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EBP1 regulates organ size through cell growth and proliferation in plants

2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês

10.1038/sj.emboj.7601362

1460-2075

Beátrix Horváth, Zoltán Magyar, Yuexing Zhang, Anne W. Hamburger, László Bakó, Richard G. F. Visser, C. Bachem, László Bögre,

Plant tissue culture and regeneration

Article5 October 2006free access EBP1 regulates organ size through cell growth and proliferation in plants Beatrix M Horváth Corresponding Author Beatrix M Horváth Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Zoltán Magyar Zoltán Magyar School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, UK Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Yuexing Zhang Yuexing Zhang Department of Pathology and Greenebaum Cancer Centre, School of Medicine, University of Maryland, Baltimore, MD, USA Search for more papers by this author Anne W Hamburger Anne W Hamburger Department of Pathology and Greenebaum Cancer Centre, School of Medicine, University of Maryland, Baltimore, MD, USA Search for more papers by this author László Bakó László Bakó Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden Search for more papers by this author Richard GF Visser Richard GF Visser Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Christian WB Bachem Christian WB Bachem Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author László Bögre László Bögre School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, UK Search for more papers by this author Beatrix M Horváth Corresponding Author Beatrix M Horváth Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Zoltán Magyar Zoltán Magyar School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, UK Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Yuexing Zhang Yuexing Zhang Department of Pathology and Greenebaum Cancer Centre, School of Medicine, University of Maryland, Baltimore, MD, USA Search for more papers by this author Anne W Hamburger Anne W Hamburger Department of Pathology and Greenebaum Cancer Centre, School of Medicine, University of Maryland, Baltimore, MD, USA Search for more papers by this author László Bakó László Bakó Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden Search for more papers by this author Richard GF Visser Richard GF Visser Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Christian WB Bachem Christian WB Bachem Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author László Bögre László Bögre School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, UK Search for more papers by this author Author Information Beatrix M Horváth 1, Zoltán Magyar2,3, Yuexing Zhang4, Anne W Hamburger4, László Bakó3,5, Richard GF Visser1, Christian WB Bachem1,‡ and László Bögre2,‡ 1Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands 2School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, UK 3Institute of Plant Biology, Biological Research Centre, Szeged, Hungary 4Department of Pathology and Greenebaum Cancer Centre, School of Medicine, University of Maryland, Baltimore, MD, USA 5Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden ‡These authors contributed equally to this work *Corresponding author. Department of Biology, Section of Molecular Genetics, Utrecht University, Padualaan 8, 3584CH Utrecht, The Netherlands. Tel.: +31 30 253 2245; Fax: +31 30 251 3655; E-mail: [email protected] The EMBO Journal (2006)25:4909-4920https://doi.org/10.1038/sj.emboj.7601362 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Plant organ size shows remarkable uniformity within species indicating strong endogenous control. We have identified a plant growth regulatory gene, functionally and structurally homologous to human EBP1. Plant EBP1 levels are tightly regulated; gene expression is highest in developing organs and correlates with genes involved in ribosome biogenesis and function. EBP1 protein is stabilised by auxin. Elevating or decreasing EBP1 levels in transgenic plants results in a dose-dependent increase or reduction in organ growth, respectively. During early stages of organ development, EBP1 promotes cell proliferation, influences cell-size threshold for division and shortens the period of meristematic activity. In postmitotic cells, it enhances cell expansion. EBP1 is required for expression of cell cycle genes; CyclinD3;1, ribonucleotide reductase 2 and the cyclin-dependent kinase B1;1. The regulation of these genes by EBP1 is dose and auxin dependent and might rely on the effect of EBP1 to reduce RBR1 protein level. We argue that EBP1 is a conserved, dose-dependent regulator of cell growth that is connected to meristematic competence and cell proliferation via regulation of RBR1 