Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1–E2F pathway activity
2010; Springer Nature; Volume: 29; Issue: 17 Linguagem: Inglês
10.1038/emboj.2010.164
ISSN1460-2075
AutoresRossana Henriques, Zoltán Magyar, Antonia Monardes, Safina Khan, Christine Zalejski, Juan Orellana, László Szabados, Consuelo de la Torre, Csaba Koncz, László Bögre,
Tópico(s)Plant Reproductive Biology
ResumoArticle3 August 2010free access Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1–E2F pathway activity Rossana Henriques Corresponding Author Rossana Henriques Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UKPresent address: Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA Search for more papers by this author Zoltán Magyar Zoltán Magyar Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Antonia Monardes Antonia Monardes Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain Search for more papers by this author Safina Khan Safina Khan Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Christine Zalejski Christine Zalejski Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Juan Orellana Juan Orellana Unidad de Genética, Departamento de Biotecnologia, ETSI Agrónomos, Universidad Politécnica de Madrid, Spain Search for more papers by this author László Szabados László Szabados Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Consuelo de la Torre Consuelo de la Torre Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain Search for more papers by this author Csaba Koncz Csaba Koncz Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Max-Planck Institut für Züchtungforschung, Carl-von-Linné-Weg 10, Köln, Germany Search for more papers by this author László Bögre Corresponding Author László Bögre Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Rossana Henriques Corresponding Author Rossana Henriques Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UKPresent address: Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA Search for more papers by this author Zoltán Magyar Zoltán Magyar Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Antonia Monardes Antonia Monardes Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain Search for more papers by this author Safina Khan Safina Khan Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Christine Zalejski Christine Zalejski Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Juan Orellana Juan Orellana Unidad de Genética, Departamento de Biotecnologia, ETSI Agrónomos, Universidad Politécnica de Madrid, Spain Search for more papers by this author László Szabados László Szabados Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Consuelo de la Torre Consuelo de la Torre Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain Search for more papers by this author Csaba Koncz Csaba Koncz Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Max-Planck Institut für Züchtungforschung, Carl-von-Linné-Weg 10, Köln, Germany Search for more papers by this author László Bögre Corresponding Author László Bögre Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK Search for more papers by this author Author Information Rossana Henriques 1,‡, Zoltán Magyar1,2,‡, Antonia Monardes3, Safina Khan1, Christine Zalejski1, Juan Orellana4, László Szabados2, Consuelo de la Torre3, Csaba Koncz2,5 and László Bögre 1 1Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, UK 2Institute of Plant Biology, Biological Research Centre, Szeged, Hungary 3Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain 4Unidad de Genética, Departamento de Biotecnologia, ETSI Agrónomos, Universidad Politécnica de Madrid, Spain 5Max-Planck Institut für Züchtungforschung, Carl-von-Linné-Weg 10, Köln, Germany ‡These authors contributed equally to this work *Corresponding authors. Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham TW20 0EX, UK. Tel.:+44 1784 443407; Fax: +44 1784 414224; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:2979-2993https://doi.org/10.1038/emboj.2010.164 Present address: Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single- and double-knockout mutations and RNA-interference (RNAi)-silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non-viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma-related 1 (RBR1)–E2FB complex and this is partly mediated by its N-terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F-dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient-limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels. Introduction Cell growth and proliferation is tightly integrated with available nutrients, cellular energy levels, developmental signals and stress factors