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First published online January 9, 2008; 10.1104/pp.107.107425 Plant Physiology 146:1155-1164 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Farnesylation Directs AtIPT3 Subcellular Localization and Modulates Cytokinin Biosynthesis in Arabidopsis1,[OA]Institute of Plant Sciences, ETH Zurich, 8092 Zurich, Switzerland (A.G., W.G.); and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 165 02 Prague 6, Czech Republic (K.H., M.K.)
Cytokinins regulate cell division and differentiation as well as a number of other processes implicated in plant development. The first step of cytokinin biosynthesis in Arabidopsis (Arabidopsis thaliana) is catalyzed by adenosine phosphate-isopentenyltransferases (AtIPT). The enzymes are localized in plastids or the cytoplasm where they utilize the intermediate dimethylallyl-diphosphate from the methylerythritolphosphate or mevalonic acid pathways. However, the regulatory mechanisms linking AtIPT activity and cytokinin biosynthesis with cytokinin homeostasis and isoprenoid synthesis are not well understood. Here, we demonstrate that expression of AtIPT3, one member of the adenosine AtIPT protein family in Arabidopsis, increased the production of specific isopentenyl-type cytokinins. Moreover, AtIPT3 is a substrate of the protein farnesyl transferase, and AtIPT3 farnesylation directed the localization of the protein in the nucleus/cytoplasm, whereas the nonfarnesylated protein was located in the plastids. AtIPT3 gain-of-function mutant analysis indicated that the different subcellular localization of the farnesylated protein and the nonfarnesylated protein was closely correlated with either isopentenyl-type or zeatin-type cytokinin biosynthesis. In addition, mutation of the farnesyl acceptor cysteine-333 of AtIPT3 abolishes cytokinin production, suggesting that cysteine-333 has a dual and essential role for AtIPT3 farnesylation and catalytic activity.
Cytokinins are involved in many aspects of plant growth and development. At the molecular level, cytokinins influence plant functions by stimulating cell division and differentiation in cooperation with other plant hormones (Haberer and Kieber, 2002  ; Howell et al., 2003; Kakimoto, 2003). Natural cytokinins are adenine derivatives carrying either an isoprene-derived molecule or an aromatic side chain at the N6 terminus (Sakakibara, 2006). In the first step of the isoprenoid cytokinin biosynthesis pathway, AMP, ADP, or ATP reacts with dimethylallyl-diphosphate (DMAPP; a central intermediate of the isoprenoid pathways) to the corresponding isopentenyl adenosine-5'-phosphates, which are subsequently metabolically converted to the monophosphate (iPRMP). Isopentenylation of adenosine phosphates is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis (Arabidopsis thaliana), IPT proteins are encoded by a family of seven genes (AtIPT1 and AtIPT3 to AtIPT8; Kakimoto, 2001; Takei et al., 2001). The remaining two IPTs (IPT2 and IPT9) catalyze isopentenylation of adenine moiety of tRNAs (Golovko et al., 2002; Miyawaki et al., 2006). Subsequent conversion of iPRMP to isopentenyl adenosine (iPR) and isopentenyl adenine (iP) is catalyzed by 5'-nucleosidase and adenosine nucleosidase (Chen and Kristopeit, 1981a, 1981b). The iP nucleotides are transformed into the corresponding trans-zeatin nucleotides by CYP735A enzymes (Chen and Leisner, 1984; Takei et al., 2004a). Cytokinin level in plant tissues is regulated by the balance of de novo biosynthesis, interconversion between the different cytokinin species, inactivation, degradation, and transport. For example, irreversible inactivation of cytokinin results from glucosylation at the N7- or the N9-position of the adenine ring, whereas O-glucosylation at the N6 side chain terminal hydroxyl of zeatins is reversible and O-glucosylated zeatins are considered storage forms of the hormone (Brzobohaty et al., 1993; Mok and Mok, 2001). In contrast, degradation of cytokinin bases and nucleosides is catalyzed by cytokinin oxidase/dehydrogenase by cleavage of the side chain (Mok and Mok, 2001; Werner et al., 2003).
