Developmentally Controlled Farnesylation Modulates AtNAP1 ...McQuibban et al., 1998; Nakagawa et...

Developmentally Controlled Farnesylation ModulatesAtNAP1;1 Function in Cell Proliferation and CellExpansion during Arabidopsis Leaf Development1

Arnaud Galichet2 and Wilhelm Gruissem*

Institute of Plant Sciences, ETH Zurich, 8092 Zurich, Switzerland

In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and celldifferentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a compo-nent of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf develop-ment. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of theprotein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development whenit promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it pro-motes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts thisdevelopmental program by promoting unscheduled cell expansion during the proliferation phase.

Genetic analysis of eukaryotic protein prenyl trans-ferases showed that modification of target proteins byfarnesyl or geranylgeranyl is critical for control of de-velopment, growth, and signaling. Prenyl transferasescovalently attach a 15-carbon farnesyl diphosphate(FPP) or the 20-carbon geranylgeranyl diphosphate(GGPP) isoprenoids to a Cys acceptor, which is part ofa C-terminal CaaX box motif in the substrate proteins.Protein farnesyl transferase (PFT) and geranylgeranyltransferase (PGGT-I) share a common a-subunit buthave distinct b-subunits. Specificity of PFT and PGGT-Iis determined by the b-subunits of each enzyme throughsequence-specific recognition of the C-terminal CaaXbox motif in substrate proteins (Yalovsky et al., 1999;Sinensky, 2000). Specific inhibitors of PFT or PGGT-Iinhibit progression of the cell cycle in plant and animalcells, indicating that prenylation is required for thefunction of proteins involved in regulating cell divi-sion (Morehead et al., 1995; Qian et al., 1996; Tamanoiet al., 2001). For example, Ras, LKB1, CENP, or TC10,which are involved in cell proliferation and differen-tiation or cytoskeletal functions, are prenylated proteins.Prenylation modulates their function by facilitatingmembrane association as well as protein-protein in-

teraction (Tamanoi et al., 2001; Hussein and Taylor,2002; Martin and St Johnston, 2003; Elam et al., 2005).Protein prenylation is conserved in animals and plants(Yalovsky et al., 1999), but, unlike mice in which PFT isessential for early embryonic proliferation (Mijimolleet al., 2005), loss of Arabidopsis (Arabidopsis thaliana)PFTb (era1) or PFT/PGGT-Ia (plp) gene functions is notlethal, although the mutants are affected in their de-velopment (Yalovsky et al., 2000a; Running et al., 2004;Mijimolle et al., 2005). This could be due to the specifictypes of proteins that are prenylated in plants, becausemost plant candidate protein prenyl transferase pro-tein substrates differ from those identified in yeast(Saccharomyces cerevisiae) and animals (Galichet andGruissem, 2003).

Among plant candidate PFT protein substrates,nucleosome assembly protein 1 (NAP1) is conservedamong eukaryotes and has been identified in soybean(Glycine max), pea (Pisum sativum), rice (Oryza sativa),and tobacco (Nicotiana tabacum) BY2 cells, human,yeast, murine, and Drosophila melanogaster cells (Ishimiet al., 1984; Ishimi and Kikuchi, 1991; Simon et al.,1994; Yoon et al., 1995; Hu et al., 1996; Ito et al., 1996;Rougeulle and Avner, 1996; Rodriguez et al., 1997; Shenet al., 2001; Dong et al., 2003). Most NAP1 proteinscontain a typical CaaX box recognition motif for PFT,and the human NAP1-like1 was shown to be in vivofarnesylated in COS-1 cells (Kho et al., 2004). NAP1was first identified from HeLa cells by its ability tofacilitate in vitro assembly of nucleosomes under phy-siological conditions. The current model suggests thatNAP1 is part of a multifactorial chromatin assemblymachinery, which mediates the ATP-facilitated assem-bly of regularly spaced nucleosomes (Ishimi et al.,1984, 1987; Ishimi and Kikuchi, 1991; Walter et al.,1995; Yoon et al., 1995; Hu et al., 1996; Ito et al., 1996;McQuibban et al., 1998; Nakagawa et al., 2001). In

1 This work was supported by the Swiss Federal Institute ofTechnology Zurich.

2 Present address: Division of Clinical Chemistry and Biochem-istry, Department of Pediatrics, University Children’s Hospital, 8032Zurich, Switzerland.

* Corresponding author; e-mail [email protected]; fax 41–44–632–1079.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Wilhelm Gruissem ([email protected]).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.088344

1412 Plant Physiology, December 2006, Vol. 142, pp. 1412–1426, www.plantphysiol.org � 2006 American Society of Plant Biologists

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addition to their proposed histone chaperone activity,NAP1 proteins may be involved in transcriptional reg-ulation through chromatin remodeling (Kawase et al.,1996; Ito et al., 2000; Shikama et al., 2000; Asaharaet al., 2002; Levchenko and Jackson, 2004; Rehtanz et al.,2004). Recent genetic studies also revealed functions ofNAP1 in the control of mitosis and during develop-ment. Xenopus and yeast NAP1 interact specificallywith B-type cyclin (Clb). Deletion of the NAP1 gene ina yeast strain that is dependent upon Clb2 functionresults in a prolonged delay of mitosis with nor-mal levels of Clb2/p34CDC28-associated kinase activity,and yeast cells are unable to induce events requiredfor assembly or proper function of the mitotic spindle.Yeast NAP1 was also shown to regulate the cell cyclein combination with the Gin4 kinase, NBP1, and SDA1(Kellogg et al., 1995; Kellogg and Murray, 1995; Altmanand Kellogg, 1997; Shimizu et al., 2000; Zimmermanand Kellogg, 2001). In addition, several mammalianNAP1 homologs appear to regulate cell proliferationand differentiation (von Lindern et al., 1992; Simonet al., 1994; Abu-Daya et al., 2005). A role of NAP1during development was revealed through the con-struction of knockout mutants in mice and Drosophila.The inactivation of Drosophila NAP1 is embryo lethal,and a null mutation of the brain-specific mouse NAP1l2gene results in embryonic lethality beginning at mid-gestation. Surviving mice mutant embryos showed ex-tensive surface ectoderm defects, open neural tubes,and exposed brain, suggesting a tissue-specific role forNAP1L2 in the regulation of neuron proliferation(Rogner et al., 2000; Lankenau et al., 2003). But despitethis extensive information, the biochemical and molec-ular functions of NAP1 proteins and the role of theprenyl modification is still poorly understood.

Arabidopsis has four NAP1-related genes whosefunction is currently not known. We investigatedArabidopsis nucleosome assembly protein 1 (AtNAP1;1),which has a predicted CaaX motif, to understand thefunction of the protein and the potential role of itsfarnesylation during Arabidopsis development. Char-acterization of gain- and loss-of-function mutantsdemonstrated that AtNAP1;1 contributes to the regu-lation of cell proliferation and cell expansion. Thedevelopmental function of AtNAP1;1 appears to beregulated by the differential farnesylation of the pro-tein during specific stages of leaf development.

