Download Developmentally Controlled Farnesylation

Developmentally Controlled Farnesylation Modulates
AtNAP1;1 Function in Cell Proliferation and Cell
Expansion during Arabidopsis Leaf Development1
Arnaud Galichet2 and Wilhelm Gruissem*
Institute of Plant Sciences, ETH Zürich, 8092 Zurich, Switzerland
In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and cell
differentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a component of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf development. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1
promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of the
protein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development when
it promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it promotes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts this
developmental program by promoting unscheduled cell expansion during the proliferation phase.
Genetic analysis of eukaryotic protein prenyl transferases showed that modification of target proteins by
farnesyl or geranylgeranyl is critical for control of development, growth, and signaling. Prenyl transferases
covalently attach a 15-carbon farnesyl diphosphate
(FPP) or the 20-carbon geranylgeranyl diphosphate
(GGPP) isoprenoids to a Cys acceptor, which is part of
a C-terminal CaaX box motif in the substrate proteins.
Protein farnesyl transferase (PFT) and geranylgeranyl
transferase (PGGT-I) share a common a-subunit but
have distinct b-subunits. Specificity of PFT and PGGT-I
is determined by the b-subunits of each enzyme through
sequence-specific recognition of the C-terminal CaaX
box motif in substrate proteins (Yalovsky et al., 1999;
Sinensky, 2000). Specific inhibitors of PFT or PGGT-I
inhibit progression of the cell cycle in plant and animal
cells, indicating that prenylation is required for the
function of proteins involved in regulating cell division (Morehead et al., 1995; Qian et al., 1996; Tamanoi
et al., 2001). For example, Ras, LKB1, CENP, or TC10,
which are involved in cell proliferation and differentiation or cytoskeletal functions, are prenylated proteins.
Prenylation modulates their function by facilitating
membrane association as well as protein-protein in1
This work was supported by the Swiss Federal Institute of
Technology Zurich.
2
Present address: Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University Children’s Hospital, 8032
Zurich, Switzerland.
* Corresponding author; e-mail [email protected]; fax 41–44–
632–1079.
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 ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.106.088344
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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 is
essential for early embryonic proliferation (Mijimolle
et al., 2005), loss of Arabidopsis (Arabidopsis thaliana)
PFTb (era1) or PFT/PGGT-Ia (plp) gene functions is not
lethal, although the mutants are affected in their development (Yalovsky et al., 2000a; Running et al., 2004;
Mijimolle et al., 2005). This could be due to the specific
types of proteins that are prenylated in plants, because
most plant candidate protein prenyl transferase protein substrates differ from those identified in yeast
(Saccharomyces cerevisiae) and animals (Galichet and
Gruissem, 2003).
Among plant candidate PFT protein substrates,
nucleosome assembly protein 1 (NAP1) is conserved
among 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 (Ishimi
et 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; Shen
et al., 2001; Dong et al., 2003). Most NAP1 proteins
contain a typical CaaX box recognition motif for PFT,
and the human NAP1-like1 was shown to be in vivo
farnesylated in COS-1 cells (Kho et al., 2004). NAP1
was first identified from HeLa cells by its ability to
facilitate in vitro assembly of nucleosomes under physiological conditions. The current model suggests that
NAP1 is part of a multifactorial chromatin assembly
machinery, which mediates the ATP-facilitated assembly 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
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Differential NAP1 Farnesylation during Leaf Development
addition to their proposed histone chaperone activity,
NAP1 proteins may be involved in transcriptional regulation through chromatin remodeling (Kawase et al.,
1996; Ito et al., 2000; Shikama et al., 2000; Asahara
et al., 2002; Levchenko and Jackson, 2004; Rehtanz et al.,
2004). Recent genetic studies also revealed functions of
NAP1 in the control of mitosis and during development. Xenopus and yeast NAP1 interact specifically
with B-type cyclin (Clb). Deletion of the NAP1 gene in
a yeast strain that is dependent upon Clb2 function
results in a prolonged delay of mitosis with normal levels of Clb2/p34CDC28-associated kinase activity,
and yeast cells are unable to induce events required
for assembly or proper function of the mitotic spindle.
Yeast NAP1 was also shown to regulate the cell cycle
in combination with the Gin4 kinase, NBP1, and SDA1
(Kellogg et al., 1995; Kellogg and Murray, 1995; Altman
and Kellogg, 1997; Shimizu et al., 2000; Zimmerman
and Kellogg, 2001). In addition, several mammalian
NAP1 homologs appear to regulate cell proliferation
and differentiation (von Lindern et al., 1992; Simon
et al., 1994; Abu-Daya et al., 2005). A role of NAP1
during development was revealed through the construction of knockout mutants in mice and Drosophila.
The inactivation of Drosophila NAP1 is embryo lethal,
and a null mutation of the brain-specific mouse NAP1l2
gene results in embryonic lethality beginning at midgestation. Surviving mice mutant embryos showed extensive surface ectoderm defects, open neural tubes,
and exposed brain, suggesting a tissue-specific role for
NAP1L2 in the regulation of neuron proliferation
(Rogner et al., 2000; Lankenau et al., 2003). But despite
this extensive information, the biochemical and molecular functions of NAP1 proteins and the role of the
prenyl modification is still poorly understood.
Arabidopsis has four NAP1-related genes whose
function is currently not known. We investigated
Arabidopsis nucleosome assembly protein 1 (AtNAP1;1),
which has a predicted CaaX motif, to understand the
function of the protein and the potential role of its
farnesylation during Arabidopsis development. Characterization of gain- and loss-of-function mutants
demonstrated that AtNAP1;1 contributes to the regulation of cell proliferation and cell expansion. The
developmental function of AtNAP1;1 appears to be
regulated by the differential farnesylation of the protein during specific stages of leaf development.