level. Introduction Morphogenesis in plants is largely postembryonic, and along with organ growth is influenced by environmental factors, such as light or nutrient availability (Ingram and Waites, 2006). Moreover, plant growth is intimately connected to the capacity of source organs to produce assimilates. This regulatory mechanism determines the yield potential for harvested organs in agricultural crops. In potato (Solanum tuberosum), the separation of source and sink organs illustrates the long-distance regulation of organ growth through the interplay of assimilates such as sucrose and other growth factors produced in the source and sink organs (Bologa et al, 2003). The capacity for growth of plant organs is determined by zones of proliferating cells, called meristems. When cells leave the meristematic zone they begin to exit the cell cycle and undergo differentiation that is accompanied by increases in cell size. Cell expansion is facilitated by the loosening of crosslinks between cell wall polymers accompanied by water uptake to vacuoles, and frequently by endoreduplication of DNA (Sugimoto-Shirasu and Roberts, 2003). Therefore, the timing of the transition between proliferative growth and cell expansion/differentiation, largely determines the final cell number and so the size potential of the organ. Differences in organ size between closely related species, such as rapeseed and Arabidopsis or differential responses to environmental conditions tend to reflect cell number rather than cell size variation (Beemster et al, 2003). The control of cell size is best understood in yeast where the attainment of a cell size threshold triggers the initiation of cell division. This coordination is thought to be regulated through the translational machinery which, in turn, is determined by the nutritional state (Jorgensen and Tyers, 2004). In plants, remarkably uniform cell sizes in both meristematic regions and young developing organs also indicate the existence of an intricate regulation of cell growth and cell division. However, their coordination is poorly understood (Beemster et al, 2003). A mechanism for such coordination could occur via the TCP transcription factors that coregulate the expression of genes coding for the translational apparatus and cell cycle genes (Ingram and Waites, 2006). In multicellular organisms, it is debated whether the factors determining growth impose their influence on organs as a whole or whether they regulate growth and proliferation at the cellular level (Tsukaya and Beemster, 2006). Moreover, cells in multicellular organisms do not proliferate logarithmically. Their growth and proliferation requires coordination with other parts of the organism mediated by growth and mitogenic factors that may impinge on growth regulation (ribosome biogenesis) and cell cycle control (DNA replication) separately (Conlon and Raff, 1999). The nucleolus is the main site of ribosome biosynthesis. In humans, EBP1, the ErbB-3 epidermal growth factor receptor binding protein has been shown to be a nucleolar dsRNA binding protein; forming part of the ribonucleoprotein complexes via association with different rRNA species (Squatrito et al, 2004). EBP1 was also shown to associate with mature ribosomes and to block the stress-induced phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and thus presumably sustain protein translation (Squatrito et al, 2006). Furthermore, EBP1 is a nuclear cell survival factor that together with the protein kinases, Akt and PKC inhibit apoptosis (Ahn et al, 2006). Surprisingly, ectopic expression of EBP1 decreased proliferation rate and induced cellular differentiation in cultured human breast carcinoma cell lines (Lessor et al, 2000). As the effect on proliferation was linked to its nucleolar localisation in human fibroblasts, it was suggested that EBP1 may represent a new link between ribosome biosynthesis and cell proliferation (Squatrito et al, 2004). Studying tuber organogenesis in potato upon sucrose induction, we have identified StEBP1, the potato homologue of the human EBP1 gene. We show, via modulating the level of EBP1 in potato and Arabidopsis, that this gene regulates plant organ growth, effects the expression of different cell cycle genes and influences RBR1 protein level. Furthermore, we demonstrate that auxin regulates the protein stability of EBP1 through which it may influence plant growth. Results Induction of gene expression during potato tuber initiation Potato tubers are formed at the termini of stolons as a result of internal and external cues such as light period and assimilate supply. We have used an in vitro system to study the molecular mechanisms that control tuber development and growth (Bachem