RESULTS

AtNAP1;1 Is a Farnesylated Protein

The presence of the CKQQ motif at the C-terminalend of AtNAP1;1 suggested that the protein is a sub-strate of PFT. To test if AtNAP1;1 can be prenylatedby PFT, we incubated the purified protein with re-combinant Arabidopsis PFT and [3H]FPP. AtNAP1;1was labeled strongly in the presence of both PFT and[3H]FPP but not with PGGT-I using either [3H]FPP or[3H]GGPP (Fig. 1A). AtNAP1;1 was also labeled

weakly by PFT using [3H]GGPP, because the enzymeis somewhat promiscuous for GGPP (Trueblood et al.,1993; Lane and Beese, 2006). Mutation of the con-served Cys farnesyl acceptor in the CKQQ motif to Ser(AtNAP1;1C369S) confirmed that farnesylation requireda functional farnesylation motif. Among the three otherArabidopsis NAP1 proteins, AtNAP1;2 and AtNAP1;3have a similar CKQQ motif and are likely prenylatedas well. Similarly, NAP1 is encoded by small genefamilies in tobacco and rice (Dong et al., 2003). The twoOsNAP1 and three of the NtNAP1 proteins have CaaXboxes that can be prenylated in vitro by ArabidopsisPFT (A. Galichet and W. Gruissem, unpublished data).

To substantiate that AtNAP1;1 is a substrate for PFT,protein extracts from wild-type or era1-1 flowers weretested as a source for the enzyme (Fig. 1B). AtNAP1;1but not AtNAP1;1C369S was labeled in the presenceof protein extracts from wild-type flowers, confirmingthat the protein extract had PFT activity and thatAtNAP1;1 could be correctly farnesylated at the Cysacceptor in the CKQQ prenylation motif. The proteinextract from era1-1 flowers was unable to farnesylateAtNAP1;1, confirming the lack of PFT activity in themutant and establishing that AtNAP1;1 is a substrateof PFT.

To confirm that AtNAP1;1 is also farnesylated invivo, we expressed green fluorescent protein (GFP)-AtNAP1;1 and GFP-AtNAP1;1C369S in tobacco BY-2cells under control of the cauliflower mosaic virus(CaMV) 35S promoter. Soluble proteins from cells la-beled with [3H]mevalonic acid were extracted andseparated on SDS-polyacrylamide gels, which werethen used either for immunoblot analysis with a poly-clonal NAP1 antibody or for fluorography to detectlabeled GFP-AtNAP1;1 (Fig. 1C). GFP-AtNAP1;1 andGFP-AtNAP1;1C369S were expressed to a similar levelin BY-2 cells. A labeled protein corresponding to thesize of GFP-AtNAP1;1 was detected only in extractsfrom cells expressing GFP-AtNAP1;1 but not in cellsexpressing GFP-AtNAP1;1C369S or in control BY-2 cells.Together, these results establish that AtNAP1;1 isefficiently farnesylated in vivo as well.

AtNAP1;1 Subcellular Localization during the Cell

Cycle Is Regulated But Does Not Depend onFarnesylation in Heterologous Cell Systems

Most farnesylated proteins in yeast and animal cellsare targeted to the plasma membrane (Sinensky, 2000),although in plants, farnesylated APETALA1 has also beenfound in the nucleus (Yalovsky et al., 2000b). To inves-tigate the subcellular localization of AtNAP1;1, GFP-AtNAP1;1 and GFP-AtNAP1;1C369S were transientlyexpressed in onion (Allium cepa) epidermal cells. GFPalone was distributed uniformly in the nucleus andthe cytoplasm, whereas GFP-AtNAP1;1 and GFP-AtNAP1;1C369S were restricted to the cytoplasm (Fig.2A). As controls, we also expressed GFP-CaM53 andGFP-CaM53mS. Their previously reported respec-tive localization to the membrane and the nucleus

Differential NAP1 Farnesylation during Leaf Development

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(Rodriguez-Concepcion et al., 1999) was also observedin onion epidermal cells, suggesting that expression ofPFT substrate proteins using the 35S promoter did notexceed the prenylation capacity of the cell that couldresult in a nonspecific localization of the protein.

To obtain further insights into the potential role offarnesylation for AtNAP1;1 subcellular localization,GFP-AtNAP1;1 and GFP-AtNAP1;1C369S were stablyexpressed in transgenic tobacco BY-2 cells (Fig. 2B).GFP-AtNAP1;1 was localized in both the cytoplasmand the nucleoplasm of interphase cells. During mito-sis, GFP-AtNAP1;1 was colocalized with the phragmo-plast in telophase. Localization of GFP-AtNAP1;1C369S

was similar (data not shown). Together, we concludethat the farnesylation status of AtNAP1;1 has noimmediate role in the subcellular localization of theprotein during the cell cycle.

Farnesylation Is Necessary for AtNAP1;1 Functionin Yeast

We examined the physiological role of AtNAP1;1farnesylation by functional complementation assaysusing the yeast nap1 mutant strain DK213. This mutanthas a reduced growth rate at 37�C and a delay in mitosis,resulting in cells that are elongated and form clumps(Kellogg et al., 1995). Transformants of DK213 express-

ing AtNAP1;1, AtNAP1;1C369S, or ScNAP1 were analyzedfor their growth and their cell phenotypes at 37�C(Fig. 3, A and B). ScNAP1 and AtNAP1;1, but notAtNAP1;1C369S, rescued the elongated bud phenotypeand the temperature sensitivity, demonstrating thatAtNAP1;1 could complement the yeast nap1 mutation.Western-blot analysis of yeast protein lysates with ananti-NAP1 antibody revealed that the three NAP1 pro-teins were expressed at similar levels (Fig. 3C). There-fore, the farnesyl-Cys acceptor of AtNAP1;1 is requiredfor its function during cell cycle progression in yeast.

