G. W. M. Barendse

An Ethylene-Mediated Increase in Sensitivity to Auxin Induces Adventitious Root Formation in Flooded Rumex palustris Sm

The hormonal regulation of adventitious root formation induced by flooding of the root system was investigated in the wetland species Rumex palustris Sm. Adventitious root development at the base of the shoot is an important adaptation to flooded conditions and takes place soon after the onset of flooding. Decreases in either endogenous auxin or ethylene concentrations induced by application of inhibitors of either auxin transport or ethylene biosynthesis reduced the number of adventitious roots formed by flooded plants, suggesting an involvement of these hormones in the rooting process. The rooting response during flooding was preceded by increased endogenous ethylene concentrations in the root system. The endogenous auxin concentration did not change during flooding-induced rooting, but a continuous basipetal transport of auxin from the shoot to the rooting zone appeared to be essential in maintaining stable auxin concentrations. These results suggest that the higher ethylene concentration in soil-flooded plants increases the sensitivity of the root-forming tissues to endogenous indoleacetic acid, thus initiating the formation of adventitious roots.

Submergence-Induced Ethylene Synthesis, Entrapment, and Growth in Two Plant Species with Contrasting Flooding Resistances.

Submergence-induced ethylene synthesis and entrapment were studied in two contrasting Rumex species, one flood-resistant (Rumex palustris) and the other flood-sensitive (Rumex acetosa). The application of a photoacoustic method to determine internal ethylene concentrations in submerged plants is discussed. A comparison with an older technique (vacuum extraction) is described. For the first time ethylene production before, during, and after submergence and the endogenous concentration during submergence were continuously measured on a single intact plant without physical perturbation. Both Rumex species were characterized by enhanced ethylene concentrations in the shoot after 24 h of submergence. This was not related to enhanced synthesis but to continued production and physical entrapment. In R. palustris, high endogenous ethylene levels correlated with enhanced petiole and lamina elongation. No dramatic change in leaf growth rate was observed in submerged R. acetosa shoots. After desubmergence both species showed an increase in ethylene production, the response being more pronounced in R. palustris. This increase was linked to the enhanced postsubmergence growth rate of leaves of R. palustris. Due to the very rapid escape of ethylene out of desubmerged plants to the atmosphere (90% disappeared within 1 min), substantial underestimation of internal ethylene concentrations can be expected using more conventional vacuum extraction techniques.

Ethylene enhances gibberellin levels and petiole sensitivity in flooding-tolerant Rumex palustris but not in flooding-intolerant R. acetosa

Abstract. The role of gibberellin (GA) and ethylene in submergence-induced petiole elongation was studied in two species of the genus Rumex. Analysis of endogenous GAs in the flooding-tolerant Rumex palustris Sm. and the intolerant Rumex acetosa L. by gas chromatography-mass spectrometry showed for both species the presence of GA1, GA4, GA9, GA19, GA20 and GA53. Gas chromatography-mass spectrometry analysis of R. palustris petiole tissue of submerged plants showed an increase in levels of 13-OH GAs, especially GA1, compared with drained plants. This effect could be mimicked by application of 5 μL L−1 ethylene. In R. acetosa, no differences between levels of GAs in drained or submerged plants were found. In R. palustris, both submergence and ethylene treatment sensitized petioles to exogenous gibberellic acid (GA3). In R. acetosa the effect was opposite, i.e. submergence and ethylene de-sensitized petioles to GA3. Our results demonstrate the dual effect of ethylene in the submergence response related to flooding tolerance, i.e. in the flooding-tolerant R. palustris ethylene causes an increased concentration of and sensitivity to GA with respect to petiole elongation while in the intolerant R. acetosa ethylene reduces growth independent of GAs.

Formation of bound gibberellins inPharbitis nil

SummaryDeveloping seeds ofPharbitis nil accumulate free as well as bound gibberellins until a maximum level is reached at approximately 25 days after anthesis. Seeds from CCC-treated parent plants have a strongly reduced level of free as well as bound gibberellins. When different spray reagents were used it was found that trichloroacetic acid in particular was suitable to locate non-hydrolysed bound GA fractions on thin-layer plates. Chromatography showed two major bound GA fractions, determined with spray reagents as well as by means of hydrolysis.3H-GA1 applied to youngPharbitis plants was converted to two water-soluble compounds present in the aqueous phase. The rate of conversion was significantly enhanced when3H-GA1 and14C-glucose were applied to the same plants. Chromatography indicated that one of the conversion products of3H-GA1 became at least partly associated with the applied14C-glucose (or its products). This suggestion was also supported by the fact that mild acid hydrolysis of the aqueous fraction resulted in the reappearance of3H-GA1 and a conversion product of3H-GA1, including a14C-radioactivity peak cochromatographing with14C-glucose. However, the conversion products obtained with3H-GA1 applied to plants appeared to be chromatographycally different from any of the bound-GA fraction established by means of hydrolysis or spray reagents in developing seeds.

The metabolism of applied gibberellic acid in Pharbitis nil choisy: tentative identification of its sole metabolite as gibberellic acid glucoside and some of its properties.

