JXB Advance Access originally published online on February 17, 2006
Journal of Experimental Botany 2006 57(4):985-996; doi:10.1093/jxb/erj084
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RESEARCH PAPER |
Ectopic over-expression of the maize ß-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco
meral1
í Malbeck3
ková3etislav Brzobohat
1,
1Institute of Biophysics AS CR, Královopolská 135, CZ-61265, Brno, Czech Republic
2Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic
3Institute of Experimental Botany AS CR, Rozvojová 135, CZ-16502 Prague, Czech Republic
4Department of Plant Physiology, Charles University, Faculty of Science, Vininá 5, CZ-12844 Prague 2, Czech Republic
To whom correspondence should be addressed. E-mail: brzoboha{at}ibp.cz
Received 26 August 2005; Accepted 2 December 2005
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Abstract |
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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 ß-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 Km 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.
Key words: ß-glucosidase, chloroplast, cytokinin metabolism, ectopic expression, hormone conjugation, zeatin-O-glucoside
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Cytokinins (CKs) are a class of plant hormones with the ability to trigger cell division in conjunction with auxin in tissue culture. They are also involved in regulating a wide range of developmental processes such as chloroplast differentiation, nutrient assimilation and translocation, seed germination, leaf expansion, flowering, and senescence (for reviews see Binns, 1994
; Mok, 1994; Haberer and Kieber, 2002). Dozens of compounds, both natural and synthetic, have been assigned a measure of CK activity spanning the entire range from completely inactive to highly active (Letham and Palni, 1983; Mok and Mok, 2001).
Most of the studies elucidating CK action have used the application of exogenous hormone to various plants and/or tissues and observing subsequent changes. Isolation of mutants with increased levels of CKs (Chaudhury et al., 1993), expression of the Agrobacterium isopentenyltransferase gene to increase endogenous levels of CKs (Li et al., 1992) and, recently, the over-expression of genes encoding CK oxidase/dehydrogenase to reduce endogenous levels (Werner et al., 2003) have been employed to study the effects of changed CK levels. While these approaches have allowed observation of the effects of gross changes in hormone levels, they have not been able to shed much light on the regulation of many of the subtler metabolic conversions.
Metabolic regulation of hormone levels in the plant fulfils two basic requirements: (i) the production of speedy and significant changes in active hormone (either in absolute concentrations or relative to other hormones) in response to an environmental and/or other stimulus; and (ii) maintenance of hormone homeostasis at a particular stage of development and/or in a particular part of the plant.
Conjugation, the addition of low molecular weight compounds, represents a mechanism to regulate the cellular level of ‘active’ hormones by generating products with little biological activity (for a review see Brzobohat et al., 1994). trans-Zeatin is a major and ubiquitous CK in higher plants, comprising an adenine with a hydroxylated isoprenoid substituent at the N6 position. t-Zeatin can undergo conjugation on the purine ring at the N7 and N9 positions as well as on the OH-group on the isoprenoid substituent. The N9 atom can be conjugated with ribose (with subsequent phosphorylation to form the ribotide), glucose, and amino acids such as alanine. Also known are oxidative degradation by the action of CK oxidase/dehydrogenase, reduction to dihydrozeatin, and isomerization to cis-zeatin (Mok and Mok, 2001). Metabolic inactivation of zeatin has long been focused on degradative reactions catalysed by CK oxidase/dehydrogenase and conjugation to monosaccharides like glucose and xylose. Understanding the regulation of this complex network of metabolic conversions will contribute to a better insight into the processes leading to hormone homeostasis as well as the corresponding physiological and developmental effects.
t-Zeatin-O-glucoside (ZOG) is resistant to CK oxidase/dehydrogenase-mediated breakdown and is readily converted into the active hormone by the action of ß-glucosidases. It has also been found in xylem sap and is therefore considered to be the transport form of the hormone (Armstrong, 1994; Letham, 1994). ZOG accumulation when CK accumulates in tissues and its decrease during phases of active growth has been taken as evidence of a storage role (Letham and Palni, 1983). For example, in young maize seedlings, CKs are apparently not synthesized during germination and early seedling development. Instead, ZOG and DHZOG were found to be transported from the endosperm to the embryo, where it was activated by a ß-glucosidase to supplement the developing embryo with active CK (Smith and van Staden, 1978). It has been demonstrated that the ß-glucosidase Zm-p60.1 first isolated from maize coleoptiles (Campos et al., 1992; Esen, 1992) fulfils all the requirements for a ß-glucosidase involved in regulation of growth in early seedling development by releasing Z from ZOG in maize (Brzobohat et al., 1993). It was shown to be localized to plastids/chloroplasts as predicted by the N-terminal signal peptide (Esen and Stetler, 1993; Kristoffersen et al., 2000). In parallel, ZMGlu1 (an enzyme coded by the same genetic locus as Zm-p60.1) was shown to hydrolyse 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucoside (DIMBOA-Glc; Cicek and Esen, 1999) in a manner similar to a ß-glucosidase purified from maize seedlings (Babcock and Esen, 1994). Based on these findings, it has been suggested that ZMGlu1 is involved in defence against pathogens by releasing the toxic aglycone (DIMBOA) from its storage form, DIMBOA-Glc. Alhough this function is well supported by the analysis of substrate specificity and substrate-enzyme structure (Czjzek et al., 2000), it does not appear to be fully consistent with the Zm-p60.1 expression pattern, enzyme abundance, and immunolocalization. The transcript is highly abundant in 3-d-old etiolated seedlings except for the primary leaf, but is hardly detectable in the major vegetative tissues (Brzobohat et al., 1993) and the distribution of both transcript and protein at the cellular level is highly specific (Kristoffersen et al., 2000). Further, strong accumulation of the protein in maize leaves in response to drought stress (Riccardi et al., 1998) indicates other roles for the enzyme besides its involvement in pest resistance. No direct experimental evidence confirming that ZMGlu1 is actually involved in pathogen defence response in planta has been published. The exact biological role(s) of the enzyme will be decided when a maize knockout line without any functional ZMGlu1 allele can be characterized. Plant genomes encode large families of ß-glucosidases (Xu et al., 2004) with varying substrate specificities. Until now, besides Zm-p60.1, the ability to release CKs from CK-O-glucosides in vitro has been demonstrated only for a Brassica napus ß-glucosidase (Falk and Rask, 1995). However, the significance of this activity was not investigated in planta. Functional genomics projects are expected to identify novel ß-glucosidases involved in the regulation of CK activity by reversible conjugation. Thus, currently Zm-p60.1 represents the best characterized enzyme available as a valuable molecular tool to understand the biological significance of CK O-glucosylation in planta.