Cell Proliferation Is Reduced in Atnap1;1 Leaves

To gain further insight into AtNAP1;1 function, weidentified two independent Atnap1;1 mutant alleles inthe SALK T-DNA insertion collection (SALK_013610and SALK_095311; Alonso et al., 2003). These alleleshave a T-DNA insertion in the seventh or the 10th exonof AtNAP1;1 that we named Atnap1;1-1 and Atnap1;1-2(Fig. 4A). Western-blot analysis of extracts from thetwo Atnap1;1 mutant alleles and control plants togetherwith extracts from Atnap1;2 and Atnap1;3 mutants(T-DNA insertion lines SALK_002892 [T-DNA inser-tion in the second intron] and SAIL_373_H11 [T-DNAinsertion in the 10th exon], respectively; Sessionset al., 2002; Alonso et al., 2003) established the lack of

Figure 1. AtNAP1 is prenylated in vitro and in vivo.A, Wild type (containing an intact CKQQ CaaX box)and C369S (CKQQ CaaX box mutated to SKQQ)versions of AtNAP1;1 are used as substrates for plantprotein prenyltransferases. Symbols 1 and 2 indicatethe presence or absence of purified AtNAP1;1, puri-fied prenyltransferase, FPP, or GGPP. After electro-phoresis and fluorography, exposure was carried outfor 5 d. B, Wild-type and era1-1 Arabidopsis flowersoluble extracts were used as prenyltransferasesources. Prenylation reactions were carried out with50 mg of crude extract, and 1 and 2 symbols in-dicate the presence or absence of AtNAP1;1,AtNAP1;1C369S, and FPP. Exposure was carried outfor 15 d. C, In vivo prenylation assay. Fluorographyand immunoblots of protein extracts from to-bacco BY-2 cells expressing GFP-AtNAP1;1 or GFP-AtNAP1;1C369S fusion proteins and labeled with3H-mevelonic acid. Protein extracts were separatedby SDS-PAGE in 10% gel, followed by westernblotting. Fluorography was carried out for 3 weeks.

Galichet and Gruissem

1414 Plant Physiol. Vol. 142, 2006



AtNAP1;1 in Atnap1;1-1 and Atnap1;1-2 (Fig. 4B). Thiswas consistent with quantitative reverse transcription(qRT)-PCR data that showed no accumulation of thedifferent NAP1 mRNAs in the respective mutants (datanot shown). The results also confirmed that the ma-jor protein detected by the antibody corresponds toAtNAP1;1, but that the antibody cross reacted withAtNAP1;2 and AtNAP1;3 as well. The observed differ-ences in band intensity could result from a difference inexpression of the AtNAP1 proteins or from the higherspecificity of the antibody for AtNAP1;1.

Homozygous Atnap1;1-1 and Atnap1;1-2 plants wereenlarged compared to wild type early in developmentbut then became reduced in size (Fig. 4, C and D). Incontrast, Atnap1;2 and Atnap1;3 plants had no obviousphenotype (data not shown). Interestingly, by day 7,Atnap1;1-1 and Atnap1;1-2 leaves had more than twiceas many cells compared to wild type (Fig. 4E). Similarly,9-d-old Atnap1;1-1 and Atnap1;1-2 cotyledons were en-larged with more cells (data not shown). Between day 7and 15, cell proliferation slowed in mutant leaves, whilecell number continued to increase in wild-type leaves,therefore resulting in approximately 17% fewer cells inmature mutant leaves (Fig. 4E). In contrast, cell size wasnot significantly different in the two mutant lines com-pared to wild type during leaf development (data notshown). Together, these results suggest that loss ofAtNAP1;1 function alters the normal cell proliferationduring leaf development.

Gain of Function of AtNAP1;1 Alters Leaf Growthand Size

To examine the effects of AtNAP1;1 gain of functionand the potential role of AtNAP1;1 farnesylation in

vivo, we generated Arabidopsis transgenic plants inwhich the AtNAP1;1 or AtNAP1;1C369S cDNA were ex-pressed under the control of the constitutive CaMV35S promoter. Nine AtNAP1;1 and 11 AtNAP1;1C369S T1hygromycin-resistant plants with single T-DNA inser-tions were selected for further analysis. Six indepen-dent T3 homozygous AtNAP1;1 and seven AtNAP1;1C369S

lines were analyzed by western blot using the anti-NAP1antibody. Except one AtNAP1;1 line, all transgenic lineshighly expressed the transgene (Fig. 5A) without anydetectable change in the RNA levels expressed fromthe endogenous members of the AtNAP1 gene family(Fig. 5B). Lines AtNAP1;1 3.5.3 (AtNAP1;1-OE) andAtNAP1;1C369S 2.2.5 (AtNAP1;1C369S-OE) had similarlevels of transgene expression and were therefore se-lected for a full analysis (Fig. 5A, lanes 7 and 10). Therewas no difference in germination and in the timingof leaf primordia initiation between AtNAP1;1-OE,AtNAP1;1C369S-OE, and wild-type lines. Plants expressingAtNAP1;1 developed smaller leaves and cotyledons,whereas these organs were enlarged in plants expressingAtNAP1;1C369S (Fig. 5C). In contrast, there was no dif-ference in petal size, indicating that AtNAP1;1 over-expression preferentially affected the development ofvegetative organs. Because the difference in leaf size wasthe most striking phenotype, we analyzed leaf develop-ment in more detail in the two gain-of-function mutants.

AtNAP1;1 Gain of Function Modulates Cell Number

and Size during Leaf Development

A change in organ size can reflect an alteration incell size or number or both. To address these possibil-ities, we performed a kinematic analysis of the firstleaf using the method reported by De Veylder and

Figure 2. Farnesylation does not affect subcellu-lar localization of AtNAP1;1. A, GFP-AtNAP1;1,GFP-AtNAP1;1C369S, GFP-CaM53, GFP-CaM53mS,and GFP proteins were detected by green fluores-cence using confocal laser scanning microscopyin epidermal onion cells 38 h after bombardment.Bars correspond to 50 mm. B, Localization ofGFP-AtNAP1;1 and GFP- AtNAP1;1C369S fusionproteins in tobacco BY-2 tobacco cells. Trans-genic suspension of BY-2 tobacco cells wereestablished and used for localization.





colleagues (De Veylder et al., 2001). Between inceptionand maturity, Arabidopsis leaf development can becategorized into three stages: proliferation (until day12 after sowing), expansion (days 12–19), and maturity(after day 19; Beemster et al., 2005). The first leaf ofAtNAP1;1-OE, AtNAP1;1C369S-OE, and control lineswas harvested between 7 and 27 d after sowing andaverage leaf area and adaxial palisade cell size weredetermined (Fig. 6, A and B). From these data, theaverage palisade cell number was calculated (Fig. 6C).We found that 9 d after sowing the AtNAP1;1-OEplants, first leaf size was reduced 65%, which wasmainly the result of decreased cell size (63%). In 15-d-old AtNAP1;1-OE plants, the first leaf had approxi-mately 20% more cells than the wild-type leaf. Thesecells were 50% reduced in size, which reduced the leafblade area by approximately 40%. This trend contin-ued in AtNAP1;1-OE mature first leaf, resulting in a

35% decrease of leaf size after 26 d. In contrast, the 9-d-old AtNAP1;1C369S-OE first leaf had an increased cellsize and number (55% and 16%, respectively), whichresulted in an enlarged leaf area of approximately78% relative to wild-type control plants. After 15 d,the AtNAP1;1C369S-OE first leaf blade size was still36% larger than control leaves, but at 26 d theAtNAP1;1C369S-OE first leaf was comparable in size towild-type first leaves. We then calculated the relativecell division rate, and the results in Figure 6D showthat the cell division rate in AtNAP1;1C369S-OE andwild-type plants increased until day 9 and then rap-idly declined during the following 5 d. In contrast, therate of cell division was increased in AtNAP1;1-OE.Based on these results, we calculated the average cellcycle duration as the inverse of the relative cell divi-sion rate (Beemster et al., 2005). On day 9, the averagecell cycle duration in the first leaf was 32.7 h in wild