SummaryRadioactive gibberellic acid ([14C]GA3) applied to seedlings of Pharbitis nil strain Violet is converted to one single metabolite (R-[14C]GA3), which has been tentatively identified as GA3-glucoside. As with authentic GA3-glucoside, R-[14C]GA3 can be hydrolysed to some extent with cellulase and β-glucuronidase, but hardly at all with β-glucosidase. Acid hydrolysis, which is more effective than enzymatic hydrolysis, yielded GA3 as well as a biological inactive compound. The latter represents a degradation product of GA3 due to the sensitivity of GA3 to acidic conditions.The R-[14C]GA3, like authentic GA3-glucoside, possesses little or no biological activity. R-[14C]GA3 applied to developing seeds is partly hydrolysed during imbibition of the mature seed but is, however, reconverted to R-[14C]GA3 during subsequent germination. Applied R-[14C]GA3 is strongly accumulated in the cotyledons of Pharbitis seedlings, to a greater extent than [14C]GA3. However, unlike [14C]GA3 it is not accumulated in the apical regions of the hypocotyl. Furthermore no competition was observed between R-[14C]GA3 and [14C]GA3, which suggests that they do not compete for the same sites.

Isolation and molecular characterization of gibberellin-regulated H1 and H2B histone cDNAs in the leaf of the gibberellin-deficient tomato.

After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B. The histones leH1 and leH2B, which show no tissue specificity, are developmentally expressed in the leaf. The mRNA accumulation was higher in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. In the leaf of the gib-1 mutant we demonstrated GA-enhanced histone leH1 and leH2B expression which was not observed in the wild type. GAs of the early-13-hydroxylated pathway (GA1 and GA3) caused most enhanced transcription compared to GAs of the early-non-hydroxylation pathway (GA4 and GA9). Application of GA to the mutant increased histone expression that could correlate with enhanced DNA replication in leaf tissue. Increased chromosome replication may indicate that there is a higher rate of cell division and/or increase of endopolyploidy which both may be dependent on cell elongation induced by GAs.

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.

Role of Benzyladenine in Flower Bud Regeneration in vitro: Timing of Hormone Action

Summary Benzyladenine was used to induce flower buds on pedicel explants of tobacco ( Nicotiana tabacum L.). The first buds appeared after 7 days and development was completed in 14 days. At the optimal concentration of 1 μmol L -1 , exposure to the hormone for only 2 days was sufficient to induce the same number of buds as were formed upon continuous incubation at the same concentration. The role of benzyladenine in flower bud formation is thus restricted to the induction phase of development unless a physiologically active concentration is maintained within the tissue after removal of the cytokinin from the medium. During the 2-day initiation period the concentration of free benzyladenine in the tissue was 0.5 μmol kg -1 , which is 50% lower than the medium concentration. More than 90% of the total benzyladenine in the tissue was bound in various conjugates. When explants that had been incubated on 1 μmol L -1 3 [H]benzyladenine were shifted to a medium without radioactivity, the internal concentration of free radiolabeled benzyladenine dropped 50-fold within 10 h irrespective of the cytokinin concentration in the chase medium. This reduction was a combined effect of conjugation and leakage of benzyladenine from the tissue. The results indicate that in explants induced to form flower buds by a 2-day exposure to l μmol L -1 benzyladenine, the hormone concentration rapidly drops to approximately 0.01 μmol kg -1 upon transfer of the tissue to hormone-free medium. This concentration presumably is too low to be physiologically active.

The diffusion of gibberellins into agar and water during early germination of Pharbitis nil Choisy

Agar diffusion of imbibed seeds yielded significant amounts of diffusible Gibberellin-like substances. An analysis of the extractable and diffusible gibberellin-like substance, including an analysis of the remaining imbibition water of the seeds, indicated that a significant part of these gibberellin-like substances could be attributed to a net biosynthesis of these substances in the imbibing seeds. At the same time it was found that water diffusion yielded considerably more gibberellin-like activities than comparable agar diffusions i.e. 10 to 12 fold in general.Agar as well as water diffusion showed a temperature effect with regard to the yield of gibberellin-like substances particularly during the first 6 h of diffusion. The yield of these substances is lower at 10°C, and remains lower as shown with consecutive diffusions, in comparison with the yields at 20°C or 30°C.With both agar and water diffusion the sum of activities obtained with consecutive diffusions is always higher, often considerably higher, than equal periods of continuous diffusion which is probably due to inactivation and/or interference of inhibitory substances with the bioassay responses. Finally, water diffusates of both seeds and seedlings of the normal growing cv. Violet of Japanese morning glory contained considerably more gibberellin-like activities than those of the dwarf cv. Kidachi which indicated that normals synthesize more gibberellins than dwarfs.

Uptake and metabolism of NAA and BAP in explants of tobacco in relation toin vitro flower bud formation

In vitro flower bud initiation and development depend on the presence of two hormones in the culture medium—auxin (NAA) and cytokinin (BAP). The uptake of both NAA and BAP by the explants was shown to be proportional to the concentrations supplied in the medium over a period of 4 days after the onset of culture. However, when supplied at equal concentrations for 24 h, the NAA uptake was up to 10-fold higher than the BAP uptake. Both hormones are rapidly metabolized by the explants. Nevertheless, the concentrations of free hormones inside the explants appeared to be high and in the case of NAA exceeded the concentration in the medium by more than 1 order of magnitude within 24 h. Apparently flower bud initiation in tobacco explants requires relatively high concentrations free NAA and BAP in the tissue maintained by a continuous supply in the medium. There are at present no indications that the products of hormone metabolism are directly involved in bud formation.