ENDOGENOUS CYTOKININS IN THREE GENERA OF MICROALGAE FROM THE CHLOROPHYTA1

Nine axenic microalgal (Chlorophyta) strains from three genera (Protococcus, Chlorella, and Scenedesmus) were analyzed for endogenous cytokinins. Cytokinin‐like activity was detected using the excised cucumber cotyledon bioassay. Five strains showed no cytokinin‐like activity and four strains, low cytokinin‐like activity. Ethanolic extracts of the microalgae containing a mixture of deuterium‐labeled standards were purified using a combined DEAE‐Sephadex octadecysilica column and immunoaffinity column based on wide‐range specific mon‐oclonal antibodies and analyzed by HPLC linked to a micromass single quadrupole mass spectrometer with an electrospray interface and a photodiode array detector. There were similar trends in cytokinin profiles for the nine microalgal strains investigated, although concentrations did vary. Both isopentenyladenine and isopentenyladenosine were detected in all nine strains. cis‐Zeatin and cis‐zeatin riboside occurred at higher concentrations than the trans isomers, whereas trans‐zeatin‐O‐glucoside and trans‐zeatin riboside‐O‐glucoside were dominant over the cis isomers. Dihydrozeatin and its conjugates were not detected in any significant amounts. The aromatic benzyladenine always occurred at higher concentrations than benzyladenosine. The topolins were well represented with all three isomers (ortho, meta, and para) being detected, with ortho‐topolin and ortho‐topolin riboside occurring at higher concentrations than the other isomers. However, for the O‐glucosides, the meta isomers (meta‐topolin‐O‐glucoside and meta‐topolin riboside‐O‐glucoside) occurred at higher concentrations than the other isomers. No N‐glucosides were detected (isopentenyladenine‐9‐glucoside, zeatin‐9‐glucoside, dihydrozeatin‐9‐glucoside, benzyladenine‐9‐glucoside, ortho‐topolin‐9‐glucoside, and meta‐topolin‐9‐glucoside). Generally, zeatin and topolin conjugates were the dominant forms of isoprenoid and aromatic cytokinins, respectively. There was no distinct trend in the proportions of isoprenoid to aromatic cytokinins.     

Auxin and cytokinin relationships in 24 microalgal strains1

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection‐41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC‐712 the lowest biomass. The auxins, indole‐3‐acetic acid (IAA) and indole‐3‐acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49 nmol IAA · g^?1 DW and 0.18 to 99.83 nmol IAM · g^?1 DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29 nmol · g^?1 DW in Klebsormidium flaccidum MACC‐692 to 21.40 nmol · g^?1 DW in Stigeoclonium nanum MACC‐790. The general trend was that cis‐zeatin types were the predominant cytokinins; isopentenyladenine‐type cytokinins were present in moderate concentrations, while low levels of trans‐zeatin‐type and very low levels of dihydrozeatin‐type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O‐glucosides were low. Only one N‐glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2‐ to 4‐fold higher than the cytokinin content.     

ENDOGENOUS CYTOKININS,AUXINS, AND ABSCISIC ACID IN RED ALGAE FROM BRAZIL1

Endogenous cytokinins, auxins, and abscisic acid (ABA) were identified and quantified in 11 red algae collected from the Brazilian coast. Field materials and two isolates cultured in the laboratory were extracted with various solvents and buffers containing a mixture of appropriate internal standards, purified by solid‐phase extraction followed by immunoaffinity chromatography, and analyzed by liquid chromatography–tandem mass spectrometry. Isoprenoid cytokinins (free and conjugated forms of isopentenyladenine [iP], cis‐zeatin [cZ], and trans‐zeatin [tZ]) were detected in all species with concentrations of cZ and iP forms being higher than tZ forms. Dihydrozeatin (DHZ) and its metabolites were only detected at very low levels in nine of the studied species. Aromatic cytokinins (6‐benzylaminopurine [BA], ortho‐ and meta‐topolin [oT and mT]) were not detected in any of the samples. The cytokinin profile of Chondracanthus teedei (Mert. ex Roth) Kütz. was distinct in comparison to other species with para‐topolin (pT) derivatives detected in low concentrations. The main auxins present in all species were free indole‐3‐acetic acid (IAA) and indole‐3‐acetamide (IAM). Indole‐3‐ethanol (IEt), indole‐3‐acetyl glutamic acid (IAGlu), and indole‐3‐acetyl leucine (IALeu) were detected in a few species at low concentrations. ABA was present in all species analyzed except for Hypnea nigrescens Grev. ex J. Agardh. No ABA conjugates were detected in any species. These results confirm that cytokinins, auxins, and ABA were common constituents in red seaweeds, with this being the first report of the occurrence of ABA in Rhodophyta. The complexity of the hormone profiles suggests that plant hormones play a role in regulating physiological processes in Rhodophyta.     

