Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.     

Does Cytokinin Transport from Root-To-Shoot in the Xylem Sap Regulate Leaf Responses to Root Hypoxia?

We have examined the hypothesis that cytokinins transportedfrom roots to shoots affects leaf growth, stomatal conductance,and cytokinin concentration of leaves of Phaseolus and a hybridpoplar (Populus trichocarpa x Populus deltoides) with hypoxicroots. Because cytokinins may interact with other substances,potassium and calcium concentrations were determined in xylemsap of Populus plants with hypoxic and aerated roots while gibberellin(GA) concentrations were measured in shoot tissues. Root hypoxiadecreased leaf growth and closed stomata in both species. Inboth species, fluxes of cytokinins out of the roots were reduced,but no differences in bulk leaf concentrations were measuredbetween the hypoxic and aerated plants. Shoots with aeratedroots contained slightly higher concentrations of GA₁ and GA₃than shoots from hypoxic plants. There were no differences incalcium or potassium concentrations in xylem sap between aerationtreatments. Exogenously applied cytokinins did not alleviatethe growth or stomatal responses caused by root hypoxia. Informationon the site(s) and mechanism(s) of cytokinin action and theways in which cytokinins are compartmentalized within plantcells will be required to understand the physiological significanceof cytokinin transport in the transpirational stream. Key words: Cytokinins, hypoxia, Populus, Phaseolus     

Evidence in support of biosynthesis de novo of free cytokinins

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, ( ²-isopentenyl)adenosine and 2-methylthio-N⁶-( ²-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N⁶-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.     

Changes in the level of endogenous cytokinin-like substances in Acer pseudoplatanus embryos during stratification and germination

The bioassay used to detect and quantify cytokinin activity was the Amaranthus test. Free cytokinin-like substances in embryos of Acer pseudoplatanus L. fruits increased during the first 20 d of fruit stratification at 5°C, but subsequently fell rapidly to values well below the amounts present in the embryos of freshly harvested fruits. These lower levels persisted throughout the remainder of a 60 d stratification period. Bound cytokinins fell during stratification from the highest detected levels present in freshly harvested material to values which were lower by about one third. No peaks of bound cytokinin activity were detected at any stage during stratification. In embryos from fruits stored at 17°C and unable to germinate, both free and bound cytokinins remained at a very low level throughout the 60 d period. Embryos from fruits previously stratified for 60 d showed increases in both free and bound cytokinins during the first 24 h of their incubation at 20°C in light, but after longer incubation periods up to 72 h, cytokinin concentrations decreased again to levels similar to those present at the commencement of the incubation period. Determinations conducted in 1979 and 1980 showed quantitative differences, but similar qualitative changes were observed in the two years. Most of the cytokinin activity was associated with compound(s) that co-chromatographed with zeatin and zeatin riboside.     

Efficient shoot regeneration from internodal explants of Populus angustifolia, Populus balsamifera and Populus deltoids

In the present study, interactions between the duration of treatment with auxin and different cytokinins and their effect on shoot regeneration were evaluated with the aim to establish a rapid and efficient in vitro regeneration method applicable to a variety of Populus species. Three different species, Populus angustifolia, P. balsamifera, and P. deltoids, were chosen for that purpose. We were successful in regenerating plantlets from stem and petiole explants from all three chosen species using a four-step simple procedure. The first step was callus induction when the explants were exposed to an auxin-rich medium for 0-20 days. During the second step, they were transferred onto a cytokinin-rich medium for shoot bud induction. In the third step, the shoots regenerated were transferred onto a medium with reduced levels of cytokinins to promote shoot proliferation and elongation; finally, in the fourth step, the shoots were rooted and acclimated. A short period (6-10 days) of time of exposure to auxin was sufficient for shoot regeneration. A culture time longer than ten days in callus induction medium drastically reduced the efficiency of shoot regeneration. Besides, cytokinin type and concentration also affected the frequency of shoot induction. A 0.2 mg/l concentration of 2,4-D for callus induction followed by 0.02 mg/l of Thidiazuron for shoot formation proved to be the best treatment for adventitious shoot bud multiplication, generating a maximum of 10-13 shoots of P. balsamifera and P. angustifolia in ten weeks. In contrast, for P. deltoids, a combination of 1.1mg/l 2,4-D, 1.0mg/l NAA, 0.1mg/l zeatin for callus induction followed by a combination of 1mg/l zeatin plus 1.0mg/l BA for shoot bud induction was found to be the most effective, generating on average 15 shoots over a period of ten weeks.     