RESULTS
AtNAP1;1 Is a Farnesylated Protein
The presence of the CKQQ motif at the C-terminal
end of AtNAP1;1 suggested that the protein is a substrate of PFT. To test if AtNAP1;1 can be prenylated
by PFT, we incubated the purified protein with recombinant Arabidopsis PFT and [3H]FPP. AtNAP1;1
was 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 enzyme
is somewhat promiscuous for GGPP (Trueblood et al.,
1993; Lane and Beese, 2006). Mutation of the conserved Cys farnesyl acceptor in the CKQQ motif to Ser
(AtNAP1;1C369S) confirmed that farnesylation required
a functional farnesylation motif. Among the three other
Arabidopsis NAP1 proteins, AtNAP1;2 and AtNAP1;3
have a similar CKQQ motif and are likely prenylated
as well. Similarly, NAP1 is encoded by small gene
families in tobacco and rice (Dong et al., 2003). The two
OsNAP1 and three of the NtNAP1 proteins have CaaX
boxes that can be prenylated in vitro by Arabidopsis
PFT (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 were
tested as a source for the enzyme (Fig. 1B). AtNAP1;1
but not AtNAP1;1C369S was labeled in the presence
of protein extracts from wild-type flowers, confirming
that the protein extract had PFT activity and that
AtNAP1;1 could be correctly farnesylated at the Cys
acceptor in the CKQQ prenylation motif. The protein
extract from era1-1 flowers was unable to farnesylate
AtNAP1;1, confirming the lack of PFT activity in the
mutant and establishing that AtNAP1;1 is a substrate
of PFT.
To confirm that AtNAP1;1 is also farnesylated in
vivo, we expressed green fluorescent protein (GFP)AtNAP1;1 and GFP-AtNAP1;1C369S in tobacco BY-2
cells under control of the cauliflower mosaic virus
(CaMV) 35S promoter. Soluble proteins from cells labeled with [3H]mevalonic acid were extracted and
separated on SDS-polyacrylamide gels, which were
then used either for immunoblot analysis with a polyclonal NAP1 antibody or for fluorography to detect
labeled GFP-AtNAP1;1 (Fig. 1C). GFP-AtNAP1;1 and
GFP-AtNAP1;1C369S were expressed to a similar level
in BY-2 cells. A labeled protein corresponding to the
size of GFP-AtNAP1;1 was detected only in extracts
from cells expressing GFP-AtNAP1;1 but not in cells
expressing GFP-AtNAP1;1C369S or in control BY-2 cells.
Together, these results establish that AtNAP1;1 is
efficiently farnesylated in vivo as well.
AtNAP1;1 Subcellular Localization during the Cell
Cycle Is Regulated But Does Not Depend on
Farnesylation in Heterologous Cell Systems
Most farnesylated proteins in yeast and animal cells
are targeted to the plasma membrane (Sinensky, 2000),
although in plants, farnesylated APETALA1 has also been
found in the nucleus (Yalovsky et al., 2000b). To investigate the subcellular localization of AtNAP1;1, GFPAtNAP1;1 and GFP-AtNAP1;1C369S were transiently
expressed in onion (Allium cepa) epidermal cells. GFP
alone was distributed uniformly in the nucleus and
the cytoplasm, whereas GFP-AtNAP1;1 and GFPAtNAP1;1C369S were restricted to the cytoplasm (Fig.
2A). As controls, we also expressed GFP-CaM53 and
GFP-CaM53mS. Their previously reported respective localization to the membrane and the nucleus
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Galichet and Gruissem
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 plant
protein prenyltransferases. Symbols 1 and 2 indicate
the presence or absence of purified AtNAP1;1, purified prenyltransferase, FPP, or GGPP. After electrophoresis and fluorography, exposure was carried out
for 5 d. B, Wild-type and era1-1 Arabidopsis flower
soluble extracts were used as prenyltransferase
sources. Prenylation reactions were carried out with
50 mg of crude extract, and 1 and 2 symbols indicate the presence or absence of AtNAP1;1,
AtNAP1;1C369S, and FPP. Exposure was carried out
for 15 d. C, In vivo prenylation assay. Fluorography
and immunoblots of protein extracts from tobacco BY-2 cells expressing GFP-AtNAP1;1 or GFPAtNAP1;1C369S fusion proteins and labeled with
3
H-mevelonic acid. Protein extracts were separated
by SDS-PAGE in 10% gel, followed by western
blotting. Fluorography was carried out for 3 weeks.
(Rodriguez-Concepcion et al., 1999) was also observed
in onion epidermal cells, suggesting that expression of
PFT substrate proteins using the 35S promoter did not
exceed the prenylation capacity of the cell that could
result in a nonspecific localization of the protein.
To obtain further insights into the potential role of
farnesylation for AtNAP1;1 subcellular localization,
GFP-AtNAP1;1 and GFP-AtNAP1;1C369S were stably
expressed in transgenic tobacco BY-2 cells (Fig. 2B).
GFP-AtNAP1;1 was localized in both the cytoplasm
and the nucleoplasm of interphase cells. During mitosis, GFP-AtNAP1;1 was colocalized with the phragmoplast in telophase. Localization of GFP-AtNAP1;1C369S
was similar (data not shown). Together, we conclude
that the farnesylation status of AtNAP1;1 has no
immediate role in the subcellular localization of the
protein during the cell cycle.
