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.
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In addition to growth regulators such as auxin and cytokinin, nutrient availability is an important factor limiting cell proliferation 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 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–cytoplasmic 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 possesses 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, nucleolin-like proteins from alfalfa (Medicago sativa; Bögre et al., 1996), pea (Pisum sativum; Tong et al., 1997; Reichler et al., 2001) and Arabidopsis (Sáez-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 (Bögre et al., 1996). Light induces the expression of nucleolin in alfalfa and pea, which is probably mediated by phytochrome (Bögre 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 carbohydrate 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.
Nucleolin genes of Arabidopsis thaliana
Of the sugar-induced genes that we identified by microarray 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 (Bögre et al., 1996) and pea (Tong et al., 1997). The Arabidopsis genome also contains another gene for nucleolin, At3g18610. The proteins encoded by At1g48920 and At3g18610 are identical with AtNuc-L1 and AtNuc-L2, respectively, described previously (Sáez-Vasquez et al., 2004). The AtNuc-L1 and AtNuc-L2 genes are located on segment 1, a large duplicated segment between chromosomes 1 and 3, respectively (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 expressing 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 occasionally 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 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 μm ABA, were ineffective in inducing AtNuc-L1 mRNA. Similar results were obtained by treatment of seedlings with sugars. Trehalose metabolism plays important roles in sugar sensing and plant development (Müller 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 modification 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-uridylation 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-methylation 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.
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 approximately 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, AtNuc-L2 and AtNop56-2 (At1g56110) did not give significant 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 immunoblotting with an anti-AtNuc-L1 antibody (Sáez-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 (Sáez-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 ΔAtNuc-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 ΔAtNuc-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 μg 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 ΔAtNuc-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 ΔAtNuc-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 ΔAtNuc-L1-1 than in Col plants.
Growth and developmental phenotypes of ΔAtNuc-L1-1 plants
The ΔAtNuc-L1-1 plants showed various growth and developmental phenotypes. The growth of roots of ΔAtNuc-L1-1 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 stunted (Figure 5b). The ΔAtNuc-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 ΔAtNuc-L1-1 seedlings had a narrow pointed shape in contrast to the round shape of Col leaves (Figure 5b), and showed abnormal vascular patterns (Figure 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, Arabidopsisminute-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 ΔAtNuc-L1-1 plants were shorter and irregular in size and shape compared with those of Col plants (Figure 5g). Furthermore, the stalks of the ΔAtNuc-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 (Nishimura et al., 2005). These results suggest that ΔAtNuc-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 ΔAtNuc-L1-1 plants
Because ΔAtNuc-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 ΔAtNuc-L1-1 seedlings treated with 5% Suc or H2O. Similar results were obtained in three independent experiments for Col plants and two independent experiments for ΔAtNuc-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 ΔAtNuc-L1-1 plants (Figure 6a). For example, the mRNAs for AtRPL34A, AtRPL27A, AtRPL27C, 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 ΔAtNuc-L1-1 plants (Figure 6b-1). The signal values for RP transcripts in H2O-treated seedlings were not noticeably different between Col and ΔAtNuc-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 ΔAtNuc-L1-1 plants.
Comparison of the scatter plots for Col and ΔAtNuc-L1-1 plants (Figure 6a) suggests that there was a general reduction in the magnitude of sugar-responsive changes in the mRNA level in ΔAtNuc-L1-1 plants. Nevertheless, expression of typical sugar-inducible genes such as β-amylase (Atβ-Amy; Mita et al., 1995), ADP-glucose pyrophosphorylase S, 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 β-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 (Figure 6b-3). These results suggest that the reduction in sugar-inducible expression of RP genes in ΔAtNuc-L1-1 is selective.
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 identified in recent proteomic analyses of the Arabidopsis nucleolus (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 protein. This induction of AtNuc-L1 by sugar occurs in conjunction 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 involved in pre-mRNA splicing. These results suggest that sugar regulation of AtNuc-L1 expression is closely associated with the regulation of de novo ribosome synthesis.
Sugar induction of AtNuc-L1 and its involvement in ribosome synthesis are consistent with the selective expression of the nucleolin gene in the meristem region (Bögre 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 Arabidopsis 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 expression 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 Physcomytrella patens, however, suggested that sugar regulation of CycD2 is more closely related to the developmental progression 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 ΔAtNuc-L1-1 null mutant showed an increased steady-state level of pre-rRNA relative to mature 25S rRNA compared to Col. This difference 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 ΔAtNuc-L1-1 plants have a shortage of ribosomes is supported by its phenotypes. In addition to reduced growth rate, ΔAtNuc-L1-1 plants exhibited various developmental phenotypes that overlap with those of previously 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 pointed leaves, abnormal vascular patterning, and defective gynoecium development. Thus, the synthesis of ribosomes might be limited in both ΔAtNuc-L1-1 and RP mutants.
The ΔAtNuc-L1-1 plants showed reduced growth rate and lived longer than Col plants. The reduced growth rate of ΔAtNuc-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 ΔAtNuc-L1-1 plants showed outgrowth of axillary organs and bushy growth. A reduced rate of organ development may alter the distribution of photoassimilates among meristems of ΔAtNuc-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.
The sugar-enhanced expression of RP genes was severely diminished in ΔAtNuc-L1-1 plants compared with Col plants. Although we observed a general diminution in the sugar-induced changes of the transcript levels in ΔAtNuc-L1-1 plants, expression of typical sugar-inducible or -repressible genes, e.g. Atβ-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 ΔAtNuc-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 ΔAtNuc-L1-1 is probably due to effects on transcription. 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 ΔAtNuc-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 (Sáez-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 polymerase 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 facilitates 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.