through the Target of rapamicin (TOR) kinase signalling pathway (Wullschleger et al, 2006; Diaz-Troya et al, 2008; Ma and Blenis, 2009). One downstream effector of TOR is the ribosomal protein S6 kinase (S6K), a master regulator of growth that tunes the translational capacity of cells through the phosphorylation of ribosomal protein S6 (RPS6) (Meyuhas, 2008). Knockout mutations in the S6K genes in mice and Drosophila indeed resulted in drastic reduction of cell sizes (Montagne et al, 1999; Pende et al, 2004), but surprisingly in mice this was not paralleled with a compromised protein synthesis (Pende et al, 2004). Similarly, mutations of the S6K phosphorylation sites on RPS6 affected cell size, but not protein synthesis, suggesting that S6K regulates cell size checkpoint independent of translation (Pende et al, 2004; Ruvinsky et al, 2005). The inhibition of TOR kinase through specific drugs also identified both cell cycle and cell growth regulation downstream of TOR (Feldman et al, 2009; Thoreen et al, 2009). How TOR can regulate cell size was first identified in fission yeast, where it was shown that TOR restrains the entry into mitosis by regulating the inhibitory phosphorylation of Cdc2 by Wee1 kinase (Petersen and Nurse, 2007; Hartmuth and Petersen, 2009). The involvement of TOR and S6K in cell size checkpoint seems to be conserved. In Drosophila cells, the activation of TOR signalling can delay the entry into mitosis and thus increase cell size (Wu et al, 2007), whereas silencing of S6K1 resulted in a reduced cell size through increasing the rate cells enter into mitosis (Bettencourt-Dias et al, 2004). In budding yeast, the homologue of S6K, Sch9 was also shown to regulate cell size, as well as nutrient signalling and ageing (Jorgensen et al, 2004; Urban et al, 2007; Steffen et al, 2008). Sch9 also has important functions to reprogram gene expression between growth and stress conditions (Roosen et al, 2005; Pascual-Ahuir and Proft, 2007; Smets et al, 2008). S6Ks are members of the AGC family (PKA, PKG, PKC) of serine/threonine kinases and are also present in plants (Bögre et al, 2003). In Arabidopsis, there are two S6K genes, S6K1 and S6K2, having highly similar sequence and arranged in tandem duplication on chromosome 3. It was shown that Arabidopsis S6K2 is able to carry out conserved signalling functions, because it could be activated by the growth hormone, insulin, in a TOR-dependent manner, when introduced into human cells (Turck et al, 1998, 2004). Correspondingly, as in other organisms, the Arabidopsis S6K functions in a complex with RAPTOR, it is activated by PDK1 and can phosphorylate RPS6 (Mahfouz et al, 2006; Otterhag et al, 2006). RPS6 phosphorylation in plants also leads to the selective recruitment of ribosomal mRNAs to polysomes and thus regulates the switch of translational capacity between growth promoting and stress conditions (Turck et al, 2004). The growth hormones, auxin and cytokinin enhance RPS6 phosphotylation in cell culture (Turck et al, 2004), whereas stress factors, such as heat and oxidative stress rapidly block it (Williams et al, 2003). In agreement with reduced RPS6 phosphorylation upon stress, osmotic stress was shown to inactivate the Arabidopsis S6K1 that was dependent on RAPTOR levels, and S6K1 over-expression resulted in an increased sensitivity to osmotic stress (Mahfouz et al, 2006). Plant growth is the result of cell proliferation within meristems and cell enlargement outside the proliferative zone. The tor mutant in Arabidopsis has an arrested embryo development at a stage when cell elongation takes place, indicating that AtTOR might not be required for early proliferative but for cell elongation-driven growth (Menand et al, 2002). Cell proliferation in the tor mutant is also unaffected during endosperm development, but there are defects in cytokinesis, suggesting that TOR might have mitotic functions also in plants (Menand et al, 2002). S6K could also regulate elongation growth, as suggested by the over-expression of a lily S6K (LS6K1) gene in Arabidopsis that resulted in decreased cell elongation in flower organs (Tzeng et al, 2009). AtTOR expression was correlated with active cell proliferation and growth (Menand et al, 2002). S6K1 is also expressed in meristematic regions both in Arabidopsis (Zhang et al, 1994) and in lilly (Tzeng et al, 2009), as well as in cells that are actively elongating within the root (Zhang et al, 1994). The transition from cell proliferation to cell differentiation is regulated by the Retinoblastoma-related 1 (RBR1)–E2F pathway in higher plants (Magyar, 2008), although in