Figure 3. AtNAP1;1 complements the yeast nap1mutant. DK213 yeast nap1 cells were transformedwith pJR1133 vector alone (1), ScNAP1 (:),AtNAP1-1 (j), or AtNAP1;1C369S (d), and cells werearrested in G2/M and then released in fresh media atthe permissive temperature (30�C) or at the restrictivetemperature (37�C). A, Cell proliferation was moni-tored at 37�C by measuring the OD600 nm of theculture over a period of 24 h. B, Nomarski imagesafter 16 h at 30�C. C, Immunoblot analysis of wild-type DK213 yeast cells (1) or DK213 cells expressingScNAP1 (2), AtNAP1;1 (3), or AtNAP1;1C369S pro-teins. Protein extracts were separated by SDS-PAGEin 10% gel followed by western blotting.





type, 33.8 h in AtNAP1;1C369S-OE, and 19.2 h inAtNAP1;1-OE plants.

The kinematic analysis of leaf development revealedthat ectopic expression of wild-type and mutant NAP1resulted in alterations of cell size and number inthe first leaf. Interestingly, ectopic expression ofAtNAP1;1C369S primarily increased cell size, but thecomparable size of mature AtNAP1;1C369S-OE andwild-type first leaves suggested that a compensatorymechanism corrects for the increased cell size inAtNAP1;1C369S-OE. Such compensation was not appar-ent in AtNAP1;1-OE plants, suggesting that the farne-sylation of AtNAP1;1 has a profound effect on thefunction of the protein in this mechanism.

Timing of Cell Differentiation Is Altered by EctopicAtNAP1;1C369S Expression

Cell proliferation, expansion, and differentiation areclosely linked during leaf development (Beemsteret al., 2005). Since the ectopic expression of AtNAP1;1

or AtNAP1;1C369S affected cell division rate and cellexpansion, we investigated the effect of the wild-typeand mutant proteins on cell differentiation. We firstanalyzed DNA endoreplication as an early marker ofleaf cell differentiation (Melaragno et al., 1993) by mea-suring DNA content of nuclei from primary leavesof wild-type, AtNAP1;1-OE, and AtNAP1;1C369S-OEplants beginning at day 9 after sowing until day 25.In wild-type leaves, all leaf nuclei had a DNA contentof 2C and 4C at day 9 (Fig. 6E, left), but beginningat day 11, the population of cells with 8C nucleiincreased, reaching a maximum of 34% of the cellson day 25. Cells with 16C nuclei were first detected atday 20, and this population of cells increased to 6% oftotal leaf cells after 25 d. Together, the results confirm acorrelation between DNA endoreplication and cellexpansion (Beemster et al., 2005). Leaves of plantsexpressing AtNAP1;1 had an increased population ofcells with 2C nuclei after 9 d (78% compared to 64% inwild type), which suggests that the G2 phase inAtNAP1;1-OE plants may be shortened relative to G1

Figure 4. Arabidopsis Atnap1;1 mutant alleles are affected in cell proliferation during leaf development. A, Atnap1;1 genestructure and location of T-DNA insertions in the Atnap1;1-1 and Atnap1;1-2 mutant alleles. B, Western-blot analysis of AtNAP1proteins in wild type (lane 1), Atnap1;1-1 (lane 2), Atnap1;1-2 (lane 3), Atnap1;2 (lane 4), and Atnap1;3 (lane 5) mutant alleles.Samples were collected from 9-d-old plants, 25 mg of total protein was loaded per well, and membrane was probed withpolyclonal AtNAP1 antibody. C, Nine-day-old sterile-grown plants showing the differences in leaf and cotyledon size. D,Atnap1;1-1 and Atnap1;1-2 average first leaf blade area and average number of cells (E). Twenty individual leaves and 40palisade mesophyll cells were analyzed in duplicate.





during the proliferation phase (Fig. 6E, middle). Inaddition, cells with 8C and 16C nuclei appeared laterin AtNAP1;1-OE plants, suggesting that DNA en-doreplication was delayed. The ploidy of nuclei inAtNAP1;1C369S-OE leaves at day 9 was similar to wild-type leaves (Fig. 6E, right), indicating that the mu-tant protein had little effect during the proliferationphase. In contrast, after the proliferation phase, cellswith 8C and 16 C nuclei were detectable earlier inAtNAP1;1C369S-OE plants, and their population was sig-nificantly increased at day 25, suggesting that theincreased size of AtNAP1;1C369S-OE leaf cells (Fig. 6B)was correlated with earlier and increased DNA endo-replication.

We next examined the effects of ectopic AtNAP1;1and AtNAP1;1C369S expression on leaf structure andcell differentiation. Dicotyledonous leaves have a dis-tinct tissue organization with specific cell types

between the adaxial and abaxial leaf surfaces. Thepalisade layer below the adaxial epidermis consists oftightly packed elongated cells arranged with their longaxes perpendicular to the leaf surface. The spongymesophyll cells between the palisade cell layer and theabaxial epidermis are smaller and more rounded(Donnelly et al., 1999). Primary 9-d-old AtNAP1;1-OEleaves did not show the typical dorsoventral or-ganization of the leaf parenchyma into palisade andspongy mesophyll observed in wild-type leaves(Fig. 6F, top) but instead had a larger number ofsmaller spherical cells (Fig. 6F, center). Palisade andspongy mesophyll layers became more distinct inolder AtNAP1;1-OE first leaves, but they remainedsmaller than cells in wild-type leaves (data notshown). Microscopic analysis of the AtNAP1;1C369S-OE first leaf at day 9 revealed that all tissue layershad significantly larger and morphologically more

Figure 5. Phenotypic analysis of AtNAP1;1-OE and AtNAP1;1C369S-OE plants. A, Western-blot analysis of 20-d-old wild-type(lanes 1 and 8), AtNAP1;1-OE (lanes 2–7), and AtNAP1;1C369S-OE (lanes 9–15) T3 homozygous plant crude extracts. Twenty-fivemicrograms of total protein was loaded per well and membrane was probed with polyclonal AtNAP1 antibody. B, Expressionanalysis of AtNAP1;1 genes was monitored in wild-type (lane 1), AtNAP1;1-OE (lane 2), and AtNAP1;1C369S-OE (lane 3) 20-d-oldplants using semiquantitative RT-mediated PCR. C, Nine-day-old sterile-grown AtNAP1;1-OE and AtNAP1;1C369S-OE plantsshow altered plant organ size.





