Download - Sugar-inducible expression of the nucleolin-1 gene of Arabidopsis thaliana and its role in ribosome synthesis, growth and development

Sugar-inducible expression of the nucleolin-1 gene ofArabidopsis thaliana and its role in ribosome synthesis,growth and development

Hisae Kojima1, Takamasa Suzuki1, Takenori Kato1, Ken-ichi Enomoto1, Shusei Sato2, Tomohiko Kato2, Satoshi Tabata2,

Julio Saez-Vasquez3, Manuel Echeverrıa3, Tsuyoshi Nakagawa4, Sumie Ishiguro1 and Kenzo Nakamura1*

1Laboratory of Biochemistry, Department of Biological Functions and Mechanisms, Graduate School of Bioagricultural

Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan,2Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan,3Laboratoire Genome et Developpement des Plantes, UMR CNRS-IRD 5096, Universite de Perpignan, 66860 Perpignan Cedex,

France, and4Research Institute of Molecular Genetics, Shimane University, Matsue, Shimane 690-8504, Japan

Received 23 July 2006; revised 21 October 2006; accepted 10 November 2006.*For correspondence (fax þ81 52 789 4094; e-mail [email protected]).

Summary

Animal and yeast nucleolin function as global regulators of ribosome synthesis, and their expression is tightly

linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 is the

predominant if not only form of the protein found in most tissues, and GFP–AtNuc-L1 fusion proteins were

targeted to the nucleolus. Expression of AtNuc-L1 was strongly induced by sucrose or glucose but not by non-

metabolizable mannitol or 2-deoxyglucose. Sucrose also caused enhanced expression of genes for subunits of

C/D and H/ACA small nucleolar ribonucleoproteins, as well as a large number of genes for ribosomal proteins

(RPs), suggesting that carbohydrate availability regulates de novo ribosome synthesis. In sugar-starved cells,

induction of AtNuc-L1 occurred with 10 mM glucose, which seemed to be a prerequisite for resumption of

growth. Disruption of AtNuc-L1 caused an increased steady-state level of pre-rRNA relative to mature 25S

rRNA, and resulted in various phenotypes that overlap those reported for several RP gene mutants, including a

reduced growth rate, prolonged lifetime, bushy growth, pointed leaf, and defective vascular patterns and pod

development. These results suggest that the rate of ribosome synthesis in the meristem has a strong impact

not only on the growth but also the structure of plants. The AtNuc-L1 disruptant exhibited significantly

reduced sugar-induced expression of RP genes, suggesting that AtNuc-L1 is involved in the sugar-inducible

expression of RP genes.

Keywords: nucleolin, ribosome synthesis, ribosomal protein genes, snoRNP, sugar.

Introduction

In addition to growth regulators such as auxin and cytokinin,

nutrient availability is an important factor limiting cell pro-

liferation in plants. Sucrose (Suc) and glucose (Glc) induce

the expression of CycD2, CycD3 and CycD4 in Arabidopsis,

and cytokinin induction of CycD3 only occurs in the presence

of Suc (Murray et al., 1998; de Veylder et al., 1999, Riou-

Khamlichi et al., 2000). During cell division, cell proliferation

(increase in cell number) must be accompanied by cell

growth (increase in cell mass), which is primarily determined

by the protein synthetic activity of the cell, a process that uses

a great deal of energy and is tightly regulated by the

nutritional status of the cell. In yeast and animals, the protein

synthetic activity of the cell is regulated by the ‘target of

rapamycin’ (TOR) kinase-mediated pathway, which adjusts

the translational activity of pre-existing ribosomes and the

synthesis of ribosomes according to nutritional availability

(reviewed by Schmelzle and Hall, 2000; Raught et al., 2001).

The TOR gene in Arabidopsis is expressed in primary

meristem and is essential for growth (Menand et al., 2002).

The synthesis of functional ribosomes requires the

coordinated assembly of 70–80 different ribosomal proteins

(RPs) and four species of rRNA, yielding mature 40S and 60S

ª 2006 The Authors 1053Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 49, 1053–1063 doi: 10.1111/j.1365-313X.2006.03016.x

ribosomal subunits. Most steps of ribosome synthesis take

place in the nucleolus, which contains many non-ribosomal

RNAs and proteins that assist in ribosome synthesis. One of

these proteins, nucleolin, plays important roles in various

steps of ribosomal synthesis, such as the transcription of

rDNA repeats, the modification and processing of pre-rRNA,

the assembly of pre-ribosomal particles, and nuclear–cyto-

plasmic transport of RPs and ribosomal subunits (reviewed

by Tuteja and Tuteja, 1998; Ginisty et al., 1999; Srivastava

and Pollard, 1999). The N-terminal part of nucleolin from

various eukaryotes contains variable numbers of acidic

stretches that are similar to those of nuclear high-mobility

group proteins. This N-terminal region interacts with non-

transcribed spacer regions in rDNA repeats and histone H1

to influence rDNA transcription. The middle of the nucleolin

sequences includes RNA-binding domains called RNA

recognition motifs (RRMs). Animal nucleolin possesses four

RRMs, whereas yeast homologs possess two. Nucleolin

interacts with the stem–loop structure of RNA through its

RRM and participates in the modification and processing of

pre-rRNA. The C-terminal part of nucleolin contains glycine-

and arginine-rich (GAR) domains that are implicated in

ribosomal assembly and nuclear import of RPs.

Nucleolin is also involved in processes other than the

ribosome synthesis. Nucleolin possesses DNA helicase

activity and interacts with replication protein A, suggesting

that it participates in DNA unwinding and replication (Kim

et al., 2005; Nasirudin et al., 2005). Nucleolin in animals

interacts with various transcription factors and nuclear

components, and is involved in the regulation of RNA

polymerase II-dependent gene expression (Masumi et al.,

2006; Huddleson et al., 2006). Remarkably, nucleolin pos-

sesses a histone chaperone activity that activates chromatin

remodeling complexes and facilitates transcription through

the nucleosomes (Angelov et al., 2006).

In animals and yeast, expression and activity of nucleolin is

coordinated with the expression of genes for rRNA and RPs,

and correlates with the proliferative activity of the cell

(Srivastava and Pollard, 1999). Like yeast nucleolin, nucleo-

lin-like proteins from alfalfa (Medicago sativa; Bogre et al.,

1996), pea (Pisum sativum; Tong et al., 1997; Reichler et al.,

2001) and Arabidopsis (Saez-Vasquez et al., 2004) possess

two RRMs, and expression of pea nucleolin cDNA in a yeast

mutant deficient in nucleolin rescues the reduced level of

rRNA and the growth rate (Reichler et al., 2001). Regulation of

expression of the nucleolin gene in plants has been studied in

only a few cases. Expression of the alfalfa gene for nucleolin,

nucMs1, occurs predominantly in meristematic tissues,

where its expression is limited to cells actively engaged in

cell division (Bogre et al., 1996). Light induces the expression

of nucleolin in alfalfa and pea, which is probably mediated by

phytochrome (Bogre et al., 1996; Tong et al., 1997).

Recent global gene expression analyses in Arabidopsis

indicate that the expression of a large number of genes

involved in protein synthesis is regulated by the carbohy-

drate and nitrogen nutritional status (Price et al., 2004; Li

et al., 2006). Our microarray analysis also indicated that

genes involved in protein synthesis are significantly

enriched among genes that are upregulated by Suc (Yoine

et al., 2006). In particular, the nucleolin gene was one of

the genes that was induced the most strongly by Suc. In the

present study, we examine the role of nucleolin in the

regulation of ribosome synthesis and in the growth and

development of plants.