the algae Chlamydomonas all components of the RB pathway, including RB, E2F and DP, control cell size (Fang et al, 2006). The retinoblastoma protein is known to inhibit the transcription factor activity of E2Fs by masking their transactivation domains and by globally repressing promoters through the recruitment of chromatin remodelling enzymes (Magyar, 2008; van den Heuvel and Dyson, 2008). In Arabidopsis, RBR1 is the single homologue of Retinoblastoma, and from the six identified E2F-related genes, three are able to form complexes with RBR1, but they differently regulate the expression of genes involved in cell proliferation: E2FC is a transcriptional repressor (del Pozo et al, 2006), whereas E2FA and E2FB are activators (de Veylder et al, 2002; Magyar et al, 2005). The RBR1-bound or -free forms of E2F complexes constitute a regulatory network for the control of cell proliferation and exit to differentiation. In this work, we studied the function of Arabidopsis S6K1 and S6K2 by analysing single mutants, a hemizygous s6k1s6k2/++ double mutant and s6k1(XVE-RNA interference (RNAi))-silenced plants. Homozygous s6k1s6k2 mutants were not recovered, probably because of the requirement of both S6K1 and S6K2 in male gametophyte development. Plants with reduced S6K1 and S6K2 levels had increased chromosome number and became aneuploid. Arabidopsis S6K1 can interact with the RBR1–E2F pathway and inhibit cell proliferation. In agreement with this notion, we found that depletion of RBR1, ectopic over-expression of E2FA together with DPA and elevation of E2FA expression under its own promoter control also lead to increased ploidy, similarly to depletion of S6K1 and S6K2 in the s6k1s6k2/++ and s6k1(XVE-RNAi) plants. Our data suggest that, in plants, S6K negatively regulates cell division as part of a signalling pathway connected to the RBR1–E2F transcriptional switch and its deregulation can influence the incident rate of polyploidization and lead to chromosome instability. Results s6k1s6k2/++ mutants show size variation, reduced fertility and increase in ploidy level The S6K1 (ATPK6, At3g08730) and S6K2 (ATPK19, At3g08720) genes of Arabidopsis are organized in a tandem direct repeat arrangement in chromosome 3. On the basis of NASCARRAY data, S6K1 is highly expressed in mature pollen (five times more than in microspores) and in sperm cells, whereas S6K2 shows limited expression in pollen, but higher expression (it remains to 25% of S6K1) in sperm cells (nucleus in G1). Furthermore, S6K1 is induced by UV, oxidative and genotoxic stresses specifically in shoot, and co-expressed with genes involved in circadian rhythm (e.g. CCA1; Supplementary Table I). S6K2 is expressed in developing seeds and induced by ABA and salt treatment in the root. S6K2 is strongly co-expressed with genes involved in stress responsive regulation of plant growth, such as BONZAI1 (Yang and Hua, 2004). Although with some overlap, AtTOR, S6K1 and S6K2 have distinct domains of expression in the root. AtTOR highest expression is in the meristem, whereas the S6K1 transcript accumulates in the elongation zone, and the S6K2 in the differentiation zone, indicating that S6K1 and S6K2 might regulate the exit from proliferative growth (Supplementary Figure 1A) (Winter et al, 2007). Despite their distinct expression patterns, single insertion mutants for S6K1 (s6k1-1, Salk_148694) or S6K2 (s6k2-1, Salk_128183 and s6k2-2, Salk_13334) are still viable and have no readily discernable phenotypes (Supplementary Figure 2). However, the homozygous s6k2-2 mutant did show seed abortion (∼30%) (Supplementary Figure 2F), and the screening of homozygous s6k2-1 individual plants for DNA content by flow cytometry and for abnormal trichome branching allowed us to identify a line with a mixture of both diploid and aneuploid DNA content (Supplementary Figure 2G), suggesting that reduced S6K2 expression can lead to chromosome instability. We have also identified a double s6k1s6k2 mutation in our library of Arabidopsis T-DNA insertion lines by systematic sequencing of T-DNA insert boundaries (Szabados et al, 2002). In the mutant line A199L, we found a T-DNA integration event that affected both S6K1 and S6K2 by generating a deletion of ∼2 kb. Sequencing of PCR fragments spanning the T-DNA insert borders and genomic DNA junctions showed that the deletion removed coding sequences of the C-terminal part of S6K1 (last exon), the N-terminal part of S6K2 (first two exons) and the intergenic region between the two S6K genes (Supplementary Figure 3A and C). Southern blotting with T-DNA right and left border probes indicated the