Elisabeth Tillberg

Cytokinin production by Paenibacillus polymyxa

Abstract The production of hormones has been suggested to be one of the mechanisms by which plant growth-promoting rhizobacteria (PGPR) stimulate plant growth. To evaluate whether the free-living soil bacterium, Paenibacillus polymyxa, releases the hormone group cytokinins and, if so, their identity, the content of cytokinins in the growth media, before and after cultivation of this bacterium, was determined by immunoaffinity chromatography (IAC). This method allows the isolation of almost all known cytokinins and their metabolites. Separation and characterization were done by high performance liquid chromatography (HPLC) with on-line ultraviolet (UV) detection, and final identification was by gas chromatography-mass spectrometry. Iso-pentenyladenine (iP) was identified in the two defined media used for the cultivation of P. polymyxa, but not earlier than at its late stationary growth. A third medium, supplemented with yeast extract, contained iso-pentenyladenine riboside (iPR) and some additional cytokinin-like substances before inoculation. When the same medium was sampled after the cultivation of P. polymyxa up to its logarithmic growth phase, the cytokinin concentration had decreased. After prolonged cultivation of P. polymyxa, small amounts of iP appeared in all three media, and iPR had disappeared from the yeast-containing medium, which indicates that the bacterium can metabolize cytokinins.

A rapid increase in cytokinin levels and enhanced ethylene evolution precede Al3+-induced inhibition of root growth in bean seedlings (Phaseolus vulgaris L.)

The role of plant hormones in aluminium (Al3+)-inducedinhibition of root growth was investigated in roots of Phaseolusvulgaris L. cv. Strike. Changes in ethylene evolution and changes inthe content and composition of cytokinins (CKs), at intervals up to 150min after treatment with Al3+, were determined by gaschromatography (GC) and combined gas chromatography-mass spectrometry (GC-MS),respectively. Seedlings were cultivated in a continuously aerated nutrientsolution at pH 4.5 with and without Al3+. The growth rate ofAl3+-treated roots was significantly decreased after 360min. Ethylene evolution from excised root tips doubled after 15min of Al3+ treatment and reached a maximum 30min after treatment. Levels of CK nucleotides declined 60 to 80%after only 5 min of Al3+ treatment whereas the zeatin(Z) content increased six-fold. The increase in Z continued over the entire 150min-sampling period and reached a level 80 times higher than thatin roots not exposed to Al3+. These results show thatAl3+-induced inhibition of root growth is preceded by significantchanges in CK content and composition and enhanced ethylene evolution. Since CKcan induce ethylene production, the rapid increase in CK, particularly Z anddihydrozeatin (dZ), may contribute to inhibition of root-growth either directlyor indirectly by affecting plant hormone homeostasis.

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.

Substrates of diacylglycerol acyltransferase in microsomes from developing oil seeds

Abstract Selectivities for diacylglycerol and acyl-CoA species by oil seed diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) were studied in enzyme assays with microsomal enzymes from developing seeds of castor bean ( Ricinus communis ), Cuphea procumbens and sunflower ( Helianthus annuus ). Seeds of castor bean and Cuphea procumbens accumulate storage lipids with acyl groups that are excluded from membrane lipids (ricinoleoyl and caproyl moieties, respectively), whereas sunflower seeds accumulate only acyl groups that are common for both membrane and storage lipids. Cuphea procumbens DAGAT showed pronounced selectivities for diacylglycerols and acyl-CoAs containing caproyl groups and the castor bean enzyme showed selectivities for substrates containing ricinoleate. These results are in accordance with the suggested role for DAGAT in the selective channelling of the unusual acyl groups into triacylglycerols in these seeds. Cuphea procumbens and sunflower DAGAT showed a selective acylation of acyl-CoA species from mixtures of palmitoyl-, stearoyl- and oleoyl-CoA. Thus, the activity of their DAGATs will also influence the molecular species of ‘common’ triacylglycerol which will be formed.

Characterization of UDP-glucose:solanidine glucosyltransferase and UDP-galactose:solanidine galactosyltransferase from potato tuber

Abstract Upon differential fractionation of potato tuber homogenate, the soluble enzym uridine-5′-diphospho-glucose(UDP-glucose):solanidine glucosyltransferase remained for the main part in the supernatants, whereas the membrane-bound UDP-glucose:β-sitosterol glucosyltransferase activity was found both in a 13 000-g and 100 000-g pellet. The-soluble fraction glycosylated not only solanidine, but also the steroidal alkaloids solasodine and tomatidine, using either UDP-glucose or UDP-galactose as sugar donor. The K m -values for the sugar donors in the solanidine glycosylating reactions were similar: UDP-glucose, 22 μM and UDP-galactose, 29 μM. UDP-galactose was a competitive inhibitor in the solanidine glucosylating reaction ( K i 1.1 mM), whereas UDP-glucose showed non-competitive inhibition in the galactosylation reaction ( K i 21 μM). After separations on gelfiltration, ion-exchange or chromatofocusing columns,glucosylating and galactosylating activities were recovered in the same fractions, but with loss of most of the galactosyltransferase activity in the two latter types of separation. The apparent molecular weight of the solanidine glycosylating enzyme(s) was 40 000 and the isoelectric point 4.8.