ZOG1, an enzyme catalysing the O-glucosylation of zeatin was isolated from Phaseolus lunatus (Martin et al., 1999). When over-expressed in tobacco, transgenic ZOG1 explants required a 10-fold higher concentration of exogenous zeatin for the initiation of callus formation (Martin et al., 2001).
Light signalling and signalling through the CK pathway are known to interact with each other (Neff et al., 2000). Chloroplasts are photosynthetic organelles that differentiate from colourless proplastids in the presence of light in a process called photomorphogenesis. Exogenous CKs as well as increased endogenous steady-state levels of CKs can partially replace the light signal leading to precocious photomorphogenesis (Chaudhury et al., 1993; Chory et al., 1994). Despite a large body of research into the photosynthetic capability of chloroplasts, as well as transport of various kinds of molecules into and out of them, little is known about the occurrence and/or transport of CKs into and out of the chloroplast. It has been shown that a wide range of CKs occurs in intact isolated chloroplasts (Benková et al., 1999). It has recently been recognized that the isoprenoid precursor for the biosynthesis of t-zeatin is primarily plastid-derived (Kasahara et al., 2004). Since Zm-p60.1 was localized in chloroplasts there was an interest in characterizing the effects of Zm-p60.1 over-expression in transgenic tobacco and in analysing its role in perturbing CK metabolism by means of estimating the in situ enzyme activity in intact chloroplasts. It is shown here that over-expression of Zm-p60.1 leads to a perturbance in zeatin homeostasis in intact transgenic plants, thus rendering them hypersensitive to exogenous zeatin.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Plant materials and growth conditions
Tobacco (Nicotiana tabacum, cv. Petit Havana SR1) plants harbouring the Zm-p60.1 cDNA under the CaMV 35S promoter were obtained by Agrobacterium-mediated leaf-disc transformation. Two homozygous lines (labelled T4 and T5) bearing the Zm-p60.1 cDNA including the N-terminal plastid-targeting signal peptide, were selected for analysis on medium supplied with zeatin.
Adult plants from an independent transgenic line were selected with methotrexate (0.5 mg l–1) added to solid MS medium (Murashige and Skoog, 1962) supplemented by sucrose (15 g l–1). Transgenic plants grew slower than control plants on methotrexate-free medium. Therefore to select plants for hormone analyses, a spectrophotometric assay was used. The enzyme activity was assayed using the chromogenic substrate p-nitrophenyl-ß-D-glucopyranoside according to Rotrekl et al. (1999). Leaves and internodes used for quantitation of hormones and for isolation of chloroplasts for Km determination experiments were taken from transgenic plants cultivated in a growth chamber (16/8 h photoperiod at 150 µmol photons m–2 s–1, 26/20 °C) for 7–8 weeks.
Zm-p60.1 cloning, transformation, and RT-PCR analysis
Standard procedures were used for DNA cloning and analysis (Sambrook et al., 1989). The methotrexate-resistant binary vector pM001 (Reiss et al., 1994) was used for Agrobacterium-mediated transformation. The CaMV 35S::Zm-p60.1::pA cassette containing the complete Zm-p60.1 coding region was excised from pRT101::Zm-p60.1 (Brzobohat et al., 1993) using HindIII and inserted into pM001 cut by HindIII yielding pM001::Zm-p60.1. In pM001::Zm-p60.1, CaMV 35S::Zm-p60.1::pA was obtained in two orientations. To minimize possible effects of genomic sequences on Zm-p60.1 expression, a clone, pM001::1.3, carrying the cassette oriented with pA close to the right border and CaMV 35S facing the ß-lactamase gene employed as an ampicillin resistance marker within the T-DNA was chosen for plant transformation.
The binary vectors were transformed into Agrobacterium tumefaciens strain GV3101::pMP90RK (Koncz and Schell, 1986) and transgenic lines were generated in Nicotiana tabacum cv. Petit Havana SR1 by the leaf disc method. The primary transformants were selected on 0.5 mg l–1 methotrexate. In tissue culture, plants were grown on MS medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose and 0.8% agar. Plants were grown with a 16/8 h light/dark regime at a daytime temperature of 24 °C.