et al, 1996). In the course of this work, a transcript-derived fragment was identified (TDF1044) that showed a transient elevation in abundance during early stages of tuberisation (Supplementary Figure 1A). The expression pattern of the gene represented by TDF1044 was confirmed by microarray data (Supplementary Figure 1B; Kloosterman et al, 2005). Northern analysis was carried out on tissues of different developmental stages using the TDF1044 as a probe (Supplementary Figure 1C). Its expression is correlated with growth and cell division activities of a range of organs. This ubiquitous expression profile suggests that this gene is functionally not limited to tuber development, but correlates with actively growing tissues. TDF1044 is homologous to the human EBP1, a conserved cell proliferation and cell growth-related protein In order to identify the full-length potato mRNA corresponding to TDF1044, a cDNA library derived from swelling stolon tips was screened (Taylor et al, 1992). The isolated 1.5 kb full-length cDNA codes for a 43 kDa protein and shows sequence similarity (69%) to the human EBP1 (Yoo et al, 2000). The gene was named StEBP1 accordingly. Two major groups of ESTs were identified in the current potato TIGR-EST database; one is showing 100% homology (TC126314), whereas the other (TC128561) shares 87% similarity with StEBP1. It is not clear whether the second group of ESTs are derived from an allele within the tetraploid potato or from another gene family member. The StEBP1 protein shows high similarity (89%) to the Arabidopsis G2p protein encoded by a single copy gene at locus At3g51800 (unigene10184), that we name AtEBP1. The structurally and functionally conserved amino-acid sequences are shown in Supplementary Figure 2. The functional conservation of StEBP1 was tested by studying the effect of its overexpression on colony formation (Figure 1A and B) and on E2F-dependent gene expression (Figure 1C) in the MCF-7 human breast cancer cell line. Cells were transfected with increasing concentrations (0.25, 0.5 and 1 μg) of plasmid coding for the full-length protein (Figure 1A) or a truncated version of the protein starting from the second in-frame ATG (Figure 1B). As a control, 1 μg of the vector p3xFLAG-CMV10 was used. The full-length StEBP1 significantly (P<0.05) reduced colony formation in a dose-dependent manner when compared to the vector alone (Figure 1A). The colony inhibition with StEBP1 is similar to that found with the human EBP1 (Lessor et al, 2000). Interestingly, transfection with the truncated StEBP1 resulted in a more pronounced inhibition of colony formation (Figure 1B). Figure 1.StEBP1 inhibits colony formation and represses E2F-mediated transcription in MCF7 human cell line. Colony formation was inhibited when cells were transfected with varying amounts (A) of the full-length StEBP1 and (B) the truncated Δ-StEBP1 plasmids and compared to the control CMV-10 construct. (C) Expression of StEBP1 or the Δ-StEBP1 repressed the activity of the E2F1 promoter-luciferase reporter when compared to the CMV-10 control. The data are shown as a ratio between the firefly and the control cotransfected Renilla luciferase activities; relative luciferase unit (RLU). Error bars represent standard deviations. (D) StEBP1 protein expression in transfected human cells was detected with the FLAG-M2 monoclonal antibody. Molecular mass (50 kDa) is indicated. Download figure Download PowerPoint To test whether StEBP1 can repress the expression of E2F-dependent genes, human cells were cotransfected with the StEBP1 constructs together with an E2F1-luciferase reporter (Cress and Nevins, 1996). The truncated version of StEBP1 was significantly more potent in suppressing E2F1 promoter activity than the full-length version (Figure 1C). The presence of the StEBP1 proteins was verified using Western blot analysis (Figure 1D). From these results, we conclude that StEBP1 is conserved both structurally and functionally with the human EBP1 protein. EBP1 regulates plant growth in a dose-dependent manner To elucidate the function of the EBP1 gene, its expression was altered in potato and Arabidopsis plants. From around 100 antisense Stebp1(as) potato lines, 11 showed growth retardation. Three independent lines, chosen for further characterisation, were smaller in their final height, as well as the tuber yield was lower than in the nontransformed control (Supplementary Figure 3A, B). Of 51 regenerated RNA interference lines, Stebp1(RNAi), 11 showed a similar but more pronounced phenotype than the antisense lines. Plants such as the Stebp1-12(RNAi) (Figure 2A and E) were severely dwarfed and showed hardly any growth on soil. Lines, such as Stebp1-13, -14 and -67(RNAi) had