Results

Nucleolin genes of Arabidopsis thaliana

Of the sugar-induced genes that we identified by micro-

array analysis, a gene encoding nucleolin (At1g48920) was

one of the most strongly induced by Suc within 6 h. This

gene encodes a protein with structural similarities to Nsr1

of Saccharomyces cerevisiae (Kondo and Inouye, 1992)

and Gar2 of Schizosaccharomyces pombe (Gulli et al.,

1995), as well as nucleolins from alfalfa (Bogre et al., 1996)

and pea (Tong et al., 1997). The Arabidopsis genome also

contains another gene for nucleolin, At3g18610. The pro-

teins encoded by At1g48920 and At3g18610 are identical

with AtNuc-L1 and AtNuc-L2, respectively, described pre-

viously (Saez-Vasquez et al., 2004). The AtNuc-L1 and

AtNuc-L2 genes are located on segment 1, a large dupli-

cated segment between chromosomes 1 and 3, respect-

ively (Arabidopsis Genome Initiative, 2000). Although both

AtNuc-L1 and AtNuc-L2 contain two RRMs, the AtNuc-L1

gene is interrupted by 14 introns (Figure 1a), whereas the

AtNuc-L2 gene contains 17 introns. AtNuc-L1 mRNA was

detected in various organs of Col plants by RT-PCR

(Figure 1b). In contrast, little AtNuc-L2 mRNA was detected

in organs other than flower buds.

Genes encoding fusion proteins with GFP at either the

C-terminus (AtNuc-L1–GFP) or N-terminus (GFP–AtNuc-L1)

of AtNuc-L1 were placed downstream of the CaMV 35S

promoter and used to generate stably transformed tobacco

BY-2 cells (Matsuoka and Nakamura, 1991). In cells expres-

sing AtNuc-L1–GFP or GFP–AtNuc-L1, strong fluorescent

GFP signals were detected in nucleoli and absent from the

cytoplasm (Figure 1c). Weak GFP fluorescence was also

detected in the nucleoplasm, where fluorescence occasion-

ally appeared as spots. In contrast, signals were absent in

nucleoli of cells expressing free GFP. In roots of Arabidopsis

plants transformed with these fusion genes, strong GFP

fluorescence appeared in nucleoli (Figure 1d).

Sugar-inducible expression of AtNuc-L1

Figure 2(a) shows the time course of changes in the level of

AtNuc-L1 mRNA after treatment of leaves of Arabidopsis Col

1054 Hisae Kojima et al.

ª 2006 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 1053–1063

seedlings with 175 mM (6% w/v) Suc or H2O in the dark. The

level of AtNuc-L1 mRNA started to increase after 1 h of Suc

treatment, and reached a maximum after 6 h, whereas it did

not change after treatment with H2O. A 6 h treatment

with 146 mM Glc also caused an increase in the level of

AtNuc-L1 mRNA (Figure 2b); however, non-metabolizable

3-O-methyl-D-glucose and 2-deoxy-D-glucose, as well as

146 mM mannitol and 50 lM ABA, were ineffective in indu-

cing AtNuc-L1 mRNA. Similar results were obtained by

treatment of seedlings with sugars. Trehalose metabolism

plays important roles in sugar sensing and plant develop-

ment (Muller et al., 1999). Neither germination of seeds on

medium containing trehalose nor treatment of seedlings with

trehalose affected the level of AtNuc-L1 mRNA. Under the

conditions employed, trehalose induced ApL3 mRNA for

starch synthesis as reported previously (Wingler et al., 2000).

We also examined the expression of AtNuc-L1 in

suspension-cultured Arabidopsis T87 cells (Axelos et al.,

1992). Exponentially growing cells were starved of sugars

for 24 h, after which the cells were cultured in medium

containing various concentrations of Glc for 6 h. As shown

in Figure 2(c), 10 mM Glc resulted in induction of a nearly

maximal level of AtNuc-L1 mRNA but did not affect the

level of ACT2 mRNA. For sugar-starved cells, 10 mM Glc

was sufficient to induce the resumption of growth, but

the growth rates were higher at 50 and 100 mM Glc

(Figure 2d).

Sugar-induced expression of other genes involved in pre-

rRNA processing

Animal and yeast nucleolin are involved in correct modifi-

cation and processing of pre-rRNA (reviewed by Tuteja and

Tuteja, 1998; Ginisty et al., 1999; Srivastava and Pollard,

1999). The site-specific cleavage and base pseudo-uridyla-

tion of pre-rRNA is mediated by the H/ACA small nucleolar

ribonucleoprotein (snoRNP) complex, which contains, in the

case of yeast S. cerevisiae, Gar1, Nap57, Nhp2, Nop10 and

snoRNAs, while the site-specific cleavage and 2¢-O-methy-

lation of pre-rRNA requires C/D snoRNP, which is composed

of Nop1 (fibrillarin), Nop58, Nop56, Snu13 and snoRNAs

(reviewed by Filipowicz and Pogacic, 2002; Meier, 2005). If

sugar-induced expression of AtNuc-L1 plays a role in the

enhanced processing and base modification of pre-rRNA,

the expression of genes for the subunits of H/ACA and C/D

snoRNPs should also be upregulated by sugar.

Figure 1. Structure and expression of AtNuc-L1.

(a) In the upper diagram showing the structure of AtNuc-L1, the white and

yellow-green boxes represent the untranslated and coding regions of exons,

respectively. The position of the T-DNA insertion in the DAtNuc-L1-1 mutant is

shown. In the lower diagram showing the structure of AtNuc-L1 protein, the

positions of acidic stretches, RRM domains and GAR domains are indicated.

(b) The same amount of RNA from various organs was used to detect AtNuc-

L1 and AtNuc-L2 mRNAs by RT-PCR. ACT2 mRNA served as a control, and

PCR reaction with chromosomal DNA as a template was also performed to

examine PCR efficiency. L, leaves of 3-week-old plants; S, shoots of 3-day-old

seedlings; F, flowers; Fb, floral buds; R, roots of liquid-cultured plants.

(c) AtNuc-L1–GFP and GFP–AtNuc-L1 fusion proteins expressed in tobacco

BY-2 cells. Expression of GFP alone served as a control.

(d) Localization of AtNuc-L1–GFP fusion protein in roots of Arabidopsis. Roots

were stained with propidium iodide and observed by confocal microscopy.

The inset shows an enlarged image without propidium iodide staining.

Figure 2. Sugar-induced expression of AtNuc-L1 in excised leaves and

cultured cells.

(a) Leaves of 3-week-old Col seedlings were treated with 6% Suc (175 mM) or

H2O for the indicated periods of time. Levels of AtNuc-L1 mRNA were

determined by real-time RT-PCR using ACT2 mRNA as an internal standard,

and the level of AtNuc-L1 mRNA at time 0 was set as 1. Results represent the

mean of two representative experiments �SD.

(b) Leaves were treated for 6 h with water (H2O), 146 mM sucrose (Suc),

146 mM glucose (Glc), 14.6 mM 3-O-methylglucose (3OMG), 14.6 mM 2-

deoxy-D-glucose (2DG), 146 mM mannitol (Mtl) or 100 lM ABA (ABA). Levels

of AtNuc-L1 mRNA were determined as in (a), and the level in H2O-treated

leaves was set as 1. Results represent the mean of two representative

experiments �SD.

(c) Arabidopsis T87 cells were starved of sugars for 24 h and then grown in

fresh medium containing 0, 1, 10, 50 or 100 mM Glc for 12 h. Levels of AtNuc-

L1 and ACT2 mRNAs were determined by real-time RT-PCR using 18S rRNA as

an internal standard. The level of each mRNA at time 0 was set as 1, and the

means of two representative experiments �SD are shown.

