Relationship between cell division and endogenous auxin in synchronously-cultured tobacco cells

In the cultured tobacco cell, we succeeded in obtaining a partial synchronization of cell division by a combination of pre-starvation, rhythmic light-dark pre-treatment and air tight pre-conditioning. The mitotic index increased during the light period according to the time interval after the end of pre-treatment, and reached its maximum (max=12%) at about 2.5 hr of irradiation, and about 80% of cells completed division 1.5 hr thereafter. During this period, IAA was biosynthesized in the cells, though these cells had been cultured in Murashige and Skoog's medium with 1 mg/l of 2,4-D as a growth substance. IAA was identified by paper chromatography, followed byAvena curvature test and combined gas chromatography-mass spectrometry. The time course of the increase and decrease in the amount of free IAA was parallel to that of the mitotic index. On the other hand, bound IAA increased later and decreased gradually after the end of cell division. Free IAA may have an important role in mitosis.     

Cytokinins in cut carnation flowers. II. Relationship between endogenous ethylene and cytokinin levels in the petals

Tentative identification using HPLC and RIA techniques indicated the presence of zeatin-O-glucoside, zeatin, ribosylzeatin, dihydrozeatin, iso-pentenyladenine and iso-pentenyladenosine in the petals of carnation flowers. Dihydrozeatin is apparently responsible for most of the biological activity. Within the petals most activity was detected in the basal parts which also senesced much slower than the upper parts of the petals. Treatment with AOA extended petal longevity and reduced ethylene production. This was associated with higher cytokinin-like activity in the basal parts of the petals.These higher levels of cytokinins were not observed in the petals of ACC treated flowers or in the detached control flowers. It is suggested that cytokinin transport and/or metabolism may play an important role in regulating ethylene production in cut carnations.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Conjugates of auxin and cytokinin

Plant growth and developmental processes as well as environmental responses require the action and cross talk of phytohormones including auxins and cytokinins. Active phytohormones are changed into multiple forms by acylation, esterification or glycosylation, for example. It seems that conjugated compounds could serve as pool of inactive phytohormones that can be converted to active forms by de-conjugation reactions. The concept of reversible conjugation of auxins and cytokinins suggests that under changeable environmental, developmental or physiological conditions these compounds can be a source of free hormones. Phytohormones metabolism may result in a loss of activity and decrease the size of the bioactive pool. All metabolic steps are in principle irreversible, except for some processes such as the formation of ester, glucoside and amide conjugates, where the free compound can be liberated by enzymatic hydrolysis. The role, chemistry, synthesis and hydrolysis of conjugated forms of two classes of plant hormones are discussed.     

Two effects of cytokinin on the auxin requirement of tobacco callus cultures

Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3-methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin.     

Calmodulin-binding drugs affect responses to cytokinin, auxin, and gibberellic Acid

Trifluoperazine, a phenothiazine tranquilizer, and tetracaine, a local anesthetic, have been found to inhibit a variety of plant hormone responses at concentrations compatible with their known inhibition of Ca²⁺-calmod-ulin-dependent enzyme activities. Among these responses are cytokinin-dependent betacyanin synthesis and increase in fresh weight in Amaranthus tricolor cotyledons, auxin-dependent increase in length of wheat coleoptile segments and gibberellic acid-dependent induction of α-amylase synthesis in barley aleurone layers. The reversibility of some of these inhibitory effects has been demonstrated, indicating that, up to a point, a generalized membrane destruction can be ruled out. The evidence, taken in conjunction with numerous examples from the literature showing calcium involvement in the action of all of the plant hormones, support a unifying theory of hormone action.     

Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves

As many processes are regulated by both light and plant hormones, evaluation of diurnal variations of their levels may contribute to the elucidation of the complex network of light and hormone signal transduction pathways. Diurnal variation of cytokinin, auxin, and abscisic acid levels was tested in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under a 16/8 h photoperiod. The main peak of physiologically active cytokinins (cytokinin bases and ribosides) was found after 9 h of light, i.e. 1 h after the middle of the light period. This peak coincided with the major auxin peak and was closely followed by a minor peak of abscisic acid. Free abscisic acid started to increase at the light/dark transition and reached its maximum 3 h after dark initiation. The content of total cytokinins (mainly N-glucosides, followed by cis-zeatin derivatives and nucleotides) exhibited the main peak after 9 h of light and the minor peak after the transition to darkness. The main, midday peak of active cytokinins was preceded by a period of minimal metabolic conversion of tritiated trans-zeatin (less than 30%). The major cytokinin-degrading enzyme, cytokinin oxidase/dehydrogenase (EC 1.5.99.12), exhibited maximal activity after the dark/light transition and during the diminishing of the midday cytokinin peak. The former peak might be connected with the elimination of the long-distance cytokinin signal. These cytokinin oxidase/dehydrogenase peaks were accompanied by increased activity of beta-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of cytokinin O-glucosides and/or in fine-tuning of active cytokinin levels at their midday peak. The achieved data indicate that cytokinin metabolism is tightly regulated by the circadian clock.     

Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate

The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a specific expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell files in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young inflorescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These findings are discussed in relation to the role of cytokinins in plant development.     