a medium phenotype and were stunted and grew slower during the entire course of their development, when compared to control plants (Figure 2A and E). Other lines, such as Stebp1-65(RNAi) did reach a similar height to the wild type (Figure 2E). Figure 2.Alteration in expression of the StEBP1 effects growth in S. tuberosum transgenic lines. (A) Silencing of the StEBP1 inhibited growth in height of the Stebp1(RNAi) lines; i-12, i-13, i-14, i-67 whereas (B) overexpression of StEBP1 in Stebp1(oe) lines, OE-3, OE-5, OE-16 led to increased height compared to the control wild-type (wt) under greenhouse conditions. (C) Silencing of StEBP1 mRNA level in the antisense Stebp1 lines (as-23, as-78, as-81) and Stebp1(RNAi) lines (i-12, i-13, i-14, i-65, i-67) was determined by qRT-PCR. The level of expression is shown as a ratio compared to the wild type given the value of 1. (D) The elevated level of StEBP1 was detected using the myc-antibody in protein extracts of the overexpression lines (OE-3, OE-5 and OE-16). Molecular mass (55 kDa) is indicated. (E) Potato plants with strong (i-12), intermediate (i-13) and a weak phenotype (i-65) of the Stebp(RNAi) lines are compared to the wt and to the Stebp1(oe) line (OE-5) grown in the climate chamber. (F) The morphology of the apical area at different developmental stages and (G) the leaf morphology of the Stebp1-13(RNAi) is compared to the control (wt). (H) Average weight of tubers per plant in the Stebp(RNAi) lines. (I) Tuber yield for four representative plants of wt and i-13 are shown. Error bars represent standard deviations. Bars on F and I=10 cm. Download figure Download PowerPoint Quantitative real-time PCR (qRT-PCR) was carried out to determine whether these alterations to the phenotype were directly correlated to the reduction of the corresponding mRNA levels. As shown in Figure 2C and Supplementary Figure 3, the level of the StEBP1 mRNA was 1.5–2-fold less in the Stebp1(as) lines, whereas it decreased between 8 and 12-fold compared to the endogenous wild-type level in the different Stebp1(RNAi) lines. Comparison between the Stebp1(as) and Stebp1(RNAi) lines should, however, be drawn independently as the plants were grown at different times and environmental conditions. Leaf size and morphology were altered both in the Stebp1(as) and the Stebp1(RNAi) lines. Wild-type potato has a compound leaf structure (Figure 2F and G). Leaves of the Stebp1(RNAi) lines have a reduced number of leaflet pairs, more comparable to younger leaves in the control plants. The total surface area of the leaves was reduced compared to the wild type, although the individual leaflets from the top nodes were somewhat larger (Figure 2F and G and Figure 4A). The wild-type leaf lamella is smooth, whereas in contrast, the morphology of the individual leaflet in the silenced lines was convex with edges curling downwards, giving a folded, compact structure (Figure 2G, i-13). At later stages of development, the tops of the plants were foreshortened and deformed resulting in zigzag internodal stem growth with unopened curled leaves turning towards the stem (Figure 2F, i-13). The size of individual tuber and tuber yield per plant were also reduced and tuber morphology was abnormal (Figure 2H and I). Plants with elevated expression of StEBP1 showed normal development, but reached a greater final height compared to the control (Figure 2B and E). Overexpressed StEBP1 protein in the different Stebp1(oe) lines was detected on Western blots via the myc-epitope (Figure 2D). The increase in StEBP1 transcript level is shown in Supplementary Figure 3D. EBP1 was also silenced and overexpressed in Arabidopsis. Eighteen out of 80 kanamycin-resistant T1 Atebp1(RNAi) lines displayed distorted growth, 10 of these did not reach maturity and only three produced homozygous T1 seeds. In a similar way, we also obtained three homozygous Atebp1(oe) lines. As shown in Figure 3A, the reduction of endogenous AtEBP1 mRNA resulted in smaller plants, whereas the increase in EBP1 led to enlarged plant size compared to the wild-type Columbia control. Images of three parallel plants from each transgenic line and measurements of the canopy area convincingly show the size differences (Supplementary Figure 4). At the seedling stage, a delay in leaf initiation and distorted leaf shape were characteristic of the silenced lines (Figure 3B). Using qRT-PCR, we found a four-fold reduction of AtEBP1 level in a representative Atebp-1(RNAi) line (Figure 3C) whereas the Atebp1(oe) lines contained significantly higher levels of both EBP1 mRNA and protein than the control (Figure 3D and E, respectively). These results are in good agreement with the data obtained for the potato Stebp1 transgenic