Changes in Endogenous Cytokinins During Germination and Seedling Establishment of <Emphasis Type="Italic">Tagetes minuta</Emphasis> L.

Endogenous cytokinins were quantified and identified in germinating achenes and developing seedlings of Tagetes minuta L. incubated at 25 °C over a 144 h period. The process of germination (radicle emergence) was completed 38 h after commencement of imbibition. Subsequent growth was considered to cover seedling establishment. Eighteen isoprenoid cytokinins, belonging to the zeatin (9), dihyrozeatin (5) and isopentenyladenine (4) groups and one aromatic cytokinin, benzyladenine, were identified. The total isoprenoid cytokinin concentration increased upon imbibition, reached a peak by 48 h and subsequently decreased with seedling development. The individual cytokinin groups and the respective derivatives within each group did, however, not follow such a consistent trend. During the course of the experiment, the ribotides and ribosides were present in the highest concentrations, reaching a peak at 48 h and decreasing thereafter. The free bases and O-glucoside remained at low levels throughout the experiment. Isopentenyladenine-9-glucoside increased dramatically in the developing seedlings and after 144 h was the predominate cytokinin. Benzyladenine was the only aromatic cytokinin detected throughout the experiment. It was present in high concentrations in the dry achenes and declined rapidly upon imbibition.     

Optimizing the micropropagation protocol for the endangered <Emphasis Type="Italic">Aloe polyphylla</Emphasis>: can <Emphasis Type="Italic">meta</Emphasis>-topolin and its derivatives serve as replacement for benzyladenine and zeatin?

Benzyladenine (BA) is the most widely used cytokinin in the micropropagation industry due to its effectiveness and affordability. It, however, has disadvantages such as genetic alteration and abnormal growth in some plants. Naturally occurring zeatin on the other hand is not as widely used as BA and is far more expensive. The use of meta-topolin and its derivatives as alternatives to BA and zeatin, both of which frequently have negative effects in tissue culture was investigated. In vitro grown Aloe polyphylla (an endangered medicinal and ornamental aloe) were cultured on full strength Murashige and Skoog basal medium with different concentrations of cytokinins and solidified with 1% Bacteriological Agar (Oxoid No. 1). mT was the preferred cytokinin both in terms of multiplication rate and rooting. The optimum concentration that induced regeneration and rooting is 5.0 μM. The problem of hyperhydricity was totally controlled. Plants rooted spontaneously in multiplication medium, thus avoiding the extra rooting step of the protocol. More than 91% of the plants transferred to ex vitro conditions were successfully acclimatized.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

Spatial and temporal changes in endogenous cytokinins in developing pea roots

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5′-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ⁶ -(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.     

The aromatic cytokinins

After the discovery of kinetin (Miller et al. 1956, J. Am. Chem. Soc. 78: 1345–1350) there was a flurry of syntheses that led to the finding of 6-benzylaminopurine (BA), an active and easily obtainable cytokinin. Much research into cytokinin physiology was subsequently done with this substance. Further, the isolation and unequivocal identification of natural BA and the high biological activity of its meta -hydroxylated analogues stimulated the search for other natural aromatic cytokinins. Screening was accomplished by ELISA of HPLC fractions using antisera against ortho - and meta -hydroxybenzyladenosine. Subsequent isolation and decisive identification by mass spectrometry led to discovery of a broad spectrum of endogenous plant growth substances structurally similar to a highly active compound, meta -topolin (6-[3-hydroxybenzyl-amino]purine), and to its less active analogue, ortho -topolin (6-[2-hydroxybenzyl-amino]purine). The structures of such aromatic cytokinins suggest considerably different biosynthetic pathways from that of zeatin and related isoprenoid cytokinins. From a physiological viewpoint, aromatic cytokinin metabolism can be classified under four main headings analogous to isoprenoid cytokinins: interconversion, hydroxylation, conjugation, and oxidative degradation. This review attempts to put into context what is known about 9-alkyl-BAs and compares their metabolism in regard to the practical use of cytokinins in agriculture and biotechnology. The recently discovered unusual specificity of additionally C2,N9-disubstituted aromatic cytokinins toward cell cycle kinases, suggests that these cytokinin-derived growth regulators may selectively inhibit certain steps of the cell cycle. The functional overlap of the aromatic cytokinins with those of their isoprenoid counterparts and cytokinin inhibitors, in relation to growth and developmental processes in plants, has yet to be determined.     

