Aromatic Cytokinins Research Articles

Effect of a novel aromatic cytokinin derivative on phytochemical levels and antioxidant potential in greenhouse grown Merwilla plumbea

In an attempt to elucidate the carry-over effect of cytokinins (CKs) on phytochemical and antioxidant activity of acclimatized plants, tissue culture-derived Merwilla plumbea supplemented with three CK types at four (0.25, 0.5, 1 and 2 µM) concentrations were grown for 6 months ex vitro. Phenolic acids including the hydroxybenzoic and hydroxycinnamic acid derivatives in M. plumbea were quantified using ultra performance liquid chromatography while the antioxidant activity was evaluated using oxygen radical absorbance capacity (ORAC). Different concentrations of gallic acid, protocatechuic acid, p-hydroxybenzoic acid and salicylic acid were observed with all the treatments with the exception of non-treated plants. Most phytochemicals (for example, gallic acid, ferulic acid protocatechuic acid and caffeic acid) were highest in plants obtained from 0.25 µM meta-topolin riboside (mTR). Likewise, plants derived from 2 µM mTR had the highest ORAC (684 µmol g−1 trolox equivalents) activity. Bearing in mind that therapeutic effects of medicinal plants are often associated to their phytochemical content, the current results are an indication on how the intricate in vitro environment (CK type and concentration in this case) affects the growth and general physiology of micropropagated plants especially after acclimatization.

Cytokinins affect the stomatal conductance and CO2 exchange of in vitro apple leaves

Effects of different cytokinin supplies including two types of aromatic cytokinins, such as benzyl-adenine, and 3-hydroxybenzyladenine applied at two different concentrations (2.0 and 6.0 μM) were studied on water and gas exchange parameters in in vitro apple leaves of ‘Royal Gala’ and ‘Freedom’ scions after 3 weeks of culture. Cytokinin supply affected the stomatal conductance of water vapour, transpiration rate and the sub-stomatal CO2 concentration of leaves. Effect of cytokinin depended on its applied type and concentration, moreover on the apple scion. According to the results, the rate of CO2 exchange itself is not usable for characterization of function of photosynthetic apparatus of in vitro leaves. However, measurements of stomatal conductance of water vapour and transpiration rate seemed to be good indicators for stomatal behaviour of in vitro apple leaves.

How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea?

Merwilla plumbea (Lindl.) Speta is a popular and highly sought after South African medicinal plant with diverse therapeutic values. Using Ultra performance liquid chromatography (UPLC), the effect of five cytokinins (CKs) [either isoprenoid = N 6-isopentenyladenine (iP) or aromatic = benzyladenine, meta-topolin (mT), meta-topolin riboside (mTR), and 6-(3-methoxybenzylamino)-9-tetrahydropyran-2-ylpurine] MemTTHP on growth and level of endogenous CKs during micropropagation and acclimatization stages was evaluated. Aromatic CK (mT/mTR) elicited the highest shoot proliferation (7–8 shoots per explant) during in vitro culture. Following acclimatization, iP-treated and the control plants were healthier with longer leaves, roots and higher fresh weight when compared to aromatic CKs. A total of 37 (22 isoprenoid and 15 aromatic) CK variants were quantified in both in vitro and acclimatized plants. Based on their metabolic function, these were grouped into five types including free bases, ribosides, ribotides, O- and 9-glucosides. In addition to enhancing our understanding of the hormone physiology in M. plumbea, the current findings are discussed in line with the effect of the exogenously applied CK on the observed differences in growth before and after the important stage of acclimatization. The observed dynamics in endogenous CK provide an avenue to manipulate in vitro growth and development of investigated species.