Among Arabidopsis IPTs, AtIPT3 is expressed in the vasculature throughout the plant, and its expression is increased by nitrate (Miyawaki et al., 2004
Modification of target proteins by farnesyl or geranylgeranyl is critical for the control of development, growth, and signaling (Yalovsky et al., 1999 We investigated AtIPT3 to determine if the protein is farnesylated and to understand the potential role of AtIPT3 farnesylation during Arabidopsis development as well as for cytokinin biosynthesis. Here, we show that AtIPT3 is effectively farnesylated in vivo and that farnesylation regulates AtIPT3 subcellular localization, which affects cytokinin biosynthesis. In addition, AtIPT3 expression in E. coli, yeast, and Arabidopsis also demonstrates a role of Cys-333 for AtIPT3 catalytic properties. Characterization of AtIPT3 gain-of-function mutants demonstrated that increased cytokinin production affects plant development and contributes to the regulation of cell proliferation.
AtIPT3 Is a Farnesylated Protein The presence of the CLVA motif at the C-terminal end of AtIPT3 suggested that the protein is a substrate of PFT. To test this hypothesis, we first incubated purified AtIPT3 with recombinant Arabidopsis PFT and [3H]FPP. AtIPT3 was labeled in the presence of both PFT and [3H]FPP (Fig. 1A ). In addition, mutation of the conserved Cys farnesyl acceptor in the CLVA motif to Ser (AtIPT3C333S) confirmed that farnesylation required a functional farnesylation motif. In contrast, no prenylation was observed with PGGT-I using [3H]GGPP or [3H]FPP (data not shown).
To confirm that AtIPT3 is also farnesylated in vivo, we generated Arabidopsis transgenic plants in which the AtIPT3 and AtIPT3C333S cDNA were expressed under the control of the cauliflower mosaic virus 35S promoter. Seven AtIPT3 and six AtIPT3C333S T1 hygromycin-resistant plants with single T-DNA insertions were selected for further analysis. Five independent T3 homozygous AtIPT3 lines and seven AtIPT3C333S lines were analyzed by western blot using a polyclonal anti-AtIPT3 antibody. Except one AtIPT3C333S line, all transgenic lines expressed the transgene (Fig. 1B). Proteins from AtIPT3 and AtIPT3C333S plants labeled with [3H]mevalonic acid (MVA) were extracted and separated on SDS-polyacrylamide gels, which were then used either for immunoblot analysis with the anti-AtIPT3 antibody or for fluorography to detect labeled AtIPT3. AtIPT3 and AtIPT3C333S were expressed to a similar level in the transgenic plants (Fig. 1C). A labeled protein corresponding to the size of AtIPT3 was detected only in extracts from plants expressing AtIPT3 but not in plants expressing AtIPT3C333S or in wild-type plants (Fig. 1C). Together, these results establish that AtIPT3 is efficiently farnesylated in vivo as well.
