days 9–18), free ABA appears to be released from a bound form as a consequence of decreased synthetic activity and increased tissue deterioration. The remarkable increase in the rate of the dry weight loss which immediately follows each peak of ABA suggests the involvement of ABA in the senescence of the cotyledons by speeding up the translocation of nutrients to the developing axis.     

The biosynthesis of abscisic acid in Cercospora rosicola

1′-Deoxyabscisic acid (1′-deoxy-ABA) has been isolated from cultures of Cercospora rosicola which are actively synthesizing abscisic acid (ABA)     

A simplified purification method for the analysis of abscisic acid

A fast and convenient method is given for the purification of abscisic acid (ABA) in plant extracts. Using a dual pH elution system on Sep-Pak C18 reverse phase prepacked cartridges, abscisic acid appears in the third eluent (32% methanol pH 8.0). The cartridges can be regenerated for multiple reuse.     

Stimulation of chlorophyll synthesis by benzyladenine and potassium in excised and intact cucumber cotyledons

Both benzyladenine (BA) and potassium (K) stimulated chlorophyll synthesis in cucumber ( Cucumus sativus L. cv. National Pickling) cotyledons. However, differences existed between the effects of BA and K. Stimulation of chlorophyll synthesis by BA (1 mg l⁻¹, 4.4 μ M ) was observed in excised cotyledons after 4 and 8 h of illumination but not after 24 h, whereas the stimulation caused by K (40 m M ) continued. In contrast to BA, K was unable to eliminate the lag phase of chlorophyll production, and it also required light for its stimulation of cotyledon expansion. Both BA and K were required to maximize cotyledon expansion and chlorophyll production. In intact plants, K was not limiting for chlorophyll synthesis since foliar or soil pretreatments with K did not markedly stimulate greening. Foliar pretreatment with BA stimulated chlorophyll levels in intact plants, whereas soil pretreatment with BA inhibited chlorophyll production, probably because BA was not readily transported from the roots to the shoot and created a "sink" effect. Inhibitor studies showed that stimulation by K of greening did not depend on RNA or chloroplastic protein synthesis to the extent that has been reported for BA. Thus it appears that BA and K stimulate chlorophyll synthesis via different mechanisms, although both cytokinins and K are essential for maximum rates of greening.     

ABA对水稻幼苗叶片光抑制的光保护作用

经ABA处理的水稻幼苗叶片和对照相比 ,PSII光化学效率 (Fv/Fm)和非光化学猝灭系数 (qN)显著受抑制。经高光处理 1h后 ,ABA处理的水稻幼苗叶片光抑制程度比对照小 ,这暗示ABA对高光光抑制具有一定的光保护作用 ,且间接表明ABA提高水稻幼苗抗光抑制的能力与叶黄素循环密切相关。     

Effects of abscisic acid on CAM in Portulacaria afra

Water stress induces Crassulacean acid metabolism (CAM) in Portulacaria afra as manifested by day stomatal closure, organic acid fluctuation, and night CO₂ uptake. We now have evidence that abscisic acid treatment of leaves causes partial stomatal closure that is accompanied by the induction of CAM in a manner similar to water stress. There appears to be an inverse relationship between exogenous CO₂ uptake and decarboxylation of organic acids in that organic acids remain high during the day providing stomata are open. When stomata close, there is consumption of organic acids by decarboxylation. The hypothesis is that stomatal opening controls CAM in this species.This material is based upon work supported by the Science and Education Administration of the USDA under Competitive Grant No. 5901-0410-8-0018-0.     

Incorporation of α-ionylidene ethanol and α-ionylidene acetic acid into abscisic acid by Cercospora rosicola

²H-Labelled α-ionylidene ethanol and α-ionylidene acetic acid are converted in high yield to 1′-deoxy-abscisic acid (1′-deoxy-ABA) and absc     

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)⁺ RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient electrophoresis-SDS polyacrylamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis, both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1–2 h). Among them, three were repressed markedly     

GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata

Abscisic acid, phaseic acid and 4′-dihydrophaseic acid were identified by GC-MS of derivatized (Me, MeTMSi) extracts from immature fruits of Vigna unguiculata. The fruits also contained some other ABA-related compounds, one of which might be epi-4′-dihydrophaseic acid while another was tentatively identified as 6′-hydroxymethylabscisic acid.