Bacterial Cytokinin Research Articles

Effect of Bacterial Strains with Different Ability to Synthesize Auxins and Cytokinins on the Growth and Water Relations of Wheat Plants

The content of cytokinins, auxins, chlorophyll, transpiration, content and relative content of water, water and osmotic potentials, hydraulic conductivity, morphological parameters of durum wheat (Triticum durum Desf.) plants after the introduction into their rhizosphere of cytokinin-producing strains (Bacillus subtilis IB-22) or auxin-producing (Pseudomonas mandelii IB-Ki14) bacteria was evaluated. The experiments were carried out in laboratory conditions in vessels with agrochernozem at an optimal level of illumination. It was shown that the cytokinin-producing strain stimulated plant growth, accumulation of chlorophyll, and increased transpiration to a greater extent compared to plants treated with the auxin-producing strain. Plants under the influence of the B. subtilis IB-22 strain lost more water during transpiration, while there was no decrease in the level of water content and turgor. It is concluded that bacterial cytokinins affect the water metabolism of wheat plants by increasing hydraulic conductivity, and bacterial auxins - to a greater extent due to osmotic regulation. A more significant stimulation of plant growth by bacteria of the B. subtilis IB-22 strain is also explained by the ability of cytokinins to influence the chlorophyll content to a greater extent than auxins.

Stable and reproducible expression of bacterial ipt gene under the control of SAM-specific promoter (pKNOX1) with interference of developmental patterns in transgenic Peperomia pellucida plants.

Reproducible and stable transgene expression is an important goal in plant basic research and applications. Hence, we report the first stable expression of bacterial transgenes in medicinal plant, Peperomia pellucida (L.) Kunth. Two key elements relevant to the dynamic expression of the bacterial cytokinin biosynthesis gene, ipt (isopentenyltransferase) were examined. First, by designing a specific expression cassette driven by a tissue-specific promoter for the required levels of gene expression in the particular function of development, and second by using P. pellucida as a model plant due to its short developmental cycle that supported expedient tracking of transgene expression in the progeny. Transgenic frequencies of ipt gene obtained from different expression cassettes of pKNOX1 for tissue-specific promoter in shoot apical meristem were compared with the cauliflower mosaic virus (CaMV35S) promoter (p35S), a constitutive promoter investigated for T3 generation. It was clearly shown that transgenic plants with pKNOX1 showed percentage of survivals in T3 at about 2.2 folds more than those of p35S-transgenic. Transgenic P. pellucida under controllable expression of pKNOX1 showed increased leaf and seed size with a high percentage of fertile seed, whereas transgenic plants with p35S showed phenotypic features of bushy and small leaves, sterile pollen and lower reproductive fitness. Quantitative examination of ipt-positive gene expression in T3 generation of transformants with pKNOX1 were 100% (line k-14) and 50% (line k-20), while 33.3% was observed in transgenic line c-11 with p35S. Interestingly, the endogenous cytokinin biosynthesis gene (ipt3) was significantly upregulated (2-3 folds higher) in pKNOX1-transformants. The overall relative mRNA expression of bacterial ipt gene and overproducing of cytokinin contents (t-ZR and 2-iP) detected in p35S-transformants caused abnormality and low percentages of transgene reproducible Interestingly, pKNOX1-transgenic plants tended to maintain chlorophyll contents 4-5 folds and extending the developmental cycle to 12.4 weeks (wk), which was 2 folds more than wildtype (5.8 wk) and p35S-transformants (7.4 wk). The promotor effect on stable and reproducible transgene-expressions demonstrated prominent features of P. pellucida and also empowered further omics studies.

