Petre I. Dobrev

Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction .

A method for separation of cytokinins from auxin and abscisic acid, which allows further separation of cytokinin ribotides from cytokinin bases, ribosides and glucosides and their purification on a single Oasis MCX column was developed. Due to the mixed reversed-phase and cation-exchange mode of the Oasis MCX sorbent the cationic cytokinin bases, ribosides and glucosides as well as the anionic auxin, abscisic acid and cytokinin ribotides are retained and can be sequentially eluted by solvents containing different concentrations of methanol and ammonium hydroxide. Characteristics of the method are high recoveries of analyzed phytohormones and their sufficient purity for quantification by HPLC-ELISA (RIA) or HPLC-MS.

Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.

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.

Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content

In order to test the possibility of improving tolerance to heat and drought (alone and in combination) by elevation of the osmoprotectant proline (Pro) content, stress responses were compared in tobacco plants constitutively over-expressing a gene for the Pro biosynthetic enzyme Δ(2)-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41) and in the corresponding wild-type. Significantly enhanced Pro production in the transformant coincided with a more negative leaf osmotic potential (both at control conditions and following stress) and enhanced production of protective xanthophyll cycle pigments. Heat stress (40 °C) caused a transient increase in the level of bioactive cytokinins, predominantly N(6)-(2-isopentenyl)adenosine, accompanied by down-regulation of the activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (EC 1.4.3.18/1.5.99.12). No significant differences were found between the tested genotypes. In parallel, a transient decrease of abscisic acid was observed. Following drought stress, bioactive cytokinin levels decreased in the whole plants, remaining relatively higher in preferentially protected upper leaves and in roots. Cytokinin suppression was less pronounced in Pro transformants. Exposure to heat stress (40 °C for 2h) at the end of 10-d drought period strongly enhanced the severity of the water stress, as indicated by a drop in leaf water potential. The activity of cytokinin oxidase/dehydrogenase was strongly stimulated in upper leaves and roots of stressed plants, coinciding with strong down-regulation of bioactive cytokinins in whole plants. Combined heat and drought stress resulted in a minor decrease in abscisic acid, but only in non-wilty upper leaves. Both stresses as well as their combination were associated with elevation of free auxin, indolylacetic acid, in lower leaves and/or in roots. Auxin increase was dependent on the stress strength. After rehydration, a marked elevation of bioactive cytokinins in leaves was observed. A greater increase in cytokinin content in Pro transformants indicated a mild elevation of their stress tolerance.

Senescence‐Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.

Complex phytohormone responses during the cold acclimation of two wheat cultivars differing in cold tolerance, winter Samanta and spring Sandra

Hormonal changes accompanying the cold stress (4°C) response that are related to the level of frost tolerance (FT; measured as LT50) and the content of the most abundant dehydrin, WCS120, were compared in the leaves and crowns of the winter wheat (Triticum aestivum L.) cv. Samanta and the spring wheat cv. Sandra. The characteristic feature of the alarm phase (1 day) response was a rapid elevation of abscisic acid (ABA) and an increase of protective proteins (dehydrin WCS120). This response was faster and stronger in winter wheat, where it coincided with the downregulation of bioactive cytokinins and auxin as well as enhanced deactivation of gibberellins, indicating rapid suppression of growth. Next, the ethylene precursor aminocyclopropane carboxylic acid was quickly upregulated. After 3-7 days of cold exposure, plant adaptation to the low temperature was correlated with a decrease in ABA and elevation of growth-promoting hormones (cytokinins, auxin and gibberellins). The content of other stress hormones, i.e., salicylic acid and jasmonic acid, also began to increase. After prolonged cold exposure (21 days), a resistance phase occurred. The winter cultivar exhibited substantially enhanced FT, which was associated with a decline in bioactive cytokinins and auxin. The inability of the spring cultivar to further increase its FT was correlated with maintenance of a relatively higher cytokinin and auxin content, which was achieved during the acclimation period.

Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins.

Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N6-(Δ2-isopentenyl)adenosine-5′-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N6-benzyladenosine (BAR), N6-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N6-(Δ2-isopentenyl)adenine (iP) and N6-(Δ2-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5′-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N6-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.

The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity

We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1. In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS). We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression. We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.

Quantification of Abscisic Acid, Cytokinin, and Auxin Content in Salt-Stressed Plant Tissues

Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope with the stress, e.g., suppression of growth or mobilization of plant metabolism. This chapter describes a procedure enabling such quantification. Due to the high variability of these hormones in plant tissues, it is advantageous to determine their content in the same sample. Reverse phase and ion exchange chromatography allows separation of the individual hormone fractions. Hormones as well as their metabolites can be identified and quantified by LC/MS.