Steady-State Levels of Cytokinins and Their Derivatives May Serve as a Unique Classifier of Arabidopsis Ecotypes.

We determined steady-state (basal) endogenous levels of three plant hormones (abscisic acid, cytokinins and indole-3-acetic acid) in a collection of thirty different ecotypes of Arabidopsis that represent a broad genetic variability within this species. Hormone contents were analysed separately in plant shoots and roots after 21 days of cultivation on agar plates in a climate-controlled chamber. Using advanced statistical and machine learning methods, we tested if basal hormonal levels can be considered a unique ecotype-specific classifier. We also explored possible relationships between hormone levels and the prevalent environmental conditions in the site of origin for each ecotype. We found significant variations in basal hormonal levels and their ratios in both root and shoot among the ecotypes. We showed the prominent position of cytokinins (CK) among the other hormones. We found the content of CK and CK metabolites to be a reliable ecotype-specific identifier. Correlation with the mean temperature at the site of origin and the large variation in basal hormonal levels suggest that the high variability may potentially be in response to environmental factors. This study provides a starting point for ecotype-specific genetic maps of the CK metabolic and signalling network to explore its contribution to the adaptation of plants to local environmental conditions.

Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis.

The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta. CK metabolite levels were measured in 2-wk-old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans-zeatin (tZ) metabolites upon application of N6 -(Δ2 -isopentenyl)adenine (iP), we observed no conversions between the individual CK-types - iP, tZ, dihydrozeatin (DHZ) and cis-zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ-type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N-glucosides, those of iP were not converted back to iP while tZ N-glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large-scale study of short-term CK metabolism kinetics and show that tZ N7- and N9-glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.

Retargeting a maize ß-glucosidase to the vacuole--evidence from intact plants that zeatin-O-glucoside is stored in the vacuole.

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize ß-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

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.

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.

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis.

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

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.