Farnesylation Is Necessary for AtNAP1;1 Function
in Yeast
We examined the physiological role of AtNAP1;1
farnesylation by functional complementation assays
using the yeast nap1 mutant strain DK213. This mutant
has a reduced growth rate at 37C 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 analyzed
for their growth and their cell phenotypes at 37C
(Fig. 3, A and B). ScNAP1 and AtNAP1;1, but not
AtNAP1;1C369S, rescued the elongated bud phenotype
and the temperature sensitivity, demonstrating that
AtNAP1;1 could complement the yeast nap1 mutation.
Western-blot analysis of yeast protein lysates with an
anti-NAP1 antibody revealed that the three NAP1 proteins were expressed at similar levels (Fig. 3C). Therefore, the farnesyl-Cys acceptor of AtNAP1;1 is required
for its function during cell cycle progression in yeast.
Cell Proliferation Is Reduced in Atnap1;1 Leaves
To gain further insight into AtNAP1;1 function, we
identified two independent Atnap1;1 mutant alleles in
the SALK T-DNA insertion collection (SALK_013610
and SALK_095311; Alonso et al., 2003). These alleles
have a T-DNA insertion in the seventh or the 10th exon
of AtNAP1;1 that we named Atnap1;1-1 and Atnap1;1-2
(Fig. 4A). Western-blot analysis of extracts from the
two Atnap1;1 mutant alleles and control plants together
with extracts from Atnap1;2 and Atnap1;3 mutants
(T-DNA insertion lines SALK_002892 [T-DNA insertion in the second intron] and SAIL_373_H11 [T-DNA
insertion in the 10th exon], respectively; Sessions
et al., 2002; Alonso et al., 2003) established the lack of
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Differential NAP1 Farnesylation during Leaf Development
Figure 2. Farnesylation does not affect subcellular localization of AtNAP1;1. A, GFP-AtNAP1;1,
GFP-AtNAP1;1C369S, GFP-CaM53, GFP-CaM53mS,
and GFP proteins were detected by green fluorescence using confocal laser scanning microscopy
in epidermal onion cells 38 h after bombardment.
Bars correspond to 50 mm. B, Localization of
GFP-AtNAP1;1 and GFP- AtNAP1;1C369S fusion
proteins in tobacco BY-2 tobacco cells. Transgenic suspension of BY-2 tobacco cells were
established and used for localization.
AtNAP1;1 in Atnap1;1-1 and Atnap1;1-2 (Fig. 4B). This
was consistent with quantitative reverse transcription
(qRT)-PCR data that showed no accumulation of the
different NAP1 mRNAs in the respective mutants (data
not shown). The results also confirmed that the major protein detected by the antibody corresponds to
AtNAP1;1, but that the antibody cross reacted with
AtNAP1;2 and AtNAP1;3 as well. The observed differences in band intensity could result from a difference in
expression of the AtNAP1 proteins or from the higher
specificity of the antibody for AtNAP1;1.
Homozygous Atnap1;1-1 and Atnap1;1-2 plants were
enlarged compared to wild type early in development
but then became reduced in size (Fig. 4, C and D). In
contrast, Atnap1;2 and Atnap1;3 plants had no obvious
phenotype (data not shown). Interestingly, by day 7,
Atnap1;1-1 and Atnap1;1-2 leaves had more than twice
as many cells compared to wild type (Fig. 4E). Similarly,
9-d-old Atnap1;1-1 and Atnap1;1-2 cotyledons were enlarged with more cells (data not shown). Between day 7
and 15, cell proliferation slowed in mutant leaves, while
cell number continued to increase in wild-type leaves,
therefore resulting in approximately 17% fewer cells in
mature mutant leaves (Fig. 4E). In contrast, cell size was
not significantly different in the two mutant lines compared to wild type during leaf development (data not
shown). Together, these results suggest that loss of
AtNAP1;1 function alters the normal cell proliferation
during leaf development.
Gain of Function of AtNAP1;1 Alters Leaf Growth
and Size
To examine the effects of AtNAP1;1 gain of function
and the potential role of AtNAP1;1 farnesylation in
vivo, we generated Arabidopsis transgenic plants in
which the AtNAP1;1 or AtNAP1;1C369S cDNA were expressed under the control of the constitutive CaMV
35S promoter. Nine AtNAP1;1 and 11 AtNAP1;1C369S T1
hygromycin-resistant plants with single T-DNA insertions were selected for further analysis. Six independent T3 homozygous AtNAP1;1 and seven AtNAP1;1C369S
lines were analyzed by western blot using the anti-NAP1
antibody. Except one AtNAP1;1 line, all transgenic lines
highly expressed the transgene (Fig. 5A) without any
detectable change in the RNA levels expressed from
the endogenous members of the AtNAP1 gene family
(Fig. 5B). Lines AtNAP1;1 3.5.3 (AtNAP1;1-OE) and
AtNAP1;1C369S 2.2.5 (AtNAP1;1C369S-OE) had similar
levels of transgene expression and were therefore selected for a full analysis (Fig. 5A, lanes 7 and 10). There
was no difference in germination and in the timing
of leaf primordia initiation between AtNAP1;1-OE,
AtNAP1;1C369S-OE, and wild-type lines. Plants expressing
AtNAP1;1 developed smaller leaves and cotyledons,
whereas these organs were enlarged in plants expressing
AtNAP1;1C369S (Fig. 5C). In contrast, there was no difference in petal size, indicating that AtNAP1;1 overexpression preferentially affected the development of
vegetative organs. Because the difference in leaf size was
the most striking phenotype, we analyzed leaf development 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 in
cell size or number or both. To address these possibilities, we performed a kinematic analysis of the first
leaf using the method reported by De Veylder and
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Galichet and Gruissem
Figure 3. AtNAP1;1 complements the yeast nap1
mutant. DK213 yeast nap1 cells were transformed
with pJR1133 vector alone (1), ScNAP1 (:),
AtNAP1-1 (j), or AtNAP1;1C369S (d), and cells were
arrested in G2/M and then released in fresh media at
the permissive temperature (30C) or at the restrictive
temperature (37C). A, Cell proliferation was monitored at 37C by measuring the OD600 nm of the
culture over a period of 24 h. B, Nomarski images
after 16 h at 30C. C, Immunoblot analysis of wildtype DK213 yeast cells (1) or DK213 cells expressing
ScNAP1 (2), AtNAP1;1 (3), or AtNAP1;1C369S proteins. Protein extracts were separated by SDS-PAGE
in 10% gel followed by western blotting.
