miRNA expression profiling and zeatin dynamic changes in a new model system of in vivo indirect regeneration of tomato.

Callus formation and adventitious shoot differentiation could be observed on the cut surface of completely decapitated tomato plants. We propose that this process can be used as a model system to investigate the mechanisms that regulate indirect regeneration of higher plants without the addition of exogenous hormones. This study analyzed the patterns of trans-zeatin and miRNA expression during in vivo regeneration of tomato. Analysis of trans-zeatin revealed that the hormone cytokinin played an important role in in vivo regeneration of tomato. Among 183 miRNAs and 1168 predicted target genes sequences identified, 93 miRNAs and 505 potential targets were selected based on differential expression levels for further characterization. Expression patterns of six miRNAs, including sly-miR166, sly-miR167, sly-miR396, sly-miR397, novel 156, and novel 128, were further validated by qRT-PCR. We speculate that sly-miR156, sly-miR160, sly-miR166, and sly-miR397 play major roles in callus formation of tomato during in vivo regeneration by regulating cytokinin, IAA, and laccase levels. Overall, our microRNA sequence and target analyses of callus formation during in vivo regeneration of tomato provide novel insights into the regulation of regeneration in higher plants.

CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola.

We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.

Identification of Potential Genes Responsible for Thermotolerance in Wheat under High Temperature Stress.

Wheat, a major worldwide staple food crop, is relatively sensitive to a changing environment, including high temperature. The comprehensive mechanism of heat stress response at the molecular level and exploitation of candidate tolerant genes are far from enough. Using transcriptome data, we analyzed the gene expression profiles of wheat under heat stress. A total of 1705 and 17 commonly differential expressed genes (DEGs) were identified in wheat grain and flag leaf, respectively, through transcriptome analysis. Gene Ontology (GO) and pathway enrichment were also applied to illustrate the functions and metabolic pathways of DEGs involved in thermotolerance of wheat grain and flag leaf. Furthermore, our data suggest that there may be a more complex molecular mechanism or tighter regulatory network in flag leaf than in grain under heat stress over time, as less commonly DEGs, more discrete expression profiles of genes (principle component analysis) and less similar pathway response were observed in flag leaf. In addition, we found that transcriptional regulation of zeatin, brassinosteroid and flavonoid biosynthesis pathways may play an important role in wheat's heat tolerance. The expression changes of some genes were validated using quantitative real-time polymerase chain reaction and three potential genes involved in the flavonoid biosynthesis process were identified.

Methylation effect on IPT5b gene expression determines cytokinin biosynthesis in apple rootstock.

The molecular mechanism of dwarfing in the apple rootstock is poorly understood, and has been attributed to low root cytokinin biosynthesis levels. Here we identified differences in root trans-zeatin content and expression of cytokinin metabolic pathway genes between dwarfing and vigorous rootstocks. Specifically, a stable low expression of IPT5b was identified in the dwarfing rootstocks. Bisulfite sequencing showed that two CpG islands (CpG2 and CpG4) in the IPT5b promoter region showed higher methylation levels in the M9 rootstock (dwarfing) compared to the Mr (vigorous) rootstock. Furthermore, IPT5b expression increased when M9 rootstocks were treated with 5-azaC, a methylation blocker, indicating that methylation levels influence IPT5b expression. In conclusion, we found low IPT5b expression with high level methylations in promoter region, leading to poor root trans-zeatin biosynthesis in the M9 rootstock, which may induce dwarfing.

Changes in cytokinin form and concentration in developing kernels correspond with variation in yield among field-grown barley cultivars.

The aim of the present study was to determine if relationships between cytokinin (CK) profiles and corresponding enzymatic regulation were consistent with differences in kernel yield among commercial barley (Hordeum vulgare L.) cultivars, differing in parameters relating to productivity and grown under agronomically-relevant field conditions. Quantification of the CKs at six distinct stages of kernel development by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) revealed a significant increase in CKs, especially trans-zeatin (tZ), during the stage when grain filling occurs, with a greater CK concentration observed in the cultivar with the greatest number of kernels per spike, which was also the highest yielding cultivar. High levels of the cis-zeatin (cZ) CK form were observed at the first developmental stage, indicating a possible role in early embryo development and viability. Cytokinin oxidase (CKX; EC 1.5.99.12) activity was evaluated at each stage through a colorimetric assay, as the enzyme provides a primary mechanism for the irreversible degradation and, thus, regulation of CKs. However, no peaks in CKX activity were observed and no differences were detected at the particular stages examined. Isopentenyl transferase (IPT) gene expression was also examined and suggests that biosynthesis contributes to regulation of CK concentrations in developing barley kernels.

G-quadruplex DNA aptamers for zeatin recognizing.