presence of three tandem T-DNA copies within the S6K1 and S6K2 locus (Supplementary Figure 3B, D, E and F). Genotyping the M3 offspring of line A199L with T-DNA and gene-specific primers revealed the absence of homozygous knockout plants. Upon self-pollination, heterozygous A199L plants yielded about 1:1 segregation of hygromycin resistant and sensitive offspring. To assess the source of distorted segregation, we performed reciprocal crosses between wild-type (Col-0) and 29 hemizygous s6k1s6k2/++ plants and determined the segregation of the T-DNA-encoded hygromycin resistance marker and the presence of T-DNA sequences in the adjacent S6K1 and S6K2 loci by PCR. Fertilization of wild type with s6k1s6k2/++ pollen produced very few seeds (274 seeds/29 crosses, 130 non-viable) and revealed a dramatic reduction of recovery of male-derived double-knockout allele in the viable progeny (3.47%; 5 HygR:139 HygS; Supplementary Table II). In the reciprocal cross, fertilization of s6k1s6k2/++ plants with wild-type pollen provided normal seed yield and an expected 1:1 segregation of wild-type and mutant S6K alleles (465 HygR:439 HygS). According to this result, we found very little viable pollen in s6k1s6k2/++ anthers (Figure 1B). Although pollen amounts might not be limiting for fertility, this seemed not to be the case, as we found 23 to 91% of aborted seeds in siliques in different individual plants (Figure 1C). Figure 1.Developmental abnormalities in s6k1s6k2/++ plants. (A) Flowers of WT and s6k1s6k2/++ plants. Scale bar, 1 mm. (B) Anthers from s6k1s6k2/++ and WT plants stained with Alexander dye. Arrows point to pollen. Note the different scale bars, both representing 50 μm. (C) Dissected siliques from WT and s6k1s6k2/++ plants. Arrows point to aborted seeds. Scale bar, 1 mm. (D) Chromosome number in cells of WT leaf epidermis cell, WT pollen meiocyte, s6k1s6k2/++ leaf epidermis cell, s6k1s6k2/++ pollen meiocyte and WT trichomes. Upper row: DAPI staining of chromocentres (CCs) showing a diploid cell (1), meiotic diploid cell in metaphase I (2), tetraploid somatic cell (3), meiotic tetraploid cell in anaphase I (4) and polytenic trichome cell (5). Middle row: FISH with a centromere-specific probe. Lower row: FISH with a specific probe for chromosome 1 pericentromeric regions. Small bars show pairs of chrom.1. A similar increase in chromosome numbers was also found in petal epidermal cells of both s6k1s6k2/++ and s6k1(XVE-RNAi) line 3 plants. Scale bars, 2.5 μm. (E) Flow cytometry measurements of DNA content from flower cells of wild-type (WT-2n), tetraploid wild-type (WT-4n) and s6k1s6k2/++ seedlings. (F) DNA content measurements of leaf no. 1 and 2, 15 DAG. (G) Scanning electron micrographs and corresponding drawings from epidermal leaf surface (third leaf at day 30) of WT and s6k1s6k2/++ plants. Scale bars, 50 μm. (H) Distribution of leaf epidermal cell sizes of s6k1s6k2/++ and WT plants. Variation in cell sizes within classes were analysed from multiple leaf samples and areas (n=299 cells for s6k1s6k2/++ mutants and n=265 cells for WT). Download figure Download PowerPoint To found the cause for aborted male gametophytes, we analysed meiosis in anthers of wild-type and s6k1s6k2/++ plants by DAPI staining and determined the number of chromocentres (CCs; heterochromatin aggregates that correspond to centromeres in mitotic stages; Fransz et al, 2002) in pollen meiocytes. Surprisingly, in s6k1s6k2/++ mutants, we found an increased (mostly doubled) number of CCs in these cells (Figure 1D). Wild-type male meiocytes have five bivalent chromosome pairs in metaphase I, rather than 10 as found in the s6k1s6k2/++ anthers, which is specifically obvious during late anaphase I, when the chromosome pairs segregate (Figure 1D). This suggests that s6k1s6k2/++ plants possessed an increased chromosome number already before meiosis. Labelling of the same male meiocytes with centromere- and chromosome 1-specific probes by fluorescence in situ hybridization (FISH) further confirmed this result. We found 10 pairs of segregating chromosomes and 2 pairs of chromosome 1 during late anaphase I of s6k1s6k2/++ pollen meiocytes (Figure 1D), but were unable to detect any chromosome segregation abnormalities during meiosis I and II (Supplementary Figure 4). To clarify whether this ploidy increase also affected somatic cells, we performed similar analysis in epidermal cells of leaves and petals. DAPI staining and FISH analysis revealed a similar increase in ploidy levels in the s6k1s6k2/++ mutant when compared with wild type (Figures 1D, 2E and F). Furthermore, flow cytometry measurement