Total RNA was isolated from entire 3-week-old seedlings grown on MS medium using Trizol (Invitrogen, Germany) according to the manufacturer's recommendations. 5 µg of total RNA was used as a template for cDNA synthesis using SuperScript II RNase H– reverse transcriptase (Invitrogen, Germany) and the oligo dT primer RTP3 (CGT TCG ACG GTA CCT ACG TTT TTT TTT TTT TTT TT) according to the supplier's protocol. 4 µl of the 40 µl reaction mix were then subjected to PCR to amplify Zm-p60.1 using primers p60(+)4: (5'-TCA AGG ACG AGC AGA AGG-3') and p60(-)1: (5'-TCT TCT TGC TGG GCT TCT-3'). Actin was amplified using the primers Nt-Act-5'ex: (5'-ATT GTG (C/T)T(G/T) GA(C/T) TCT GGT GAT GGT G-3'); Nt-Act-3'a: (5'-ATC CAG ACA CT(A/G) TAC TT(C/T) CTC TC-3') and Nt-Act-3'b: (5'-TCC A(A/G)A C(A/G)C TGT A(C/T)T TCC TCT C-3').
Growth on medium containing zeatin
Ten seeds each from wild-type tobacco and from transgenic lines were sown on MS medium supplemented with 15 g l–1 sucrose and 2.5 µM zeatin, solidified with 0.8% agar. The plates were incubated vertically in a controlled-environment growth chamber (Percival) under a 8/16 h, 21/19 °C. light/dark regime with a photon fluence rate of approximately 80 µmol photons m–2 s–1. Seedlings were collected at 14, 21, 28, and 32 d after sowing (DAS), the fresh weight determined in batches of five or 10 seedlings, and then processed for further analysis (ß-glucosidase staining, CK extraction).
ß-Glucosidase staining
ß-Glucosidase was detected by histochemical staining using 5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside (X-glc; Sigma) as a substrate. The staining was done in 0.4 M citrate-phosphate buffer (pH 5.6). The reaction contained 500 µM each of potassium hexacyanoferrate II and III and 0.1 mg ml–1 X-glc. The seedlings were infiltrated under vacuum for three 5 min bursts, and incubated at 30 °C for 4 h. The seedlings were washed in excess buffer immediately after incubation and decoloured before photography.
Chloroplast isolation
Intact chloroplasts were isolated and purified according to Benková et al. (1999). Leaves were deribbed, cut, and mixed with a homogenization medium (0.33 M sorbitol, 50 mM TRIS/HCl, pH 7.8, 0.4 mM KCl, 0.04 mM Na2EDTA, 0.1% (w/v) bovine serum albumin, 1% (w/v) polyvinylpyrrolidone, 5 mM isoascorbic acid) in a semi-frozen state, homogenized with a homogenizer, and filtered through a sandwich of cotton wool between eight layers of muslin. The chloroplast fraction was recovered by centrifugation (1000 g; 2 min; 4 °C), washed with a resuspension medium (RM: 0.33 M sorbitol, 2 mM Na2EDTA, 1 mM MgCl2, 1 mM MnCl2, 50 mM HEPES, pH 7.6) and resuspended in RM. This suspension was layered on a Percoll density gradient (40% and 80% (v/v) Percoll solution in RM) and centrifuged for 15 min at 1000 g. Intact chloroplasts were collected at the interface of the gradient, diluted with RM and centrifuged for 3 min at 1000 g. The pellet was resuspended in RM. All the procedures were done at 4 °C. The isolated chloroplasts were used immediately.
Chlorophyll determination
Chlorophyll was extracted into 80% (v/v) acetone. The total chlorophyll a+b content was calculated from the absorbance at 652 nm of the clear extract after centrifugation (500 g, 5 min) according to Arnon (1949).
Extraction and purification of IAA, ABA and CK
IAA, ABA, and CKs were extracted overnight at –20 °C with Bieleski solvent (methanol:chloroform:water:acetic acid, 12:5:2:1, by vol; Bieleski, 1964) from plant tissue ground under liquid nitrogen. [3H] IAA and [3H] ABA (Sigma, USA) for 2D-HPLC analysis and deuterium-labelled CKs ([2H5]Z, [2H5]ZR, [2H5]Z-7G, [2H5]Z-9G, [2H5]Z-OG, [2H5]ZR-OG, [2H3]DZ, [2H3]DZR, [2H6]iP, [2H6]iPR, [2H6]iP-7G, [2H6]iP 9G; Apex Organics, UK) for MS quantification were added as internal standards. After centrifugation, the extracts were purified using Sep-Pak C18 cartridges (Waters Corporation, Milford, MA, USA) and evaporated to water phase. After acidifying with HCOOH, hormones were trapped on an Oasis MCX mixed mode, cation exchange, reverse-phase column (150 mg, Waters) (Dobrev and Kamínek 2002). After a wash with 1 M HCOOH, IAA and ABA were eluted with 100% MeOH and evaporated to dryness. Further, CK phosphates (CK nucleotides) were eluted with 0.34 M NH4OH in water and CK bases, ribosides, and glucosides were eluted with 0.34 M NH4OH in 60% (v/v) MeOH. The latter eluate was evaporated to dryness. NH4OH was evaporated from the eluted fraction with CK nucleotides. 0.1 M TRIS (pH 9.6) was added to samples and after treatment with alkaline phosphatase (30 min at 37 °C), CK nucleotides were analysed as their corresponding ribosides. After neutralization, the solution was passed through a C18 Sep-Pak cartridge. CKs were eluted with 80% (v/v) methanol and evaporated to dryness. Evaporated IAA, ABA, and CK samples were stored at –20 °C until further analysis. IAA and ABA were separated and quantified by 2D-HPLC according to Dobrev et al. (2005).