Proteomic Identification of a Candidate Sequence of Wheat Cytokinin-Binding Protein 1

Our knowledge on the principle mechanisms of cytokinin action has been significantly deepened over the last years, but several weakly explored areas still remain on the map of cytokinin cellular physiology. Cytokinin-binding proteins could also be included in this pending field of cytokinin research. Probably, the best explored representative of this group is the wheat cytokinin-binding protein 1 (CBP-1). The role of this germ-allocated protein as a presumable regulator of free aromatic cytokinin levels during grain germination has been discussed intensively. To dig deeper into this interesting protein, this study was aimed at the identification of the CBP-1 amino acid sequence. A combination of in silico BLAST search, classical biochemical CBP-1 purification based on isoelectric point precipitation and ion-exchange chromatography, and proteomic analysis of the isolated protein by ultra-high resolution tandem mass spectrometry allowed us to uncover and validate two CBP-1 subunit candidate sequences with molecular masses of 56.2 and 55.0 kDa, respectively. Interestingly, we found the latter sequence alternated in two amino acids in the putative cytokinin-binding motive in comparison to the composition of this domain reported in the original studies. A BLAST search for the amino acid sequence of the binding region among plant proteomes revealed several highly related protein sequences, all originating from the Poaceae family. This piece of information could give support to the elucidation of the role of CBP-1 in physiological processes mediated by aromatic cytokinins.

A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase

We have developed a N 6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N 6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N 6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM−1cm−2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N 6-(Δ2-isopentenyl) adenine, N 6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

Effects of cytokinins on antioxidant enzymes in in vitro grown shoots of Pelargonium hortorum L. H. Bayley

<p>The aim of this study was to determine the influence of <em>meta</em>-topolin (<em>m</em>T) and 6-benzyl-aminopurine (BAP) on the hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) level and antioxidant enzymes activities in relation to the shoot formation and senescence process in <em>Pelargonium hortorum</em> cultivars, which differ in their susceptibility to leaf yellowing under <em>in vitro</em> conditions.</p><p>In an early senescing cultivar ‘Grand Prix’, the addition of an aromatic cytokinin <em>m</em>T to abscisic acid (ABA)-enriched Murashige and Skoog (MS) basal medium more efficiently inhibited leaf yellowing than BAP. In both genotypes, <em>meta</em>-topolin was also the most effective in shoot formation. It was found that <em>Pelargonium</em> species varying in their susceptibility to senescence differ in H<sub>2</sub>O<sub>2</sub> production and antioxidant enzymes activities. Generally, <em>meta</em>-topolin more effectively enhanced H<sub>2</sub>O<sub>2 </sub>production and POD activity than BAP and control medium, but its effect depended on genotype. The highest H<sub>2</sub>O<sub>2</sub> production stimulated by <em>m</em>T was observed on day 5 of subculture in late senescing cv. ‘Bergpalais’. In both geranium genotypes, superoxide dismutase (SOD) and catalase (CAT) levels were highest at the beginning of the subculture period, during the initiation of shoot formation. SOD showed the highest activity on day 5 of subculture on the medium without cytokinin and generally being higher in cv. ‘Bergpalais’ than in cv. ‘Grand Prix’. CAT activity was positively regulated by both cytokinins. POD activity was most effectively enhanced by <em>m</em>T, but on different days of subculture - on the 2<sup>nd</sup> day of subculture in cv. ‘Bergpalais’ and on the 22<sup>nd</sup> day of subculture in cv. ‘Grand Prix’. The enhanced activity of POD in the presence of <em>m</em>T, 4-fold higher than on control medium, at the end of subculture in <em>P. hortorum </em>‘Grand Prix’ coincided with the inhibition of leaf senescence.</p>

Endogenous cytokinin profiles of tissue-cultured and acclimatized ‘Williams’ bananas subjected to different aromatic cytokinin treatments

Endogenous cytokinin (CK) levels of in vitro-cultured and greenhouse-acclimatized ‘Williams’ bananas treated with six aromatic CKs were quantified using UPLC–MS/MS. The underground parts had higher endogenous CK levels than the aerial parts. Control plantlets had more isoprenoid CKs while the aromatic-type CKs were predominant in all other regenerants. Following acclimatization of the control and 10μM CK regenerants, there was a rapid decline in both isoprenoid and aromatic CK in the greenhouse-grown plants. Apart from the control and 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP) treatment with higher level of isoprenoid CK, aromatic CK remain the predominant CK-type across all CK treatments. The most abundant CK forms were meta-topolin (mT) and benzyladenine (BA) in the micropropagated and acclimatized plants, respectively. Micropropagated plantlets had cis-Zeatin (cZ) as the major isoprenoid CK-type which was in turn replaced by isopentenyladenine (iP) upon acclimatization. On a structural and functional basis, 9-glucoside, a deactivation/detoxicification product was the most abundant and mainly located in the underground parts (micropropagation and acclimatization). The results establish the wide variation in metabolic products of the tested aromatic CKs during micropropagation and acclimatization. The findings are discussed with the possible physiological roles of the various CK constituents on the growth and development of banana plants.