Most farnesylated proteins in yeast and animal cells are targeted to the plasma membrane (Sinensky, 2000
AtIPT3 Gain of Function Increases Cytokinin Production To examine the effects of AtIPT3 gain of function on cytokinin production and the potential role of its farnesylation in vivo, AtIPT3 was also expressed in era1-1 plants by crosses between six independent AtIPT3 homozygous plants that express a high level of the transgene and era1-1 (Fig. 3A ). Cytokinin production was investigated in several independent AtIPT3 and era1-1AtIPT3 plants. All analyzed lines had elevated cytokinin accumulation in leaves compared to their respective wild type and era1-1 control lines. The lines AtIPT3 3.2.5 (AtIPT3-OE) and era1-1AtIPT3 3.7 (era1-1AtIPT3-OE) were therefore selected for full analysis. As shown in Table I , AtIPT3-OE plants had a 3.4-fold increase of total cytokinin. This accumulation was associated with a nearly 10-fold increase of iP ribotides [N6-(2-isopentenyl)adenosine 9-riboside 5'-phosphates; iPRPs], indicating that cytokinins are synthesized via the iPRP-dependent pathway. In addition, AtIPT3 overexpression resulted in a preferential production of iP-type cytokinins (11.2-fold), while the content of zeatin-type cytokinins was increased to a much lesser extent (1.5-fold). This accumulation of iP-type cytokinins was mainly due to the 12-fold increase of N6-(2-isopentenyl)adenine 7-glucoside (iP7G), which represented 53% of the pool of total cytokinin as compared to 15% in the wild type. In contrast, the content of trans-zeatin 7-glucoside (Z7G), the most abundant cytokinin present in wild-type plants, was only 1.5-fold increased in AtIPT3-OE plants, and its participation to the pool of total cytokinin decreased from 42% to 19% (Table I). Interestingly, era1-1 plants accumulated 2.2-fold more cytokinins than the wild type (Table I). Although iPRP content was 4-fold increased in era1-1 leaves, zeatin-type cytokinins predominantly accumulated in era1-1 with a 2-, 4-, 10-, and 15-fold increase of trans-zeatin 9-riboside (ZR), trans-zeatin O-glucoside (ZOG), trans-zeatin 9-riboside O-glucoside (ZROG), and dihydrozeatin 7-glucoside (DHZ7G), respectively. In contrast, the content of Z7G and trans-zeatin 9-glucoside decreased to 32% and 7% of the total pool of cytokinin compared to 42% and 13.6%, respectively, in the wild type. Similar to the situation in AtIPT3-OE plants, the increase in cytokinin production in era1-1AtIPT3-OE leaves was preferentially due to an accumulation of iP-type cytokinins (5.3-fold increase) rather than an increase of zeatin-type (1.7-fold increase) compared to era1-1 leaves. Cytokinin accumulation in era1-1AtIPT3-OE leaves was associated with a nearly 6-fold increase of iPRPs compared to era1-1, indicating that cytokinins are also synthesized via the iPRMP-dependent pathway in era1-1AtIPT3-OE plants. Furthermore, iP7G, like in AtIPT3-OE plants, was the predominant cytokinin (Table I). More importantly, ZR and ZROG content was 5.5- and 2-fold increased, respectively, in era1-1AtIPT3-OE leaves compared to era1-1. Overexpression of AtIPT3 in wild-type Arabidopsis had no effect on the content of cis-zeatin-type cytokinins, whereas a 2- and a 5-fold increase in cis-zeatin 9-riboside 5'-monophosphate (c-ZRMP) and cis-zeatin 9-riboside O-glucoside (c-ZROG), respectively, was measured in era1-1 leaves. Additionally, cis-zeatin riboside monophosphate content was 2-fold higher in era1-1AtIPT3-OE leaves than in era1-1. Surprisingly, cytokinin overproduction was almost completely abolished in AtIPT3C333S-OE plants. Replacement of the farnesyl Cys acceptor by Ser strongly reduced (2.7-fold) the accumulation of total cytokinins compared to AtIPT3-OE plants and only slightly increased cytokinin content (1.2-fold) compared to the wild type, indicating that the Cys might be important for AtIPT3 catalytic activity (Table I).
The high accumulation of cytokinin N-glucosides [IP7G and N6-(2-isopentenyl)adenine 9-glucoside (abbreviated IP9G)] in AtIPT3-OE and era1-1AtIPT3-OE plants indicated that the conversion of the primary products of the cytokinin biosynthesis to IP7G and IP9G could represent an important pathway involved in the maintenance of cytokinin homeostasis and induced in AtIPT3 plants. Degradation of cytokinins by cytokinin oxidase/dehydrogenase also occurs in Arabidopsis (Werner et al., 2003 ). However, cytokinin oxidase/dehydrogenase activity was altered neither in AtIPT3-OE nor in era1-1AtIPT3-OE plants compared to their corresponding controls (Table II
). Together, these results show that the lack of protein farnesylation in era1-1 strongly affects cytokinin biosynthesis or/and metabolism. In addition, although the overall cytokinin content was very similar in AtIPT3-OE and era1-1AtIPT3-OE plants, the increased accumulation of zeatin-type cytokinins in era1-1AtIPT3-OE plants suggests that farnesylation affects IPT3-mediated cytokinin biosynthesis.