Quantitative analysis of CKs
Purified CK samples were analysed by LC-MS system consisting of HTS PAL autosampler (CTC Analytics, Switzerland), Rheos 2000 quaternary pump (FLUX, Switzerland) with Csi 6200 Series HPLC Oven (Cambridge Scientific Instruments, England) and LCQ Ion Trap mass spectrometer (Finnigan, USA) equipped with an electrospray. 10 µl of sample were injected onto a C18 column (AQUA, 2 mmx250 mmx5 µm, Phenomenex, USA) and eluted with 0.0005% acetic acid (A) and acetonitrile (B). The HPLC gradient profile was as following: 5 min 10% B, then increasing to 17% within 10 min, and to 46% within further 10 min at a flow rate of 0.2 ml min–1. The column temperature was kept at 30 °C. The effluent was introduced in mass spectrometer being operated in the positive ion, full-scan MS/MS mode. Quantification was performed using a multilevel calibration graph with deuterated CKs as internal standards. As standards of cis-zeatin-glucosides and cis-zeatin-9-riboside-O-glucoside were not available, the amounts of these compounds were estimated only from the calibration graphs of the corresponding trans-isomers.
Determination of kinetic constant in isolated chloroplasts
The reaction mixture contained 0.64 µCi (23.5 kBq) of substrate ([3H] ZOG, radioactively labelled on position 8 of the purine ring by Dr Jan Hanu
, Institute of Experimental Botany, Prague), 1.1–281.7 µM unlabelled substrate ZOG and freshly isolated chloroplasts (0.9 mg chlorophyll equivalent to 1.6 mg of total protein content) in RM in a final volume of 500 µl. The assays were carried out in quadruplicates at seven different substrate concentrations, incubated at 30 °C for 10 min at 75 rpm shaking and terminated by the addition of Bieleski solution. ZOG and released zeatin were purified using C18 SPE columns. After elution with 80% (v/v) methanol samples were concentrated and analysed by HPLC using column Luna C18 (2) (150x4.6 mm, 3 µm, Phenomenex, Torrance, CA, USA); flow rate: 0.6 ml min–1; mobile phase: A: 40 mM formic acid adjusted to pH 4.1 with ammonium hydroxide and B: acetonitrile:methanol, 1:1, v/v); gradient: 0 min, 10% B; 2 min, 15% B; 11 min, 20% B; 11.1 min, 34% B; 19 min, 45% B; 21 min, 100% B; 23 min, 100% B; 25 min, 10% B; detection at 270 nm. The Km values were estimated from a Lineweaver–Burk plot.
Determination of kinetic constant in vitro
Zeatin and glucose were assayed independently to follow enzyme activity of purified Zm-p60.1 with ZOG as a substrate. The purification scheme was essentially as described (Zouhar et al., 1999). The enzymatic reaction was started by adding the purified enzyme preparation to ZOG diluted to the individual concentrations in 50 and 100 mM citrate/phosphate buffer pH 5.5, the reaction mix was incubated at 30 °C and aliquots (50 µl) were withdrawn for glucose and zeatin determination, respectively. The reaction mix was kept at 30 °C for not more than 10 min and 0.3 min, and aliquots were withdrawn at 1 min and 3 s intervals, respectively, when Z and glucose were assayed as the reaction product. The short incubation intervals when glucose was assayed were chosen to prevent any significant conversion of ß- to 
-glucose. The reaction buffer was supplemented with 20% PEG as appropriate. For zeatin determination, the reaction was stopped by mixing the aliquots with 1 M gluconolactone (75 µl) and the samples were kept on ice. The released zeatin was assayed using ELISA as described previously (Faiss et al., 1997). The released glucose was assayed using the glucose oxidase-peroxidase-coupled reaction (P Mazura et al., unpublished results). In brief, glucose was oxidized by glucose oxidase (Fluka) which results in the generation of D-gluconolactone and hydrogen peroxide. In the presence of horseradish peroxidase (HRP Sigma), the hydrogen peroxide then reacts with Amplex UltraRed reagent (Molecular Probes) which is converted to the fluorescent resorufin. Thus, aliquots withdrawn from the reaction mix were automatically injected into detection mix (50 µl, containing glucose oxidase, HRP and AmplexUltraRed) in 96-well plates. A fluorescence reader (BMG Fluostar Galaxy) was used to measure resorufin fluorescence (excitation 544 nm and emission/detection 590 nm). The software package ORIGIN (OriginLab Corp., Northampton, USA) was used to determine Km values.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Construction of transgenic tobacco plants over-expressing Zm-p60.1
To analyse effects of constitutive over-expression of Zm-p60.1 on CK metabolism and action in whole plants, the transcription cassette CaMV 35S::Zm-p60.1::pA (Brzobohat
et al., 1993) was cloned into a plant transformation vector, and the resulting construct, pM001::Zm-p60.1 was used to generate transgenic tobacco plants. Over 20 independent rooting primary transformants (T0) were regenerated following Agrobacterium-mediated transformation of tobacco leaf discs with the pM001::Zm-p60.1 vector. The primary transformants were scored for the levels of Zm-p60.1 expression by northern and western blot analysis (data not shown). Single locus homozygous lines were identified in T2 and propagated for several generations without any consistent phenotype alteration compared with the wild type. Leaf protoplasts derived from several of these lines were shown to use CK-O- and N3-glucosides to initiate cell division (Brzobohat et al., 1993). Two independent single locus homozygous lines (designated T4 and T5) were selected for analysis outlined below based on the stability of transmission of ß-glucosidase activity through several generations. Over-expression of Zm-p60.1 in these lines was confirmed by RT-PCR (Fig. 1A). Adult transgenic plants over-expressing Zm-p60.1 showed no apparent morphological deviations from the wild type although there were differences in phytohormone levels (see below).