Figure 6. Increased AtNAP1;1 level in Arabidopsis leaves influences cell proliferation, cell size, and differentiation. A to D,Kinematic analysis of AtNAP1;1-OE and AtNAP1;1C369S-OE leaf development. Kinematic analysis was performed on the first leafpair of wild-type (¤), AtNAP1;1-OE (n), and AtNAP1;1C369S-OE (h) plants. Twenty individual leaves and 40 palisade mesophyllcells were analyzed in duplicate. A, Average leaf blade area; B, average cell area; C, average number of cells; and D, divisionrate. E, Quantification of ploidy distribution as mean 6 SD of two independent measurements involving different sets of plants. F,Transverse sections through the central part of 9-d-old plastic-embedded first leaves stained with toluidine blue. Scale bars are50 mm. G, Expression analyses of CYCB1 (top) and EXP5 (bottom) genes by qRT-PCR in AtNAP1;1-OE and AtNAP1;1C369S-OE 9-,15-, and 25-d-old first leaves. Results of quantification using GAPDH as a reference are relative to wild-type expression level.




differentiated cells than the wild-type first leaf (Fig. 6F,bottom). Together, the results indicate that ectopicexpression of AtNAP1;1 or AtNAP1;1C369S had oppos-ing effects on cell differentiation and elongation,which correlated with the changes in nuclear ploidy.

Ectopic Expression of AtNAP1;1 Affects the Expression

of Cell Division and Cell Expansion-Related Genes

To begin to understand how ectopic AtNAP1;1 ex-pression may modulate cell proliferation, expansion,and differentiation at the molecular level, we analyzedthe accumulation of mRNAs for cell cycle and cellexpansion-related genes during leaf development.qRT-PCR revealed that the CycB1;1 mRNA level wasincreased in AtNAP1;1-OE leaves at day 9 but was notsignificantly changed in AtNAP1;1C369S-OE leaves com-pared to wild type (Fig. 6G, top). No difference wasfound in the expression of CycD1;1 and CycD1;3 genes(data not shown). EXP5 represents a member of thegene family for expansin proteins that induce extensionof the plant cell wall. The mRNA of this gene wasstrongly increased in leaves expressing AtNAP1;1C369S,consistent with the increased cell size (Fig. 6G, bottom).Interestingly, EXP5 mRNA also accumulated to higher

levels in leaves expressing AtNAP1;1 despite theirsmaller cell size, suggesting that EXP5 function is notentirely restricted to cell elongation.

Farnesylation of AtNAP1;1 Is Specific to the CellProliferation Phase during Leaf Development

Given the effects of ectopic AtNAP1;1 or AtNAP1;1C369S

expression at the cellular and molecular levels, wenext asked whether posttranslational farnesylation ofAtNAP1;1 was altered during leaf development, whichin turn could modulate the function of AtNAP1;1 to affectcell proliferation, expansion, and differentiation. Forthis work, we also took advantage of the era1-1 mutant,which lacks PFTactivity (Yalovsky et al., 2000a). Immu-noblot analysis of protein extracts isolated from wild-type and era1-1 Arabidopsis primary leaves duringdevelopment using the anti-NAP1;1 antibody revealedthree bands, with AtNAP1;1 being the major band(Figs. 7A and 4B). In the wild-type first leaf, NAP1 pro-teins were highly expressed during the cell prolifera-tion phase but declined later in development (Fig. 7A,lanes 1–4). Attachment of farnesyl increases proteinmobility in SDS-polyacrylamide gels (Zhu et al., 1993).Interestingly, AtNAP1;1 migrated faster at day 9 as

Figure 7. Stage-specific farnesylation and subcellu-lar localization of AtNAP1;1 directs its functions. A,Western-blot analysis of AtNAP1 proteins duringwild-type and era1-1 primary leaf development.Samples were collected after 9, 14, 19, and 25 dfor wild type (lanes 1–4) and 11, 16, 21, and 27 d forera1-1 (lanes 5–8). The difference of harvesting timeoriginates from a 2-d delay in era1-1 germination.Twenty-five micrograms of total protein was loadedper well and separated by SDS-PAGE in 7.5% gelfollowed by western blotting. Membrane was probedwith polyclonal AtNAP1 antibody. B, In vivo prenyl-ation assay of AtNAP1;1 during leaf development.The 9- and 15-d-old wild-type (lane 1), AtNAP1;1-OE(lane 2), and AtNAP1;1C369S-OE (lane 3) first leaveswere labeled with [3H]mevalonic acid, and proteinextracts were subjected to immunoblot analysis andfluorography. Fluorography was carried out for 3weeks. C, Subcellular fractionation of 9- and 15-d-old wild-type, AtNAP1;1-OE, and AtNAP1;1C369S-OEfirst leaf protein extracts. Twenty micrograms of totalprotein (T) and equivalent volumes of cytoplasmic (C)and nuclear (N) fractions were loaded. D, Subcellularfractionation of 9- and 15-d-old wild-type and 11-and 17-d-old era1-1 primary leaf protein extracts.The experiment was carried out as described for C. Ascontrol, 50 mg of total protein and equivalent vol-umes of the two other fractions were used for detec-tion using the antibody raised against the MSI nuclearprotein.





compared to days 15 and 21 (Fig. 7A, lanes 2 and 3) or toAtNAP1;1 protein in era1-1 protein extract (Fig. 7A,lanes 5–8). The shift in mobility was reproducible andcould indicate that AtNAP1;1 expressed later in devel-opment does not become farnesylated or that farnesylis removed from the existing protein. To investigatethis possibility, we incubated primary leaves isolatedfrom 9- and 15-d-old AtNAP1;1-OE and AtNAP1;1C369S-OE plants with [3H]mevalonic acid and analyzed thelabeled proteins. Figure 7B shows that AtNAP1;1, butnot AtNAP1;1C369S, was actively farnesylated in 9-d-oldprimary leaves. AtNAP1;1 farnesylation was not de-tectable in 15-d-old leaves, although the protein accu-mulated to similar levels at both developmental stages.No labeled protein was detected in wild-type leaves ateither developmental stage. This was not unexpectedconsidering the high levels of AtNAP1;1 expression inthe transgenic line that was needed to detect labeledprotein and the low efficiency of farnesylation duringthe short labeling time.

Because era1-1 lacks PFT activity, NAP1;1 is notfarnesylated in this mutant (Fig. 1, A and B) or mod-ified by PGGT-I, which is somewhat promiscuous forthe CaaX motif (Trueblood et al., 1993; Lane and Beese,2006). Interestingly, AtNAP1 proteins accumulated tosignificantly higher levels in the era1-1 first leaf early indevelopment (Fig. 7A, lanes 5 and 6) and remained athigher levels in older leaves (Fig. 7A, lanes 7 and 8) aswild type at comparable stages. This result suggeststhat the lack of AtNAP1 farnesylation in era1-1 mayaffect the regulation of NAP1 gene expression. Alter-natively, nonfarnesylated NAP1;1 may be more stablein era1-1.

