The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid     

Coaction of three factors controlling chlorophyll and anthocyanin synthesis

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, P _fr) and an excision factor. It was found that the three factors operate additively provided that the P _fr level is high enough. When the P _fr level is below approximately 1 per cent (<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low P _fr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.Abbreviations Chl chlorophyll(ide) a - P _fr far-red absorbing form of phytochrome - P _fr/P _tot ratio at photoequilibrium - RL red light - FR far-red light - GL green light - RG9 light long wavelength far-red light - WL white light     

Increase in anthocyanin yield from wild-carrot cell cultures by a selection system based on cell-aggregate size

Wild-carrot (Daucus carota L.) cell cultures were screened to yield small (less than 63 m) or large (greater than 170 m) cell aggregates which were then subcultured. Cultures of the small-size class had a higher, those of the large-size class a lower anthocyanin yield than the unscreened culture. This relationship became accentuated with an increasing number of passages with screening prior to subculture. At the end of six months (12 passages), the pigment yield of the small-size class was triple that of the unscreened cells. Following this selection period, the tendency of the small-size fraction to increase in clump size when subcultured without screening was much less than that of freshly isolated cell aggregates of the same size. These observations may be explainable on the basis of a differential distribution of cytokinin between aggregates of different sizes. High levels of cytokinin inhibit anthocyanin accumulation and inhibit cell separation; these effects result in large cell aggregates having low levels of anthocyanin. In support of this hypothesis, it is shown that addition of kinetin to cultures of small cell aggregates causes an increase in the size of cell aggregates and a parallel decrease in anthocyanin yield.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine     

The influence of growth regulators absorbed by the root on sex expression in hemp plants

Application, through the root system, of growth regulators to hemp (Cannabis sativa L.) plants having 2–3 pairs of visible leaves caused pronounced shifts of sex expression in the adult individuals. Treatment with gibberellic acid (25 mg/l) resulted in more than 80% of the plants being male, i.e. having staminate flowers (controls, ca. 30%). Treatment with 6-benzylaminopurine and with indole-3-acetic acid (in either case, 15 mg/l) resulted in all plants being either female (pistillate flowers) or intersexes (bisexual flowers); treatment with abscisic acid (10 mg/l) had a similar but somewhat less pronounced effect.     

Regulators of cell division in plant tissues

[³H]zeatin was supplied through the transpiration stream to de-rooted lupin (Lupinus angustifolius L.) seedlings. The following previously known metabolites were identified chromatographically: 5-phosphates of zeatin riboside and dihydrozeatin riboside, adenosine-5-phosphate, zeatin riboside, zeatin-7-glucopyranoside, zeatin-9-glucopyranoside, adenine, adenosine and dihydrozeatin. Five new metabolites were purified; four of these contain an intact zeatin moiety. Two were identified unequivocally, one as l--[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]alanine, a metabolite now termed lupinic acid, and the second as O--d-glucopyranosylzeatin. These two compounds were the major metabolites formed when zeatin solution (100 M) was supplied to the de-rooted seedlings. The radioactivity in the xylem sap of intact seedlings, supplied with [³H]zeatin via the roots, was largely due to zeatin, dihydrozeatin and zeatin riboside. When [³H]zeatin (5 M) was supplied via the transpiration stream to de-rooted Lupinus luteus L. seedlings, the principal metabolite in the lamina was adenosine, while in the stem nucleotides of zeatin and adenine were the dominant metabolites. O-Glucosylzeatin and lupinic acid were also detected as metabolites. The level of the latter varied greatly in the tissues of the shoot, and was greatest in the lower region of the stem and in the expanding lamina. Minor metabolites also detected chromatographically were: (a) dihydrolupinic acid, (b) a partially characterized metabolite which appears to be a 9-substituted adenine (also formed in L. angustifolius), (c) glucosides of zeatin riboside and/or dihydrozeatin riboside, and (d) O-glucosyldihydrozeatin. While lupinic acid supplied exogenously to L. luteus leaves underwent little metabolism, chromatographic studies indicated that O-glucosylzeatin was converted to its riboside, the principal metabolite formed, and also to adenosine, zeatin and dihydrozeatin. A thinlayer chromatography procedure for separating zeatin, dihydrozeatin, zeatin riboside and dihydrozeatin riboside is described.Abbreviations Me₃Si trimethylsilyl - TLC thin-layer chromatography - UV ultraviolet XXIV=Gordon et al., 1975     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - PPi-PFK pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) This study was supported by grants from the Danish Research Counsil (SJVF 13-4148 and 13-4547 to P.U. SJVF 13-4146 and 13-4494 to T.H.N.) and from The Research Center for Plant Biotechnology to P.U.