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Morphological effects of cultivation on medium supplemented with exogenous zeatin
Transgenic seedlings growing on MS medium supplemented with 2.5 µM zeatin showed a significant increase in fresh weight at 21 DAS compared with controls growing on the same plate (Fig. 1B). The difference became increasingly significant at the later time points (28 and 32 DAS). The morphology of transgenic seedlings remained indistinguishable from the wild type until about 21 DAS. At 28 DAS ectopic outgrowths were seen forming at the base of the hypocotyl (Fig. 1). They resembled leaves in that they were blade-like structures that frequently turned green and developed trichomes (Fig. 1E, F). Similar structures form at the base of the hypocotyl of wild-type seedlings when the medium contains 10.0 µM zeatin (see supplementary Fig. 1 at JXB online).
Histochemical staining for ß-glucosidase in seedlings grown on exogenous zeatin
An indigogenic histochemical staining procedure using 5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside was optimized for use with seedlings over-expressing Zm-p60.1. The incubations were shortened to minimize false signals from endogenous ß-glucosidases (see Materials and methods). Staining in transgenic seedlings grown on medium containing zeatin was restricted to the base of the hypocotyl and to patches in the ectopic structures themselves (Fig. 1K, L).
Kinetic analysis of Zm-p60.1 activity
The wild type full-length Zm-p60.1 cDNA used in pM001::Zm-p60.1 encodes a nascent polypeptide including the N-terminal plastid-targeting signal peptide. In cell fractionation experiments performed with leaves of the transgenic plants, Zm-p60.1 enzyme activity co-purified with the chloroplast fraction (data not shown) indicating that Zm-p60.1 is located exclusively in chloroplasts. Kinetic analysis was performed to determine whether Zm-p60.1 is likely to act on CK-O-glucosides at physiologically relevant levels that were determined in tobacco chloroplasts earlier (Benková et al., 1999). Under standard in vitro assay conditions, release by purified Zm-p60.1 of zeatin and glucose from ZOG, was monitored using ELISA and the glucose oxidase/peroxidase-coupled reaction, respectively. Both in vitro assays yielded comparable, though unexpectedly high values for Km (Table 1). To assess the possible influence of the native chloroplast micro-environment on Zm-p60.1 catalytic properties, subsequent kinetic analysis was performed using a highly purified fraction of freshly isolated, intact chloroplasts from fully expanded leaves of transgenic plants. 3H-labelled ZOG was taken up rapidly into and converted in the chloroplasts (see supplementary Results and supplementary Table 2 at JXB online). The ZOG conversion was almost completed after a 4 h incubation period in chloroplasts isolated from the transgenic plants while more than 90% of ZOG remained unconverted in wild-type chloroplasts. These results enabled us to establish an assay to determine the apparent Km of Zm-p60.1 in isolated chloroplasts with 3H-labelled ZOG as a substrate.
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The apparent Km in these chloroplasts using 3H-labelled ZOG as a substrate was more than ten times lower compared with the apparent Km from the in vitro measurements. Thus, the Km value in chloroplasts is within the physiological range for CK-O-glucoside levels. The inclusion of an inert hydrophilic polymer (polyethylene glycol, PEG) in the standard in vitro assay buffer resulted in a more than 3-fold decrease in Km with ZOG as a substrate (Table 1), suggesting a strong dependence of Zm-p60.1 catalytic properties on water content in the reaction environment. Intact chloroplasts isolated from wild-type and transgenic tobacco were incubated with ZOG and its uptake and conversion followed. Only 1% of ZOG remained unconverted at the end of a 4 h incubation period with chloroplasts isolated from the transgenic plants. When chloroplasts isolated from wild-type were used, 91% of initial ZOG remained unconverted under the same incubation conditions. ZOG uptake into chloroplasts was temperature dependent (not shown). However, the extent of uptake (a maximum of 2.5% of total radioactivity) does not indicate ZOG enrichment in the chloroplasts.
