Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4.

The CRE1/AHK4 cytokinin receptor is an important component of plants' hormone signaling systems, and compounds that can alter its activity have potential utility for studying the receptor's functions and/or developing new plant growth regulators. A high throughput method was developed for screening compounds with agonist or antagonist properties toward the CRE1/AHK4 cytokinin receptor in a single experiment using the Nanodrop II liquid handling system and 384-well plates. Potential ligands are screened directly, using a reporter system in which receptor signaling activity triggers expression of ß-galactosidase in Escherichia coli. This enzyme generates a fluorescent product from a non-fluorescent substrate, allowing the agonistic/antagonistic behavior of tested compounds to be assayed in relation to that of an internal standard (here the natural ligand, trans-zeatin). The method includes a robust control procedure to determine false positive or false negative effects of the tested compounds arising from their fluorescent or fluorescent-quenching properties. The presented method enables robust, automated screening of large libraries of compounds for ability to activate or inhibit the Arabidopsis thaliana cytokinin receptor CRE1/AHK4.

An automated method to evaluate the enzyme kinetics of ß-glucosidases.

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize ß-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of ß-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

Combining rational and random strategies in ß-glucosidase Zm-p60.1 protein library construction.

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in ß-glucosidase Zm-p60.1, which is highly conserved among ß-glucosidases. Unexpectedly, ß-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

Engineering the cytokinin-glucoside specificity of the maize ß-D-glucosidase Zm-p60.1 using site-directed random mutagenesis.

The maize ß-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-ß-D-glucopyranoside versus the trans-zeatin-O-ß-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-ß-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.

Focused directed evolution of ß-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening.

Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired ß-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize ß-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of ß-glucosidases for either fundamental studies or potential biotechnological applications.

Functional analysis of the aglycone-binding site of the maize beta-glucosidase Zm-p60.1.

Beta-glucosidases such as Zm-p60.1 (Zea mays) and Bgl4:1 (Brassica napus) have implicated roles in regulating plant development by releasing biologically active cytokinins from O-glucosides. A key determinant of substrate specificity in Zm-p60.1 is the F193-F200-W373-F461 cluster. However, despite sharing the same substrates, amino acids in the active sites of Zm-p60.1 and Bgl4:1 differ dramatically. In members of the Brassicaceae we found a group of beta-glucosidases sharing both high similarity to Bgl4:1 and a consensus motif A-K-K-L corresponding to the F193-F200-W373-F461 cluster. To study the mechanism of substrate specificity further, we generated and analyzed four single (F193A, F200K, W373K and F461L) and one quadruple (F193A-F200K-W373K-F461L) mutants of Zm-p60.1. The F193A mutant showed a specific increase in affinity for a small polar aglycone, and a deep decrease in k(cat) compared with the wild-type. Formation of a cavity with decreased hydrophobicity, and significant consequent alterations in ratios of reactive and non-reactive complexes, revealed by computer modeling, may explain the observed changes in kinetic parameters of the F193 mutant. The large decrease in k(cat) for the W373K mutant was unexpected, but the findings are consistent with the F193-aglycone-W373 interaction playing a dual role in the enzyme's catalytic action; influencing both substrate specificity, and the catalytic rate by fixing the glucosidic bond in a favorable orientation for attack by the catalytic pair. Investigation of the combined effects of all of the mutations in the quadruple mutant of Zm-p60.1 was precluded by extensive alterations in its structure and almost complete abolition of its enzymatic activity.

A new, sensitive method for enzyme kinetic studies of scarce glucosides.

The maize beta-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates. We developed a glucose assay using a system comprising three enzymes beta-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-beta-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.

Ectopic over-expression of the maize beta-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco.

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.