Physiological effects of a novel aromatic cytokinin analogue in micropropagated Aloe arborescens and Harpagophytum procumbens

Globally, there is a continuous effort geared toward improving micropropagation protocols with much emphasis on the type of cytokinins (CKs). We investigated the effect of meta-topolin tetrahydropyran-2-yl [mTTHP—a novel derivative of the aromatic cytokinin meta-topolin (mT)] on shoot proliferation, photosynthetic pigment content, phytochemicals and antioxidant activity of two widely used medicinal plants, Aloe arborescens and Harpagophytum procumbens. In terms of shoot proliferation, besides mTTHP and mT (at equimolar level) showing comparable effects, both topolins were significantly better than the control and benzyladenine riboside (BAR) in micropropagated A. arborescens. In H. procumbens, mT-treated cultures were the most responsive treatment at 2.5 μM when compared to the control. At 5.0 μM concentration, mTTHP and meta-topolin riboside (mTR) demonstrated a comparable effect on shoot proliferation. To a certain extent (particularly at low concentrations), mTTHP had a better rooting stimulatory effect when compared to the other CKs in both investigated species. In micropropagated A. arborescens, 2.5 μM mTTHP showed the best antioxidant activity (ferric reducing power) which was significantly higher than the control and all BAR-treated plants. Although 2.5 μM mTTHP exhibited a significant antioxidant activity (in β-carotene based assay) when compared to mTR at equimolar level in H. procumbens, it was not significantly different from the treatment with 2.5 μM mT and the control. As evidenced in the current study, the efficacy of the novel cytokinin may be species-specific and beneficial for few physiological parameters. It is conceivable that mTTHP is another viable alternative topolin with the extra advantage of inducing rooting at a low concentration.

Effect of aromatic cytokinins and explant position on shoot multiplication of Asparagus offi cinalis L.

Asparagus offi cinalis has been widely studied, but little information is available about its in vitro response to exogenous cytokinin during shoot multiplication. To study the effects of different cytokinins on shoot multiplication of A. offi cinalis ‘Grolim’, in vitro culture was initiated from shoot segments cultured on media with Murashige and Skoog medium. Effects of different aromatic cytokinins (6-benzylaminopurine, 6-benzylaminopurine riboside and meta-topolin) applied in four concentrations (0.5, 1.0, 1.5, 2.0 mg/l) on shoot multiplication of ‘Grolim’ were tested. Effect of explant position (vertically or horizontally) on the shoot multiplication outcome was also studied. Both the length and the number of newly developed shoots were signifi cantly affected by explant position and cytokinin content of the medium. The highest numbers of shoots (4.9) were produced in the presence of 0.5 mg l-1 6-benzylaminopurine riboside when explants were paced horizontally onto the medium. Although the longest shoots (41.5 mm) developed on explants placed vertically onto medium supplemented with 2.0 mg l-1 meta-topolin, the lengths of shoots developed on medium with 0.5 mg l-1 6-benzylaminopurine riboside were also adequate in both explant position (29.5 and 33.6 mm placed horizontally and vertically, respectively).

Proteome Analysis in Arabidopsis Reveals Shoot- and Root-Specific Targets of Cytokinin Action and Differential Regulation of Hormonal Homeostasis

