A procedure for quantification of cytokinins as free bases involving scintillation proximity immunoassay

Cytokinins occur in a diversity of forms and determination of their individual levels requires extensive purification. However, determination of the total level of each major base in free, riboside and nucleotide forms would often be adequate. Hence, a methanolysis procedure which releases cytokinin bases from 9-ribosyl derivatives was developed and applied to plant extracts. A simple procedure, involving low pressure column chromatography, for purification of the cytokinin bases in treated extracts, and a scintillation proximity immunoassay for their quantification, were developed. The total level of each cytokinin base [N⁶-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribosylated forms determined by these methods is reported for several plant tissues and the results are compared with those obtained after additional purification by HPLC. Values for zeatin were not changed by HPLC but isopentenyl-adenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay. Modifications to the above purification method to quantify O-glucosyl cytokinins are also described.
The methods described facilitate the quantification of the total amount of each cytokinin base in forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation.     

Use of Lethal Control to Reduce Habituation to Blank Rounds by Scavenging Birds

Abstract Scavenging bird deterrence frequently fails due to habituation. We demonstrated such habituation by gulls and corvids to blank rounds used in a dawn-to-dusk regime at a landfill site in southern England. We then combined blank rounds with live rounds and shot birds whenever they attempted to land. Gull numbers declined significantly despite only 1.9% of the population being shot. Corvid numbers returned to precontrol levels despite 52.7% of the population being shot. We suggest that shooting reduces gull habituation to blank rounds but is ineffective at reducing habituation by corvids.     

Topical application of a plant extract to different life stages of Trichoplusia ni fails to influence feeding or oviposition behaviour

We have previously determined that larval feeding experience with a feeding/oviposition deterrent modified the feeding responses of larvae and oviposition responses of subsequent moths. These behavioural changes were attributed to learning, but the possibility of chemical legacy could not be ruled out. In the present study, we have topically applied a feeding/oviposition deterrent plant extract from Hoodia gordonii (Masson) Sweet ex Decne (Asclepiadaceae) to larvae, pupae, and adults of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) to determine whether the feeding response of larvae and oviposition response of subsequent female moths is similarly modified by chemicals applied to the external surface of the insect. Our results indicate that traces of the extract that may be present internally or externally on the larvae do not reduce the feeding deterrent response of larvae. Furthermore, traces of the extract in or on larvae, pupae, or adult moths did not alter oviposition choice of female moths, leading us to discount the role of experience through topical application in this study. The fact that feeding/oviposition choice was only influenced by prior feeding experience of the larvae and not by topical administration suggests that habituation via sensory stimulation through mouthpart chemosensilla is likely a central phenomenon. Continuous exposure of adult moths to the extract over a period of 7 days did not affect the oviposition response of the female moths, ruling out the role of adult experience on host-plant selection in T. ni . To the best of our knowledge, this is the first study to examine the role of experience via topical application of chemicals onto all life stages of the insect except the egg. Chemical legacy may not be playing a role in influencing the oviposition choices of female T. ni moths.     

Metabolism of zeatin riboside in a hormone autonomous genetic tumour line of tobacco

The metabolic fate of externally applied [³H]-zeatin riboside ([9R]Z) was studied in a cultured genetic tumour line of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grows on auxin and cytokinin free medium. Metabolism by 3.5-week-old tissues showed enhanced stability of supplied [9R]Z; unmetabolized [9R]Z accounted for 48.7 and 37.5% of extracted radioactivity following 8 and 24 h incubation, respectively; tissues of different ages (1–10 weeks following subculture) also indicated high cytokinin stability following 8 h incubation (unmetabolized [9R]Z accounted for 32.5–53.0% of extracted radioactivity). All analyses were performed by thin layer chromatography (TLC) and the results subsequently confirmed by high performance liquid chromatography (HPLC). Side-chain cleavage and modification of the purine ring were the major forms of metabolism; metabolites with an intact cytokinin moiety included zeatin (Z), [9R]Z nucleotides and glucosyl derivatives. Detailed analysis of metabolites carried out in the experiments using 3.5-week-old tissues indicated that both dihydro-derivatives as well as cis isomers of Z and [9R]Z were not formed. Adenine, adenosine and its nucleotide(s) were the main degradative metabolites; in 3.5-week-old tissues these metabolites accounted for about 5.9 and 7.8% (of ³H extracted) following 8 and 24 h incubation, respectively. In tissues of different ages (1–10 weeks following subculture), these metabolites accounted for about 7.6–22.9% of the extracted ³H. Some metabolites (zeatin, adenine and adenosine) were also detected in the staled incubation media. The observed high [9R]Z stability in this tissue may reflect low levels of cytokinin oxidase activity and/or some form of compartmentation.     