Both epiphytic and endophytic strains of Rhodococcus fascians influence transporter gene expression and cytokinins in infected Pisum sativum L. seedlings

Some strains of the soil bacterium Rhodococcus fascians maintain an epiphytic life style while others become endophytic. Virulent, endophytic strains cause multiple shoot growth and inhibit root growth of seed-inoculated Pisum sativum L. We were interested in assessing, at the molecular level, the impact of strains of contrasting niche on the emerging shoots and roots of inoculated seeds. The presence of R. fascians was monitored microscopically, endogenous cytokinin and chlorophyll levels were measured, and the expression of genes monitored by RT-qPCR. The expression of the pea sugar transporter genes (SWEET and SUT), amino acid (AAP) transporters and cell wall invertase gene family members, as well as expression of plant and bacterial cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) genes were monitored. Both the virulent strain and the epiphytic strain affected the expression of the transporter genes, with less obvious differences between the strains on the shoot compared with the effect on the root. Strong expression of the R. fascians genes, RfIPT, RfLOG and RfCKX, in pea seedlings at 15 days post inoculation was mirrored by increased expression of transporter gene family members in the plant. However, the elevated levels of isopentenyl adenine-type and zeatin-type cytokinins were not consistently associated with the virulent strain. In conclusion, while both the virulent strain and the epiphytic strain impacted the expression of transporter genes in the shoots and roots, only the virulent strain affected morphology. The inhibited root growth, the greening of the roots, and the expression of the pea response regulators in the infected roots are indicative of a response to cytokinin, but a role for the ‘classical’ cytokinins as virulence determinants was not established.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana.

Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures-including the strigolactone response-in reaction to bacterial cytokinins to constrain the activity of R. fascians.

The leafy gall syndrome induced byRhodococcus fascians

The Actinomycete Rhodococcus fascians causes the leafy gall syndrome, an infectious plant disease that affects a wide range of plants, primarily dicotyledonous herbs. The syndrome is associated with delayed senescence, loss of apical dominance, activation of dormant axillary meristems, and formation of multiple inflorescences, leading to a stunted and bushy plant appearance. A major breakthrough in the elucidation of the virulence strategy of this pathogen was the discovery of a linear virulence plasmid, pFiD188 for R.fascians strain D188. Upon perception of a compatible host plant, an autoregulatory mechanism mediated by the att operon directs a switch in the bacterial life style from a harmless epiphyte into a pathogenic endophyte and, concomitantly, activates gene expression of the fas operon that encodes a cytokinin biosynthesis pathway. A mixture of five cytokinins determines the cytokinin activity of R.fascians that directly affects plant responses and development. Moreover, the bacterial cytokinins stimulate the host to produce auxins and polyamines, that function as accessory signals to aid in symptom development. The plant reacts against the developmental hijacking by R.fascians by activating a set of counteracting measures that ultimately results in a delicate balance, allowing a long-lasting biotrophic interaction.

Plant‐derived auxin plays an accessory role in symptom development upon Rhodococcus fascians infection

The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.

Interactions of bacterial cytokinins and IAA in the rhizosphere may alter phytostimulatory efficiency of rhizobacteria

Phytohormones from rhizobacterial origin have been linked to their phytostimulation potential. However, while studying the efficacy of plant growth promoting bacteria, focus has always been on a single hormone. The role of plant hormones often overlay and they mutually modulate their effect. In current study focus was on the role of two hormones (cytokinins and indole acetic acid) in phytostimulation by rhizobacteria. Endogenous rhizosphere bacteria were isolated and screened for the presence of phytohormones. Bacterial strains from three different genera (Pseudomonas, Bacillus and Azospirillum) were screened positive for cytokinins and IAA. Phytohormones were simultaneously determined in SPE purified bacterial extract by ultra performance liquid chromatography (UPLC) coupled to a tandem mass spectrometer through electrospray interface. Cytokinins and IAA were determined in positive and negative mode, respectively with MRM scan. Zeatin, zeatin riboside and dihydrozeatin riboside were detected and quantified in the selected strains. Significant positive correlation between cytokinins and IAA in bacterial culture and plant endogenous hormones (r = 0.933 and r = 0.983; P = 0.01, respectively) was observed. However, strains with high IAA to cytokinins ratio could hardly enhance in-planta cytokinins, indicating antagonistic relation between the two hormones. Significant correlation of cytokinin with shoot length (r = 0.797; P = 0.01), fresh weight (r = 0.685; P = 0.01) and dry weight (r = 0.704; P = 0.01) was reported under axenic conditions. Bacterial IAA was correlated negatively to root length (r = 0.853; P = 0.01) and positively correlated to the number of roots (r = 0.964; P = 0.01). In natural conditions maximum increase in spike length (33%), number of tillers (71%) and weight of seeds (39%) was documented at final harvest in bacterially inoculated plants.