Hormone quantification in adult transgenic plants in the absence of exogenous zeatin
CK quantification from plants grown in the absence of exogenous zeatin revealed a higher level of active CKs (free bases and ribosides) in the top portion (leaf numbers 1–4 and internode numbers 1–4) of plants over-expressing Zm-p60.1 than in the corresponding parts of wild-type plants (Table 2; Fig. 2A). However, no consistent trend was observed in levels of storage and inactivated forms of CKs (see supplementary Table 1 at JXB online). Auxin (free IAA) measurements revealed that transgenic tobacco over-expressing Zm-p60.1 has lower levels of IAA in the apex and the first two internodes than in the corresponding controls (Table 2). The gradient in leaf IAA levels from high to low from the apex downward was steeper than wild-type in plants over-expressing Zm-p60.1 (Fig. 2B). ABA levels were measured in plants over-expressing Zm-p60.1. In older leaves (numbers 5–10 from the apex) as well as in older internodes (numbers 5–8), ABA levels were higher than the wild type in plants over-expressing Zm-p60.1 (Table 2; Fig. 2C).
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CK quantification analyses of seedlings grown in the presence of exogenous zeatin
CK quantification was carried out on seedlings grown on medium containing zeatin. The changes in CK levels were restricted to the zeatin-type and the iP-type metabolites were not affected. The most significant pattern was seen in the levels of active zeatin metabolites. The transgenic seedlings accumulated Z, ZR, and ZRP to levels higher than the wild type (Fig. 3A). Seedlings over-expressing Zm-p60.1 were able to accumulate the substrate ZOG to levels higher than their wild-type counterparts in a time-dependent manner (Fig. 3B). The same was also true, to a greater extent, of Z7G, a terminal inactivation conjugate (Fig. 3C).
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Presentation of the CK quantification results from seedlings grown on exogenous zeatin
Fresh weight is most commonly used to normalize CK quantification data. However, in this case, transgenic seedlings were significantly heavier than the wild type (Fig. 1B) and so the quantification data were normalized to number of seedlings. This also has the advantage of providing an average picture in whole seedlings of steady-state levels of individual CK metabolites. The y-axis in Fig. 3 represents the number obtained when the value for transgenic seedlings was subtracted from the corresponding wild-type value. In addition, a table showing the CK accumulations in transgenic seedlings as percentages of wild-type values is presented (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Transgenic tobacco plants over-expressing Zm-p60.1, a maize ß-glucosidase capable of releasing active CK from O- and N3- glucosides, were analysed in order to understand the regulation of zeatin metabolism in planta. Although the plants showed no apparent phenotype alterations, they had substantial differences in phytohormone levels. The apparent Km value for Zm-p60.1 in isolated chloroplasts with ZOG as a substrate was close to physiological levels of CK-O-glucosides. Unexpectedly, the value was 10-fold lower than the apparent Km value determined in vitro for the purified enzyme suggesting a higher affinity of the enzyme for ZOG in its native compartment. When grown on exogenous zeatin, the transgenic seedlings displayed a hypersensitivity to zeatin. The study revealed a perturbation in zeatin homeostasis, and that the chloroplast is not the storage organelle for zeatin-O-glucoside. It is proposed that Zm-p60.1 can be used as a molecular tool in investigating the biological role(s) of CK-O-glucosylation.
Morphological changes in seedlings over-expressing Zm-p60.1 grown on exogenous zeatin
Juvenile hypocotyls are known to be competent for in vitro bud formation in woody species ‘recalcitrant’ to bud regeneration like Eucalyptus globulus (Azmi et al., 1997a). Shoot formation in callus with numerous shoots was correlated with high levels of Z and ZR in shooty tumours compared with non-shooty tumours (Azmi et al., 1997b). In addition, the shooty capacity of the tumour was not associated with an overall increase in CK levels but only with the presence of small transformed areas highly provided with CKs, while the buds themselves were provided with a moderate CK signal. The restriction of histochemical staining for Zm-p60.1 to the ectopic structures suggests that the enzyme is involved in increasing the local availability of active CKs. In this respect these results recall the pattern of CK localization in E. globulus calli (Azmi et al., 2001). The hypocotyl–root junction is one of the sites of auxin accumulation (Ni et al., 2001; Dr Alena Kuderová, personal communication), and the contemporaneous availability of active CKs might lead to an additive effect (Rashotte et al., 2005) on its development. This might be a reason why the ectopic structures arise in this region. Zm-p60.1 expression is driven by the CaMV 35S promoter that is known to be active to higher levels in vascular tissues. The ectopic structures contain a high proportion of vasculature and thus are enriched in Zm-p60.1. Increased levels of Zm-p60.1 in these structures might cause a kind of autocatalytic effect (where the high proportion of vasculature allows further accumulation of Zm-p60.1) with respect to perturbance of zeatin metabolism.
It is known that exogenously applied CKs (specifically zeatin at micromolar concentrations) can induce D-type cyclins in responsive cells (Riou-Khamlichi et al., 1999). Further, the over-expression of a D-type cyclin accelerates cell-cycle progression from G-phase to S-phase and Nicta;CycD3;2 controls the G1/S transition in tobacco cells (Nakagami et al., 2002). Boucheron et al. (2002) have shown CK involvement in the redifferentiation of xylem and phloem tissues in tobacco explants growing on medium containing exogenous CKs. These results strongly suggest that the increased availability of active CK metabolites like ZR and ZRP leads to the formation of the ectopic structures at the base of the hypocotyl.