colleagues (De Veylder et al., 2001). Between inception
and maturity, Arabidopsis leaf development can be
categorized into three stages: proliferation (until day
12 after sowing), expansion (days 12–19), and maturity
(after day 19; Beemster et al., 2005). The first leaf of
AtNAP1;1-OE, AtNAP1;1C369S-OE, and control lines
was harvested between 7 and 27 d after sowing and
average leaf area and adaxial palisade cell size were
determined (Fig. 6, A and B). From these data, the
average palisade cell number was calculated (Fig. 6C).
We found that 9 d after sowing the AtNAP1;1-OE
plants, first leaf size was reduced 65%, which was
mainly the result of decreased cell size (63%). In 15-dold AtNAP1;1-OE plants, the first leaf had approximately 20% more cells than the wild-type leaf. These
cells were 50% reduced in size, which reduced the leaf
blade area by approximately 40%. This trend continued in AtNAP1;1-OE mature first leaf, resulting in a
35% decrease of leaf size after 26 d. In contrast, the 9-dold AtNAP1;1C369S-OE first leaf had an increased cell
size and number (55% and 16%, respectively), which
resulted in an enlarged leaf area of approximately
78% relative to wild-type control plants. After 15 d,
the AtNAP1;1C369S-OE first leaf blade size was still
36% larger than control leaves, but at 26 d the
AtNAP1;1C369S-OE first leaf was comparable in size to
wild-type first leaves. We then calculated the relative
cell division rate, and the results in Figure 6D show
that the cell division rate in AtNAP1;1C369S-OE and
wild-type plants increased until day 9 and then rapidly declined during the following 5 d. In contrast, the
rate of cell division was increased in AtNAP1;1-OE.
Based on these results, we calculated the average cell
cycle duration as the inverse of the relative cell division rate (Beemster et al., 2005). On day 9, the average
cell cycle duration in the first leaf was 32.7 h in wild
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Differential NAP1 Farnesylation during Leaf Development
Figure 4. Arabidopsis Atnap1;1 mutant alleles are affected in cell proliferation during leaf development. A, Atnap1;1 gene
structure and location of T-DNA insertions in the Atnap1;1-1 and Atnap1;1-2 mutant alleles. B, Western-blot analysis of AtNAP1
proteins 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 with
polyclonal 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 40
palisade mesophyll cells were analyzed in duplicate.
type, 33.8 h in AtNAP1;1C369S-OE, and 19.2 h in
AtNAP1;1-OE plants.
The kinematic analysis of leaf development revealed
that ectopic expression of wild-type and mutant NAP1
resulted in alterations of cell size and number in
the first leaf. Interestingly, ectopic expression of
AtNAP1;1C369S primarily increased cell size, but the
comparable size of mature AtNAP1;1C369S-OE and
wild-type first leaves suggested that a compensatory
mechanism corrects for the increased cell size in
AtNAP1;1C369S-OE. Such compensation was not apparent in AtNAP1;1-OE plants, suggesting that the farnesylation of AtNAP1;1 has a profound effect on the
function of the protein in this mechanism.
Timing of Cell Differentiation Is Altered by Ectopic
AtNAP1;1C369S Expression
Cell proliferation, expansion, and differentiation are
closely linked during leaf development (Beemster
et al., 2005). Since the ectopic expression of AtNAP1;1
or AtNAP1;1C369S affected cell division rate and cell
expansion, we investigated the effect of the wild-type
and mutant proteins on cell differentiation. We first
analyzed DNA endoreplication as an early marker of
leaf cell differentiation (Melaragno et al., 1993) by measuring DNA content of nuclei from primary leaves
of wild-type, AtNAP1;1-OE, and AtNAP1;1C369S-OE
plants beginning at day 9 after sowing until day 25.
In wild-type leaves, all leaf nuclei had a DNA content
of 2C and 4C at day 9 (Fig. 6E, left), but beginning
at day 11, the population of cells with 8C nuclei
increased, reaching a maximum of 34% of the cells
on day 25. Cells with 16C nuclei were first detected at
day 20, and this population of cells increased to 6% of
total leaf cells after 25 d. Together, the results confirm a
correlation between DNA endoreplication and cell
expansion (Beemster et al., 2005). Leaves of plants
expressing AtNAP1;1 had an increased population of
cells with 2C nuclei after 9 d (78% compared to 64% in
wild type), which suggests that the G2 phase in
AtNAP1;1-OE plants may be shortened relative to G1
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Galichet and Gruissem
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-five
micrograms of total protein was loaded per well and membrane was probed with polyclonal AtNAP1 antibody. B, Expression
analysis of AtNAP1;1 genes was monitored in wild-type (lane 1), AtNAP1;1-OE (lane 2), and AtNAP1;1C369S-OE (lane 3) 20-d-old
plants using semiquantitative RT-mediated PCR. C, Nine-day-old sterile-grown AtNAP1;1-OE and AtNAP1;1C369S-OE plants
show altered plant organ size.