(d) T87 cells starved of sugar for 24 h were re-incubated in medium containing

various concentrations of Glc, and cell fresh weights per unit volume of

culture were measured at the indicated times. The means of two represen-

tative experiments �SD are shown.

Sugar-inducible expression of nucleolin gene 1055


Among the subunits of H/ACA snoRNP, an Arabidopsis

homolog of Nap57 has been characterized (Maceluch et al.,

2001). The predicted Arabidopsis gene (At3g03920) encodes

a 202 amino acid Gar1-like protein that has 65% amino acid

identity with the 205 amino acid Gar1 protein. In addition,

At5g18180 is also predicted to encode a 189 amino acid

protein that has 50% amino acid identity with Gar1. The

predicted gene At5g08180 encodes a 156 amino acid protein

that has 42% amino acid identity with the 173 amino acid

Nhp2 protein, while At2g20490 is predicted to encode a 64

amino acid protein that has 58% amino acid identity with

Nop10. Furthermore, the Arabidopsis genome includes two

genes for fibrillarin, AtFib1and AtFib2 (Barneche et al., 2000;

Pih et al., 2000). Although a third gene, AtFib3, can encode a

protein that has 67% identity with AtFib1 and AtFib2, this

gene does not appear to be expressed. Two predicted genes,

At3g05060 and At5g27120, are predicted to encode 533

amino acid proteins that have 55% identities with the 511

amino acid protein Nop58. Similarly, two predicted genes,

At3g12860 and At1g56110, encode proteins of 477 and 522

amino acids, respectively, both of which share approxi-

mately 52% identity with the 504 amino acid Nop56. The

Arabidopsis genome also contains three putative genes

(At5g20160, At4g22380, and At4g12600) that encode 128

amino acid proteins that share 66% identity with the 126

amino acid Snu13.

We examined our microarray data to determine whether

sugar induces the expression of genes encoding subunits of

the H/ACA and C/D snoRNP complexes. In these analyses,

we analyzed three independently isolated pairs of RNA

samples from Col seedlings that had been treated with 5%

Suc or H2O for 6 h in the dark using Agilent Arabidopsis-1

and -2 oligo microarrays. For each pair of RNAs, the array

data after dye swapping of Cy3 and Cy5 labeling were

averaged. The results from two representative arrays are

shown in Figure 3(a). Among the predicted genes analyzed,

Figure 3. Sucrose-induced expression of genes

encoding subunits of snoRNP.

(a) Signal levels of transcripts of nucleolin genes

and genes encoding subunits of H/ACA and C/D

snoRNP and those for snRNP in Col seedlings as

determined by microarray analysis. Signal val-

ues for each transcript in seedlings treated with

H2O (blue bars) or 5% Suc (red bars) for 6 h from

two representative experiments (one each) are

shown. Black circles indicate the mean � SD fold

increase of transcript in Suc-treated seedlings

relative to H2O-treated seedlings.

(b) Levels of transcripts in leaves treated with

water (blue bars) or 5% Suc (red bars) for 6 h as

determined by real-time RT-PCR. The levels of

each mRNA in H2O-treated seedlings were set as

1, and the results represent the means for two

independent experiments �SD.

(c) Proteins extracted from leaves treated with

H2O (H) or 5% Suc (S) for 24 h were separated on

SDS–PAGE, and the bands for nucleolin, fibrilla-

rin and Nop58 were detected with anti-AtNuc-L1,

anti-AtFib and anti-NtMARBP61, respectively.

The arrowhead indicates the major cross-react-

ing band.



AtNuc-L2 and AtNop56-2 (At1g56110) did not give signifi-

cant signals. One of the two genes for Gar1-like protein,

At5g18180, and AtFib3 showed signals that did not vary

between Suc- and H2O-treated plants. However, all the other

genes showed Suc-induced increases in the mRNA level. In

addition to AtNuc-L1, Suc caused an approximately 10-fold

increase in the level of AtFib2 mRNA. Other genes showed

1.8–5.5-fold increases by Suc treatment. To confirm the

microarray data, we examined the levels of several mRNAs

by quantitative real-time RT-PCR. In addition to an 11-fold

induction of AtNuc-L1 mRNA by Suc, the levels of mRNAs

for AtNap57, AtNhp2 and AtNop10 were 3–10-fold higher in

seedlings treated with Suc than those treated with H2O

(Figure 3b). In contrast to genes encoding subunits of H/ACA

and C/D snoRNP complexes, Suc did not increase the mRNA

levels for the 14 putative genes encoding the subunits of U1,

U2, U4/U6 and U5 snRNP complexes, which are involved in

intron splicing of pre-mRNAs.

To determine whether the induction of AtNuc-L1 mRNA by

sugar is accompanied by an increase in the level of protein,

we extracted proteins from seedlings that had been treated

with 5% Suc or H2O for 24 h and analyzed them by immu-

noblotting with an anti-AtNuc-L1 antibody (Saez-Vasquez

et al., 2004). The anti-AtNuc-L1 antibody reacted with an

approximately 78 kDa polypeptide, which is larger than the

expected molecular mass of 58.8 kDa (Figure 3c). In addition,

the antibody revealed a weaker band of approximately

140 kDa. The intensities of both bands were stronger in

extracts from Suc-treated seedlings than those from H2O-

treated seedlings. We also examined levels of fibrillarin using

an anti-AtFib antibody (Saez-Vasquez et al., 2004) and those

of the putative Nop58 protein using an antibody against a

tobacco homolog of Nop58 (NtMARBP61; Fujiwara et al.,

2002). The anti-AtFib antibody detected a single band with an

apparent molecular mass of 32 kDa. Despite the induction of

AtFib1 and AtFib2 mRNAs by Suc, the intensity of this 32 kDa

band did not differ between the H2O- and Suc-treated

seedlings. The anti-NtMARBP61 antibody reacted with a

single band with an apparent molecular mass of 58 kDa that

appeared to be increased in the Suc-treated plants.

Reduced processing of rRNA precursors in a disruptant of

AtNuc-L1

We searched for T-DNA insertion lines of AtNuc-L1 in the

Kazusa T-DNA tag-line collection by PCR screening of

pooled chromosomal DNA, and identified a line in which

T-DNA was inserted in the second intron (Figure 1a). In this

DAtNuc-L1-1 mutant in the Col background, we detected

neither AtNuc-L1 mRNA by RT-PCR (Figure 4a) nor AtNuc-L1

protein by immunoblotting with an anti-AtNuc-L1 antibody

(Figure 4b).

The 45S pre-rRNA contains a 5¢ external transcribed

sequence (ETS), internal transcribed sequences 1 and 2

(ITS1 and ITS2), and a 3¢ ETS, which are removed during a

complex series of maturation steps (reviewed by Fromont-

Racine et al., 2003). To examine the effects of AtNuc-L1

deficiency on the processing of 45S pre-rRNA, we compared

the levels of corresponding RNA sequences between Col

and DAtNuc-L1-1 plants by real-time RT-PCR using primers

specific to various regions of the 45S pre-rRNA (Figure 4c).

To quantify ETS and ITS sequences, 1 lg of RNA was used

as a template for the first-strand cDNA synthesis, whereas

0.2 ng of RNA was used for quantification of mature rRNA

sequences. We normalized the signal values for each PCR

product against the values for 25S rRNA and then compared

the relative values for Col and DAtNuc-L1-1 plants. We found

that the relative levels of the 5¢ ETS, ITS1, ITS2 and 3¢ ETS

sequences were 1.7–4-fold higher in DAtNuc-L1-1 plants

than in Col plants (Figure 4c), suggesting that the steady-

state level of pre-rRNA relative to mature 25S rRNA is higher

in DAtNuc-L1-1 than in Col plants.