The plant hormones cytokinins (CKs) regulate multiple developmental and physiological processes in Arabidopsis (Arabidopsis thaliana). Responses to CKs vary in different organs and tissues (e.g. the response to CKs has been shown to be opposite in shoot and root samples). However, the tissue-specific targets of CKs and the mechanisms underlying such specificity remain largely unclear. Here, we show that the Arabidopsis proteome responds with strong tissue and time specificity to the aromatic CK 6-benzylaminopurine (BAP) and that fast posttranscriptional and/or posttranslational regulation of protein abundance is involved in the contrasting shoot and root proteome responses to BAP. We demonstrate that BAP predominantly regulates proteins involved in carbohydrate and energy metabolism in the shoot as well as protein synthesis and destination in the root. Furthermore, we found that BAP treatment affects endogenous hormonal homeostasis, again with strong tissue specificity. In the shoot, BAP up-regulates the abundance of proteins involved in abscisic acid (ABA) biosynthesis and the ABA response, whereas in the root, BAP rapidly and strongly up-regulates the majority of proteins in the ethylene biosynthetic pathway. This was further corroborated by direct measurements of hormone metabolites, showing that BAP increases ABA levels in the shoot and 1-aminocyclopropane-1-carboxylic acid, the rate-limiting precursor of ethylene biosynthesis, in the root. In support of the physiological importance of these findings, we identified the role of proteins mediating BAP-induced ethylene production, METHIONINE SYNTHASE1 and ACC OXIDASE2, in the early root growth response to BAP.

Endogenous cytokinin profiles and their relationships to between-family differences during adventitious caulogenesis in Pinus pinea cotyledons

Caulogenesis in mature stone pine (Pinus pinea L.) cotyledons is promoted, to varying degrees depending on genotype, by exogenous application of the cytokinin (CK) benzyladenine (BA). In the present study, endogenous CK profiles of cotyledons from open-pollinated plants and two families of stone pine with widely differing organogenic capacities were monitored during caulogenesis and linked to previously characterized BA uptake and induction phases. Changes in levels of free bases, ribosides, ribotides and glucosides of both isoprenoid and aromatic CKs were followed. Before BA application, the pool of endogenous CKs in all sets of cotyledons was dominated by isoprenoid ribotides, but 1h after BA exposure, aromatic CKs (mainly active free bases and ribosides of topolins) accounted for more than 90% of the pool. BA N-glucosides were also observed, levels of which (and topolins) rose from 2d until the end of the (six-day) culture period. The CK profiles of the two selected pine families also differed, although the general trends were similar. During the first 6h, levels of BA and meta-topolin were highest in cotyledons from the family with the strongest caulogenic responses, while levels of ribotides and aromatic glucosides were highest in cotyledons from the other family.

In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill

Aloe species are highly-prized for their ornamental value and have been utilized for centuries in traditional medicine. Due to their habitat loss and exploitation for medicinal and ornamental plant trade, many species in this genus have become threatened. One of the most important globally rated medicinal species in Aloe genus is A. arborescens. The current study evaluated the roles of different aromatic cytokinin types and concentrations on direct organogenesis, in vitro bioactive secondary metabolite production and antioxidant activity of regenerated shoots of A. arborescens. There was an increase in the number of adventitious shoots produced per explant with an increase in concentration in cultures treated with meta-topolin (mT), meta-topolin riboside (mTR), meta-methoxytopolin (MemT) and benzyladenine riboside (BAR), reaching an optimum at either 5.0 or 7.5 μM. Overall, the treatment with 5.0 μM mT gave the largest number of transplantable shoots (regenerated shoots with length greater than 10 mm). Rooted shoots were successfully acclimatized after 8 weeks with a survival frequency above 90 % and no observable morphological abnormalities. Variable amounts of total iridoids, phenolics, flavonoids and condensed tannins were detected in regenerated shoots from all the cytokinin treatments. An increased free-radical scavenging activity with an increase in concentration was recorded in regenerated shoots from mT and mTR treatments, reaching an optimum at 7.5 μM concentration. The present study shows that the choice of cytokinin type and concentration exogenously supplied during tissue culture markedly influences not only shoot proliferation but also the in vitro production of bioactive secondary metabolites.

Novel Cytokinin Derivatives Do Not Show Negative Effects on Root Growth and Proliferation in Submicromolar Range

BackgroundWhen applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N6-benzylaminopurine were further tested in planta.Methodology/Principal FindingsThe main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants.Conclusions/SignificanceTetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.

Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana

The effects of five topolins (meta-Topolin, mT; meta-Topolin riboside, mTR; meta-Methoxy topolin, MemT; meta-Methoxy topolin riboside, MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl, MemTTHP) on the phenolic content and subsequent acclimatization potential of micropropagated ‘Williams’ bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30 μM of the above aromatic cytokinins (CKs) for 42 days. Phenolic contents were quantified spectrophotometrically. Growth parameters and photosynthetic pigments of the greenhouse-acclimatized plants were determined after 5 months. Total phenolic levels were highest in 10 μM mT-treated plantlets within the aerial parts and 30 μM MemTTHP for the underground parts. In the underground parts, 10 μM mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. Furthermore, 10 μM MemTTHP-treated plantlets had significantly higher total flavonoids (30.1 ± 0.24 mg CE/g DW) within the aerial parts. Plantlets regenerated using MemT, MemTR and MemTTHP had significantly longer roots and better shoot/root ratios compared to the control and BA-treated plants. In terms of root fresh weight, it was significantly higher in MemT-treated plantlets than in the control and BA treatments. Chlorophyll a/b ratio was significantly improved with the use of MemT, mTR and mT compared to control. Current findings indicate the potential of topolins in stimulating the accumulation of phenolic compounds in micropropagated plantlets. In view of the importance of plant secondary metabolites, their substantial accumulation probably enhanced the acclimatization and subsequent ex vitro survival of the micropropagated plantlets. Topolins, particularly, the new derivative MemTTHP could be an alternative CK for the micropropagation of plant species based on their stimulatory effect on ex vitro rooting that inevitably enhances acclimatization competence. Furthermore, topolins are demonstrated as potential elicitors in micropropagation.

The role of cytokinins during micropropagation of wych elm

We have compared the influence of two aromatic cytokinin derivatives, N 6-benzyladenine (BA) and meta-topolin (mT), on the in vitro multiplication and senescence of wych elm (Ulmus glabra Huds.). After 3 months of cultivation, the micropropagation rate was higher (approx. six times more shoots developed) on Murashige and Skoog (MS) medium supplemented with mT than on MS supplemented with BA. Quantification of 50 endogenous cytokinins, using a recently developed UPLC-ESI(+)-MS/MS method, showed significant differences in the cytokinin metabolites (especially different glucosides) in explants cultivated on media supplemented with BA or mT. Differences in efficiency of photosystem II was also detected.

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N6-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N6-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.

Platinum-Modified Adenines: Unprecedented Protonation Behavior Revealed by NMR Spectroscopy and Relativistic Density-Functional Theory Calculations

Two novel Pt(IV) complexes of aromatic cytokinins with possible antitumor properties were prepared by reaction of selected aminopurines with K(2)PtCl(6). The structures of both complexes, 9-[6-(benzylamino)purine] pentachloroplatinate (IV) and 9-[6-(furfurylamino)purine] pentachloroplatinate (IV), were characterized in detail by using two-dimensional NMR spectroscopy ((1)H, (13)C, (15)N, and (195)Pt) in solution and CP/MAS NMR techniques in the solid state. We report for the first time the X-ray structure of a nucleobase adenine derivative coordinated to Pt(IV) via the N9 atom. The protonation equilibria for the complexes in solution were characterized by using NMR spectroscopy (isotropic chemical shifts and indirect nuclear spin-spin coupling constants) and the structural conclusions drawn from the NMR analysis are supported by relativistic density-functional theory (DFT) calculations. Because of the presence of the Pt atom, hybrid GGA functionals and scalar-relativistic and spin-orbit corrections were employed for both the DFT calculations of the molecular structure and particularly for the NMR chemical shifts. In particular, the populations of the N7-protonated and neutral forms of the complexes in solution were characterized by correlating the experimental and the DFT-calculated NMR chemical shifts. In contrast to the chemical exchange process involving the N7-H group, the hydrogen atom at N3 was determined to be unexpectedly rigid, probably because of the presence of the stabilizing intramolecular interaction N3-H···Cl. The described methodology combining the NMR spectroscopy and relativistic DFT calculations can be employed for characterizing the tautomeric and protonation equilibria in a large family of transition-metal-modified purine bases.

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N 6-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N 6-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.