during the proliferation phase (Fig. 6E, middle). In
addition, cells with 8C and 16C nuclei appeared later
in AtNAP1;1-OE plants, suggesting that DNA endoreplication was delayed. The ploidy of nuclei in
AtNAP1;1C369S-OE leaves at day 9 was similar to wildtype leaves (Fig. 6E, right), indicating that the mutant protein had little effect during the proliferation
phase. In contrast, after the proliferation phase, cells
with 8C and 16 C nuclei were detectable earlier in
AtNAP1;1C369S-OE plants, and their population was significantly increased at day 25, suggesting that the
increased size of AtNAP1;1C369S-OE leaf cells (Fig. 6B)
was correlated with earlier and increased DNA endoreplication.
We next examined the effects of ectopic AtNAP1;1
and AtNAP1;1C369S expression on leaf structure and
cell differentiation. Dicotyledonous leaves have a distinct tissue organization with specific cell types
between the adaxial and abaxial leaf surfaces. The
palisade layer below the adaxial epidermis consists of
tightly packed elongated cells arranged with their long
axes perpendicular to the leaf surface. The spongy
mesophyll cells between the palisade cell layer and the
abaxial epidermis are smaller and more rounded
(Donnelly et al., 1999). Primary 9-d-old AtNAP1;1-OE
leaves did not show the typical dorsoventral organization of the leaf parenchyma into palisade and
spongy mesophyll observed in wild-type leaves
(Fig. 6F, top) but instead had a larger number of
smaller spherical cells (Fig. 6F, center). Palisade and
spongy mesophyll layers became more distinct in
older AtNAP1;1-OE first leaves, but they remained
smaller than cells in wild-type leaves (data not
shown). Microscopic analysis of the AtNAP1;1C369SOE first leaf at day 9 revealed that all tissue layers
had significantly larger and morphologically more
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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 leaf
pair of wild-type (¤), AtNAP1;1-OE (n), and AtNAP1;1C369S-OE (h) plants. Twenty individual leaves and 40 palisade mesophyll
cells were analyzed in duplicate. A, Average leaf blade area; B, average cell area; C, average number of cells; and D, division
rate. 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 are
50 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.
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differentiated cells than the wild-type first leaf (Fig. 6F,
bottom). Together, the results indicate that ectopic
expression of AtNAP1;1 or AtNAP1;1C369S had opposing effects on cell differentiation and elongation,
which correlated with the changes in nuclear ploidy.
levels in leaves expressing AtNAP1;1 despite their
smaller cell size, suggesting that EXP5 function is not
entirely restricted to cell elongation.
Ectopic Expression of AtNAP1;1 Affects the Expression
of Cell Division and Cell Expansion-Related Genes
Given the effects of ectopic AtNAP1;1 or AtNAP1;1C369S
expression at the cellular and molecular levels, we
next asked whether posttranslational farnesylation of
AtNAP1;1 was altered during leaf development, which
in turn could modulate the function of AtNAP1;1 to affect
cell proliferation, expansion, and differentiation. For
this work, we also took advantage of the era1-1 mutant,
which lacks PFT activity (Yalovsky et al., 2000a). Immunoblot analysis of protein extracts isolated from wildtype and era1-1 Arabidopsis primary leaves during
development using the anti-NAP1;1 antibody revealed
three bands, with AtNAP1;1 being the major band
(Figs. 7A and 4B). In the wild-type first leaf, NAP1 proteins were highly expressed during the cell proliferation phase but declined later in development (Fig. 7A,
lanes 1–4). Attachment of farnesyl increases protein
mobility in SDS-polyacrylamide gels (Zhu et al., 1993).
Interestingly, AtNAP1;1 migrated faster at day 9 as
To begin to understand how ectopic AtNAP1;1 expression may modulate cell proliferation, expansion,
and differentiation at the molecular level, we analyzed
the accumulation of mRNAs for cell cycle and cell
expansion-related genes during leaf development.
qRT-PCR revealed that the CycB1;1 mRNA level was
increased in AtNAP1;1-OE leaves at day 9 but was not
significantly changed in AtNAP1;1C369S-OE leaves compared to wild type (Fig. 6G, top). No difference was
found in the expression of CycD1;1 and CycD1;3 genes
(data not shown). EXP5 represents a member of the
gene family for expansin proteins that induce extension
of the plant cell wall. The mRNA of this gene was
strongly increased in leaves expressing AtNAP1;1C369S,
consistent with the increased cell size (Fig. 6G, bottom).
Interestingly, EXP5 mRNA also accumulated to higher
Farnesylation of AtNAP1;1 Is Specific to the Cell
Proliferation Phase during Leaf Development
Figure 7. Stage-specific farnesylation and subcellular localization of AtNAP1;1 directs its functions. A,
Western-blot analysis of AtNAP1 proteins during
wild-type and era1-1 primary leaf development.
Samples were collected after 9, 14, 19, and 25 d
for wild type (lanes 1–4) and 11, 16, 21, and 27 d for
era1-1 (lanes 5–8). The difference of harvesting time
originates from a 2-d delay in era1-1 germination.