lines. Figure 3.Altered expression level of EBP1 results in changes in growth habit in Arabidopsis transgenic lines. (A) Silencing of the AtEBP1 causes growth retardation in Atebp-1(RNAi), referred as Ati-1, whereas overexpression of the StEBP1 in Atebp-12(oe) line, labelled as AtOE12 leads to larger plants compared to the control Columbia. The plants were 3 weeks old. (B) Silencing of AtEBP1 delays leaf initiation and alters leaf morphology, as shown by representatives of Atebp1-1(RNAi), and Atebp1-5(RNAi) lines compared to the wild type photographed 10 days after germination. (C) Silencing of the AtEBP1 expression is shown for the representative line Atebp1-1(RNAi) and (D) the elevated expression of StEBP1 is shown in the overexpression lines (AtOE-12, AtOE-19, AtOE-43). RNA for qRT-PCR was isolated from the second to fourth leaves of the same set of plants shown in (A). The expression level of EBP1 is standardised to the level of the Arabidopsis actin2 gene. The relative expression is given as a ratio compared to the endogenous EBP1 transcript, set to unit 1 in the control. (E) The presence of EBP1 in Atebp1(oe) lines (AtOE-12, AtOE-19, AtOE-43) was confirmed with Western blot analysis using myc antibody. Molecular mass (55 kDa) is indicated. Error bars represent standard deviations. Asterisk indicates a cross-reacting protein band with the myc antibody. As a loading control, amido-black staining of the corresponding membrane is shown. Download figure Download PowerPoint Figure 4.Leaf size, cell size, total cell number per leaflet (cell number index) and cell shape factor in the Stebp1 transgenic lines. (A) Stebp1(RNAi) lines (i-13, i-65, i-67) and (B) Stebp1(oe) lines (OE-3 and OE-5) compared to the wild type (wt). Columns represent values for each successive leaflet from the 6th, 8th, 10th and 12th nodes and coloured in shades of grey. The error bars refer to standard deviations of the values except in the cell number index, where the standard error was calculated. (C) Representative images from the adaxial epidermal cell layer of the sixth leaflet and (D) of the 12th leaflet from the Stebp1-13(RNAi), wild type and Stebp1-5 (oe) are shown for illustration. Bar=100 μM. Download figure Download PowerPoint StEBP1 regulates both cell number and cell size in developing leaves Leaves positioned along the potato stem represent consecutive developmental stages, and therefore provide a suitable experimental system to study the developmental regulation of organ growth. The size of the leaflets in the wild-type plant gradually increases from the sixth to the 12th node. In the Stebp1(RNAi) lines, leaflets at the sixth node were slightly larger than the equivalent control. At subsequent developmental stages, leaf growth ceased between leaf nodes 8 and 12 in lines Stebp1-13(RNAi) and Stebp1-67(RNAi), whereas leaves in Stebp1-65(RNAi) continued to grow, although their size stayed behind the equivalent wild type (Figure 4A). To determine the basis of the observed organ size differences, cell size was measured in the leaves of these lines (Figure 4, Supplementary Table 2). The cell size increased gradually between sixth and 12th leaves in the wild type. In the sixth nodal leaf of the RNAi lines, the cell size was ∼30% larger. In 8–12th node leaves, the size of the pavement cells remained smaller in the Stebp1-13(RNAi) and Stebp1-67(RNAi) lines compared to the wild type, but was similar in line Stebp1-65(RNAi), correlating with the degree of silencing (Figure 2C). In summary, in young developing leaves, cell size becomes larger in the Stebp1 silenced lines, whereas at later stages, during expansion growth, the cell size falls behind the wild type. Using statistical analysis, the differences in cell size were significant here and in subsequent data sets. Numerical presentation of the data is summarised in Supplementary Table 2. In order to better understand the relationship between leaf and cell size, an index for the total cell number in the investigated leaflet was calculated by dividing the leaflet surface area with the average cell size. We were aware of the fact that the size of cells in different areas of the leaves is variable, but we standardised our sampling procedure as much as possible. The total cell number per leaflet gradually increased with the developmental stages in the wild type. The comparison of several data sets revealed that the total cell number per leaflet generally reaches a plateau of around 106 cells per leaflet. We thus assume that after this number, no more cell division occurs. Leaf growth then continues through cell expansion (Figure 4A and B). In contrast, in Stebp1-13(RNAi) and Stebp1-67(RNAi) lines there was no increase in the