Quantitative analysis of protein changes during meristem intiation and bud development in protoplast-derived Petunia hybrida callus

Small calluses from Petunia hybrida leaf protoplasts differentiated into meristems and then vegetative buds in the presence of high cytokinin concentrations. Total proteins from calluses grown at two cytokinin concentrations, of which only the highest induced bud formation, were analysed for molecular markers of cytokinin action by two-dimensional electrophoresis. The protein patterns were also compared to those of differentiated shoots and roots and to those of calluses grown under various hormonal conditions. Five percent of the total proteins detected by this method varied significantly in their level of expression according to the treatment. Quantification by image analysis demonstrated the occurrence of five groups of polypeptides with different expression patterns. Polypeptides from group 1 marked the development of buds and were abundant in young photosynthetic organs from whole plants. Polypeptides from group 5 increased only in calluses grown on control medium, i.e. in the presence of the lowest cytokinin concentration. No protein increased specifically during meristem formation. The possible action of cytokinin on regeneration is discussed in relation to these results.     

An integrated physiological and genetic approach to the dynamics of FtsZ targeting and organisation in a moss, Physcomitrella patens

Plant FtsZ (filamentous temperature-sensitive Z) proteins are regarded as descendants of prokaryotic cell division proteins. We could show previously that four FtsZ isoforms of the moss Physcomitrella patens assemble into, and interact in, distinct structures inside the chloroplasts and in the cytosol. Their organisation and localisation patterns indicate an involvement in chloroplast and cell division and in the maintenance of chloroplast shape and integrity. The cellular processes of chloroplast division and maintenance of chloroplast shape were disturbed either by application of the beta-lactam antibiotic ampicillin or by a mutation that presumably affects signal transduction of the plant hormone cytokinin. When cells of these plants were analysed microscopically, there was no indication that cytosolic functions of FtsZ proteins were affected. Furthermore, FtsZ proteins continued to build three-dimensional plastoskeleton networks, even in considerably enlarged or malformed chloroplasts. On the other hand, macrochloroplast formation promoted the localisation of FtsZ proteins in filaments that emanate from the plastids and, therefore, most likely represent stromules. Annular FtsZ structures that are regarded as essential components of the division apparatus were absent from macrochloroplasts of ampicillin-treated cells. Thus, the distribution of FtsZ proteins after inhibition of chloroplast division further strengthens our hypothesis on the functions of distinct isoforms. In addition, the results provide further insight into the regulation of protein targeting and dynamics of plastoskeletal elements.     

Somatic embryogenesis in Agave tequilana Weber cultivar azul

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.     

An estimation of the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn

The aim of the experiment was to determine the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn grown under controlled conditions. The experiment was to check whether an application of exogenous hormones during vegetative and generative stages of the plant had an effect on above-ground mass development, on nitrate reductase activity and on plastid pigments content. Experiment factor was synthetic auxin and cytokinin and the date of their application. Auxin was applied in the form of a synthetic indole-3-butyric acid, while cytokinin was sprayed as synthetic 6-benzylaminopurine. The control plants were treated with distilled water. Depending on the experimental variant, spraying was applied at the sixth true leaf stage and at the first flower bud stage. The research showed that the response of the alfalfa plants to the application of cytokinin and auxin was not uniform. It seems that the most effective was the application of a mixture of them both but only during the vegetative stage.Additionally, cytokinin caused an increase in plastid pigments content in alfalfa leaves. On the other hand, a mixture of auxin and cytokinin triggered the highest nitrate reductase activity in alfalfa roots and raised the ratio of total chlorophyll content to carotenoids. Synthetic auxin caused the decrease of the levels of most parameters compared to the control.     

Nonlinearities in Meerkat Alarm Calls Prevent Receivers from Habituating

Despite the fact that nonlinearities are present in the calls of a number of different species, their adaptive function has received little empirical investigation. Previous studies have demonstrated that playbacks of nonlinear calls evoke a more extreme behavioural response and lead to an increase in responsiveness compared with control playbacks without nonlinearities. Consequently, it has been suggested that nonlinearities might prevent receivers from habituating, by increasing the unpredictability of the call (‘unpredictability hypothesis’). In this study, we tested the unpredictability hypothesis, specifically whether nonlinearities prevent receivers from habituating, by means of a playback experiment using meerkat (Suricata suricatta) alarm calls. We found that in meerkats, playbacks of naturally occurring nonlinear alarm calls take longer to habituate to than alarm calls without any nonlinear intrusions. These data provide important empirical support for the hypothesis that nonlinearities are not just an irrelevant by‐product of the vocal production system, but indeed function adaptively.     

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N⁶-benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.     