Growth and developmental phenotypes of DAtNuc-L1-1

plants

The DAtNuc-L1-1 plants showed various growth and devel-

opmental phenotypes. The growth of roots of DAtNuc-L1-1

Figure 4. Reduced pre-rRNA processing in DAtNuc-L1-1.

(a) Detection of AtNuc-L1 mRNA by RT-PCR in the same amounts of RNA from

Col and DAtNuc-L1-1 plants (D). Tubulin mRNA (TUA3/5) served as a control.

(b) Total proteins (15 lg) extracted from Col and DAtNuc-L1-1 (D) seedlings

were separated by SDS–PAGE and analyzed by immunoblotting with an anti-

AtNuc-L1 antibody. The arrowhead indicates the 78 kDa band (see Figure 3c).

(c) Comparison of the relative levels of the various regions of pre-rRNA in Col

and DAtNucL1-1 plants. Primer sets specific to various regions of 45S pre-

rRNA (upper panel) were used for synthesis of the first-strand cDNA and

determination of the levels of corresponding RNA sequences by real-time RT-

PCR. Signal values for each PCR product were normalized against the values

for 25S mature rRNA, and then compared between Col and DAtNuc-L1-1

plants (lower panel). Black bars represent ETS and ITS sequences, and white

bars represent 16S, 5.8S and 25S vRNAs. The values represent the

means � SD for two independent isolations of RNAs.



germinated on vertically placed agar plates was 60–80%

slower than that of Col seedlings (Figure 5a), and the above-

ground parts of seedlings grown on agar plates were stun-

ted (Figure 5b). The DAtNuc-L1-1 plants also grew more

slowly than Col plants on vermiculite (Figure 5c). They often

showed outgrowth of organs from axillary buds (Figure 5d),

bushy growth with many stems, and a longer life duration

compared with the Col plants (Figure 5e).

The leaves of young DAtNuc-L1-1 seedlings had a narrow

pointed shape in contrast to the round shape of Col leaves

(Figure 5b), and showed abnormal vascular patterns (Fig-

ure 5f). In addition to the reduced growth rate, these

phenotypes are similar to those commonly observed for

mutants with disruptions of RP genes, such as pointed first

leaf(pfl), in which AtRPS18A (pfl1; van Lijsebettens et al.,

1994) or AtRPS13A (pfl2; Ito et al., 2000) is disrupted,

Arabidopsis minute-like 1 (aml1), in which AtRPS5A is

disrupted (Weijers et al., 2001), and short valve 1 (stv1), in

which AtRPL24B is disrupted (Nishimura et al., 2005). In

addition, pods of DAtNuc-L1-1 plants were shorter and

irregular in size and shape compared with those of Col

plants (Figure 5g). Furthermore, the stalks of the DAtNuc-L1-

1 pods were longer than those of Col pods (Figure 5g;

arrowheads). A long stalk is a characteristic feature of the

pods from the stv1 mutant as well as those from mutants of

auxin response factors ETTIN and MONOPTEROS, which are

defective in gynoecium development (Nishimura et al.,

2005). AtRPL24, encoded by STV1, is specifically involved

in translation re-initiation of polycistronic mRNAs, and

disruption of AtRPL24B affects the expression of ETTIN

and MONOPTEROS genes, which contain upstream open

reading frames in their 5¢ untranslated sequences (Nishim-

ura et al., 2005). These results suggest that DAtNuc-L1-1

plants contain a reduced amount of RPs due to defective

ribosome synthesis and suffer from a shortage of ribosomes

in proliferating cells.

Expression of RP genes in DAtNuc-L1-1 plants

Because DAtNuc-L1-1 plants seemed to contain a reduced

amount of ribosomes and RPs, we examined the expression

of RP genes in these plants. Figure 6(a) shows the scatter

plots of representative microarray data for transcript levels

of Col and DAtNuc-L1-1 seedlings treated with 5% Suc or

H2O. Similar results were obtained in three independent

experiments for Col plants and two independent experi-

ments for DAtNuc-L1-1 plants. In Arabidopsis, each of the 80

RPs are encoded by a small family of 2–7 members (Barakat

et al., 2001). In Figure 6(a), the 217 RP genes are indicated by

pink triangles. In Col plants, the mRNA levels for 85% of the

RP genes were increased more than twofold in Suc-treated

plants compared with H2O-treated plants. In contrast, the

mRNA level for none of the RP genes was increased more

than twofold by Suc in the DAtNuc-L1-1 plants (Figure 6a).

For example, the mRNAs for AtRPL34A, AtRPL27A, At-

RPL27C, AtRPS3aA and AtRPS11A were increased two to

fivefold in Col plants treated with 5% Suc compared to plants

treated with H2O, whereas there was no noticeable induction

by Suc in DAtNuc-L1-1 plants (Figure 6b-1). The signal val-

ues for RP transcripts in H2O-treated seedlings were not

noticeably different between Col and DAtNuc-L1-1plants,

suggesting that the nucleolin deficiency affected the sugar-

induced increase in RP mRNAs. On average, Suc treatment

caused a 2.8-fold increase in the levels of the 217 RP mRNAs

in Col plants but only a 1.3-fold difference in DAtNuc-L1-1

plants.

Comparison of the scatter plots for Col and DAtNuc-L1-1

plants (Figure 6a) suggests that there was a general reduc-

tion in the magnitude of sugar-responsive changes in the

mRNA level in DAtNuc-L1-1 plants. Nevertheless, expression

of typical sugar-inducible genes such as b-amylase (Atb-

Amy; Mita et al., 1995), ADP-glucose pyrophosphorylase S,

Figure 5. Phenotypes of the DAtNuc-L1-1 mutant.

(a) Length of roots of Col (hatched bar) and DAtNuc-L1-1 plants (black bar) that

had been grown on vertically placed plates containing 0% or 1% Suc for 8 or

14 days. The results represent the means for 10–20 seedlings �SD.

(b, c) Seeds of Col and DAtNuc-L1-1 were germinated and grown on plates

containing 2% Suc for 3 weeks (b) or on vermiculite for 48 days (c).

(d) Axillary organ development in DAtNuc-L1-1.

(e) Representative Col and DAtNuc-L1-1 plants grown for 60 and 90 days,

respectively. Bar ¼ 1 cm.

(f) Vascular patterns in young leaves of Col and DAtNuc-L1-1 plants.

(g) Flowers and pods from the top (left) to the bottom (right) of one stem of Col

and DAtNuc-L1-1 are shown. The stalks of a pod from Col and DAtNuc-L1-

1plants are indicated by open and closed arrowheads, respectively.

Bar ¼ 0.5 cm.



starch branching enzyme 2, 3-keto-acyl CoA thiolase 2 and

the bZIP transcription factor ATB2 (Rook et al., 1998) was not

noticeably affected by the disruption of AtNuc-L1 (Figure 6b-

2). Similarly, sugar repression of mRNAs for a putative

b-xylosidase, peroxidase 2, glycolate oxidase, SEN1 (Oh

et al., 1996) and a putative peptide transporter were not

noticeably affected by the disruption of AtNuc-L1-1 (Fig-

ure 6b-3). These results suggest that the reduction in sugar-

inducible expression of RP genes in DAtNuc-L1-1 is selective.