total cell number between sixth and 12th leaves, indicating that leaf growth in these lines is attributed to cell expansion across all the developmental stages. In line Stebp1-65(RNAi), the total cell number initially increased from leaves sixth to eighth, after which it levelled off. Interestingly, when Stebp1-13(RNAi) was grown under greenhouse conditions, these plants showed similar stability in the total cell number, but an abnormal cell expansion led to a larger leaf size (Supplementary Figure 5). The phenomenon of counteracting the block in cell proliferation by cell expansion is known as compensation (Horiguchi et al, 2006). Our experiments show that compensation can be environmentally dependent. During differentiation, pavement cells gain a more complex shape, becoming longitudinally expanded featuring a lobed structure. The shape complexity was quantified by calculating the ‘shape factor’ (4π area/perimeter2), which is defined as 1 for a perfect circle and decreases for more complex shapes. During development, wild-type cells adopt more complex shapes as they differentiate (Figure 4A and B). Pavement cells in the Stebp1(RNAi) lines were arrested in cell division and although they expanded, the complexity of their shape did not increase (Figure 4C and D, sixth and 12th leaves, respectively). We analysed leaves of Stebp1(oe) lines in order to learn whether StEBP1 is sufficient to drive cell and organ growth. These lines had a larger leaflet surface area when compared to the corresponding leaflets in the controls (Figure 4B). Surprisingly, the cell size of the overexpression lines in the youngest sixth leaf was around half of the size of the wild-type cells whereas at later stages cell sizes surpassed the wild type, in parallel with the accelerated organ growth. The total cell number per leaflet reached a higher level at an earlier stage than in the wild type, indicating that the switch between meristematic and expansion growth is shifted to an earlier developmental stage (Figure 4B). Overexpression of the StEBP1 also brought forward differentiation (Figure 4B and D). In summary, elevated StEBP1 level leads to an increase in the number of cells at early stages of leaf development, ceasing later on in development when further organ growth occurs via a boost in cell expansion and differentiation. EBP1 regulates RBR1 protein level and the expression of cell cycle regulators in an auxin-dependent manner To understand how cell division is arrested during leaf development when the StEBP1 mRNA level is reduced, the expression of critical cell cycle regulators of the G1 to S and G2 to M transitions were followed. QRT-PCR was carried out using primers for the potato homologues of the Arabidopsis CYCD3;1 (Dewitte et al, 2003), RNR2 (ribonucleotide reductase) (Chaboute et al, 2000), CDKB1;1 (Boudolf et al, 2004) genes. Two independent lines, Stebp1-13 and -67(RNAi) and a control plant were chosen to detect the expression in the apex (pool of the meristem, the first and second nodal leaves) and in leaves from the sixth and tenth nodes. The expression of different genes was quantified as the difference in the cycle numbers (ΔCT) in the qRT-PCR experiments between the gene of interest and the constitutive Ubiquitine gene (Figure 5A). In the apex of young, developing plants, no significant difference was detected in the expression level of the CDKB1;1 and RNR2 genes, whereas the level of CYCD3;1 mRNA was somewhat lower in the Stebp1(RNAi) plants compared to the control (Figure 5A). However, in young developing leaves, the abundance of all the three cell cycle regulators was reduced compared to the control (Figure 5A). Although the expression of these cell cycle genes naturally diminishes as leaves develop, their relative levels in the Stebp1(RNAi) lines remained below the control even in the fully developed leaves (Figure 5A). Thus, the effect of StEBP1 gene silencing reduces the expression of cell cycle regulators in a developmentally dependent manner. Figure 5.Altered level of StEBP1 affects the expression of cell-cycle-related genes. (A) The expression levels of CYCD3, CDKB1:1 and RNR2 mRNAs were determined by qRT-PCR in the apex (meristem, first and second leaves sampled together) and in the sixth and 10th leaves taken from the Stebp1-13(RNAi), Stebp1-67(RNAi) and the wild-type control. The expression levels are given as a difference in cycle numbers during qRT-PCR of the genes of interest and Ubiquitin (ΔCT). (B) The expression levels of these cell-cycle-related transcripts in the Stebp1-5(oe) line and the wild type were similarly determined as in (A) in the meristem sampled together with the first leaf primordium (M+1), and in the second, third, fifth and