Twenty-five micrograms of total protein was loaded
per well and separated by SDS-PAGE in 7.5% gel
followed by western blotting. Membrane was probed
with polyclonal AtNAP1 antibody. B, In vivo prenylation 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 leaves
were labeled with [3H]mevalonic acid, and protein
extracts were subjected to immunoblot analysis and
fluorography. Fluorography was carried out for 3
weeks. C, Subcellular fractionation of 9- and 15-dold wild-type, AtNAP1;1-OE, and AtNAP1;1C369S-OE
first leaf protein extracts. Twenty micrograms of total
protein (T) and equivalent volumes of cytoplasmic (C)
and nuclear (N) fractions were loaded. D, Subcellular
fractionation of 9- and 15-d-old wild-type and 11and 17-d-old era1-1 primary leaf protein extracts.
The experiment was carried out as described for C. As
control, 50 mg of total protein and equivalent volumes of the two other fractions were used for detection using the antibody raised against the MSI nuclear
protein.
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Differential NAP1 Farnesylation during Leaf Development
compared to days 15 and 21 (Fig. 7A, lanes 2 and 3) or to
AtNAP1;1 protein in era1-1 protein extract (Fig. 7A,
lanes 5–8). The shift in mobility was reproducible and
could indicate that AtNAP1;1 expressed later in development does not become farnesylated or that farnesyl
is removed from the existing protein. To investigate
this possibility, we incubated primary leaves isolated
from 9- and 15-d-old AtNAP1;1-OE and AtNAP1;1C369SOE plants with [3H]mevalonic acid and analyzed the
labeled proteins. Figure 7B shows that AtNAP1;1, but
not AtNAP1;1C369S, was actively farnesylated in 9-d-old
primary leaves. AtNAP1;1 farnesylation was not detectable in 15-d-old leaves, although the protein accumulated to similar levels at both developmental stages.
No labeled protein was detected in wild-type leaves at
either developmental stage. This was not unexpected
considering the high levels of AtNAP1;1 expression in
the transgenic line that was needed to detect labeled
protein and the low efficiency of farnesylation during
the short labeling time.
Because era1-1 lacks PFT activity, NAP1;1 is not
farnesylated in this mutant (Fig. 1, A and B) or modified by PGGT-I, which is somewhat promiscuous for
the CaaX motif (Trueblood et al., 1993; Lane and Beese,
2006). Interestingly, AtNAP1 proteins accumulated to
significantly higher levels in the era1-1 first leaf early in
development (Fig. 7A, lanes 5 and 6) and remained at
higher levels in older leaves (Fig. 7A, lanes 7 and 8) as
wild type at comparable stages. This result suggests
that the lack of AtNAP1 farnesylation in era1-1 may
affect the regulation of NAP1 gene expression. Alternatively, nonfarnesylated NAP1;1 may be more stable
in era1-1.
Different Subcellular Localization of AtNAP1;1 Is
Correlated with the Different Phases of
Leaf Development
Subcellular analysis of AtNAP1;1 expressed in BY-2
tobacco cells had revealed that the protein was located
in the cytoplasm or the nucleus depending on stage of
the cell cycle, but this was not depending on farnesylation (Fig. 2). Considering the opposing effects of
ectopically expressed AtNAP1;1 and AtNAP1;1C369S on
cell proliferation and expansion (Fig. 6), it was still
possible that the farnesylation status of NAP1;1 during
leaf development (Fig. 7B) was important to direct the
subcellular localization of the protein. We therefore
analyzed the subcellular localization of AtNAP1 proteins during development of the first leaf. In leaves
from control and AtNAP1;1-OE lines, the AtNAP1
proteins detected by the antibody were found exclusively in the nuclear fraction on day 9, whereas they
were equally distributed between the nuclear and
cytoplasmic fractions on day 15 (Fig. 7C). In contrast,
AtNAP1 proteins were equally distributed between
the nucleus and the cytoplasm in leaves of
AtNAP1;1C369S expressing lines at both 9 and 15 d. A
parallel analysis of the first leaf of era1-1 showed that
AtNAP1 proteins were found both in the nucleus and
cytoplasm at day 11, whereas they were exclusively
present in the cytoplasm after 17 d (Fig. 7D). Together,
we conclude that farnesylation of AtNAP1;1 directs
the protein to the nucleus early in leaf development,
perhaps in concert with other localization mechanisms. The results also suggest that farnesylation and
nuclear localization of AtNAP1;1 facilitate cell proliferation during early leaf development.
DISCUSSION
The analysis of mutants has provided significant
new insights into leaf development during the last
several years. Typically, leaf growth is tightly regulated by the control of cell number, cell size, and
differentiation. The genetic and biochemical networks
that integrate these cellular processes, however, are
still largely unknown. Our analysis established that
AtNAP1;1 function is required for correct cell proliferation control during Arabidopsis leaf development
and that this function is dependent on the temporal
farnesylation of the protein.