Discussion

Although the Arabidopsis genome contains two genes for

plant-type nucleolin, expression of only AtNuc-L1 was

detected in most of the tissues examined. An AtNuc-L1-l-

GFP fusion protein localized mostly in the nucleolus, and

AtNuc-L1 protein but not AtNuc-L2 protein have been iden-

tified in recent proteomic analyses of the Arabidopsis nuc-

leolus (Pendle et al., 2005). These results indicate that

AtNuc-L1 is the predominant, if not exclusive, form of

nucleolin in most tissues of Arabidopsis.

Sugar induction of AtNuc-L1 is associated with ribosome

synthesis and linked to cell proliferation

In the present study, we showed that sugar causes a rapid

increase in the level of AtNuc-L1 mRNA and AtNuc-L1 pro-

tein. This induction of AtNuc-L1 by sugar occurs in con-

junction with the enhanced expression of a large number of

RP genes and genes encoding subunits of the H/ACA and C/

D snoRNP complexes. In contrast, Suc did not enhance the

expression of genes encoding components of snRNPs in-

volved in pre-mRNA splicing. These results suggest that

sugar regulation of AtNuc-L1 expression is closely associ-

ated with the regulation of de novo ribosome synthesis.

Sugar induction of AtNuc-L1 and its involvement in

ribosome synthesis are consistent with the selective expres-

sion of the nucleolin gene in the meristem region (Bogre

Figure 6. Suc-responsive gene expression in

Col and DAtNuc-L1-1 plants.

(a) Scatter plot of representative results from

microarray analysis comparing transcript levels

in H2O-treated versus 5% Suc-treated seedlings

of Col and DAtNuc-L1-1 plants. Transcripts for

217 RP genes are indicated by pink triangles.

(b) Signal values for (1) transcripts of five repre-

sentative RP genes, i.e. AtRPL34A (At1g26880),

AtRPL27A (At2g32220), AtRPL27C (At4g15000),

AtRPS3aA (At3g04840) and AtRPS11A

(At3g48930), (2) typical sugar-inducible genes,

i.e. Atb-Amy (At4g15210), AGPS (At5g48300),

SBE2.2 (At5g03650), 3-KACT (At5g48880) and

ATB2 (At4g34590), and (3) typical sugar-repress-

ible genes, i.e. b-xylosidase (At5g64570), Prx2

(At4g37520), glycolate oxidase (At3g14415),

SEN1 (At4g35770) and putative peptide trans-

porter (At5g46050), in H2O-treated (blue) and 5%

Suc-treated (red) Col (W) or DAtNuc-L1-1 (D)

seedlings in two independent microarrays (1

and 2).



et al., 1996). Expression of Arabidopsis RP genes, such as

AtRPS18A (van Lijsebettens et al., 1994) and AtRPS5A

(Weijers et al., 2001), also occurs predominantly in cells

actively engaged in cell division in the meristem. In Arabid-

opsis suspension-cultured cells that had been starved of

sugars, inclusion of 10 mM Glc in the medium was sufficient

to induce the maximum level of expression of AtNuc-L1 and

to cause the resumption of growth. On the other hand, the

growth rate increased with the concentration of Glc (up to

100 mM), suggesting that the induction of AtNuc-L1 expres-

sion by 10 mM Glc is prerequisite for the resumption of

growth rather than the expression of AtNuc-L1 being

controlled by the growth rate. Sugar induces the expression

of a variety of genes involved in nutrient storage, such as

genes for vegetative storage proteins and for the synthesis

of starch (reviewed by Koch, 1996; Rolland et al., 2006).

Sugar induction of expression of genes for reserve synthesis

generally occurs at much higher concentrations of sugars

and in quiescent cells.

Relationship of sugar regulation of ribosome synthesis with

cell division

The availability of sugars affects cell division in the meristem,

for which cell proliferation must be accompanied by cell

growth. In the early developmental stage of Vicia fabaseeds,

where cell proliferation and differentiation predominate, the

spatial distribution of Glc, rather than Suc, correlates well

with the mitotic index (Borisjuk et al., 1998). During these

stages, transported Suc is cleaved into Glc and fructose by

cell-wall-bound invertase, generating a high Glc/Suc ratio. In

the later stages of seed development, the Suc/Glc ratio is

high because of a decline in cell-wall-bound invertase, and

Suc synthase plays a predominant role in the utilization of

Suc for reserve synthesis (Borisjuk et al., 1998, 2004; ). In

Arabidopsis cells starved for sugars, the expression of CycD2

and CycD3 is induced by 10 mM Glc (Riou-Khamlichi et al.,

2000). Under similar conditions, the induction of expression

of AtNuc-L1 and other genes involved in ribosome synthesis

also occurred with 10 mM Glc. CycD2, but not CycD3, is

induced by 2-deoxy-D-glucose (2DG), suggesting that CycD2

expression is under the control of hexokinase sensor-

dependent sugar signaling, wherein hexokinase functions as

a Glc sensor (Jang et al., 1997). A recent study with Physc-

omytrella patens, however, suggested that sugar regulation

of CycD2 is more closely related to the developmental pro-

gression in response to nutrient availability than the control

of cell division (Lorenz et al., 2003). Similar to CycD3, the

expression of AtNuc-L1 and RP genes is not induced by 2DG,

suggesting that a metabolic signal derived from Glc is

required for the expression of genes related to cell division

and growth; however, in itself, the absence of a response to

2DG does not exclude the involvement of hexokinase sensor-

dependent signaling in the regulation.

Deficiency of AtNuc-L1 leads to a shortage of ribosomes in

dividing cells

Although Nsr1 of S. cerevisiae (Kondo and Inouye, 1992)

and Gar2 of S. pombe (Gulli et al., 1995) are not essential for

cell viability, their null mutants show growth defects. The

nsr1and gar2 mutants accumulate 35S pre-rRNA and have

reduced steady-state levels of the 40S ribosomal subunit,

which most likely causes the defective growth. Expression of

pea nucleolin cDNA in the nsr1 mutant rescues the reduced

amount of large subunit rRNA and the reduced growth rate

(Reichler et al., 2001). We found that the DAtNuc-L1-1 null

mutant showed an increased steady-state level of pre-rRNA

relative to mature 25S rRNA compared to Col. This differ-

ence could be due to delayed pre-rRNA processing or the

production of abnormal transcripts of pre-rRNA that are not

processed promptly. It is suggested that ribosome synthesis

is reduced in the mutant.

The idea that DAtNuc-L1-1 plants have a shortage of

ribosomes is supported by its phenotypes. In addition to

reduced growth rate, DAtNuc-L1-1 plants exhibited various

developmental phenotypes that overlap with those of pre-

viously reported Arabidopsis mutants defective in specific

RP genes (van Lijsebettens et al., 1994; Ito et al., 2000;

Weijers et al., 2001; Nishimura et al., 2005), including poin-

ted leaves, abnormal vascular patterning, and defective

gynoecium development. Thus, the synthesis of ribosomes

might be limited in both DAtNuc-L1-1 and RP mutants.

The DAtNuc-L1-1 plants showed reduced growth rate and

lived longer than Col plants. The reduced growth rate of

DAtNuc-L1-1 plants is probably due to reduced cell division

as a result of a shortage of ribosomes, and therefore an

inability to meet the demands for active protein synthesis.

The DAtNuc-L1-1 plants showed outgrowth of axillary

organs and bushy growth. A reduced rate of organ devel-

opment may alter the distribution of photoassimilates

among meristems of DAtNuc-L1-1 plants. These results

suggest that the rate of ribosome synthesis in the meristem

has a strong impact on the growth and the structure and

architecture of plants.