Zeatins, a major type of cytokinin, are ubiquitous in higher plants, and involve in regulating a wide range of developmental processes. The development of highly specific ligands to zeatins would be very useful in plant biological research. Here we describe a group of oligonucleotide ligands (aptamers) generated against trans-zeatin. The optimized aptamers possess strong affinity to trans-zeatin and trans-zeatin riboside (Kd=3-5 µM), and relatively weak affinity (Kd=27-30 µM) to cis-zeatin and dihydrozeatin. These aptamers adopt a hairpin-G-quadruplex structure for binding to zeatin. A fluorescence turn-on aptasensor based on graphene oxide (GO) was developed for the recognition of zeatins. The specificity assay of this aptasensor shows good response to zeatins, and no response to the adenine derivatives (analog of zeatins) abundantly existing in biological samples. These results show the great potential of these aptamers in chemical analysis and biological investigation of zeatins.

Physiological response to drought in radiata pine: phytohormone implication at leaf level.

Pinus radiata D. Don is one of the most abundant species in the north of Spain. Knowledge of drought response mechanisms is essential to guarantee plantation survival under reduced water supply as predicted in the future. Tolerance mechanisms are being studied in breeding programs, because information on such mechanisms can be used for genotype selection. In this paper, we analyze the changes of leaf water potential, hydraulic conductance (K(leaf)), stomatal conductance and phytohormones under drought in P. radiata breeds (O1, O2, O3, O4, O5 and O6) from different climatology areas, hypothesizing that they could show variable drought tolerance. As a primary signal, drought decreased cytokinin (zeatin and zeatin riboside-Z + ZR) levels in needles parallel to K(leaf) and gas exchange. When Z + ZR decreased by 65%, indole-3-acetic acid (IAA) and abscisic acid (ABA) accumulation started as a second signal and increments were higher for IAA than for ABA. When plants decreased by 80%, Z + ZR and K(leaf) doubled their ABA and IAA levels, the photosystem II yield decreased and the electrolyte leakage increased. At the end of the drought period, less tolerant breeds increased IAA over 10-fold compared with controls. External damage also induced jasmonic acid accumulation in all breeds except in O5 (P. radiata var. radiata × var. cedrosensis), which accumulated salicylic acid as a defense mechanism. After rewatering, only the most tolerant plants recovered their K(leaf,) perhaps due to an IAA decrease and 1-aminocyclopropane-1-carboxylic acid maintenance. From all phytohormones, IAA was the most representative 'water deficit signal' in P. radiata.

Adenosine kinase contributes to cytokinin interconversion in Arabidopsis.

Purine salvage enzymes have been implicated, but not proven, to be involved in the interconversion of cytokinin (CK) bases, ribosides, and nucleotides. Here, we use Arabidopsis (Arabidopsis thaliana) lines silenced in adenosine kinase (ADK) expression to understand the contributions of this enzyme activity to in vivo CK metabolism. Both small interfering RNA- and artificial microRNA-mediated silencing of ADK led to impaired root growth, small, crinkled rosette leaves, and reduced apical dominance. Further examination of ADK-deficient roots and leaves revealed their irregular cell division. Root tips had uneven arrangements of root cap cells, reduced meristem sizes, and enlarged cells in the elongation zone; rosette leaves exhibited decreased cell size but increased cell abundance. Expression patterns of the cyclinB1;1::ß-glucuronidase and Arabidopsis Response Regulator5::ß-glucuronidase reporters in the ADK-deficient background were consistent with altered cell division and an increase in CK activity, respectively. In vivo feeding of ADK-deficient leaves with radiolabeled CK ribosides of isopentenyladenosine and zeatin showed a decreased flux into the corresponding CK nucleotides. Comprehensive high-performance liquid chromatography-tandem mass spectrometry analysis detected significantly higher levels of active CK ribosides in both sense ADK and artificial microADK. Taken together, these metabolic and phenotypic analyses of ADK-deficient lines indicate that ADK contributes to CK homeostasis in vivo.

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants.

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

[Cytokinin content and immunolocalization in wheat primary root cells].

Immunohystochemical localization of cytokinins in cells of different root zones of wheat plants showed intensive immunostaining of zeatin in the apical root zone and its subsequent decline with the increase in the distance from the root tip. More intensive labeling of metaxylem and parenchyma cells of the root central cylinder was observed on the sections of the zone where root hairs appeared. Above this zone the decline in immunostaining of the cells of the central cylinder was paralleled by the signs if finalization of differentiation of the xylem vessels shown by lignin deposition. The data of immunohystochemical staining were confirmed by the results of enzyme immunoassay of different cytokinin forms. Likely sources of zeatin accumulation are considered. Possibility of additional (alongside with that in apical root zone) synthesis of cytokinins in the vascular tissues of root and the role of cytokinins in stimulation oflignification are discussed.

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.

Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci.

The nucleotide sequence of a Pseudomonas trans-zeatin producing gene (ptz) from the pCK1 plasmid of Pseudomonas syringae pv. savastanoi strain 1006 has been determined. This gene confers upon E. coli the ability to synthesize and secrete several cytokinins including trans-zeatin, iso-pentenyladenine and their respective N9-ribosyl derivatives. Sequence analysis indicates an open reading frame encoding a protein of 234 amino acids with a molecular weight of 26,816. Significant sequence homology is found between ptz and both the tzs and tmr genes from Agrobacterium tumefaciens. The results suggest a close relationship between the cytokinin biosynthetic pathways in P. savastanoi and A. tumefaciens.