Cytokinin Differences in In Vitro Cultures and Inflorescences from Normal and Mantled Oil Palm (Elaeis guineensis Jacq.)

The mantled abnormality phenotype of the oil palm affects fruit development and thus jeopardizes oil yield. Cytokinins have been implicated in the development of the mantled phenotype. Endogenous cytokinin levels in the normal and mantled phenotypes were compared to determine whether levels of specific cytokinins are associated with mantling. Endogenous cytokinins were identified and quantified in in vitro cultures and inflorescences from normal and mantled oil palms. Twenty-two isoprenoid cytokinins, comprising the zeatin, dihydrozeatin, and isopentenyladenine types, were quantified. Total cytokinin levels, particularly of trans-zeatin and isopentenyladenine types, increased during the in vitro culture process, with the highest levels detected at the proliferating polyembryoid stages. The cytokinins were present mainly in their inactive 9-glucoside forms during in vitro culture. On the other hand, the predominant trans-zeatin cytokinins in inflorescences were present mainly in their ribotide forms, suggesting a metabolic pool of cytokinins for conversion to biologically active free bases or ribosides. Levels of specific cytokinins were significantly different in tissues at different stages. Mantled developed inflorescences contained higher levels of isopentenyladenine 9-glucoside compared with normal inflorescences. Mantled-derived callus tissues had higher isopentenyladenine levels but significantly lower levels of trans-zeatin 9-glucoside, dihydrozeatin riboside, and dihydrozeatin riboside 5′-monophosphate cytokinins compared with normal-derived callus. It would be of considerable interest to verify these specific cytokinin differences in more callus cultures and clones.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

Effect of the physical nature of the culture medium on the metabolism of benzyladenine and endogenous cytokinins in Actinida deliciosa tissues cultured in vitro

Endogenous cytokinin and benzyladenine (BA) metabolites were studied in kiwifruit explants grown on solidified and liquid Murashige and Skoog media supplemented with BA. The same proliferation rate was observed in both media. However, in the liquid medium the release from apical dominance was faster and the growth rate was higher than on solid medium. At the same time the number of vitrified shoots increased considerably in the liquid cultures. Exogenous BA was transformed into 9-β-D-glucopyranosyl-BA ([9G]BA), 7-β-D-glucopyranosyl-BA ([7G]BA). 7-β-D-ribofura-nosyl-BA ([9R]BA) and the 5¹-monophosphate of [9R]BA ([9R-5¹P]BA). The proportion of active forms (BA, [9R-5¹P]BA and [9R]BA) in respect to total BA metabolites, was generally higher in shoots from liquid than from solid media. An exception was found at day 20 when this balance was inverted. Endogenous cytokinins in kiwifruit shoots were measured by enzyme-linked immuno sorbent assay (ELISA). The content of natural cytokinins was influenced by the levels of active forms of BA.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

植物体内的芳环细胞分裂素

芳环细胞分裂素包括6—苄基氨基嘌呤(BA),6—(3—羟苄基氨基)—嘌呤(mT),6—(2—羟苄基氨基)—嘌吟(oT)和它们的衍生物。它的代谢具有相互转换、羟基化作用、结合作用和氧化降解四个特征。本文从信号的感受,转导和应答阐明细胞分裂素在细胞和分子水平上的功能。     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teresand of Zea mays by Dreschslera maydis were characterized by green island formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin >dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies,where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.     

Specific photoaffinity labelling of a thylakoid membrane protein with an azido-cytokinin agonist

A cytokinin-binding peptide (CBP) of 46 kDa (Thy46) has been identified in thylakoid membranes of pea chloroplasts, by photoaffinity labelling with tritiated 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ([³H]azidoCPPU), a urea-type cytokinin agonist. The labelled peptide is also detected in Nicotiana plumbaginifolia, Nicotiana tabacum and spinach thylakoid membranes, but is absent in thylakoid membranes of Chlamydomonas reinhardtii. A pharmacological study of the interaction of this peptide with different cytokinin agonist molecules has been achieved. Urea derivatives are the most efficient competitors of photolabelling, and this efficiency is in good agreement with the cytokinin activity of these compounds. A quantitative analysis of the displacement of the photoaffinity labelling of the peptide by increasing concentrations of CPPU indicates an apparent dissociation constant of 1 M for this ligand. Purine-type cytokinins are weaker competitors than urea-type molecules, but the efficiency of the competition is also correlated to their respective cytokinin activity. A partial purification of Thy46 by a protocol involving ion exchange chromatography and 2D-gel electrophoresis is described.