AtNuc-L1 deficiency affects gene expression pattern

The sugar-enhanced expression of RP genes was severely

diminished in DAtNuc-L1-1 plants compared with Col plants.

Although we observed a general diminution in the sugar-

induced changes of the transcript levels in DAtNuc-L1-1

plants, expression of typical sugar-inducible or -repressible

genes, e.g. Atb-Amy (Mita et al., 1995), ATB2 (Rook et al.,

1998) or SEN1 (Oh et al., 1996), was not noticeably affected.

The nucleolin deficiency seems to selectively affect the ability

of sugar to induce the expression of RP genes because the

signal values for transcripts in H2O-treated seedlings were

not noticeably different between Col and DAtNuc-L1-1plants.



Because sugar does not affect the stability of several RP

mRNAs (K. Enomoto, H. Kojima and K. Nakamura,

unpublished results), the reduced ability of sugar to activate

RP genes in DAtNuc-L1-1 is probably due to effects on tran-

scription. Most sugar-induced changes in transcript levels in

Arabidopsis require de novo protein synthesis (Price et al.,

2004). Although a reduction in protein synthesis may explain

the general reduction in the magnitude of sugar-induced

changes in transcript levels in DAtNuc-L1-1, it does not

account for the selective effect of nucleolin deficiency on the

sugar-inducible expression of RP genes.

Nucleolin binds to spacer regions in rDNA and histone H1

through its N-terminal acidic stretches, and participates in

transcription of rDNA by RNA polymerase I (Ginisty et al.,

1999; Srivastava and Pollard, 1999). Nuclear factor D purified

from the inflorescence of cauliflower (Brassica oleracea)

binds to rDNA and contains nucleolin-like protein, fibrillarin

and snoRNAs, suggesting that snoRNP may link rDNA

transcription and pre-rRNA maturation (Saez-Vasquez et al.,

2004). In addition, nucleolin in animals interacts with various

transcription factors and nuclear components (Ginisty et al.,

1999; Srivastava and Pollard, 1999), and is required for

transcriptional regulation of several genes by RNA polym-

erase II (Masumi et al., 2006; Huddleson et al., 2006). Animal

nucleolin has been shown to exhibit histone chaperone

activity and enhances the remodeling of nucleosomes by

SWI/SNF and ATP-dependent chromatin-assembly factor

(Angelov et al., 2006). In particular, nucleolin promotes the

remodeling of nucleosomes that contain histone variant,

which are otherwise resistant to remodeling, and it facili-

tates passage of RNA polymerase II through nucleosomes.

In yeast, the TOR pathway regulates expression of rDNA and

RP genes by chromatin-mediated mechanisms in response

to nutrient availability (Rohde and Cardenas, 2003; Tsang

et al., 2003). It seems worth examining the possibility that

AtNuc-L1 regulates the transcription of RP genes through a

chromatin-mediated mechanism.

Experimental procedures

Plant materials and treatment with sugars

Seeds of Arabidopsis thaliana (L.) Heynh. (ecotype Col-0) weresurface-sterilized, kept at 4�C for 3 days in sterile water, and sown on0.3% gellan gum plates containing Murashige and Skoog medium(pH 5.7), 100 mg l)1 myo-inositol, 10 mg l)1 thiamine-HCl, 1 mg l)1

nicotinic acid, 1 mg l)1 pyridoxine HCl and 2% w/v Suc. Plates wereincubated in a growth chamber at 22�C under continuous light of65 lmol m)2 sec)1. Mature leaves of the 3-week-old plants wereexcised with a sharp razor blade, and the cut edges of petioles wereimmersed in a sterile solution of sugar or water and incubated at22�C in the dark (Mita et al., 1995). Plants were also grown on ver-miculite at 22�C under continuous light. Plants were watered withHoagland’s nutrient solution every week and water as needed.

A primary PCR screen for a T-DNA insertion mutant of AtNuc-L1was performed on pooled chromosomal DNA from approximately

20 000 individual lines of a collection of Arabidopsis T-DNAinsertion lines (Kazusa DNA Research Institute, Kazusa, Japan)using gene-specific primers and T-DNA border primers. For geno-typing of DAtNuc-L1-1, we carried out genomic PCR with a gene-specific PCR primer and a T-DNA right border primer (Table S1).Seeds of the homozygous T-DNA insertion line that had been back-crossed to Col twice were used for further analyses.

Arabidopsis suspension-cultured cell line T87 derived from Col(Axelos et al., 1992), was obtained from the RIKEN Plant Cell Bank(Yokohama, Japan) and grown in GB5 medium (Yamada et al.,2004), pH 5.7, containing 3.3 g l)1 of Gamborg’s B5 salt mixture(Wako www.wako-chem.co.jp), 1 ml l)1 of diluted Gamborg’s vita-min solution (Sigma-Aldrich, http://www.sigmaaldrich.com/),0.5 g l)1 of 2-morpholinoethanesulfonic acid monohydrate, 1 lM

1-naphthalene acetic acid and 1.5% Suc (w/v). The 6-day-old cellswere collected by centrifugation, and washed three times with freshmedium without Suc. Cells were starved of sugars for 24 h,resuspended in fresh medium containing 0, 1, 10, 50 or 100 mM

Glc, and grown for 6 h before isolating RNA.

Localization of GFP fusion proteins of AtNuc-L1

The full-length cDNA for AtNuc-L1 was obtained by RT-PCR usingthe primer set shown in Table S1. The cDNA was cloned intopGWB5 or pGWB6 vectors containing the coding sequence for sGFPby GatewayTM cloning technology (Invitrogen, http://www.invitro-gen.com/) to produce binary Ti plasmids carrying genes encodingAtNuc-L1–GFP and GFP–AtNuc-L1 fusion proteins under the controlof the CaMV 35S promoter, respectively. These plasmids were usedto transform tobacco BY-2 cells as described previously (Matsuokaand Nakamura, 1991). Several independent transformed calli werebrought into suspension culture, and GFP fluorescence was ob-served using an FV500 confocal fluorescence microscope (Olym-pus, http://www.olympus-global.com/). The same Agrobacteriumstrains were used to transform Arabidopsis plants, and roots of T2

plants from several independent transformed lines were stainedwith propidium iodide and observed for GFP fluorescence by con-focal fluorescence microscopy.

Isolation of RNA, RT-PCR and real-time RT-PCR

Total RNA was isolated from plants or tissues using an RNeasy PlantMini Kit (Qiagen, http://www.qiagen.com) and dissolved in ribo-nuclease-free water. For detection of mRNAs, first-strand cDNA wassynthesized from 2 lg of total RNA using oligo(dT)20 primers andSuperscript III (Invitrogen) and diluted with four volumes of water.PCR was performed in a 25 ll mixture containing 2 ll of the dilutedcDNA solution and 0.4 lM of each primer. The PCR reaction cycleswere as follows: denaturation at 95�C for 30 sec, annealing at 60�C for30 sec, and extension at 72�C for 30 sec. The number of cycles wasoptimized for each mRNA. For quantitative real-time RT-PCR, PCRwas performed with iQ SYBR Green Supermix using an iCycler iQ(Bio-Rad, http://www.bio-rad.com/), and the comparative thresholdcycle method was used to determine the relative levels of mRNAs,with ACT2 mRNA as an internal reference. The primer sets for AtNuc-L1, ACT2, AtNap57, AtNhp2 and AtNop10 are listed in Table S1.