Increased Cytokinin Accumulation Affects Plant Development and Cell Proliferation
The accumulation of cytokinins in plants expressing AtIPT3 has a significant effect on root development. Primary root length was reduced in AtIPT3-OE and era1-1AtIPT3-OE plants compared to the wild type and era1-1 (Fig. 3B). In addition, the transgenic plants also developed fewer lateral roots. Root development in AtIPT3C333S-OE plants, however, was not affected. To address the effect of AtIPT3 expression in leaves, we measured the area of the first leaves, which develop synchronously compared to other leaves (De Veylder et al., 2001
The decreased cytokinin production in AtIPT3C333S-OE plants and the absence of phenotypic alterations suggested that Cys-333 may have an important function for AtIPT3 catalytic activity. To investigate the function of Cys-333 for AtIPT3-mediated cytokinin biosynthesis, AtIPT3 and AtIPT3C333S were expressed in the yeast strains CTY716, which lacks PFT activity, and SYY705, which expresses the tomato (Solanum lycopersicum) PFT (Lavy et al., 2002
The preferential formation of iP-type cytokinins in yeast, E. coli, and Arabidopsis plants expressing AtIPT3 indicates that AtIPT3-mediated cytokinin biosynthesis proceeds via the iPRP-dependent pathway that produces iPRPs as first products. These results confirmed previously reported data from Arabidopsis plants or E. coli cells expressing IPT3, which accumulated iPRPs and iP. The results are also consistent with Arabidopsis Atipt3 mutants that have decreased iPRP and iPR levels (Takei et al., 2001 ; Sakakibara et al., 2005; Miyawaki et al., 2006). Similar to other studies, our method of cytokinin nucleotide measurement did not allow us to determine if AtIPT3 uses AMP, ADP, or ATP as acceptor of the isopentenyl moiety for cytokinin biosynthesis (Kakimoto, 2001; Takei et al., 2001; Sun et al., 2003). Interestingly, both ZR and ZROG accumulated in era1-1AtIPT3-OE plants, and ZR, ZOG, DHZ7G, and ZROG levels were increased in era1-1 plants. In contrast, the content of zeatin-type cytokinins was not significantly altered in AtIPT3-OE plants, and only iP-type cytokinins accumulated in these plants. This reveals that the lack of protein farnesylation in era1-1 promotes trans-zeatin cytokinin production in Arabidopsis (Table III
). Protein farnesylation controls IPT3 subcellular localization. The nonfarnesylated AtIPT3 is localized in the plastids, and the farnesylated AtIPT3 is located in the nucleus and the cytoplasm, at least in the onion epidermal cell assay. The observed dual subcellular localization of AtIPT3 could represent a regulatory mechanism of cytokinin production as a function of the activity of the cytoplasmic isoprenoid pathway (MVA pathway) and/or PFT. Depending on the efficiency of protein farnesylation and the availability of FPP for prenylation, AtIPT3 would be localized in the plastids or the nucleus. Consequently, and depending on the localization and the efficiency of cytokinin hydroxylase activities that catalyze hydroxylation of iP-type cytokinins, AtIPT3-mediated cytokinin production would then favor iP- or zeatin-type cytokinins by using DMAPP derived from the MVA pathway or the methylerythritolphosphate pathway, respectively (Table III). Whether the lack of protein farnesylation affects cytokinin biosynthesis only by modulating AtIPT3 subcellular localization or also influences the activity/localization of other proteins is currently not known, but the high accumulation of iPRPs in era1-1 plants would suggest a role of other farnesylated proteins in cytokinin production. Most of the small GTP-binding proteins are farnesylated (Tamanoi et al., 2001; Galichet and Gruissem, 2003), and cytokinin production is increased in tobacco (Nicotiana tabacum) plants expressing the rice (Oryza sativa) rgp1 gene (Sano et al., 1994), which would indicate that protein farnesylation could also influence cytokinin biosynthesis through modulation of small GTP-binding protein farnesylation. We attempted to further substantiate our analysis of AtIPT3 dual subcellular localization using biochemical fractionation. However, because of the low affinity of the IPT3 antibody, the results were not conclusive. Our results on IPT3 subcellular localization are also partially in contradiction with previous reports showing that AtIPT3 is only localized in plastids. These results were obtained from the expression of a fusion protein between the first 55 amino acids of AtIPT3 (which can function as a TP) and GFP (TP-GFP) or from stable expression of an AtIPT3-GFP fusion protein under the control of the AtIPT3 promoter (Kasahara et al., 2004; Miyawaki et al., 2006). However, a fusion of GFP at the C terminus of AtIPT3 would block farnesylation of the protein by masking the functional CAAX box.
