ISOPENTENYL TRANSFERASE
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Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.
- MeSH
- biologická evoluce MeSH
- cytokininy metabolismus MeSH
- Escherichia coli enzymologie růst a vývoj MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny genetika růst a vývoj metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mutageneze cílená MeSH
- Nostoc enzymologie genetika MeSH
- oxidoreduktasy genetika metabolismus MeSH
- prenyltransferáza genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- rekombinantní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- tabák enzymologie růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation.
Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot-infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT-LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans-zeatin, a reaction catalysed by a CK-specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt-log. Deletion of cpipt-log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper-sporulating phenotype, implying that CKs are environmental factors influencing fungal development.
- MeSH
- alkyltransferasy a aryltransferasy metabolismus MeSH
- Claviceps genetika růst a vývoj metabolismus MeSH
- cytokininy biosyntéza MeSH
- delece genu MeSH
- geny hub genetika MeSH
- isopentenyladenosin biosyntéza MeSH
- rostlinné geny genetika MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- žito mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MAIN CONCLUSION: Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.
- MeSH
- alkyltransferasy a aryltransferasy genetika metabolismus MeSH
- Arabidopsis genetika metabolismus MeSH
- cytokininy biosyntéza MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- listy rostlin genetika metabolismus MeSH
- Populus genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.
- MeSH
- alkyltransferasy a aryltransferasy metabolismus MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- chromatografie kapalinová MeSH
- cytokininy farmakologie MeSH
- dexamethason farmakologie MeSH
- geneticky modifikované rostliny MeSH
- hmotnostní spektrometrie MeSH
- ječmen (rod) účinky léků metabolismus MeSH
- metabolom účinky léků genetika MeSH
- metabolomika MeSH
- proteiny huseníčku metabolismus MeSH
- proteom metabolismus MeSH
- proteomika MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- semenáček účinky léků genetika MeSH
- upregulace účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transfer RNA isopentenylation an adenine 37 position (A37) is a universal modification known in prokaryotes and eukaryotes. A set of highly homologous enzymes catalyse a series of reactions, leading to tRNA modifications, aimed to increase adaptation to environmental condition through the control of translation efficiency and reading frame maintenance. Transfer RNA-isopentenylation-related (TI-related) functions are well studied in bacteria, mitochondria of yeast and human, but completely unexplored in plants. Transfer RNA-isopentenylation-unrelated (TI-unrelated) functions participate in adaptation to environmental stresses via the regulation of sterol metabolism, gene silencing/suppression and amyloid fibrils formation. TI-unrelated functions are mostly studied in yeast. Finally, the degradation of A37-modified tRNA releases a set of bioactive compounds known as cis-cytokinins. Although all organisms are able to produce cis-cytokinins, its physiological role is still a matter of debates. For several species of bacteria and fungi, cis-cytokinins are known to play a crucial role in pathogenesis. In mammalian and human models cis-cytokinins have tumour-suppressing and anti-inflammation effects. This review aims to summarise current knowledge of the TI-related and TI-unrelated functions and main bioactive by-products of isopentenylated tRNA degradation.
The diverse forms of today's dominant vascular plant flora are generated by the sustained proliferative activity of sporophyte meristems at plants' shoot and root tips, a trait known as indeterminacy [1]. Bryophyte sister lineages to the vascular plants lack such indeterminate meristems and have an overall sporophyte form comprising a single small axis that ceases growth in the formation of a reproductive sporangium [1]. Genetic mechanisms regulating indeterminacy are well characterized in flowering plants, involving a feedback loop between class I KNOX genes and cytokinin [2, 3], and class I KNOX expression is a conserved feature of vascular plant meristems [4]. The transition from determinate growth to indeterminacy during evolution was a pre-requisite to vascular plant diversification, but mechanisms enabling the innovation of indeterminacy are unknown [5]. Here, we show that class I KNOX gene activity is necessary and sufficient for axis extension from an intercalary region of determinate moss shoots. As in Arabidopsis, class I KNOX activity can promote cytokinin biosynthesis by an ISOPENTENYL TRANSFERASE gene, PpIPT3. PpIPT3 promotes axis extension, and PpIPT3 and exogenously applied cytokinin can partially compensate for loss of class I KNOX function. By outgroup comparison, the results suggest that a pre-existing KNOX-cytokinin regulatory module was recruited into vascular plant shoot meristems during evolution to promote indeterminacy, thereby enabling the radiation of vascular plant shoot forms.
Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
- MeSH
- Agrobacterium tumefaciens MeSH
- cytokininy biosyntéza genetika MeSH
- exprese genu * MeSH
- fertilita genetika MeSH
- geneticky modifikované rostliny enzymologie genetika MeSH
- ječmen (rod) enzymologie genetika MeSH
- kořeny rostlin embryologie genetika MeSH
- oxidoreduktasy biosyntéza genetika MeSH
- rostlinné proteiny biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteins MiaA and MiaB catalyze sequential isopentenylation and methylthiolation, respectively, of adenosine residue in 37th position of tRNAXXA. The mia mutations were recently shown by us to affect secondary metabolism and morphology of Streptomyces. However, it remained unknown as to whether both or one of the aforementioned modifications is critical for colony development and antibiotic production. Here, we addressed this issue through analysis of Streptomyces albus J1074 strains carrying double miaAmiaB knockout or extra copy of miaB gene. The double mutant differed from wild-type and miaA-minus strains in severity of morphological defects, growth dynamics, and secondary metabolism. Introduction of extra copy of miaB gene into miaA mutant restored aerial mycelium formation to the latter on certain solid media. Hence, miaB gene might be involved in tRNA thiomethylation in the absence of miaA; either MiaA- or MiaB-mediated modification appears to be enough to support normal metabolic and morphological processes in Streptomyces.
- MeSH
- alkyltransferasy a aryltransferasy genetika MeSH
- antibakteriální látky biosyntéza MeSH
- bakteriální geny genetika MeSH
- bakteriální proteiny genetika MeSH
- fenotyp * MeSH
- genetická transkripce MeSH
- genetické testování metody MeSH
- mutace MeSH
- peroxid vodíku farmakologie MeSH
- posttranskripční úpravy RNA * MeSH
- RNA transferová metabolismus MeSH
- sekundární metabolismus účinky léků genetika MeSH
- Streptomyces účinky léků genetika růst a vývoj metabolismus MeSH
- sulfurtransferasy genetika MeSH
- Publikační typ
- časopisecké články MeSH