Regulation of Glycoalkaloid Accumulation in Potato Tuber Discs

Summary The level of glycoalkaloids present in freshly cut potato tuber discs started to increase after 24 hours of incubation. This accumulation was inhibited by the sterol synthesis inhibitor, tridemorph, and was thus due to synthesis de novo . Concomitant to the accumulation of glycoalkaloids, there was an increase in the specific activity of a glycoalkaloid-specific enzyme, UDP-glucose:solanidine glucosyltransferase (solanidine-GT, EC 2.4.1). Time-course studies of enzyme activities in the discs showed that solanidine-GT activity increased at a low rate during the first 11 hours after slicing and at the highest rate between 11 and 16 hours. Other sterol-metabolizing enzymes exhibited different time-course curves:S-adenosyl-L-methionine:cycloartenol methyltransferase (cycloartenol-MT, EC 2.1.1.41) activity increased 5 hours after slicing and peaked at 11 hours. UDP-glucose:sterol glucosyltransferase (sterol-GT, EC 2.4.1) activity was not enhanced as a result of wounding. The accumulation of glycoalkaloids was not affected by light exposure or addition of abscisic acid, gibberellic acid or (2-chloroethyl)trimethylammonium chloride to the discs. Inhibition of ethylene synthesis or action gave a higher content of glycoalkaloids than in control discs, whereas discs incubated at high levels of ethylene had a very low glycoalkaloid content and also a lower activity of solanidine-GT than control discs.

Stress-induced Ethylene Biosynthesis in Pine Needles: A Search for the putative 1 -aminocyclopropane-l-carboxylic Acid-Independent Pathway

Summary A possible involvement of an alternative, 1-aminocyclopropane-l-carboxylic acid (ACC) independent pathway of ethylene biosynthesis in ethylene evolution from stressed pine ( Pinus sylvestris L.) needles was investigated. Inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine and aminooxyacetic acid) and lipid peroxidation (selenomethionine) were applied to establish whether they can be used to distinguish between different pathways of ethylene production. Ethylene biosynthesis in detached pine needles was initiated by induction of ACC synthesis. Exogenous H 2 0 2 and Na 2 S 2 0 5 induced increases in ethylene production in detached needles in a concentration-dependent manner. Aminooxyacetic acid completely prevented an increase in ACC content in detachment-stressed needles and by 90% and 80% inhibited that in H 2 0 2 and Na 2 S 2 0 5 -treated ones. However, a significant portion of ethylene produced by stressed needles was insensitive to the inhibitor. Cycloheximide completely inhibited H 2 0 2 -induced ethylene production. Based on aminooxyacetic acid and selenomethionine experiments, it is possible to exclude a participation of an ACC-independent pathway in ethylene production by stressed pine needles, with only the exception of that induced by high concentrations of Na 2 S 2 0 5 . The data collectively indicate that the residual rate of ethylene production from plant tissues in the presence of inhibitors does not reflect an ACC-independent pathway.

Effect of light on abscisic acid content in photosensitive Scots pine (Pinus sylvestris L.) seed

Dormancy-breaking treatment of the photosensitive Scots pine (Pinus sylvestris L.) seed by white light incubation or a 15-min exposure to red light decreased the abscisic acid content prior to radicle protrusion. Incubation in the dark or exposure to red light followed by a 5-min far-red light irradiation did not cause as great a decrease in abscisic acid content nor was the dormancy relieved. The ability of the far-red light to keep the ABA level high and to prevent germination gradually disappeared as the length of the dark period between the red and far-red treatments was increased to 24 h. ABA was quantified on a gas chromatograph with an electron capture detector.

Levels of IAA, ABA and carbohydrates in source and sink leaves of Betula pendula Roth.

Young birch (Betula pendula Roth) seedlings were grown in hydroponic cultures to which nutrients were added in amounts that increased exponentially over time. The nutrient additions were adjusted to give three different suboptimal, but stable, relative growth rates (RGR). Levels of glucose, fructose, sucrose and starch and the hormones 3‐Indolyl acetic acid (IAA) and abscisic acid (ABA) were determined in immature (sink) and mature (source) leaves and related to the measured RGR of the seedlings. The results show that ABA increased and IAA decreased in the sink leaves as the RGR of the plants decreased. This occurred in concert with a decrease in soluble sugar levels and starch accumulation in the source leaves. These observations suggest that ABA and IAA may be involved in source‐sink communication in the seedlings, although such causal relationships remain to be proven.

Effect of red and far-red irradiation on ABA and IAA content in Pinus sylvestris L. seed during the escape time period from photocontrol

The time-course of ABA and IAA dynamics in the photosensitive Scots pine (Pinus silvestris L.) seed was followed during the escape time period from photocontrol, with recordings of the respective hormone immediately before and after a 15-min red irradiation (R) stimulus and after subsequent dark incubation for periods of 10 min, 30 min, 4 h, 8 h and 24 h. The control treatment was exposure to R as above immediately followed by exposure to 5-min far-red irradiation (FR), and its effect was recorded in parallel. The hormones were identified on a gas chromatograph with a mass selective detector, and quantified on a high performance liquid chromatograph with UV detector and spectrofluorophotometer, respectively. Only ABA responded differently to the two irradiation treatments. The difference in ABA content between paired treatments with R and R + FR was significant after 6 to 8 with an enhanced decrease in ABA content in seeds that received a dormancy-breaking R treatment. The IAA content in the seeds decreased during the experimental period regardless of the type of irradiation treatment.