Different Subcellular Localization of AtNAP1;1 IsCorrelated with the Different Phases ofLeaf Development

Subcellular analysis of AtNAP1;1 expressed in BY-2tobacco cells had revealed that the protein was locatedin the cytoplasm or the nucleus depending on stage ofthe cell cycle, but this was not depending on farnesy-lation (Fig. 2). Considering the opposing effects ofectopically expressed AtNAP1;1 and AtNAP1;1C369S oncell proliferation and expansion (Fig. 6), it was stillpossible that the farnesylation status of NAP1;1 duringleaf development (Fig. 7B) was important to direct thesubcellular localization of the protein. We thereforeanalyzed the subcellular localization of AtNAP1 pro-teins during development of the first leaf. In leavesfrom control and AtNAP1;1-OE lines, the AtNAP1proteins detected by the antibody were found exclu-sively in the nuclear fraction on day 9, whereas theywere equally distributed between the nuclear andcytoplasmic fractions on day 15 (Fig. 7C). In contrast,AtNAP1 proteins were equally distributed betweenthe nucleus and the cytoplasm in leaves ofAtNAP1;1C369S expressing lines at both 9 and 15 d. Aparallel analysis of the first leaf of era1-1 showed thatAtNAP1 proteins were found both in the nucleus and

cytoplasm at day 11, whereas they were exclusivelypresent in the cytoplasm after 17 d (Fig. 7D). Together,we conclude that farnesylation of AtNAP1;1 directsthe protein to the nucleus early in leaf development,perhaps in concert with other localization mecha-nisms. The results also suggest that farnesylation andnuclear localization of AtNAP1;1 facilitate cell prolif-eration during early leaf development.

DISCUSSION

The analysis of mutants has provided significantnew insights into leaf development during the lastseveral years. Typically, leaf growth is tightly regu-lated by the control of cell number, cell size, anddifferentiation. The genetic and biochemical networksthat integrate these cellular processes, however, arestill largely unknown. Our analysis established thatAtNAP1;1 function is required for correct cell prolif-eration control during Arabidopsis leaf developmentand that this function is dependent on the temporalfarnesylation of the protein.

Organ development is tightly coordinated at thecellular level by cell division, expansion, and differ-entiation such that each organ reaches its appropriatesize relative to the size of the organism. In determinateplant organs, particularly leaves, final organ cell num-ber is regulated by the initial number of cells recruitedinto the primodium, the timing of cell division, and therate of proliferation. After mitotic activity ceases, celldifferentiation and expansion establish the regular pat-tern of tissue layers in the leaf blade (Donnelly et al.,1999). In Arabidopsis, loss of AINTEGUMENTA func-tion or ectopic expression of KRP2, an inhibitor of celldivision, reduces leaf cell number, which is largelycompensated by increase in cell size (Mizukami andFischer, 2000; De Veylder et al., 2001). These observa-tions suggest that, at the organ level, both cell divisionand expansion are coordinated. They follow patternsdependent on the anatomical and developmental con-text to generate leaves of normal size and shape. Thebalanced regulation of cell division and expansionmay also ensure the completion of a basic morpho-genesis program in case either one of the processes isimpaired. Several mutations have been reported, how-ever, that affect the balance between cell number andsize, resulting in modifications of leaf growth andshape. The Arabidopsis angustifolia and rotundifoliamutants have reduced polar cell expansion but noalteration in cell number, resulting in the production oflong, narrow leaves and shorter, wider leaves, respec-tively (Kim et al., 1998, 2002). In contrast, the Arabi-dopsis struwwelpeter mutant has smaller leaves withfewer cells but maintains cell size. Overexpression ofboth E2Fa and DPa, two transcription factors that arerequired for cell cycle regulation, strongly induces cellproliferation in Arabidopsis but also severely reducescell expansion and plant growth (Autran et al., 2002;De Veylder et al., 2002). Together, cell division or cell





expansion can independently influence leaf morpho-genesis by affecting leaves to maintain compensatorymechanisms during growth.

AtNAP1;1 Is a Possible Regulatory Link That ControlsCell Proliferation during Leaf Development

The reduced leaf growth in Atnap1;1 is the conse-quence of a decreased rate of cell division. In contrast,ectopic AtNAP1;1 expression in Arabidopsis increasedcell proliferation but did not affect the time window ofcell cycle activity during leaf development. Becausethe effect of either loss or gain of AtNAP1;1 functionappears to be restricted primarily to the phase of cellproliferation, we suggest that AtNAP1;1 functions as astage-specific positive regulator of cell proliferation.The high AtNAP1 protein level that we detected inleaves during the cell proliferation phase is also con-sistent with a role of AtNAP1 in the control of cell divi-sion. In humans, HsNAP1L1 gene is expressed in alltissues but its expression is increased in proliferatingcells. Moreover, the HsNAP1L1 protein level increasesin cultured T-lymphocytes induced to proliferate (Simonet al., 1994). Similarly, mouse NAP1L2 is mainly ex-pressed in the nervous system and is necessary fornormal proliferation of neuronal precursor cells (Rogneret al., 2000). Interestingly, ectopic AtNAP1;1C369S expres-sion did not have a significant affect on cell prolifer-ation, suggesting that prenylation of NAP1;1 is requiredfor its function during the cell proliferation phase ofleaf development.

How does AtNAP1;1 influence cell proliferationrate? Our analysis has revealed that AtNAP1;1 over-expression increases the expression of CYCB1;1, whichfunctions in late G2 and M phases (Donnelly et al.,1999). Most of the targets regulated by CYCB1;1-CDKcomplexes are currently unknown, but unscheduledactivation of CYCB1;1 expression by AtNAP1;1 mayresult in a shortened G2 phase or interfere with de-velopmentally regulated exit from the cell cycle.

AtNAP1;1 Promotes Cell Proliferation and ConnectsIt to Cell Expansion

Following the cell division phase, cell enlargementand cellular differentiation contribute to the final sizeand shape of leaves. It has been long recognized that acorrelation exists between DNA endoreplication andleaf cell expansion, suggesting that DNA ploidy levelmay determine cell size (Melaragno et al., 1993; Folkerset al., 1997). Plants that overexpress KRP2, whichinhibits CDK activity, however, have leaves with sig-nificantly enlarged cells but reduced DNA endorepli-cation, suggesting a more complex link between thesetwo processes (De Veylder et al., 2001). We have foundthat ectopic expression of AtNAP1;1C369S increasedcell size and promoted cellular differentiation alreadyearly during the proliferation phase of leaf develop-ment. This activity of AtNAP1;1C369S was uncoupledfrom the promotion of DNA endoreplication, which oc-

curred late during the proliferation phase. The resultstherefore suggest that increased levels of nonfarnesy-lated AtNAP1;1 are sufficient to promote unsched-uled cell expansion. At present, we can only speculateabout the mechanism, but one possibility is thatthe lack of the farnesyl group alters the subcellulardistribution of AtNAP1;1C369S and therefore allowsinteractions between AtNAP1;1C369S and other pro-teins that would promote cell expansion only later inleaf development. This view is supported by theobservation that the level of nonfarnesylated endoge-nous AtNAP1;1 is increased later in leaf development.