Effects of foliar application of BAP on source and sink strength in four six-rowed barley (Hordeum vulgare L.) cultivars

A field experiment was conducted to investigate the effects of foliar application of a synthetic cytokinin (BAP) on source and sink strength of four different six-rowed barley (Hordeum vulgare L.) cultivars. Different spraying treatments consisting of spraying on whole plant, spraying only on leaves and spraying only on ears started at anthesis and continued for 7 days. One additional spraying was carried out on late period of grain filling. Results showed that spraying only on leaves did not affect ear weight, grain yield and 1,000-grain weight, while the two other treatments increased all above mentioned traits. Neither of treatments affected stem weight, biological yield and contribution of stem reserves in grain filling. Exogenous cytokinin did not increase photosynthetic rate and chlorophyll content in treated leaves until late period of grain filling, although there was no significant increase in final grain weight due to late application of BAP. Our results suggested that effects of foliar application of BAP were mostly due to increased sink size soon after anthesis and increased sink demand probably met by current photosynthesis of organs other than leaves, like ear green tissues. An erratum to this article is available at .     

The effects of observer presence on the behavior of Cebus capucinus in Costa Rica

We report on the responses of Cebus capucinus in the Santa Rosa Sector of the Area de Conservación Guanacaste, Costa Rica, to the presence of observers over a 4-week period. Study groups were habituated to different degrees: (1) Cerco de Piedra (CP): continuous observations began in 1984; (2) Exclosure (EX): focus of an 18-month study on males from 1998 to 1999; and (3) NBH: never studied/followed but the group frequently encounters researchers. We collected three types of data: group scans (group state was coded as calm or agitated at observer presence), focal animal data (observer-directed behaviors were recorded), and fecal cortisol levels. The two less-habituated groups (NBH and EX) differed significantly from the habituated group (CP) in their behavioral and cortisol responses, and they showed an increase in habituation over the study period (agitation and cortisol levels both dropped). Individuals in NBH also decreased their responses to observers during focal follows; however, at the end of the study the responses of the two less-habituated groups (NBH and EX) remained elevated in comparison to the habituated group (CP), suggesting the need for further habituation. Unlike capuchin groups that rarely encounter humans, NBH and EX never fled from observers and they rarely emitted observer-directed alarm calls. We suggest that the permanence of habituation and the ability to habituate animals passively through a neutral human presence are both important considerations for researchers conducting studies in areas where animal safety from poachers, etc. cannot be guaranteed.     

Arabidopsis cytokinin-resistant mutant, cnr1, displays altered auxin responses and sugar sensitivity

Based upon the phenotype of young, dark-grown seedlings, a cytokinin-resistant mutant, cnr1, has been isolated, which displays altered cytokinin- and auxin-induced responses. The mutant seedlings possess short hypocotyls and open apical hooks (in dark), and display agravitropism, hyponastic cotyledons, reduced shoot growth, compact rosettes and short roots with increased adventitious branching and reduced number of root hairs. A number of these features invariably depend upon auxin/cytokinin ratio but the cnr1 mutant retains normal sensitivity towards auxin as well as auxin polar transport inhibitor, TIBA, although upregulation of primary auxin-responsive Aux/IAA genes is reduced. The mutant shows resistance towards cytokinin in hypocotyl/root growth inhibition assays, displays reduced regeneration in tissue cultures (cytokinin response) and decreased sensitivity to cytokinin for anthocyanin accumulation. It is thus conceivable that due to reduced sensitivity to cytokinin, the cnr1 mutant also shows altered auxin response. Surprisingly, the mutant retains normal sensitivity to cytokinin for induction of primary response genes, the type-A Arabidopsis response regulators, although the basal level of their expression was considerably reduced as compared to the wild-type. The zeatin and zeatin riboside levels, as estimated by HPLC, and the cytokinin oxidase activity were comparable in the cnr1 mutant and the wild-type. The hypersensitivity to red light (in hypocotyl growth inhibition assay), partial photomorphogenesis in dark, and hypersensitivity to sugars, are some other features displayed by the cnr1 mutant. The lesion in the cnr1 mutant has been mapped to the top of chromosome 1 where no other previously known cytokinin-resistant mutant has been mapped, indicating that the cnr1 mutant defines a novel locus involved in hormone, light and sugar signalling.     

Pea PR 10.1 is a Ribonuclease and its Transgenic Expression Elevates Cytokinin Levels

The constitutive expression of a cDNA encoding a pea (Pisum sativum L.) PR 10 protein in Brassica napus leading to an enhancement of germination under saline conditions has been previously reported. In order to understand the biochemical function of this pea PR 10 protein, its cDNA has been expressed in Escherichia coli and the recombinant protein purified to homogeneity. Ribonuclease activity of the recombinant pea PR 10 protein has been demonstrated for the first time using an in-solution as well as an in-gel RNA degradation assay. Furthermore, in order to characterize the changes brought about as a result of the constitutive expression of the pea PR 10 cDNA in B. napus, we have measured the endogenous concentrations of several phytohormones. Increased cytokinin and, decreased abscisic acid (ABA) were observed in 7-day-old transgenic seedlings whereas no significant changes in the concentrations of gibberellin (GA) or indoleacetic acid (IAA) were observed at this stage of growth and development. The potential role(s) of PR 10 proteins with RNase activity and elevated cytokinins during plant stress responses as well as the possible relationship between PR 10 protein and changes in cytokinin concentrations are discussed.