New cytokinin derivatives – their discovery, development and use for micropropagation of endangered tree species

Elms (Ulmus sp.) are highly valuable trees for their great strength, tightly twisted grain, and durability, as well as for their cold and salt tolerance[1]. Wych elm (Ulmus glabra, Huds.) is a native species in Europe. In the Czech Republic, it is spread especially in mountain regions. During the years 1970–80, the second epidemic of Dutch elm disease destroyed most elm populations in Europe. The elm trees, which have survived the disease, are capable to maintain a high degree of resistance against the repeated infection. Micropropagation represents promising alternative of their vegetative reproduction. The wild service tree (Sorbus torminalis (L.) Crantz) is a slow-growing tree reaching maximum height of 20–25 m at around 80–100 years. According to the latest data, the species is also disappearing from forests of the Czech Republic and it is considered to be endangered. On the other hand, the wild service tree is rated as one of the most valuable hardwoods [2] with a great potential for wider use in forestry and forest ecology, and also for its importance in the timber industry. The in vitro induction of organogenesis, rooting and acclimatization of above mentioned elm species as well as wild service tree were optimalized and standartized. Widely used BAP is an important and affordable cytokinin routinely utilized for its effective stimulatory properties in micropropagation practice. On the other hand, BAP may negatively influence the growth, rooting and acclimatization processes in some crops [3].It is known that hydroxylated aromatic cytokinins (topolins) are more resistant to cytokinin oxidase (CKX), more stable and in some biological systems active at lower concetrations than the isoprenoid CKs. In addition, meta - topolin does not inhibit root formation which is a typical inconvenient effect of high concentrations of BAP. These properties might help to enhance the future productivity of plant tissue culture industry crops [3,4]. The development of other new CK derivatives exhibiting high morphogenetic activity might consequently be of a great practical importance in plant biotechnology.During our recent search for naturally occurring aromatic cytokinins (ARCKs) in plants, another new group of endogenous BAP derivatives –methoxytopolins – exhibiting high biological, especially anti-senescence (and surprisingly also anti-cancer) activity, was discovered. Based on these results, we synthesized several groups of their synthetic analogues that exhibited high activity in three different CK bioassays and showed the ability to activate cytokinin receptors and/or to inhibit CKX. Best compounds so far (6-(3-hydroxybenzylamino)purine (mT) and the 6-(3-methoxybenzylamino)purine-9-ribofuranoside (MeomTR)) were used for retardation of senescence during multiplication stage of micropropagation of above mentioned tree species as well as for rooting support. Subsequently, wide range of endogenous plant hormones (isoprenoid cytokinins, IAA, ethylene) were quantified (using UPLC-MS/MS and/or GC/FID, respectively) and compared in relation to cytokinin exogenously used. The results about optimal endogenous plant hormone concentrations and their dependence on different exogenous cytokinin used in the cultivation media, may improve in-vitro micropropagation efficiency as well as quality of ex vitro acclimatized plants of above mentioned elm species, wild service tree and possibly also of other tree species. This work was supported by the Ministry of Agriculture of the Czech Republic (Mze 0002070202, QI 92A247), Czech Ministry of Education (MSM 6198959216), ED0007/01/01 Centre of the Region Hana for Biotechnological and Agricultural Research.

Novel BAP degradation pathway during adventitious caulogenesis in Pinus pineaL. cotyledons

Cytokinins (CKs) are a group of phytohormones which probably regulate the growth, development, and metabolism of all plants. The aromatic CK benzyladenine (BA) has been widely applied in in vitro culture for inducing shoot organogenesis. Our study of endogenous cytokinin profiles during the caulogenic process based on mature cotyledons of stone pine (Pinus pinea L.) showed a novel metabolic pathway of aromatic cytokinins based on modification of purine skeleton. Methods Three-year-old mature seeds from two half-sibling selected families and open-pollinated trees of P. pinea were used [1] and the samples were collected following the Alonso et al.[2] procedure. Extraction and purification of cytokinins was based on the method described by Novak et al. [3], including modifications published later [4]. The samples were purified using a combination of a cation (SCX-cartridge) and anion [DEAE-Sephadex/ C18-cartridge] exchangers. Combination of high performance liquid chromatography (HPLC) with quadrupoletime of flight mass spectrometry (QqTOF) was used for accurate and sensitive identification and quantification of cytokinins.