The absence of an AtIPT3-mediated effect on cis-zeatin-type cytokinins is consistent with the model that tRNA degradation by AtIPT2 and AtIPT9 is the major source of the cis-zeatin cytokinins and that isoprenoid cytokinin biosynthesis does not influence cis-zeatin-type cytokinin formation (Kasahara et al., 2004 ; Miyawaki et al., 2006). However, the significant increase of c-ZRMP and c-ZROG in era1-1 and era1-1AtIPT3-OE plants suggests that the isomerization of trans-zeatin-type cytokinins could occur in Arabidopsis and that farnesylated proteins could play a role in this mechanism. A cis-trans-zeatin isomerase activity has been measured in Phaseolus vulgaris immature seeds (Bassil et al., 1993), but the existence of such an activity has not been demonstrated in Arabidopsis.
Cytokinin N-glucosides represented the main part of the pool of total cytokinins in AtIPT3-OE plants, and cytokinin oxidase/dehydrogenase activity was not altered in those plants. This indicates that N-glucosylation represents an important pathway of cytokinin inactivation and maintenance of cytokinin homeostasis in AtIPT3 Arabidopsis plants. Although cytokinin oxidases/dehydrogenases are involved in cytokinin degradation in many plants (Blagoeva et al., 2004
Mature wild-type and era1-1 Arabidopsis plants expressing AtIPT3 had enhanced leaf size with an increased number of cells, whereas root development was impaired. These phenotypes agree with the model that cytokinins modulate plant development through regulation of cell division (Sakakibara, 2006
Based on the cytokinin measurement in AtIPT3C333S-OE Arabidopsis plants, as well as in yeast and E. coli cells expressing AtIPT3C333S, we postulate that Cys-333 is important for AtIPT3 catalytic activity. Although the attachment of a farnesyl group to Cys-333 does not appear to inhibit enzyme activity, this Cys by itself is of particular importance for AtIPT3 function. The carboxyl-terminal end of the different AtIPTs is not well conserved, and AtIPT3 is the only AtIPT possessing a CAAX box for modification by protein farnesylation. In contrast, the putative DMAPP-binding site, located at the amino-terminal end of the proteins, is well conserved among the different members of this protein family (Takei et al., 2001
Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) plants used in our study were all derived from the Columbia accession line. Seeds were surface sterilized using 5% bleach and germinated on Murashige and Skoog medium. After 2 weeks, the seedlings were transferred to soil and grown in Conviron chambers with a 16-h-light/8-h-dark cycle at 23°C in 70% humidity.