Enzymatic activity of Zm-p60.1 in isolated chloroplasts
The apparent Km value for Zm-p60.1 in isolated chloroplasts was 10-fold lower against ZOG than the apparent Km determined in vitro for the purified enzyme against the same substrate. This value is more than 2-fold lower than against 4-methylumbelliferyl-ß-D-glucopyranoside (140 µM) and 10-fold lower than against p-nitrophenyl-ß-D-glucopyranoside (640 µM) both of which were obtained using purified enzyme in vitro (Zouhar et al., 2001).
The chloroplast is an organelle that presents a considerably different environment for proteins and it is generally believed that the water content is significantly lower than that of the cytoplasm. Given this it can be expected that chloroplast enzymes may behave very differently in vitro. Interestingly, a more than 3-fold decrease in Zm-p60.1 Km with ZOG was observed when the standard in vitro assay buffer was supplemented with 20% PEG. This represents the first direct experimental evidence suggesting that a decrease in water content might result in increased catalytic efficiency of a plastid/chloroplast enzyme. Besides this, other, more specific, mechanisms might be involved in modulation of chloroplast enzyme activity. Active transport of CK-O-glucosides into chloroplasts might contribute to the observed low value of the apparent Km. However, when wild-type chloroplasts were incubated with ZOG, significant ZOG accumulation in the chloroplasts was not observed (data not shown).
A system of reductive activation of disulphide-bond-containing enzymes has been well-characterized in chloroplasts. It involves the reduction and reformation of disulphide bridges mediated by chloroplast thioredoxin which, in turn, is activated by ferredoxin and hence by light (Buchanan et al., 2002). Zm-p60.1 has five cysteine residues. Formation of an intramolecular disulphide bridge between two of them (C205 and C211) was shown to be essential for enzyme activity through stabilization of a loop forming a part of an aglycone binding site and acquisition of the competence to assemble a catalysis-competent homodimer (Rotrekl et al., 1999; Czjzek et al., 2000; Zouhar et al., 2001). It has previously been reported that CK-O-glucosides transiently occur in chloroplasts at the end of the dark phase (Benková et al., 1999). The light-activation of glucoside cleavage could thus involve the action of a similar glucosidase.
Hormone quantification in adult transgenic plants in the absence of exogenous zeatin
CK-mediated reduction in auxin levels has been documented in plants over-expressing the CK biosynthesis gene ipt from Agrobacterium (Eklöf et al., 2000). A recent study using plants over-expressing ipt has shown that elevated CK levels lead to a long-term reduction in auxin biosynthesis rates and pool sizes. However, the authors conclude it to be an indirect effect, possibly mediated by developmental changes (Nordström et al., 2004). It was found that CK effects are partly mediated by elevation of ethylene biosynthesis (Genkov et al., 2003). Elevated ethylene biosynthesis in its turn can stimulate the oxidative decarboxylation of IAA (Winer et al., 2000). Thus, the elevated levels of active CK metabolites in plants over-expressing Zm-p60.1 could lead to lower levels of free IAA.
Since Zm-p60.1 was identified in drought-stressed maize plants (Riccardi et al., 1998), ABA levels were determined in plants over-expressing Zm-p60.1. It was found that ABA tends to accumulate in older leaves as well as in older internodes of transgenic plants compared with the wild type. Correlations between CKs and ABA levels have been reported. Increased levels of ABA were observed in potato plants with a high CK content carrying the Agrobacterium ipt gene (Macháková et al., 1997). ABA levels increased in response to benzyladenine treatment in micropropagated explants of Actinidia deliciosa (Moncaleán et al., 2003). A correlated increase in free ABA and ZOG was observed during germination of plants carrying the etr1-2 mutation (Chiwocha et al., 2005). By contrast, delayed corolla senescence in petunia flowers overproducing CKs due to the action of PSAG12-driven ipt is correlated with lower accumulation of ABA (Chang et al., 2003). Pretreatment with benzyladenine of bean and maize resulted in lower ABA accumulation during subsequent water stress (Pospíilová et al., 2005). Similarly, a contrasting role for ABA and CK in the senescence process is well documented (Panavas et al., 1998; Gan and Amasino, 1996). On the other hand, an increase in ABA levels can cause a decrease in CK levels. ABA accumulates in kernels of drought-stressed maize and is accompanied by a decrease in zeatin-type CKs (Setter et al., 2001). It is also known that ABA can induce the expression of CK oxidase/dehydrogenase. Thus, in stressed and/or senescing tissue ABA-mediated CK oxidase/dehydrogenase induction can lead to lowered CK levels. However, recent evidence has raised the possibility that the ABA-induced increase in CK oxidase/dehydrogenase activity may be important in regulating levels of CKs transiting the vascular system, but that endogenous CKs that are not transported away from their site of synthesis by vascular system may be compartmentalized away from this CK oxidase/dehydrogenase activity (Brugière et al., 2003; Yang et al., 2002).
Although leaves from adult transgenic plants over-expressing Zm-p60.1 show deviations from wild-type levels of phytohormones, the plants show no obvious morphological alterations unless they are challenged with exogenous zeatin. In tobacco over-expressing the zeatin-specific O-glucosyltrasferase ZOG1, CK-dependent explants differed significantly from the wild type in their capacity to differentiate on medium supplemented with zeatin, but few substantial changes in adult habit were observed under normal growth conditions (Martin et al., 2001).