Organ development is tightly coordinated at the
cellular level by cell division, expansion, and differentiation such that each organ reaches its appropriate
size relative to the size of the organism. In determinate
plant organs, particularly leaves, final organ cell number is regulated by the initial number of cells recruited
into the primodium, the timing of cell division, and the
rate of proliferation. After mitotic activity ceases, cell
differentiation and expansion establish the regular pattern of tissue layers in the leaf blade (Donnelly et al.,
1999). In Arabidopsis, loss of AINTEGUMENTA function or ectopic expression of KRP2, an inhibitor of cell
division, reduces leaf cell number, which is largely
compensated by increase in cell size (Mizukami and
Fischer, 2000; De Veylder et al., 2001). These observations suggest that, at the organ level, both cell division
and expansion are coordinated. They follow patterns
dependent on the anatomical and developmental context to generate leaves of normal size and shape. The
balanced regulation of cell division and expansion
may also ensure the completion of a basic morphogenesis program in case either one of the processes is
impaired. Several mutations have been reported, however, that affect the balance between cell number and
size, resulting in modifications of leaf growth and
shape. The Arabidopsis angustifolia and rotundifolia
mutants have reduced polar cell expansion but no
alteration in cell number, resulting in the production of
long, narrow leaves and shorter, wider leaves, respectively (Kim et al., 1998, 2002). In contrast, the Arabidopsis struwwelpeter mutant has smaller leaves with
fewer cells but maintains cell size. Overexpression of
both E2Fa and DPa, two transcription factors that are
required for cell cycle regulation, strongly induces cell
proliferation in Arabidopsis but also severely reduces
cell expansion and plant growth (Autran et al., 2002;
De Veylder et al., 2002). Together, cell division or cell
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Galichet and Gruissem
expansion can independently influence leaf morphogenesis by affecting leaves to maintain compensatory
mechanisms during growth.
AtNAP1;1 Is a Possible Regulatory Link That Controls
Cell Proliferation during Leaf Development
The reduced leaf growth in Atnap1;1 is the consequence of a decreased rate of cell division. In contrast,
ectopic AtNAP1;1 expression in Arabidopsis increased
cell proliferation but did not affect the time window of
cell cycle activity during leaf development. Because
the effect of either loss or gain of AtNAP1;1 function
appears to be restricted primarily to the phase of cell
proliferation, we suggest that AtNAP1;1 functions as a
stage-specific positive regulator of cell proliferation.
The high AtNAP1 protein level that we detected in
leaves during the cell proliferation phase is also consistent with a role of AtNAP1 in the control of cell division. In humans, HsNAP1L1 gene is expressed in all
tissues but its expression is increased in proliferating
cells. Moreover, the HsNAP1L1 protein level increases
in cultured T-lymphocytes induced to proliferate (Simon
et al., 1994). Similarly, mouse NAP1L2 is mainly expressed in the nervous system and is necessary for
normal proliferation of neuronal precursor cells (Rogner
et al., 2000). Interestingly, ectopic AtNAP1;1C369S expression did not have a significant affect on cell proliferation, suggesting that prenylation of NAP1;1 is required
for its function during the cell proliferation phase of
leaf development.
How does AtNAP1;1 influence cell proliferation
rate? Our analysis has revealed that AtNAP1;1 overexpression increases the expression of CYCB1;1, which
functions in late G2 and M phases (Donnelly et al.,
1999). Most of the targets regulated by CYCB1;1-CDK
complexes are currently unknown, but unscheduled
activation of CYCB1;1 expression by AtNAP1;1 may
result in a shortened G2 phase or interfere with developmentally regulated exit from the cell cycle.
AtNAP1;1 Promotes Cell Proliferation and Connects
It to Cell Expansion
Following the cell division phase, cell enlargement
and cellular differentiation contribute to the final size
and shape of leaves. It has been long recognized that a
correlation exists between DNA endoreplication and
leaf cell expansion, suggesting that DNA ploidy level
may determine cell size (Melaragno et al., 1993; Folkers
et al., 1997). Plants that overexpress KRP2, which
inhibits CDK activity, however, have leaves with significantly enlarged cells but reduced DNA endoreplication, suggesting a more complex link between these
two processes (De Veylder et al., 2001). We have found
that ectopic expression of AtNAP1;1C369S increased
cell size and promoted cellular differentiation already
early during the proliferation phase of leaf development. This activity of AtNAP1;1C369S was uncoupled
from the promotion of DNA endoreplication, which oc-
curred late during the proliferation phase. The results
therefore suggest that increased levels of nonfarnesylated AtNAP1;1 are sufficient to promote unscheduled cell expansion. At present, we can only speculate
about the mechanism, but one possibility is that
the lack of the farnesyl group alters the subcellular
distribution of AtNAP1;1C369S and therefore allows
interactions between AtNAP1;1C369S and other proteins that would promote cell expansion only later in
leaf development. This view is supported by the
observation that the level of nonfarnesylated endogenous AtNAP1;1 is increased later in leaf development.
The possibility that AtNAP1;1 can both negatively
or positively influence cell growth and expansion
dependent on its farnesylation status is also consistent
with the result that ectopic expression of AtNAP1;1
reduced normal cell growth during the cell proliferation phase of leaf development. It must be noted,
however, that increased cell division can also inhibit
cell growth (Fleming, 2002). The reduced cell size,
together with delayed cell differentiation and endoreplication that we observed in AtNAP1;1-OE plants
could therefore be a consequence of the initially increased cell proliferation triggered by AtNAP1;1 early
during leaf growth, rather than a direct effect of the
protein on cell growth. This possibility would also be
consistent with the observation that following the cell
proliferation phase in AtNAP1;1-OE leaves, cell size
remains smaller than in wild-type leaves, but the
relative increase in cell size between 11 and 21 d is
significantly higher in AtNAP1;1-OE leaves than in
wild-type leaves. In this case, the increased levels of
AtNAP1;1 later in leaf development could partially
compensate the reduced growth during the proliferation phase. This interpretation is reasonable, because
loss of AtNAP1;1 function reduces cell proliferation,
resulting in smaller leaves with fewer cells, but cell
size is not affected. It is unlikely that overexpression
of AtNAP1;1 titrates out other prenlylation substrates
and PFT. Previous experiments showed that overexpression of CaM53, a prenylated Calmodulin-related
protein, does not exceed the prenylation capacity of
the cells (Rodriguez-Concepcion et al., 1999). This view
is also supported by the localization data shown
in Figure 2. Our results therefore suggest that the
observed effects in AtNAP1;1-OE plants are only related to the overexpression of the protein. Together,
AtNAP1;1 promotes cell proliferation and participates
in connecting cell proliferation to cell expansion to
achieve the correct balance between cell number and
cell size that determines normal leaf blade size.