of the DNA content of proliferating leaves 1 and 2 at 15 days after germination (DAG) from s6k1s6k2/++ mutant compared with wild-type diploid (WT-2n) and tetraploid (WT-4n) plants revealed an increase comparable with the DNA content of leaves 1 and 2 of a tetraploid plant (Figure 1F). Similarly, fully expanded leaves and flowers of s6k1s6k2/++ plants revealed an increase in DNA content when compared with WT-2n (Figure 1E; Supplementary Figure 5). Although, we observed morphological phenotypes typical of tetraploid Arabidopsis plants, such as large flowers, increased pollen grain size (Figure 1A and B), the extent of phenotypic instability and size variation found in the s6k1s6k2/++ mutant is higher than expected from a stable tetraploid (Koornneef et al, 2003; Yu et al, 2009) (Supplementary Figure 6A and B). Moreover, some of the phenotypes identified, such as narrow leaves, were reminiscent of aneuploid swarm from a diploid to tetraploid cross (Henry et al, 2005). To clarify which of the described phenotypes are due to ploidy changes, we analysed the offspring of the s6k1s6k2/++ mutant grown in the absence of the T-DNA-derived hygromicin marker. These plants were then analysed for the presence of T-DNA in S6K1 and S6K2 loci, their trichome branching (Supplementary Figure 6C) and ploidy level (Supplementary Figure 7). As expected from their higher ploidy levels, s6k1s6k2/++ offspring had trichomes with more than three branches. However, s6k1s6k2/++ plants showed the highest trichome branching (six branches), which was not detected in the plants with wild-type S6K1S6K2 loci. Flow cytometry analysis of flowers, compared with WT-2n and WT-4n, indicated the existence of aneuploidy (Supplementary Figure 7). Moreover, we identified an individual plant (s6k1s6k2/++ #8) with a mixture of both aneuploid (close to triploid) and tetraploid DNA content within the same flower, an indication of a chimera tissue, which confirms results showing that aneuploid plants can develop largely over-branched trichomes even if not proportional to the increase in their DNA content (Yu et al, 2009). Moreover, the aneuploidy measured by flow cytometry did not necessarily correlate with the severity of morphological changes in the s6k1s6k2/++ plants. Figure 2.Reducing S6K transcripts in s6k1(XVE-RNAi) plants also leads to ploidy changes. S6K1 (A) and S6K2 (B) transcript levels in s6k1s6k2/++ mutants and the corresponding WT control determined by quantitative RT–PCR. S6K1 (C) and S6K2 (D) transcript levels in s6k1(XVE-RNAi) seedlings (line #3 and #6) and the corresponding WT control in control (−) and 5 μM β-estradiol-treated conditions (+), determined by quantitative RT–PCR. All samples were collected at day 30 after sowing. (E) Percentage of nuclei from leaf epidermal cells having <10 (% 10 CCs (%>10) from WT (n=59), 5 μM β-estradiol-treated (+) s6k1(XVE-RNAi) line 3 (n=50) plants and s6k1s6k2/++ (n=106) mutants. (F) Percentage of CCs in nuclei from petal epidermal cells in WT (n=166), in 5 μM β-estradiol-treated (+) s6k1(XVE-RNAi) line 3 plants (n=67) and in s6k1s6k2/++ mutants (n=415). (G) Scanning electron micrographs of trichomes from WT and s6k1s6k2/++ leaves. Scale bar, 200 μm. (H) Percentage of trichomes with 3,4,5 and 6 branches in XVE-RNAi empty vector control without β-estradiol (control, −) (n=227), and treated with 5 μM β-estradiol (treated, +) (n=108), s6k1(XVE-RNAi) line 3 control (−) (n=227) and β-estradiol treated (+) (n=292), WT plants (n=279) and s6k1s6k2/++ mutants (n=326). (I) Summary of flow cytometry measurements of DNA content from cells in the first and second leaves at 15 DAG of XVE-RNAi empty vector control and s6k1(XVE-RNAi) constructs. Download figure Download PowerPoint It is known that increased ploidy is accompanied with increased cell size (Galbraith et al, 1991). Therefore, we determined the cell size distribution of epidermal pavement cells from fully developed leaf 3 of wild-type and growth-arrested s6k1s6k2/++ plants, and found a higher proportion of small cells in the leaf epidermis of s6k1s6k2/++ mutants (Figure 1G and H). However, the total cell number in the leaf has not increased in the s6k1s6k2/++ leaves compared with wild type (Supplementary Figure 6D), indicating that cell elongation rather than cell proliferation was impaired. Cell elongation is accompanied by endoreduplication in Arabidopsis leaves, and our flow cytometry analysis of s6k1s6k2/++ expanding leaves 15 DAG (Figure 1F) showed that the 2C peak was missing, as expected from the higher chromosome number; however, the reduction in 16C cells, when compared with WT-4n