Chromatin reorganization and endogenous auxin/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay. Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole totipotency process of dedifferentiation and redifferentiation. We identified two phases of chromatin decondensation during the dedifferentiation and redifferentiation. At the same time, sharp increase in the ratio of indoleacetic acid (IAA), isopentenyladenosine group (iPAs) at the same stage of cell dedifferentiation and redifferentiation process serve as distinct biochemical maker of dedifferentiation and SE initiation with the unique feature. Our results suggest the two phases of chromatin reorganization associated with endogenous auxin/cytokinin dynamic activity may underlie dedifferentiation and redifferentiation during the entire SE process in cotton.     

Chromatographic analysis of a cytokinin from tissue cultures of crown-gall

Extracts from tissue cultures of crown-gall from Parthenocissus tricuspidata (Sieb. and Zucc.) Planch. exhibited cell division activity in the soybean cytokinin assay. The chromatographic migration of one component, responsible for most of the activity, is similar to that of zeatin ribonucleoside. In addition, acid hydrolysis of the active region taken from chromatograms and of an eluate from a cation-exchange resin column resulted in the production of an active free-base-like derivative. Since the derivative and the parent compound lose activity after KMnO₄ treatment, they are believed to possess an unsaturated constituent essential for biological activity. The major active factor present in crown-gall from Parthenocissus tricuspidata is therefore distinct from the nicotinamide derivative reported to be present in Vinca rosea L. tumor cells.     

Changes in endogenous cytokinin levels in partially synchronized cultured tobacco cells

During the course of partially synchronized cell divisions in cultured tobacco (Xanthi) cells the amount of endogenous cytokinins in the butanol-soluble fraction increased 5 to 10 times in 3 hours and paralleled the increase in frequency of mitosis. Among three cytokinins detected in tobacco cells, the activity corresponding to the R_F of authentic zeatin in thin layer chromatography changed in parallel with the mitotic index.     

Relationship of berberine-producing capability between Thalictrum plants and their tissue cultures

Callus cultures of Thalictrum minus showed a wide variation in the berberine content depending upon the maternal plants from which they were derived. However, no significant correlation in the berberine content was found between the maternal plants and the corresponding callus or cell suspension cultures. Thus, relatively stable and high berberine-producing cell lines could be obtained from some of the plants showing low berberine contents. On the other hand, the amount of berberine released into the liquid medium was found to be correlated with the berberine-productivity of the cell line.     

Phytochrome and cytokinin responses

Cytokinins (CKs) and light can elicit similar morphogenic and biochemical responses in a wide range of plant species. Contradictory reports have been presented that CKs and phytochrome may have independent or identical mechanisms of action in photomorphogenic processes. These reports, relating to seed dormancy and germination, seedling development and growth efficiency, pigment production, and the photoperiodic control of flowering are reviewed. Based on historical data and recent genetic approaches using Arabidopsis mutants, the possible role of CKs in physiological and biochemical pathways affected by light are discussed briefly. Together with the phytochrome system, CKs may contribute towards entrainment of circadian rhythms and thus participate in photoperiodic signalling. Both light and CKs apparently also participate in nutrient assessment pathways. Current models propose that light and CKs might act independently or sequentially through common signal transduction intermediates to control the same downstream responses. We presently have a poor understanding of the mechanism(s) whereby these signals are integrated at the molecular level and the physiological significance of the apparent overlap between the actions of phytochrome and CK cannot yet be fully appreciated.     

Effect of ethylene on the endogenous cytokinin and gibberellin levels in tuberizing potatoes

High concentrations of 2-chloroethylphosphonic acid inhibited tuberization on aged potato tubers (Solanum tuberosum) that had been predisposed to the little tuber disorder. As a result of this treatment sprouts developed which contained relatively high levels of endogenous gibberellins and which elongated normally. The endogenous cytokinin levels in the different treatments did not change appreciably. It is suggested that tuberization is prevented by ethylene either as a direct inhibition of cell division or that it prevents the endogenous cytokinins from functioning. Irrespective of the mode of action of ethylene, cell division apparently is the primary process affected, the result being that storage tissue required for the accumulation of starch is not formed.     

Different effects of two brassinosteroids on growth,auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2,3, 17-trihydroxy-5-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the yes or no type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N⁶-(²-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.Abbreviations BR(s) brassinosteroid(s) - 24-epiBR 24-epibrassinolide - THA-BR 2,33, 17-trihydroxy-5-androstan-6-one - CK(s) cytokinin(s) - iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - Z trans-zeatin - [9R]Z ribosyl-trans-zeatin - ABA abscisic acid - IAA indole-3-acetic acid - NAA naphtalene-1-acetic acid - DEAE cellulose diethylaminoethyl cellulose - HPLC high performance liquid chromatography - ELISA enzyme linked immuno-sorbent assay     

The interaction of auxin and cytokinin in the induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

The dependence of phenylalanine ammonialyase (PAL) induction in bean suspension cultures on the concentration of naphthylacetic acid (NAA) and kinetin has been investigated and the timing of the effect of each hormone has been determined. NAA was required at an optimal concentration of 1 mg l^-1 2 days prior to the increase in PAL activity. Kinetin caused a prapid stimulation of the rate of PAL induction and the total amount of PAL induced in a concentration range of 0.1–0.5 mg l^-1 when it was supplied to the cells immediately prior to the expected rise in PAL activity. The inhibitory effect of 2 mg l^-1 NAA on PAL induction was overcome by an increased concentration of kinetin.Abbreviations NAA naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3yl acetic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5.)