To examine the processing of pre-rRNA, total RNA was isolatedfrom3-week-oldColandDAtNuc-L1-1 plants.Forquantification of25S,18S and 5.8S rRNA, cDNAs were synthesized from 200 pg of total RNAwith specific primers in a 20 ll reaction, whereas 1 lg of total RNAwas used for the synthesis of cDNA with specific primers for quanti-fication of ETS and ITS sequences of pre-rRNA. Quantitative real-timeRT-PCR was carried out using the primer sets listed in Table S1.



Oligo microarray analysis

Total RNAs used for microarray analyses were prepared usingTrizol reagent (Invitrogen www.invitrogen.com) and subsequentlypurified using an RNeasy Plant Mini Kit (Qiagen). Cy3- andCy5-labeled cDNA probes were synthesized and hybridized to theAgilent Arabidopsis-1 and -2 oligo microarrays (Agilent Technol-ogies www.home.agilent.com) according to the manufacturer’sinstructions. The microarray analysis was performed with two orthree independently isolated RNA samples and assessed in eachexperiment by dye swapping as described previously (Yoine et al.,2006).

Extraction of proteins and immunological detection

Proteins were extracted from 3-week-old seedlings that had beentreated with 5% Suc or H2O for 24 h. SDS–PAGE was carried out inan 8% acrylamide gel. The proteins were transferred from the gel toa poly(vinylidene difluoride) membrane (Immobilon; Milliporewww.millipore.com), and the antigen on the membrane wasdetected with primary antibodies, followed by horseradish peroxi-dase-coupled protein A and chemiluminescence reagents (ECL kit;GE Healthcare www.gehealthcare.com). Antibodies raised againstrecombinant AtNuc-L1 (AtNuc-L1) and AtFib1 (Saez-Vasquez et al.,2004) were used, and the antibody against recombinantNtMARBP61 (Fujiwara et al., 2002) was a generous gift from DrMasayoshi Maeshima of Nagoya University.

Acknowledgements

We thank S. Ukai and T. Kawai for technical assistance, M. Mae-shima of Nagoya University for anti-NtMARBP61, and K. Shinozakiof RIKEN for T87 cells. This work was supported in part by theResearch for the Future program of the Japan Society for the Pro-motion of Science (grant number 00L01603) and the 21st CenturyCOE program from the Ministry of Education, Science, Sports andCulture of Japan to K.N.

Supplementary Material

The following supplementary material is available for this articleonline:Table S1 List of primersThis material is available as part of the online article from http://www.blackwell-synergy.com.

References

Angelov, D., Bondarenko, V.A., Almagro, S. et al. (2006) Nucleolin isa histone chaperone with FACT-like activity and assists remode-ling of nucleosomes. EMBO J. 25, 1669–1679.

Arabidopsis Genome Initiative (2000) Analysis of the genomesequence of the flowering plant Arabidopsis thaliana. Nature,408, 796–815.

Axelos, M., Curie, C., Mazzolini, L., Bardet, C. and Lescure, B. (1992)A protocol for transient gene expression in Arabidopsis thali-anaprotoplasts isolated from cell suspension cultures. PlantPhysiol. Biochem. 30, 123–128.

Barakat, A., Szick-Miranda, K., Chang, I.F., Guyot, R., Blanc, G.,

Cooke, R., Delseny, M. and Bailey-Serres, J. (2001) The organ-ization of cytoplasmic ribosomal protein genes in the Arabidopsisgenome. Plant Physiol. 127, 398–415.

Barneche, F., Steinmetz, F. and Echeverria, M. (2000) Fibrillaringenes encode both a conserved nucleolar protein and a novelsmall nucleolar RNA involved in ribosomal RNA methylation inArabidopsis thaliana. J. Biol. Chem. 275, 27212–27220.

Bogre, L., Jonak, C., Mink, M. et al. (1996) Developmental and cellcycle regulation of alfalfa nucMs1, a plant homolog of the yeastNsr1 and mammalian nucleolin. Plant Cell, 8, 417–428.

Borisjuk, L., Walenta, S., Weber, H., Mueller-Klieser, W. and Wobus,

U. (1998) High-resolution histographical mapping of glucoseconcentrations in developing cotyledons of Vicia faba in relationto mitotic activity and storage processes: glucose as a possibledevelopmental trigger. Plant J. 15, 583–591.

Borisjuk, L., Rolletschek, H., Radchuk, R., Weschke, W., Wobus, U.

and Weber, H. (2004) Seed development and differentiation: arole for metabolic regulation. Plant Biol. 6, 375–386.

de Veylder, L., de Almeida Engler, J., Burssens, S., Manevski, A.,

Lescure, B., Van Montagu, M., Engler, G. and Inze, D. (1999)A new D-type cyclin of Arabidopsis thaliana expressed duringlateral root primordial formation. Planta, 208, 453–462.

Filipowicz, W. and Pogacic, V. (2002) Biogenesis of small nucleolarribonucleoproteins. Curr. Opin. Cell Biol. 14, 319–327.

Fromont-Racine, M., Senger, B., Saveanu, C. and Fasiolo, F. (2003)Ribosome assembly in eukaryotes. Gene, 313, 17–42.

Fujiwara, S., Matsuda, N., Sato, T., Sonobe, S. and Maeshima, M.

(2002) Molecular properties of a matrix attachment region-bind-ing protein located in the nucleoli of tobacco cells. Plant CellPhysiol. 43, 1558–1567.

Ginisty, H., Sicard, H., Roger, B. and Bouvet, P. (1999) Structure andfunctions of nucleolin. J. Cell Sci. 12, 761–772.

Gulli, M.P., Girard, J.P., Zabetakis, D., Lapeyre, B., Melese, T. and

Caizergues-Ferrer, M. (1995) gar2 is a nucleolar protein fromSchizosaccharomyces pombe required for 18S rRNA and 40Sribosomal subunit accumulation. Nucleic Acids Res. 23, 1912–1918.

Huddleson, J.P., Ahmad, N. and Lingrel, J.B. (2006) Upregulation ofthe KLF2 transcription factor by fluid shear stress requiresnucleolin. J. Biol. Chem. 281, 15121–15128.

Ito, T., Kim, G.T. and Shinozaki, K. (2000) Disruption of an Arabid-opsis cytoplasmic ribosomal protein S13-homologous gene bytransposon mutagenesis causes aberrant growth and develop-ment. Plant J. 22, 257–264.

Jang, J.C., Leon, P., Zhou, L. and Sheen, J. (1997) Hexokinase as asugar sensor in higher plants. Plant Cell, 9, 5–19.

Kim, K., Dimitrova, D.D., Carta, K.M., Saxena, A., Daras, M. and

Borowiec, J.A. (2005) Novel checkpoint response to genotoxicstress mediated by nucleolin–replication protein A complex for-mation. Mol. Cell. Biol. 25, 2463–2474.

Koch, K.E. (1996) Carbohydrate-modulated gene expression inplants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 509–540.

Kondo, K. and Inouye, M. (1992) Yeast NSR1 protein that hasstructural similarity to mammalian nucleolin is involved in pre-rRNA processing. J. Biol. Chem. 267, 16252–16258.

Li, Y., Lee, K.H., Walsh, S., Smith, C., Hadingham, S., Sorefan, K.,

Cawley, G. and Bevan, M.W. (2006) Establishing glucose- andABA regulated transcription networks in Arabidopsisby microar-ray analysis and promoter classification using a Relevance VectorMachine. Genome Res. 16, 414–427.

Lorenz, S., Tintelnot, S., Reski, R. and Decker, E.L. (2003)Cyclin D-knockout uncouples developmental progression fromsugar availability. Plant Mol. Biol. 53, 227–236.

Maceluch, J., Kmieciak, M. and Szweykowska-Kulinska, Z. (2001)Cloning and characterization of Arabidopsis thaliana AtNAP57 – ahomolog of yeast pseudouridine synthase Cbf5p. Acta Bio-chim.Pol. 48, 699–709.