leaves, suggests a decrease in endoreduplication. Silencing of S6K1 and S6K2 also results in increased ploidy We could not determine the exact moment, during the mutant isolation procedure, when the s6k1s6k2/++ line A199L became polyploid. To independently evaluate whether S6K1 and S6K2 genes are linked to the observed change in ploidy, we have generated RNAi lines, in which both S6K genes are silenced through the expression of a β-estradiol-inducible S6K1 RNAi construct, s6k1(XVE-RNAi) (Zuo et al, 2000). By quantitative RT–PCR, we found that in hemizygous s6k1s6k2/++ mutant, the transcript levels were 53–84% for S6K1 and 40–70% for S6K2 compared with wild type (Figure 2A and B). This result was confirmed by northern analysis of S6K transcript levels in s6k1s6k2/++ mutants (Supplementary Figure 3G and H). Interestingly, we detected, in one individual, a truncated S6K1 mRNA, smaller than the expected S6K1 transcript before the T-DNA insert site, which probably corresponds to the N-terminal region of S6K1. We find unlikely, but cannot rule out, that this low level of short RNA could generate an S6K protein fragment that exerts a dominant negative effect over the wild-type function. In s6k1(XVE-RNAi), lines 3 and 6 grown in the absence of β-estradiol inducer, the S6K1 and S6K2 transcript levels were already lower than in wild-type plants because of leaky basal expression of the RNAi construct. However, upon β-estradiol treatment, the S6K1 and S6K2 transcript levels were further reduced in line 3 to 25% of S6K1 and to 37% of S6K2 compared with wild type. The degree of silencing was slightly less in line 6 with reduction of S6K1 to 50% and S6K2 to 57% of wild-type levels (Figure 2C and D). We have not recovered viable silenced lines with a complete loss of S6K levels. Similarly to s6k1s6k2/++ mutants, the s6k1(XVE-RNAi) lines also showed large flowers and high level of aborted seeds (Supplementary Figure 6E and I). To determine how S6K silencing affects the DNA content, s6k1(XVE-RNAi) line 3 was compared with s6k1s6k2/++ mutant and wild type by performing flow cytometry analysis of developed leaves and flowers (Figure 2I; Supplementary Figure 5). The 2C and 4C peaks of wild-type and XVE-RNAi control plants were replaced by 4C and 8C peaks both in the s6k1s6k2/++ and s6k1(XVE-RNAi) flower samples, an organ that normally remains diploid and does not show endoreduplication cycles. The DNA content also increased in leaves of s6k1s6k2/++ and s6k1(XVE-RNAi) plants compared with wild-type and XVE-RNAi control plants. Therefore, we also determined the number of chromosomes by counting CCs in the s6k1(XVE-RNAi) line 3 compared with the line transformed with empty vector, and found a similar doubling of chromosome number as in the s6k1s6k2/++ mutants both in epidermal cells of leaves and petals (Figure 2E and F). The doubled chromosome number is indicative for the occurrence of endomitosis (chromosome duplication without cell division), rather than a change in the endoreduplication cycle, where the repeated rounds of DNA replication result in unseparated sister chromatids, such as seen in wild-type trichomes, a cell type with endoreduplication (Figure 1D). Leaves from s6k1s6k2/++ and s6k1(XVE-RNAi) plants revealed an increase in trichome branching, a phenotype known to correlate with ploidy changes (Hulskamp, 2004). Correspondingly, trichomes developed on leaves of s6k1s6k2/++ plants had increased number of branches compared with the wild type (Figure 2G). The majority of trichomes on wild-type plants had three branches (n=279) and only around 10% had four, whereas about half of the trichomes on s6k1s6k2/++ (n=326) leaves developed four branches (Figure 2H). The previously screened s6k1(XVE-RNAi) lines (# 3, 6 and also line #2) had up to 70% of trichomes with four branches after β-estradiol induction of s6k1(XVE-RNAi) when compared with the empty vector control lines. Later, we found that increased trichome branching was already present without inducer, shown for the s6k1(XVE-RNAi) line 3 (n=292), but upon β-estradiol treatment, the proportion of trichomes with five branches has substantially increased (Figure 2H). This increase in DNA content found in the s6k1s6k2/++ mutant, three independent s6k1(XVE-RNAi) lines and in one offspring of s6k2-1, suggests that S6K could be required for maintenance of stable chromosome numbers. As chromosome instability accumulates in each generation, we analysed T1 primary transformants expressing the s6k1(XVE-RNAi) construct in a wild-type (Col-0; n=20) or s6k2-2 mutant (n=27) b
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