Cytokinins and flower bud formation in vitro in tobacco: role of the metabolites

Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7-glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound.     

Cytokinins in Populus x robusta (schneid): Light effects on endogenous levels

Summary Cytokinin levels in both attached and detached mature leaves of poplar (Populus x robusta) increase transiently after short periods of exposure to red light. The degree and rapidity of response seems dependent on the physiological condition of the leaves. The cytokinin, 6-(2-hydroxybenzyl)amino purine riboside, specifically increases after red light treatment. Diurnal changes of leaf cytokinins occur, with a pronounced peak of activity being present at daybreak.     

The interplay between cytokinins and light during senescence in detached Arabidopsis leaves

Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild‐type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis‐zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N⁶‐(Δ²‐isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.     

The mechanism of gall induction makes galls red

We propose that the commonly observed red coloration of insect-induced plant galls is due to the production of exogenous cytokinins by gall-inducing insects. A growing body of evidence indicates that gall-inducing insects, bacteria, and fungi produce cytokinins. We hypothesize that gall induction generally requires an exogenous source of cytokinin and auxin. Plant galls are mobilizing sinks induced by cytokinin and reinforced by transport and accumulation of sugar. Exogenous cytokinins lead to a cascade of effects including the up-regulation of anthocyanin synthesis, the source of red coloration. Experiments demonstrate that exogenous cytokinins and sugars up-regulate the phenylpropanoid and flavonoid pathways, leading to localized anthocyanin accumulation. We suggest that red coloration in plant galls is merely a consequence of the mechanism of gall induction, and therefore an example of fabricational noise rather than aposematic coloration. Only color manipulation experiments can determine whether gall color is also secondarily aposematic.     

Effects of gibberellin A3 and cytokinins on natural and post-harvest, ethylene-induced pigmentation of Satsuma mandarin peel

Natural and post-harvest ethylene-induced pigment changes in the rind of Satsuma mandarin ( Citrus unshiu Marc.) fruits respond differently to the exogenous application of growth regulators. Both gibberellin A₃ and the synthetic cytokinins N⁶-benzyladenine and kinetin opposed the ethylene-induced chlorophyll destruction, while the loss of chlorophyll during natural maturation was retarded by the gibberellin but not by the cytokinins. This different behaviour suggests that ethylene may not be playing a central role in the endogenous control of ripening. Carotenoid accumulation during natural maturation is apparently controlled through a different mechanism than chlorophyll loss since it is reduced both by the cytokinins and gibberellin A₃.
Kinetin and gibberellin A₃ increased to a similar extent the accumulation of reducing sugars and free amino acids, and reduced that of non-reducing sugars in the peel during natural maturation. Their differential effect on chlorophyll loss may not be explained through their effects on sugar accumulation.     

Hormonal Complex and Ultrastructure of Maturing Aesculus hippocastanum Seeds

The content and temporal changes in the endogenous IAA, cytokinins, gibberellin-like compounds (GLC), and ABA were determined during horse chestnut (Aesculus hippocastanum L.) seed development (the stages of embryo axis development, its active growth, and storage compound deposition). The active growth of the embryo was characterized by the highest amounts of free phytohormones. Later, by the end of seed maturation, we observed the accumulation of the bound forms of IAA and ABA and a trend to a decrease in the content of free IAA, zeatin, and GLC (butanol fraction). The electron-microscopic examination of the embryo from the mature seed demonstrated that some structural components of the cytoplasm were similar in the cells of embryo axes and cotyledons. During the entire period of maturation, the embryo cells preserved native vacuoles and protein bodies were not formed. Thus, the structure of cotyledonary and axial cells and the distribution of free and bound phytohormones in the horse-chestnut seeds are similar to those in maturing seeds characterized by exogenous dormancy.     