Farnesylation Regulates AtNAP1;1 Subcellular
Localization and Function
In plants, protein farnesylation has been shown to
affect protein function and subcellular localization
(Zhu et al., 1993; Yalovsky et al., 2000b; Suzuki et al.,
2002). Most of AtNAP1;1 is farnesylated during
the cell proliferation phase of leaf development, but
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Differential NAP1 Farnesylation during Leaf Development
AtNAP1;1 does not appear to be farnesylated during
the subsequent leaf expansion phase. Our complementation studies of the yeast nap1 mutant confirmed
that AtNAP1;1 farnesylation is necessary to complement the cell cycle phenotype of the mutant. It was
also reported that the carboxy-terminal region of
ScNAP1 is important to allow mitotic progression of
the nap1 yeast mutant in complementation experiments
(Miyaji-Yamaguchi et al., 2003). Together, these results
suggest that the farnesylation status of NAP1 is of
functional relevance. During the cell proliferation
phase of leaf development, farnesylated AtNAP1;1
localizes to the nucleus and promotes cell division.
Later in leaf development when cells are expanding,
most of the NAP1;1 appears to be nonfarnesylated
and accumulates both in the nucleus and the cytoplasm, and absence of AtNAP1;1 farnesylation results
in cellular growth. Subcellular localization analysis
of the rice and tobacco NAP1 proteins also revealed
a similar partitioning of the proteins between the
nucleus and the cytoplasm (Dong et al., 2003), although this study did not provide information on the
developmental context or farnesylation status of the
proteins.
If the farnesylation-dependent localization of
AtNAP1;1 is altered during leaf development, as was
the case during the cell proliferation phase in
AtNAP1;1C369S-OE plants, this results in premature
cell expansion. It is possible that unscheduled accumulation of AtNAP1;1 may allow the protein to engage interactions with other proteins that control cell
growth and expansion in the context of the cell cycle
but independent of AtNAP1;1. These interactions
could occur in the nucleus or the cytoplasm, but it is
currently unknown if they require NtNAP1;1 to be
farnesylated. Also, these interactions do not easily
explain how the different subcellular localizations of
AtNAP1;1 could exert effects on cell proliferation or
cell growth and expansion. Additional experiments
will be necessary to identify proteins that specifically
interact with NAP1;1 during different leaf development phases.
It is reasonable to expect that the farnesylation status
of AtNAP1;1 may allow the protein to engage in
different protein complexes, which exert specific functions during leaf development. In yeast, NAP1 was
shown to specifically interact with a set of proteins,
including histones, Clb2, Gin4, Nbp1, and p300/
CREB-binding proteins (Ishimi et al., 1987; Kellogg
et al., 1995; Altman and Kellogg, 1997; Shikama et al.,
2000; Shimizu et al., 2000). These proteins are involved
in different regulatory processes, including cell cycle
control, chromatin modifications, or transcription control. There is also considerable evidence in plants for
regulatory links between cell proliferation, cell expansion, and environmental and physiological signals
(Pyke and Lopez-Juez, 1999; Meijer and Murray, 2001;
Beemster et al., 2003). Earlier studies revealed that
protein prenylation is required during plant development and identified functional associations between
protein prenylation and specific biological processes
or 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 therefore
provide an interesting but currently little-explored
mechanism to link cell cycle and cell growth control
functions to the metabolic status of the cell. For example, during the cell proliferation phase early in leaf
development, the organ functions as a physiological
sink, whereas the cell expansion phase later in leaf
development coincides with the autotrophic functions
of the organ (Paul and Foyer, 2001; Jeong et al., 2004).
An important but still unresolved aspect is the mechanism by which cells regulate the dynamic farnesylation of AtNAP1;1. It has been reported that PFT
activity is increased during cell cycle progression in
tobacco BY-2 cells (Morehead et al., 1995). It is therefore possible that PFT activity itself is regulated during
leaf development, which could result in differential
farnesylation of AtNAP1;1. Alternatively, the pool of
FPP available for protein prenylation may be tightly
regulated during development. Finally, developmentally regulated farnesylation of AtNAP1;1 may require
additional cofactors, whose expression is also developmentally controlled. Further experiments will be
necessary 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 obtained 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 germinated 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 23C 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#-GTCGACTTACTGTTGCTTGCATTCGGG-3#, for the wild-type version of the CaaX box, CKQQ) or NAP1Rev2 (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 purified AtNAP1;1 in mouse.
Immunoblots
Nitrocellulose membranes were first blocked overnight at 4C 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 containing 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
(Köhler et al., 2003).
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Galichet and Gruissem
In Vitro and in Vivo Prenylation Assay
Histological Analysis
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).
Histological analyses were performed with samples in Technovit 7100
resin according to the manufacturer’s instructions (Kulzer & Co.). For transverse 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.
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 30C (Zimmerman and Kellogg, 2001).
GFP Constructs and Confocal and
Fluorescence 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 fragments. 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 Kinematic
Analysis 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 tumefaciens 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, 58C–63C)
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 (Köhler et al., 2003).
Subcellular Protein Fractioning
Nuclei were isolated from harvested first leaves as described (Köhler et al.,
2003).
ACKNOWLEDGMENTS
We are grateful to Johannes Fütterer 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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