Uptake and translocation of [14C]-labelled benzylaminopurine in apple shoots grown in vitro in relation to shoot development

Shoots of apple (rootstock A2, Malus sylvestris (L.) Mill var. domestica Borkh.) were grown in vitro on agar media containing 4.4 μ M benzylaminopurine (BAP) during the whole of a 36-day culture period or during the first 6 days of the period. Development of lateral shoots and an appreciable increase in fresh weight were obtained only when BAP was present during the whole period. Treatment for 6 days only was not sufficient for shoot proliferation although the transferred shoot continued its growth on fresh medium without BAP. Uptake of the cytokinin was studied by adding [8–¹⁴C]-benzylaminopurine to the medium and then following the distribution of the isotope in the shoots during the 36-day culture period. There was a steady, almost linear, uptake of ¹⁴C during the whole period. Most of the activity accumulated in the lower part of the shoot. It is concluded that BAP, due to slow translocation, is active mainly in the basal parts of the shoot where it stimulates formation and growth of buds. Its action is dependent on continuous supply of new BAP from the medium, indicating metabolism of BAP in the tissue.     

Effects of Polyamines and Kinetin on In Vitro Frond Senescence in Lemna aequinoctialis 6746

Both polyamines and kinetin could retard the loss of chlorophyll during dark-induced senescence in excised frond of Lernna aequinoctialis 6746. The effect of polyamines on retarding the chlorophyll loss was stronger than that of kinetin. Kinetin remarkably inhibited the loss of soluble proteins and the increase of protease activity, while no similar effects were observed from polyamines. An inhibitor of polyamine biosynthesis, methylglyoxal bis- (guanyl- hydrazone) (MGBG), slightly increased the loss of chlorophyll and soluble proteins. During senescience, both the increase of putrescine (Put) content and the decrease of spermidine (Spd) content were inhibited by kinetin at the concentration of 0.05 mmol/L, but the spermine (Spm) level was not affected by kinetin. The arginine decarboxylase (ADC) activity was dominant in frond of Lemna aequinoctialis 6746. Kinetin slightly increased ADC activity, while it had no marked effect on ornithine decarboxylase (ODC) and s-adenosylmethionine decarboxylase (SAMDC). The possible relationship between polyamines and cytokinins in retarding senescence was also discussed.     

Long-distance nitrate signaling displays cytokinin dependent and independent branches

The long-distance signaling network allowing a plant to properly develop its root system is crucial to optimize root foraging in areas where nutrients are available. Cytokinin is an essential element of the systemic signaling network leading to the enhancement of lateral root proliferation in areas where nitrate is available. Here,we explore more precisely:(i) which particular traits of lateral root growth(density and length of emerged lateral roots) are the targets of systemic signaling in a context of heterogeneous nitrate supply; and(ii) if the systemic signaling depends only on cytokinin or on a combination of several signalings.     

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.     

Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco

When several concentrations of brassinolide (BL) were added to a shoot induction medium (SIM) that contained only BA, redifferentiation of adventitious shoots from tobacco leaf discs was unaffected at low BL levels (10^-10~10^-8 M), but was inhibited at higher concentrations. In comparison, when BL was applied without BA, only cell expansion occurred and no shoots formed. The determination time for shoot formation was shortened at low BL concentrations, but their formation was postponed (i.e., time was lengthened) at higher concentrations. Elongation of shoots incubated for 30 d was unaffected at low BL concentrations, but was inhibited as that amount increased.NTH1, a tobacco homeobox gene that is expressed in the central zone of the tobacco shoot apex, showed greater expression levels in the SIM over time, and its expression was stronger in media treated with low concentrations of BL compared with the SIM control at the same time point. Expression ofNTH1 was postponed at higher BL concentrations. In conclusion, at low concentrations, brassinolide has no effect on shoot formation. However, it inhibits their formation at high concentrations when cytokinin is included in the media.     

Role for Cytokinins in Somatic Embryogenesis of Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)?

The effects of cytokinins on somatic embryogenesis in pepper were studied using a Murashige and Skoog-based medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid and 10% sucrose. Compared to the medium without cytokinins, N⁶-(Δ²-isopentenyl)-adenine and kinetin had no significant effect, whereas benzylaminopurine and zeatin significantly reduced somatic embryogenesis. Coconut water (10%) either had no significant effect or it reduced embryogenesis as well. Induction of somatic embryogenesis was also dependent on genotype. Appearance of somatic embryos continued after removal of the embryos that developed first, especially on cultivars with a lower initial induction rate. Although somatic embryos germinated, the apical meristem did not elongate.