植物细胞分裂素的糖苷物生物活性分析

用附加细胞分裂素及其糖苷物的MS培养基,垂直培养拟南芥并测定根的伸长,以此分析细胞分裂素及其糖苷物的生物学活性的结果表明,细胞分裂素的N-糖苷物几乎完全失去细胞分裂素活性,而D-糖苷仍具有细胞分裂素活性。推测两类糖苷物对植物体内细胞分裂素活性可能有不同的调控作用。     

亚种间杂交稻根系伤流液与地上部细胞分裂素的变化

本文以水稻亚种间杂交组合Ⅱ优2070及恢复系2070、Ⅱ优419及恢复系中419为材料．应用HPLC和ELISA法测定灌浆期间根系伤流液中根源细胞分裂素(CTKs)种类和数量以及稻叶和籽粒中细胞分裂素组分含量的变化,研究表明在亚种杂交稻及其恢复系灌浆起始阶段,玉米素(Z) 占总CTKs的比例高达62．8％-89．1％,是根系伤流液中细胞分裂素的主要成分,而二氢玉米素(di- HZ)和二氢玉米素核苷(diHZR)则在灌浆后期明显上升．这种变化动态与Ⅱ优2070和2070剑叶中ZRs(Z ZR)、diHZRs(diHZ diHZR)的波动变化是相符合的。与根系伤流液的主要细胞分裂素组成不同,IPAs在叶片和籽粒中占总CTKs量的比例最高。说明在灌浆期间,根系伤流液中CTKs的种类及其活性存在着变化,这种变化可能与细胞分裂素代谢酶活性变化及其相关酶基因表达的不同有关。讨论了强、弱势粒的CTKs组分的变化与籽粒结实的可能关系。     

亚种间杂交稻根系伤流液与地上部细胞分裂素的变化

本文以水稻亚种间杂交组合Ⅱ优2070及恢复系2070、Ⅱ优419及恢复系中419为材料．应用HPLC和ELISA法测定灌浆期间根系伤流液中根源细胞分裂素（CTKs）种类和数量以及稻叶和籽粒中细胞分裂素组分含量的变化．研究表明在亚种杂交稻及其恢复系灌浆起始阶段,玉米素（Z）占总CTKs的比例高达62．8％-89．1％,是根系伤流液中细胞分裂素的主要成分．而二氢玉米素（diHZ）和二氢玉米素核苷（diHZR）则在灌浆后期明显上升,这种变化动态与II优2070和2070剑叶中ZRs（Z＋ZR）、diHZRs（diHZ＋diHZR）的波动变化是相符合的。与根系伤流液的主要细胞分裂素组成不同,IPAs在叶片和籽粒中占总CTKs量的比例最高。说明在灌浆期间．根系伤流液中CTKs的种类及其活性存在着变化,这种变化可能与细胞分裂素代谢酶活性变化及其相关酶基因表达的不同有关。讨论了强、弱势粒的CTKs组分的变化与籽粒结实的可能关系。     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis

Plants display a number of responses to low phosphate availability, involving biochemical and developmental changes. Recently we have shown that many of these responses can be repressed in roots by exogenous addition of cytokinins. In order to understand the genetic basis to this effect of cytokinins, and its relation with the better known roles of cytokinins in the control of cell-cycle and differentiation, we have undertaken mutant screening and characterization using a transgenic line of Arabidopsis thaliana harbouring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS). One type of mutant identified displayed reduced sensitivity of AtIPS1::GUS to cytokinin repression. Several other Pi starvation response genes showed reduced cytokinin sensitivity in these lines. These mutants also showed reduced cytokinin repression of the anthocyanin accumulation induced by Pi starvation in the aerial part of the plants. Mapping and molecular characterization of these mutants showed that they were allelic of CRE1/WOL, a locus known to encode a cytokinin receptor. CRE1 is downregulated by Pi starvation and induced by cytokinins, both in the wild-type and in the cre1 mutants, in which cre1 mRNA levels are higher. These results reveal the existence of a positive feed-back loop, in addition to the already established negative feedback loop, in cytokinin signalling and indicate that the negative regulation of Pi starvation responses by cytokinins involves a two-component signalling circuitry, as it is the case of other types of cytokinin response.     

Endogenous Phytohormones and Regulation of Morphogenesis of Arabidopsis thalianaby Blue Light

We studied the effects of blue light (BL) on the levels of endogenous phytohormones (IAA, ABA, gibberellins, and cytokinins) and morphogenesis of the 7-day-old Arabidopsis thaliana(L.) Heynh seedlings of wild type (Ler) and its hy4mutant with a disturbed synthesis of cryptochrome 1 (CRY1), which is a receptor for BL. In darkness, the mutant contained considerably less free IAA and zeatin, but much more ABA as compared to the wild-type seedlings. BL retarded the hypocotyl growth in the wild-type seedlings but stimulated it in the mutant. Elongation of mutant hypocotyls was accompanied by accumulation of free IAA and a decrease in the content of free ABA; the level of cytokinins did not change. We believe that the response of the hy4hypocotyls to BL is mediated by a BL receptor distinct from cryptochrome 1. The conclusion is that light and hormonal signals interact in the control of the hypocotyl growth in A. thalianaseedlings.     

Changes in the carbohydrate and hormonal status in <Emphasis Type="Italic">Tulipa bifloriformis</Emphasis> bulbs forced into bloom in a greenhouse and in the open ground

Time-course of ABA, cytokinins, monosaccharides (MS), and water-soluble polysaccharides (WSP) contents were followed during cold dormancy period in the bulbs of tulip (Tulipa bifloriformis Vved.) that stayed over winter in the open ground or were cold-forced into bloom in a greenhouse. In both cases, the level of monosaccharides and water-soluble polysaccharides in the storage scale tissues increased, whereas the MS/WSP ratio in the bulbs planted in the open remained essentially the same and in case of forcing treatment decreased almost fivefold. In the apical bud tissues, the level of monosaccharides also rose in both cases, but the MS/WSP ratio in the open was greater throughout the whole experiment. The level of cytokinins in the apical bud tissues in the open was higher than in the forcing case, although the changes in their total content were identical following both treatments. Following the forcing treatment, the contents of free and bound ABA in the apical bud tissues increased reaching their peaks by the end of cold period. In the open, there were two peaks of free ABA: in October (when early frosts occurred) and in March (at the end of the wintering period). The winter forcing treatment resulted in rapid depletion of energy and plastic resources in T. bifloriformis and the death of 20% of embryonic flower buds (in the open, all flower buds survived). Nevertheless, plant adaptation potential ensured the development of generative shoots with 4–5 normal flowers, which makes it possible to use this species of multiflorous tulip for winter forcing in a greenhouse.     

黄土区树种抗旱性指数的研究

根据ＰＶ曲线主要水分参数测定数据,进行了黄土区６个乔,灌木树种抗旱性季节变化趋势和树种间抗旱性指数排序分析,结果表明,树种抗旱性指数在生长季（５－１０月）呈具有阶段降低的持续增大趋势,仅在雨季中后期（即树木年第２４生长高峰期）略有减小,在年生长季土壤严重干旱阶段,树种的抗旱性指数大小排序为：海红〉河北杨〉柠条〉小叶杨〉北京杨〉刺槐。