Bacterial and plant signal integration via D3-type cyclins enhances symptom development in the Arabidopsis-Rhodococcus fascians interaction.

The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

Effect on shoot water relations, and cytokinin and abscisic acid levels of inducing expression of a gene coding for isopentenyltransferase in roots of transgenic tobacco plants

Heat shock (HS) at 40 degrees C was given to the root system of Nicotiana tabacum wild type (WT) and to HSIPT transgenic plants transformed with the bacterial cytokinin biosynthesis gene isopentenyltransferase (ipt) cloned behind the heat shock 70 promoter from Drosophila melanogaster. HS increased cytokinin concentrations in roots and leaves of transgenic plants. The effect was smaller in WT plants and restricted to upper leaves. HS also increased the activity of the cytokinin-degrading enzyme cytokinin oxidase in leaves of transgenic plants. This suggests that increases in cytokinin concentration induced by HS were lessened but not eliminated by increases in cytokinin oxidase. Elevated levels of zeatin riboside (the main transportable form of cytokinin) were also found in the HS-treated roots. It is proposed that increases in leaves were the outcome of increased transport of this hormone from roots in the transpiration stream. In conjunction with increased leaf cytokinin concentration, HS treatment to the roots increased stomatal conductivity and transpiration in both transgenic and WT plants. Subsequently, increased transpiration depressed leaf relative water content. This, in turn, raised leaf abscisic acid (ABA) concentrations, resulting in stomatal closure. It is concluded that the preceding increases in leaf cytokinin concentration, stomatal opening, and faster transpiration resulting from the localized induction of ip gene expression in roots strengthens the concept of cytokinin involvement in root to shoot signalling.

Promotion of shoot development and tuberisation in potato by expression of a chimaeric cytokinin synthesis gene at normal and elevated CO2 levels

The bacterial cytokinin biosynthesis gene ipt under control of a chalcone synthase promoter (PCHS) was introduced into potato (Solanum tuberosum L.). Two transgenic lines were selected for detailed study, because in these, root development was reduced only moderately, thus, enabling the plants to be grown in pots. Expression of the PCHS-ipt gene elevated the level of zeatin cytokinins markedly in the apical bud, subapical stems and leaves. The transgenic (IPT) plants exhibited a lower and denser leaf canopy relative to wild-type (WT) plants owing to reduction in main stem length, increase in node number per stem and promotion of lateral shoot development. Main stem diameter was increased markedly due to promotion of cell division associated with activation of cyclin-dependent kinase in the subapical stem. Expression of the PCHS-ipt gene induced aerial stolons, promoted growth of underground stolons and increased tuber number but reduced tuber weight and nitrogen content. The gene expression also increased pinnae and pinnule number per leaf, increased thickness of pinnae and promoted transpiration, photosynthesis and stomatal conductance – effects monitored by gas exchange and 18O and 13C analysis. The elevation of [CO2] to 900 μmol mol–1 promoted growth of both WT and IPT plants, ameliorated the negative effect of high cytokinin on tuber weight and interacted additively with ipt gene expression to promote stem growth.