The possibility that AtNAP1;1 can both negativelyor positively influence cell growth and expansiondependent on its farnesylation status is also consistentwith the result that ectopic expression of AtNAP1;1reduced normal cell growth during the cell prolifera-tion phase of leaf development. It must be noted,however, that increased cell division can also inhibitcell growth (Fleming, 2002). The reduced cell size,together with delayed cell differentiation and endor-eplication that we observed in AtNAP1;1-OE plantscould therefore be a consequence of the initially in-creased cell proliferation triggered by AtNAP1;1 earlyduring leaf growth, rather than a direct effect of theprotein on cell growth. This possibility would also beconsistent with the observation that following the cellproliferation phase in AtNAP1;1-OE leaves, cell sizeremains smaller than in wild-type leaves, but therelative increase in cell size between 11 and 21 d issignificantly higher in AtNAP1;1-OE leaves than inwild-type leaves. In this case, the increased levels ofAtNAP1;1 later in leaf development could partiallycompensate the reduced growth during the prolifera-tion phase. This interpretation is reasonable, becauseloss of AtNAP1;1 function reduces cell proliferation,resulting in smaller leaves with fewer cells, but cellsize is not affected. It is unlikely that overexpressionof AtNAP1;1 titrates out other prenlylation substratesand PFT. Previous experiments showed that overex-pression of CaM53, a prenylated Calmodulin-relatedprotein, does not exceed the prenylation capacity ofthe cells (Rodriguez-Concepcion et al., 1999). This viewis also supported by the localization data shownin Figure 2. Our results therefore suggest that theobserved effects in AtNAP1;1-OE plants are only re-lated to the overexpression of the protein. Together,AtNAP1;1 promotes cell proliferation and participatesin connecting cell proliferation to cell expansion toachieve the correct balance between cell number andcell size that determines normal leaf blade size.

Farnesylation Regulates AtNAP1;1 SubcellularLocalization and Function

In plants, protein farnesylation has been shown toaffect protein function and subcellular localization(Zhu et al., 1993; Yalovsky et al., 2000b; Suzuki et al.,2002). Most of AtNAP1;1 is farnesylated duringthe cell proliferation phase of leaf development, but





AtNAP1;1 does not appear to be farnesylated duringthe subsequent leaf expansion phase. Our comple-mentation studies of the yeast nap1 mutant confirmedthat AtNAP1;1 farnesylation is necessary to comple-ment the cell cycle phenotype of the mutant. It wasalso reported that the carboxy-terminal region ofScNAP1 is important to allow mitotic progression ofthe nap1 yeast mutant in complementation experiments(Miyaji-Yamaguchi et al., 2003). Together, these resultssuggest that the farnesylation status of NAP1 is offunctional relevance. During the cell proliferationphase of leaf development, farnesylated AtNAP1;1localizes to the nucleus and promotes cell division.Later in leaf development when cells are expanding,most of the NAP1;1 appears to be nonfarnesylatedand accumulates both in the nucleus and the cyto-plasm, and absence of AtNAP1;1 farnesylation resultsin cellular growth. Subcellular localization analysisof the rice and tobacco NAP1 proteins also revealeda similar partitioning of the proteins between thenucleus and the cytoplasm (Dong et al., 2003), al-though this study did not provide information on thedevelopmental context or farnesylation status of theproteins.

If the farnesylation-dependent localization ofAtNAP1;1 is altered during leaf development, as wasthe case during the cell proliferation phase inAtNAP1;1C369S-OE plants, this results in prematurecell expansion. It is possible that unscheduled accu-mulation of AtNAP1;1 may allow the protein to en-gage interactions with other proteins that control cellgrowth and expansion in the context of the cell cyclebut independent of AtNAP1;1. These interactionscould occur in the nucleus or the cytoplasm, but it iscurrently unknown if they require NtNAP1;1 to befarnesylated. Also, these interactions do not easilyexplain how the different subcellular localizations ofAtNAP1;1 could exert effects on cell proliferation orcell growth and expansion. Additional experimentswill be necessary to identify proteins that specificallyinteract with NAP1;1 during different leaf develop-ment phases.

It is reasonable to expect that the farnesylation statusof AtNAP1;1 may allow the protein to engage indifferent protein complexes, which exert specific func-tions during leaf development. In yeast, NAP1 wasshown to specifically interact with a set of proteins,including histones, Clb2, Gin4, Nbp1, and p300/CREB-binding proteins (Ishimi et al., 1987; Kellogget al., 1995; Altman and Kellogg, 1997; Shikama et al.,2000; Shimizu et al., 2000). These proteins are involvedin different regulatory processes, including cell cyclecontrol, chromatin modifications, or transcription con-trol. There is also considerable evidence in plants forregulatory links between cell proliferation, cell expan-sion, and environmental and physiological signals(Pyke and Lopez-Juez, 1999; Meijer and Murray, 2001;Beemster et al., 2003). Earlier studies revealed thatprotein prenylation is required during plant develop-ment and identified functional associations between

protein prenylation and specific biological processesor signal transduction pathways (Pei et al., 1998;Rodriguez-Concepcion et al., 1999; Yalovsky et al.,2000a; Galichet and Gruissem, 2003; Running et al.,2004). The farnesylation status of NAP1 may thereforeprovide an interesting but currently little-exploredmechanism to link cell cycle and cell growth controlfunctions to the metabolic status of the cell. For exam-ple, during the cell proliferation phase early in leafdevelopment, the organ functions as a physiologicalsink, whereas the cell expansion phase later in leafdevelopment coincides with the autotrophic functionsof the organ (Paul and Foyer, 2001; Jeong et al., 2004).An important but still unresolved aspect is the mech-anism by which cells regulate the dynamic farnesyla-tion of AtNAP1;1. It has been reported that PFTactivity is increased during cell cycle progression intobacco BY-2 cells (Morehead et al., 1995). It is there-fore possible that PFT activity itself is regulated duringleaf development, which could result in differentialfarnesylation of AtNAP1;1. Alternatively, the pool ofFPP available for protein prenylation may be tightlyregulated during development. Finally, developmen-tally regulated farnesylation of AtNAP1;1 may requireadditional cofactors, whose expression is also devel-opmentally controlled. Further experiments will benecessary to distinguish between these scenarios.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants used in our study were all derived

from the Columbia (Col-0) accession line. Seeds of Atnap1;1-1 (SALK_013610),

Atnap1;1-2 (SALK_095311), and Atnap1;2 (SALK_002892) mutants were ob-

tained from the SALK T-DNA insertion collection (http://signal.salk.edu)

and Atnap1;3 (SAIL_373_H11) mutant seeds from the Syngenta Arabidopsis

insertion library. Seeds were surface sterilized using 5% bleach and germi-

nated on Murashige and Skoog medium. After 2 weeks, the seedlings were

transferred to soil and grown in Conviron chambers with a 16-/8-light/-dark

cycle at 23�C in 70% humidity.