Plant materials and treatment with sugars
Seeds of Arabidopsis thaliana (L.) Heynh. (ecotype Col-0) were surface-sterilized, kept at 4°C for 3 days in sterile water, and sown on 0.3% gellan gum plates containing Murashige and Skoog medium (pH 5.7), 100 mg l−1myo-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 were incubated in a growth chamber at 22°C under continuous light of 65 μmol m−2 sec−1. Mature leaves of the 3-week-old plants were excised with a sharp razor blade, and the cut edges of petioles were immersed in a sterile solution of sugar or water and incubated at 22°C in the dark (Mita et al., 1995). Plants were also grown on vermiculite at 22°C under continuous light. Plants were watered with Hoagland's nutrient solution every week and water as needed.
A primary PCR screen for a T-DNA insertion mutant of AtNuc-L1 was performed on pooled chromosomal DNA from approximately 20 000 individual lines of a collection of Arabidopsis T-DNA insertion lines (Kazusa DNA Research Institute, Kazusa, Japan) using gene-specific primers and T-DNA border primers. For genotyping of ΔAtNuc-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 http://www.wako-chem.co.jp), 1 ml l−1 of diluted Gamborg's vitamin solution (Sigma-Aldrich, http://www.sigmaaldrich.com/), 0.5 g l−1 of 2-morpholinoethanesulfonic acid monohydrate, 1 μm 1-naphthalene acetic acid and 1.5% Suc (w/v). The 6-day-old cells were collected by centrifugation, and washed three times with fresh medium 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 using the primer set shown in Table S1. The cDNA was cloned into pGWB5 or pGWB6 vectors containing the coding sequence for sGFP by GatewayTM cloning technology (Invitrogen, http://www.invitrogen.com/) to produce binary Ti plasmids carrying genes encoding AtNuc-L1–GFP and GFP–AtNuc-L1 fusion proteins under the control of the CaMV 35S promoter, respectively. These plasmids were used to transform tobacco BY-2 cells as described previously (Matsuoka and Nakamura, 1991). Several independent transformed calli were brought into suspension culture, and GFP fluorescence was observed using an FV500 confocal fluorescence microscope (Olympus, http://www.olympus-global.com/). The same Agrobacterium strains were used to transform Arabidopsis plants, and roots of T2 plants from several independent transformed lines were stained with propidium iodide and observed for GFP fluorescence by confocal fluorescence microscopy.
Isolation of RNA, RT-PCR and real-time RT-PCR
Total RNA was isolated from plants or tissues using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com) and dissolved in ribonuclease-free water. For detection of mRNAs, first-strand cDNA was synthesized from 2 μg of total RNA using oligo(dT)20 primers and Superscript III (Invitrogen) and diluted with four volumes of water. PCR was performed in a 25 μl mixture containing 2 μl of the diluted cDNA solution and 0.4 μm of each primer. The PCR reaction cycles were as follows: denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The number of cycles was optimized for each mRNA. For quantitative real-time RT-PCR, PCR was performed with iQ SYBR Green Supermix using an iCycler iQ (Bio-Rad, http://www.bio-rad.com/), and the comparative threshold cycle 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 isolated from 3-week-old Col and ΔAtNuc-L1-1 plants. For quantification of 25S, 18S and 5.8S rRNA, cDNAs were synthesized from 200 pg of total RNA with specific primers in a 20 μl reaction, whereas 1 μg of total RNA was used for the synthesis of cDNA with specific primers for quantification of ETS and ITS sequences of pre-rRNA. Quantitative real-time RT-PCR was carried out using the primer sets listed in Table S1.
Oligo microarray analysis
Total RNAs used for microarray analyses were prepared using Trizol reagent (Invitrogen http://www.invitrogen.com) and subsequently purified using an RNeasy Plant Mini Kit (Qiagen). Cy3- and Cy5-labeled cDNA probes were synthesized and hybridized to the Agilent Arabidopsis-1 and -2 oligo microarrays (Agilent Technologies http://www.home.agilent.com) according to the manufacturer's instructions. The microarray analysis was performed with two or three independently isolated RNA samples and assessed in each experiment 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 been treated with 5% Suc or H2O for 24 h. SDS–PAGE was carried out in an 8% acrylamide gel. The proteins were transferred from the gel to a poly(vinylidene difluoride) membrane (Immobilon; Millipore http://www.millipore.com), and the antigen on the membrane was detected with primary antibodies, followed by horseradish peroxidase-coupled protein A and chemiluminescence reagents (ECL kit; GE Healthcare http://www.gehealthcare.com). Antibodies raised against recombinant AtNuc-L1 (AtNuc-L1) and AtFib1 (Sáez-Vasquez et al., 2004) were used, and the antibody against recombinant NtMARBP61 (Fujiwara et al., 2002) was a generous gift from Dr Masayoshi Maeshima of Nagoya University.
We thank S. Ukai and T. Kawai for technical assistance, M. Maeshima of Nagoya University for anti-NtMARBP61, and K. Shinozaki of RIKEN for T87 cells. This work was supported in part by the Research for the Future program of the Japan Society for the Promotion of Science (grant number 00L01603) and the 21st Century COE program from the Ministry of Education, Science, Sports and Culture of Japan to K.N.