Masumi, A., Fukazawa, H., Shimazu, T., Yoshida, M., Ozato, K.,

Komuro, K. and Yamaguchi, K. (2006) Nucleolin is involved ininterferon regulatory factor-2-dependent transcriptional activa-tion. Oncogene, 25, 5113–5124.

Matsuoka, K. and Nakamura, K. (1991) Propeptide of a precursor toa plant vacuolar protein required for vacuolar targeting. Proc. NatlAcad. Sci. USA, 88, 834–838.

Meier, U.T. (2005) The many facets of H/ACA ribonucleoproteins.Chromosoma, 114, 1–14.

Menand, B., Desnos, T., Nussaume, L., Berger, F., Bouchez, D.,

Meyer, C. and Robaglia, C. (2002) Expression and disruption ofthe Arabidopsis TOR (target of rapamycin) gene. Proc. Natl Acad.Sci. USA, 99, 6422–6427.

Mita, S., Suzuki-Fujii, K. and Nakamura, K. (1995) Sugar-inducibleexpression of a gene for b-amylase in Arabidopsis thaliana. PlantPhysiol. 107, 895–904.

Muller, J., Wiemken, A. and Aeschbacher, R. (1999) Trehalosemetabolism in sugar sensing and plant development. Plant Sci.147, 37–47.

Murray, J.A.H., Freeman, D., Greenwood, J. et al. (1998) PlantD cyclins and retinoblastoma protein homologues. In Plant CellDivision (Francis, D., Dudits, D. and Inze, D., eds). London: Port-land Press, pp. 99–127.

Nasirudin, K.M., Ehtesham, N.Z., Tuteja, R., Sopory, S.K. and

Tuteja, N. (2005) The Gly-Arg-rich C-terminal domain of peanucleolin is a DNA helicase that catalytically translocates in the5¢- to 3¢-direction. Arch. Biochem. Biophys. 434, 306–315.

Nishimura, T., Wada, T., Yamamoto, K.T. and Okada, K. (2005) TheArabidopsis STV1 protein, responsible for translation reinitiation,is required for auxin-mediated gynoecium patterning. Plant Cell,17, 2940–2953.

Oh, S.A., Lee, S.Y., Chung, I.K., Lee, C.H. and Nam, H.G. (1996) Asenescence-associated gene of Arabidopsis thaliana is distinc-tively regulated during natural and artificially induced leaf sen-escence. Plant Mol. Biol. 30, 739–754.

Pendle, A.F., Clark, G.P., Boon, R., Lewandowska, D., Lam, Y.W.,

Andersen, J., Mann, M., Lamond, A.I., Brown, J.W. and Shaw,

P.J. (2005) Proteomic analysis of the Arabidopsis nucleolus sug-gests novel nucleolar functions. Mol. Biol. Cell, 16, 260–269.

Pih, K.T., Yi, M.J., Liang, Y.S., Shin, B.J., Cho, M.J., Hwang, I. and

Son, D. (2000) Molecular cloning and targeting of a fibrillarinhomolog from Arabidopsis. Plant Physiol. 123, 51–58.

Price, J., Laxmi, A., St. Martin, S.K. and Jang, J.-C. (2004) Globaltranscription profiling reveals multiple sugar signal transductionmechanisms in Arabidopsis. Plant Cell, 16, 2128–2150.

Raught, B., Gingras, A.-C. and Sonenberg, N. (2001) The target ofrapamycin (TOR) proteins. Proc. Natl Acad. Sci. USA, 98, 7037–7044.

Reichler, S.A., Balk, J., Brown, M.E., Woodruff, K., Clark, G.B. and

Roux, S.J. (2001) Light differentially regulates cell division andthe mRNA abundance of pea nucleolin during de-etiolation. PlantPhysiol. 125, 339–350.

Riou-Khamlichi, C., Menges, M., Healy, J.M.S. and Murray, J.A.H.

(2000) Sugar control of the plant cell cycle: differential regulation

of Arabidopsis D-type cyclin gene expression. Mol. Cell. Biol. 20,4513–4521.

Rohde, J.R. and Cardenas, M.E. (2003) The Tor pathway regulatesgene expression by linking nutrient sensing to histone acetyla-tion. Mol. Cell. Biol. 23, 629–635.

Rolland, F., Baena-Gonzalez, E. and Sheen, J. (2006) Sugar sensingand signaling in plants: conserved and novel mechanisms. Annu.Rev. Plant Biol. 57, 675–709.

Rook, F., Weisbeek, P. and Smeekens, S. (1998) The lightregulatedArabidopsis bZIP transcription factor gene ATB2 encodes a pro-tein with an unusually long leucine zipper domain. Plant Mol.Biol. 37, 171–178.

Saez-Vasquez, J., Caparros-Ruiz, D., Barneche, F. and Echeverrıa,

M. (2004) A plant snoRNP complex containing snoRNAs, fibrilla-rin, and nucleolin-like proteins is competent for both rRNA genebinding and pre-rRNA processing in vitro. Mol. Cell. Biol. 24,7284–7297.

Schmelzle, T. and Hall, M.N. (2000) TOR, a central controller of cellgrowth. Cell, 103, 253–262.

Srivastava, M. and Pollard, H.B. (1999) Molecular dissection ofnucleolin’s role in growth and cell proliferation: new insights.FASEB J. 13, 1911–1922.

Tong, C.G., Reichler, S., Blumenthal, S., Balk, J., Hsieh, H.L. and

Roux, S.J. (1997) Light regulation of the abundance of mRNAencoding a nucleolin-like protein localized in the nucleoli of peanuclei. Plant Physiol. 114, 643–652.

Tsang, C.K., Bertram, P.G., Ai, W., Drenan, R. and Zheng, X.F.S.

(2003) Chromatin-mediated regulation of nucleolar structure andRNA polymerase I localization by TOR. EMBO J. 22, 6045–6056.

Tuteja, R. and Tuteja, N. (1998) Nucleolin: a multifunctional majornucleolar phosphoprotein. Crit. Rev. Biochem. Mol. Biol. 33, 407–436.

van Lijsebettens, M., Vanderhaeghen, R., de Block, M., Bauw, G.,

Villarroel, R. and van Montagu, M. (1994) An S18 ribosomalprotein gene copy at the Arabidopsis PFL locus affects plantdevelopment by its specific expression in meristems. EMBO J. 13,3378–3388.

Weijers, D., Franke-van Dijk, M., Vencken, R.J., Quint, A., Hooykaas,

P. and Offringa, R. (2001) An Arabidopsis Minute-like phenotypecaused by a semi-dominant mutation in a RIBOSOMAL PROTEINS5 gene. Development, 128, 4289–4299.

Wingler, A., Fritzius, T., Wiemken, A., Boller, T. and Aeschbacher,

R.A. (2000) Trehalose induces the ADP-glucose pyrophosphory-lase gene, ApL3, and starch synthesis in Arabidopsis. PlantPhysiol. 124, 105–114.

Yamada, H., Koizumi, N., Nakamichi, N., Kiba, T., Yamashino, T. and

Mizuno, T. (2004) Rapid response of Arabidopsis T87 culturedcells to cytokinin through His-to-Asp phosphorelay signal trans-duction. Biosci. Biotechnol. Biochem. 68, 1966–1976.

Yoine, M., Ohto, M., Onai, K., Mita, S. and Nakamura, K. (2006)The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic chan-ges and altered sugar signaling in Arabidopsis. Plant J. 47, 49–62.