Growth habit and sugar accumulation in sugarbeet (Beta vulgaris L.) transformed with a cytokinin biosynthesis gene

Expression of a bacterial cytokinin biosynthesis gene fused to a patatin gene promoter was studied in sugarbeet (Beta vulgaris L.). Two independent transformants, Pat-ipt 1 and 2, exhibited a number of distinguishable morphological alterations commonly induced by cytokinins, i.e. less root growth, reduced leaf surface area, and increased axillary shoot development. Concentrations of the cytokinins zeatin and zeatin riboside were increased by twofold in taproots and 7- to 18-fold in leaves. Leaf sucrose and glucose concentrations were not significantly different from those in control plants except in Pat-ipt 2 where glucose levels were elevated ninefold. Since normal taproot development was severely inhibited, sucrose concentrations in the taproots were significantly reduced.

Analysis of insecticidal activity in transgenic plants carrying the ipt plant growth hormone gene

The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below 42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance.

Occurrence of plant hormones in cells of the phototrophic purple bacteriumRhodospirillum rubrum1R

Plant hormones from biomass of the purple non-sulfur bacterium Rhodospiririllum rubrum were isolated for the first time. These compounds show high physiological activities (300–330%) in the cytokinin bioassay. All three detected cytokinins are adenine derivatives, according to spectral analysis. One of them was identified as 6-(4-hydroxy-3-methyl-2-trans-2-bytenylamino)-9-β-D-ribofuranosylpurine (zeatinriboside) as shown by thin-layer chromatography and reversed-phase high-performance liquid chromatography. The possible functions of bacterial cytokinins are also discussed.

Occurrence of plant hormones in cells of the phototrophic purple bacterium Rhodospirillum rubrum 1R

Plant hormones from biomass of the purple non-sulfur bacterium Rhodospiririllum rubrum were isolated for the first time. These compounds show high physiological activities (300–330%) in the cytokinin bioassay. All three detected cytokinins are adenine derivatives, according to spectral analysis. One of them was identified as 6-(4-hydroxy-3-methyl-2-trans-2-bytenylamino)-9-β-D-ribofuranosylpurine (zeatinriboside) as shown by thin-layer chromatography and reversed-phase high-performance liquid chromatography. The possible functions of bacterial cytokinins are also discussed.

Cytokinins in Plant Pathogenic Bacteria and Developing Cereal Grains

Cytokinin analysis by immunoaffinity chromatography (IAC), high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) or enzyme-linked immunosorption assay (ELISA) has been used to study two separate topics: the role of tRNA in bacterial cytokinin biosynthesis and the changes in cytokinin concentration which occur during cereal grain development. Transfer RNA isopentenylation in the gall-forming plant pathogen Agrobacterium tumefaciens is encoded by the chromosomal miaA locus. Mutation of miaA reduces tRNA isopentenylation significantly and preliminary data suggest that turnover of isopentenylated tRNA is responsible for low level secretion of free N6-isopentenyladenine (iP) by the bacteria. However, the major route of cytokinin biosynthesis by gall-forming plant patho- genic bacteria is not via tRNA turnover but by direct biosynthesis mediated by dimethylallylpyro- phosphate: 5'-AMP transferase (DMAPP :AMP transferase) encoded by such genes as ipt, tzs (from A, tumefaciens) or ptz (from Pseudomonas savastanoi). Analysis of cytokinin levels in developing wheat and rice grains in the period immediately following pollination showed large transient increases in zeatin (Z) and zeatin riboside (ZR) which coincided with the period of maximum endosperm cell division reported by others. Detailed analyses of maize kernels, where development can be staged readily, showed that Z and ZR concentrations peaked 9 days after pollination (DAP). During the period 8-10 DAP, cytokinin oxidase underwent a significant increase in specific activity, indicating that cytokinin catabolism was enhanced as endosperm cell division ended.

Rapid Identification of Cytokinins by an Immunological Method

A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed [(3)H]adenine, the cytokinins (including (3)H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of trans-zeatin, dihydrozeatin, 1''-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances. Corynebacterium fascians, produced cis-zeatin, isopentenyladenine and isopentenyladenosine. The technique is designed for qualitative rather than quantitative studies and allows ready identification of bacterial cytokinins. It may also have utility in the study of plant cytokinins if adequate incorporation of label into cytokinin precursor pools can be achieved.