Björn Nicander

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.

Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer

tRNA-isopentenyltransferase (tRNA-IPT) catalyses the addition of N6-isopentenyladenosine (i6A) on residue 37 of tRNA molecules that bind codons starting with uridine. Post-transcriptional modifications of tRNA molecules have been demonstrated to be essential in maintaining the correct reading frame of the translational machinery, thus improving fidelity and efficiency of protein synthesis. We show here that the human tRNA-isopentenyltransferase (TRIT1) gene encodes a complex pattern of mRNA variants through alternative splicing in both normal and tumor lung tissue and that the nonsense suppressor activity of tRNA-IPT is maintained only in the full-length mRNA isoform, as revealed by gene complementation in yeast. Expression of the full-length transcript was down-regulated 6–14-fold in lung adenocarcinomas as compared to normal lung tissue. A549 lung cancer cells transfected to express the functional TRIT1 gene formed significantly smaller colonies with reduced scattering on the edges and had only limited ability to induce tumors in nude mice. Our findings raise the possibility of TRIT1 as a candidate lung tumor suppressor.

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.

Soybean (Glycine max) SWEET gene family: insights through comparative genomics, transcriptome profiling and whole genome re-sequence analysis

BackgroundSWEET (MtN3_saliva) domain proteins, a recently identified group of efflux transporters, play an indispensable role in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development. The SWEET gene family is predominantly studied in Arabidopsis and members of the family are being investigated in rice. To date, no transcriptome or genomics analysis of soybean SWEET genes has been reported.ResultsIn the present investigation, we explored the evolutionary aspect of the SWEET gene family in diverse plant species including primitive single cell algae to angiosperms with a major emphasis on Glycine max. Evolutionary features showed expansion and duplication of the SWEET gene family in land plants. Homology searches with BLAST tools and Hidden Markov Model-directed sequence alignments identified 52 SWEET genes that were mapped to 15 chromosomes in the soybean genome as tandem duplication events. Soybean SWEET (GmSWEET) genes showed a wide range of expression profiles in different tissues and developmental stages. Analysis of public transcriptome data and expression profiling using quantitative real time PCR (qRT-PCR) showed that a majority of the GmSWEET genes were confined to reproductive tissue development. Several natural genetic variants (non-synonymous SNPs, premature stop codons and haplotype) were identified in the GmSWEET genes using whole genome re-sequencing data analysis of 106 soybean genotypes. A significant association was observed between SNP-haplogroup and seed sucrose content in three gene clusters on chromosome 6.ConclusionPresent investigation utilized comparative genomics, transcriptome profiling and whole genome re-sequencing approaches and provided a systematic description of soybean SWEET genes and identified putative candidates with probable roles in the reproductive tissue development. Gene expression profiling at different developmental stages and genomic variation data will aid as an important resource for the soybean research community and can be extremely valuable for understanding sink unloading and enhancing carbohydrate delivery to developing seeds for improving yield.

Cloning of a human tRNA isopentenyl transferase.

A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.

Identification of two additional members of the tRNA isopentenyltransferase family in Physcomitrella patens.

The Physcomitrellapatens genome has seven genes apparently coding for the isopentenyltransferase type of tRNA-modifying enzyme, while other organisms have one or two. The predicted sequences have parts that differ significantly from other isopentenyltransferases. Only one of the seven (PpIPT1) has earlier been shown to be expressed. We now report expression of two more, PpIPT4 and PpIPT5. The cloned genes were able to functionally complement a yeast mutant lacking tRNA isopentenyltransferase. Sequencing showed they are related to the earlier studied PpIPT1. The sequences of the three differ mainly from each other in a tRNA-binding area and the 5′-end subcellular targeting motif area. This indicates that, after arising through gene duplication, they have evolved to enable partly different functions.

Irradiance-induced alterations of growth and cytokinins in Phaseolus vulgaris seedlings

Light is a major environmental factor affecting plant growth and development. The cytokinins have many similar effects on these processes and may be involved in photomorphogenesis. In order to study the correlation between light and endogenous cytokinins, we have examined growth parameters and endogenous cytokinins in stems, leaves and other organs of Phaseolus vulgaris, cultivated for 10 days under a range of irradiances (25, 110, 350 and 500 µmol m−2 s−1). The nucleotides isopentenyladenosine-5′-monophosphate and zeatin riboside-5′-monophosphate were the dominant cytokinins, whereas both free bases and ribosides were below the detection level (0.5 pmol g−1). Plants grown at the highest irradiance had in their stems, leaves, petioles and roots significantly higher levels of cytokinins than had plants grown at the lowest irradiance. As expected, increased light influx increased the dry weight of the root, petiole and leaf, and increased the leaf area, with concomitant increases in the cytokinins in these plant parts. However, the stem showed a different and more complex relationship with irradiance. Stem cytokinin levels increased drastically between 350 and 500 µmol m−2 s−1, but this was not correlated with any change in stem length; the light inhibition of stem elongation was mainly seen when irradiance was increased to 110 µmol m−2 s−1. Taken as a whole, the results are consistent with an effect of irradiance and cytokinins on the processes favouring biomass production.

Expression of a human tRNA isopentenyltransferase in tobacco reveals a developmental role for tRNA isopentenyladenosine

In addition to their role as plant hormones, cytokinins are also found as structural components in tRNA. Six different tRNA cytokinins have been found in plants, but most other organisms, including humans, have only one-isopentenyladenosine. In an attempt to probe if the different forms have different functionality, we attempted to alter tRNA cytokinin composition by expressing the human tRNA isopentenyltransferase gene (EC 5.1.2.8) in tobacco [Nicotiana tabacum (L.) cv. Wisconsin 38]. The resulting transgenics had ~40% more isopentenyladenosine in tRNA, and an altered phenotype characterised by reduced internode length, increased stem diameter and rigidity, greener leaves, increased axillary bud outgrowth, abnormal flower morphology, and reduced seed viability. The levels of the two other major isoprene adenines of tRNA, cis-zeatin and 2-methyltiolated cis-zeatin, were also increased, but to a lower degree. Nearly all of the increase in isopentenyladenosine was in a single tRNA species. Two quantitatively minor isopentenyladenosine-containing tRNAs had also increased strongly. IPPT: Dimethylallylpyrophosphate.