AtIPT3 cDNA (At3g63110) was amplified by PCR using the primer IPT3-For (5'-ATGATCATGAAGATATCTATGGCT-3') together with either IPT3-Rev1 (5'-GTCGACTCACGCCACTAGACACCGCGA-3', for the wild-type version of the CaaX box, CLVA) or IPT3-Rev2 (5'-GTCGACTCACGCCACTAGACTCCGCGA-3', for the C333S version, SLVA). Both fragments were cloned in the pCR 2.1-TOPO cloning vector (Stratagene), sequenced, and subsequently cloned in the pRSETa vector for protein expression in Escherichia coli. Recombinant proteins were purified on nickel-nitrilotriacetic acid agarose talon super-flow metal affinity resin (Clontech). For cytokinin analysis, E. coli colonies cultivated overnight on Luria-Bertani (LB) plates supplemented with ampicillin and chloramphenicol (both at 50 µg/mL) at 37°C were inoculated into 2 mL of LB media supplemented with ampicillin (50 µg/mL) and cultured for 4 h at 37°C. Suspension was further inoculated into 200 mL of LB for 2 h at 37°C and then 0.1 mM isopropylthio-β-galactoside (IPTG) was added. After culture for 2.5 h at 37°C, the optical density (A600) was measured, and bacterial suspension was frozen in liquid nitrogen and stored at –80°C.
Nitrocellulose membranes were first blocked for 2 h at room temperature with 4% nonfat milk and subsequently incubated overnight at 4°C with the polyclonal anti-AtIPT3 antibody (diluted 1:100), washed with Tris-buffered saline containing Tween 20, and incubated 1 h with 10,000-fold diluted secondary antibody conjugated with horseradish peroxidase for detection with an ECL kit (Amersham Pharmacia Biotech).
Both in vitro and in vivo prenylation assays were performed as previously described (Yalovsky et al., 1997
Part of the AtIPT3 gene (Met-1-Ser-55, TP) was fused in frame to the amino terminus of the GFP gene in pGFP-MRC (Rodriguez-Concepcion et al., 1999
AtIPT3 and AtIPT3C333S cDNA were cloned in the modified vector pCAMBIA 1380 containing a cauliflower mosaic virus 35S promoter (kindly provided by L. Gomez-Gomez). The constructs were introduced into Agrobacterium tumefaciens strain LBA4404. These strains were used to transform Arabidopsis Columbia plants by floral dip (Clough and Bent, 1998
RNA was extracted using Qiagen (Chatsworth) RNeasy columns according to the manufacturer's instructions. For RT-PCR analysis, 5 µg of total RNA was treated with DNase I, and DNA-free RNA was transcribed using an oligo(dT) primer and moloney murine leukemia virus reverse transcriptase (Clontech). Aliquots of the generated cDNA, which equaled 50 ng of total RNA, were used as a template for PCR with gene-specific primers.
His-AtIPT3 and His-AtIPT3C333S fusion genes were cut from pRSETa constructs and were directionally cloned in pJR1138 vector containing the LEU2 marker (Yalovsky et al., 1997
Endogenous cytokinins were extracted by methanol/formic acid/water (15/1/4, v/v/v) from 55-d-old Arabidopsis rosette leaves, homogenized in liquid nitrogen, and purified using dual-mode solid phase extraction method (Dobrev and Kaminek, 2002
Cytokinin oxidase/dehydrogenase activity was measured as previously described (Motyka and Kaminek, 1994
We thank Dr. Hitoshi Sakakibara for the AtIPT3 antibody, Dr. Shaul Yalovsky for the CTY7166 and SYY705 yeast strains, Dr. Václav Motyka for determination of cytokinin oxidase/dehydrogenase activity in Arabidopsis, and Ji  í Malbeck and Alena Trávní ková for HPLC/MS/MS. Received August 15, 2007; accepted January 2, 2008; published January 9, 2008.
1 This work was supported by ETH Zurich (to W.G.), by the Grant Agency of the Academy of Sciences of the Czech Republic (grant no. A600380507 to M.K.), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant no. LCO6034 to M.K.). 
2 These authors contributed equally to the article.
3 Present address: Division of Psychiatry Research, University of Zurich, August Forel-strasse 1, 8008 Zurich, Switzerland. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wilhelm Gruissem (wgruissem{at}ethz.ch).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.107425 * Corresponding author; e-mail wgruissem{at}ethz.ch.
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(plp) gene functions is not lethal, although the mutants are affected in their development (Yalovsky et al., 2000a