Protein Expression and Antibody Production

The Arabidopsis AtNAP1;1 cDNA (At4g26110) was amplified by PCR

using the primer NAP1-For (5#-ATGAGCAACGACAAGGATAGCTTC-3#),

together with either NAP1-Rev1 (5#-GTCGACTTACTGTTGCTTGCAT-

TCGGG-3#, for the wild-type version of the CaaX box, CKQQ) or NAP1-

Rev2 (5#-GTCGACTTACTGTTGCTTGCTTTCGGG-3#, for the C369S version,

SKQQ). The wild-type and the mS PCR fragments were cloned in the pCR

2.1-TOPO cloning vector (Stratagene) and subsequently in the pRSETa vector

for protein expression in Escherichia coli. Recombinant proteins were purified

on nickel-nitrilotriacetic acid agarose talon superflow metal affinity resin

(CLONTECH). Polyclonal anti-AtNAP1;1 was produced by injecting the puri-

fied AtNAP1;1 in mouse.

Immunoblots

Nitrocellulose membranes were first blocked overnight at 4�C with 5%

nonfat milk and subsequently incubated for 2 h at room temperature with the

AtNAP1 antibody (diluted 1:5,000), washed with Tris-buffered saline con-

taining Tween 20, and incubated 2 h with 5,000-fold diluted goat anti-mouse

secondary antibody conjugated with horseradish peroxidase for detection

with an ECL kit (Amersham Pharmacia Biotech). Immunodetection using

polyclonal antibodies raised against MSI1 was carried out as described

(Kohler et al., 2003).





In Vitro and in Vivo Prenylation Assay

In vitro prenylation assay was performed as previously described (Yalovsky

et al., 2000b). In vivo prenylation reactions were carried out as follows. Tobacco

(Nicotiana tabacum) BY-2 cells expressing GFP-AtNAP1;1 or AtNAP1;1C369S

fusion proteins were treated with 5 mM mevinolin during 12 h and then

incubated in the presence of 6 mM [3H]mevalonic acid for 24 h. Cells were

homogenized in 100 mL of 1 3 SDS-loading buffer and boiled for 5 min.

Leaves from AtNAP1;1 and AtNAP1;1C369S plants were treated as described

(Yalovsky et al., 2000b).

Yeast Complementation Assay

AtNAP1;1, AtNAP1;1C369S, and ScNAP1 genes were directionally cloned in

pJR1133 vector, containing the URA3 marker. The resulting plasmids were

used to transform the yeast (Saccharomyces cerevisiae) strain DK213 (MATa

clb3TTRP1 clb4THIS3 Dclb1 NAP1TLEU2 leu2-3, 112 ura3-52 can1-100 ade2-1

his3-11 Dbar1). Cell cycle arrest in G2/M was performed with 30 mg/mL

benomyl for 3 h at 30�C (Zimmerman and Kellogg, 2001).

GFP Constructs and Confocal andFluorescence Microscopy

Wild AtNAP1;1 and AtNAP1;1C369S cDNA were cloned in pGFP-MRC

(Rodriguez-Concepcion et al., 1999). Onion (Allium cepa) epidermal cells were

transformed as described (Scott et al., 1999). Confocal imaging was performed

using a Leica confocal laser-scanning microscope. For ectopic expression in

BY-2 cells, previous constructs in pGFP-MRC vector were digested with SphI

to isolate 35STGFP-AtNAP1;1-NOS and 35STGFP-AtNAP1;1C369S-NOS frag-

ments. These fragments were cloned in pCAMBIA 1380 vector. BY-2 cells were

transformed by particle bombardment. Transgenic cells were cultivated

in medium containing hygromycin and examined for GFP fluorescence at

520 nm by using a Zeiss Axioplan2 fluorescence microscope.

Construction of AtNAP1;1 Plants and KinematicAnalysis of Leaf Development

AtNAP1;1 and AtNAP1;1C369S cDNA were cloned in the modified vector

pCAMBIA 1380 containing a CaMV 35S promoter (kindly provided by L.

Gomez-Gomez). The constructs were introduced into Agrobacterium tumefa-

ciens strain LBA4404. These strains were used to transform Arabidopsis Col-0

plants by floral dip (Clough and Bent, 1998). The kinematic analysis of

AtNAP1;1, AtNAP1;1C369S, and Atnap1;1 mutants and wild-type Col-0 leaves

growth was performed as described by De Veylder et al. (2001) on the adaxial

palisade mesophyll cells.

RNA Isolation and qRT-PCR

RNA was extracted using Qiagen RNeasy columns according to the

manufacturer’s instructions. For RT-PCR analysis, 5 mg 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 total RNA, were used

as template for qRT-PCR. Specific primers (temperature, melting, 58�C–63�C)

were designed to generate PCR products between 150 and 350 bp. CycB1;1

(At3g11520) forward primer (5#-CCTCAACCAGTTAGAGGTGATCC-3#) and

reverse primer (5#-GTTTCCAATGTCGCCAAGAG-3#), AtExp5 (At3g29030)

forward primer (5#-GCTCATGCCACTTTTTACGG-3#) and reverse primer

(5#-TCTCCAGTCCATAACCTTGG-3#) were used and qRT-PCR of GAPDHA

(At3g26650) with forward primer (5#-CTCCCTTGGAAGGAGCTAGG-3#) and

reverse primer (5#-TTCTTGGCACCAGCTTCAAT-3#) was performed for

standardization. qRT-PCR reactions were monitored using an ABI Prism

7700 Sequence Detection system with the SYBR green PCR Mastermix

(Applied Biosystem).

Flow-Cytometry Analysis

After removal of the leaf petioles, leaf blades were chopped with a razor

blade and ploidy analysis was carried out as described (Kohler et al., 2003).

Histological Analysis

Histological analyses were performed with samples in Technovit 7100

resin according to the manufacturer’s instructions (Kulzer & Co.). For trans-

verse sections, tissue samples were cut at the center of the first leaf. For

longitudinal sections, tissue samples were cut halfway between themed rib

and leaf margin.

Subcellular Protein Fractioning

Nuclei were isolated from harvested first leaves as described (Kohler et al.,

2003).

ACKNOWLEDGMENTS

We are grateful to Johannes Futterer for tobacco BY-2 cell transformation

and GFP fluorescence analysis, to Joanna Wyrzykowska, Gerrit T.S. Beemster,

and Andrew J. Fleming for helpful discussions and for critical reading of the

manuscript. We thank Marzanna Gontarczyk for help with the histological

preparations and Chantal Ebel for useful discussions. We thank D.R. Kellog

for the DK213 yeast strain, Syngenta for the Atnap1;3 T-DNA line, the Salk

Institute Genomic Analysis Laboratory for providing the sequence-indexed

Arabidopsis T-DNA Atnap1;1-1, Atnap1;1-2, and Atnap1;2 insertion mutants,

and Arabidopsis Biological Resource Center for providing us the seeds.

Received August 15, 2006; accepted September 27, 2006; published October 13,

2006.

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