CK quantification in seedlings grown on exogenous zeatin
It is generally accepted that N7- and N9-glucosylation is irreversible while O-glucosylation is reversible and that it plays a role in storage and/or transport of zeatin. In line with this, enzymes from plants have been identified for both glucosylation (ZOG1 from Phaseolus lunatus; Martin et al., 1999) and deglucosylation (Zm-p60.1 from Zea mays; Brzobohat et al., 1993) at the hydroxyl oxygen while no enzyme has yet been identified that deglucosylates N7- and/or N9-glucosides. It has been shown that over-expression of the glucosyltransferase ZOG1 leads to a specific increase in the levels of O-glucosides and the changes are largely restricted to O-glucosylation (Martin et al., 2001). It is shown here that the over-expression of an enzyme catalysing the reverse reaction increases throughput through a larger set of zeatin conversions (conversion to N-glucosides, ribosides, and ribotides) compared with controls and, further, that seedlings over-expressing this activity are hypersensitive to exogenous zeatin. The exact tissue and temporal distribution of these metabolites is still not clear. The presence of the enzyme in and around the ectopic structures indicates that a local maximum of active CKs leads to the formation of those structures. The observation that wild-type seedlings form similar ectopic structures when incubated with higher concentrations of zeatin leads us to conclude that these seedlings are indeed hypersensitive to exogenous zeatin. It is known that moderate increases in steady-state CK levels result in a moderate increase in zeatin pool-sizes, but a large increase in the content of Z7G (Eklöf et al., 1996), as observed in the case of Zm-p60.1 transgenic seedlings grown on zeatin. iP-type CK metabolites were not affected, in line with previous observations (Lexa et al., 2003).
Both shoots and roots are now recognized to be sites of CK biosynthesis. In tobacco roots, iPA nucleotides are the major CKs detected, and the low levels of ZR and ZMP is in good agreement with results from Arabidopsis, where root-derived biosynthesis of CKs occurs mainly via the iPMP-dependent pathway (Nordström et al., 2004). The pattern of expression of AtIPT3 and AtIPT7 in root tissue is consistent with their possible involvement in such synthesis (Miyawaki et al., 2004).
Subcellular compartmentation is expected to play a significant role in CK homeostasis. The hydroxylated isoprenoid side-chain substituent for t-Z biosynthesis is derived largely from the plastid-localized methylerythritol phosphate (MEP) pathway, and four Arabidopsis IPT proteins (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) are located in the chloroplast (Kasahara et al., 2004). Further, it has been suggested that the presence of chloroplasts might be a prerequisite for the iPMP-independent pathway, which may explain the shoot-localization of t-Z biosynthesis (Nordström et al., 2004).
The ability of seedlings over-expressing Zm-p60.1 to accumulate ZOG was unexpected. This observation in transgenic seedlings supplied with exogenous zeatin strongly implies that the terminal subcellular destination of ZOG is not the chloroplast. It has been shown that CK-O-glucosides accumulate preferentially in the vacuole (Fußeder and Ziegler, 1988). Previous results have shown that ZOG accumulates transiently in chloroplasts (Benková et al., 1999) where it may fulfill specific biological function(s). Thus, in the transgenic plants over-producing Zm-p60.1 in chloroplasts, it was possible to affect only this part of the total pool of intracellular ZOG. This relatively small perturbance is probably overcome during the normal course of plant development and, therefore, the adult plant is morphologically indistinguishable from the wild type. However, this perturbance is sufficient for the phenotype manifestation on medium containing zeatin, as seen by the ability of the transgenic seedlings to accumulate active CK metabolites to a larger extent than they accumulate ZOG. Experiments are in progress in this laboratory using a recombinant Zm-p60.1 to probe the subcellular compartmentation of zeatin metabolism further.
Taken together, it is proposed that over-expression of Zm-p60.1 can be used as a powerful tool for understanding the regulation of zeatin metabolism in general and its subcellular compartmentation in particular.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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The supplementary data available at JXB online are: (i) Table 1 listing the individual measurements of CKs in leaves and internodes from adult plants grown in the absence of zeatin; (ii) a figure showing the phenotype of wild-type and transgenic seedlings grown on concentrations of zeatin in the range 1.0 to 10.0 µM; and (iii) results presenting uptake (Table 2) and conversion of ZOG in chloroplasts together with relevant materials and methods.
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Acknowledgements |
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We thank Professor Miroslav Strnad for his generous help during the initial phases of the ELISA assays and for providing us with the polyclonal anti-zeatin-riboside antibodies. We thank Professor Asim Esen for stimulating discussions on the effects of chloroplast micro-environment on Zm-p60.1 catalytic properties, and Dr Alena Kuderová for sharing unpublished information. This work was supported by grant nos LN00A081, MSM143100008, and MSM0021622415 (Ministry of Education of the Czech Republic), AVOZ50040507 and AV0Z50380511 (Academy of Sciences of the Czech Republic), IAA600380507 (Grant Agency of the Academy of Sciences of the Czech Republic), and 206/03/0369 (Grant Agency of the Czech Republic).
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* These authors contributed equally to the work presented here.
Present address: Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521-0124, USA.
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L. Polanska, A. Vicankova, M. Novakova, J. Malbeck, P. I. Dobrev, B. Brzobohaty, R. Vankova, and I. Machackova Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco J. Exp. Bot., February 1, 2007; 58(3): 637 - 649. [Abstract] [Full Text] [PDF]
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