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Post-Transcriptional Regulation of Plant Gene Expression Plant growth: the translational connection C. Robaglia*1 , B. Menand†, Y. Lei*, R. Sormani*, M. Nicolaı̈*, C. Gery‡, E. Teoulé‡, D. Deprost§ and C. Meyer§ *Laboratoire de Génétique et Biophysique des Plantes, Département d’Ecophysiologie Végétale et Microbiologie, UMR 6191 CNRS-CEA-Université de la Méditerranée, Luminy, Marseille, France, †Department of Cell and Molecular Biology, John Innes Center, Norwich, U.K., ‡Unité de Génétique, INRA, Versailles, France, and §Laboratoire de Nutrition Azotée des Plantes, INRA, Versailles, France Abstract The TOR (target of rapamycin) pathway is a phylogenetically conserved transduction system in eukaryotes linking the energy status of the cell to the protein synthesis apparatus and to cell growth. The TOR protein is specifically inhibited by a rapamycin–FKBP12 complex (where FKBP stands for FK506-binding protein) in yeast and animal cells. Whereas plants appear insensitive to rapamycin, Arabidopsis thaliana harbours a single TOR gene, which is essential for embryonic development. It was found that the product of this gene was capable of binding to rapamycin and yeast FKBP12. In-frame fusion with a GUS reporter gene shows that the TOR protein is produced essentially in proliferating zones, whereas the TOR mRNA can be detected in all organs suggesting a translational regulation of TOR. Phenotypic analysis of Arabidopsis TOR mutants indicates that the plant TOR pathway fulfils the same role in controlling cell growth as its other eukaryotic counterparts. Introduction The growth of an organism depends on the size and the number of its cells. Increase in cell numbers depends on the rate of cell division and on the number of proliferating cells. Cell size can be variably adjusted during cell division or during differentiation [1]. In plants, a large size can be reached by the process of cell expansion, where cells can enlarge post-mitotically up to 1000 times by taking up water and synthesizing a cell wall: ‘liquid growth’ [2]. This kind of cell differentiation is specific to plants; however, the provision of new cells to constitute new organs still depends on cell proliferation as in other multicellular organisms. To provide the building blocks of new organs, cell proliferation must be linked to cell growth, which accounts for the increase in mass, which will be shared by the daughter cells. This increase in mass is intimately linked to the process of protein synthesis and to the nutritional input perceived by the cell. A connection between nutrient perception, protein synthesis and growth is certainly essential for all cells [3,4], and genes that are supposed to be involved in this regulation appear to be phylogenetically conserved among eukaryotes. One of the pathways controlling growth in eukaryotes is known as the TOR (target of rapamycin) pathway. TOR is a very large protein kinase, initially discovered in yeast, which is the target of the antiproliferative drug rapamycin. A decade of research on TOR in yeast and animals has shown that this protein is at the heart of a regulatory system linking nutrient availability to the activity of the protein synthesis Key words: Arabidopsis thaliana, cell growth, cell proliferation, meristem, target of rapamycin (TOR), translation. Abbreviations used: eIF, eukaryotic initiation factor; eIF4E-BP, eIF4E-binding protein; FKBP, FK506-binding protein; TOR, target of rapamycin. 1 To whom correspondence should be addressed (email [email protected]). machinery to regulate cell proliferation in unicellular organisms and body growth in multicellular organisms [5–7]. TOR responds to various inputs linked to the energy available to the cell such as amino acids, ATP status and stresses, but also to mitosis-promoting hormones (insulin) in multicellular animals and integrates them to regulate several effectors of cell proliferation and survival such as the protein synthesis machinery, the cytoskeleton, the uptake of nutrients and autophagy (Figure 1). TOR is supposed to integrate its inputs by engaging in several large molecular mass complexes regulating its protein kinase activity towards various substrates. The rapamycin inhibitor triggers the formation of a complex with an FKBP12 (FK506-binding protein), which in turn binds TOR and blocks its activity. One of the main TOR targets is translation initiation mediated by the eIF4F (where eIF stands for eukaryotic initiation factor) translation–initiation complex. In animal cells, TOR activation releases an active eIF4E cap-binding protein by phosphorylating the repressor eIF4E-BP (eIF4E-binding protein, [8]). In yeast, a different eIF4E-BP named Eap1 is supposed to play a similar role and TOR was found to regulate cell-cycle progression by activating the translation of the mRNA for the G1 cyclin CLN3 [9,10]. In animal cells, TOR also promotes the up-regulation of translation of a class of mRNAs bearing 5 -terminal oligopyrimidine tracts (5 TOP mRNAs) encoding components of the translational apparatus, through the activation of the p70 ribosomal protein S6 kinase (p70S6k ), thereby controlling the biogenesis of the ribosome [11]. Although yeast is not known to possess the equivalent of p70S6k , TOR also controls ribosome biogenesis through regulation of the translation of the ribosomal protein mRNA as well as the transcription of rRNA and tRNA by polymerase I and polymerase III and the structure of the chromatin of rDNA genes [12]. C 2004 Biochemical Society 581 582 Biochemical Society Transactions (2004) Volume 32, part 4 Figure 1 TOR cellular roles: summary of data from yeast, Figure 2 AtTOR functions. AtTOR knockout phenotypical analysis mammalian cells, Caenorhabditis elegans and Drosophila melanogaster and expression pattern are summarized Grey areas symbolize the cytoplasm, white areas symbolize the vacuole. Features of the AtTOR gene In plants, cell proliferation occurs in meristems, dynamic structures continuously traversed by a flux of cells that divide and grow at different rates, and which are defined during embryogenesis. Although the plants are autotrophic, the meristems can be considered as heterotrophs and they depend on the rest of the plant for their supply of nutrients to sustain growth. As plant development and morphogenesis are highly adaptive, cell proliferation in the meristems has to be integrated all through a plant’s life with the various environmental stimuli, including nutrient supply [13]. Investigation of the Arabidopsis genome reveals that plants possess a probable orthologue of the TOR gene. This gene is located on the lower arm of chromosome 1 and encodes a 2481-amino-acid protein with all the attributes of a TOR protein including the FKBP-rapamycin-binding domain, the C-terminal kinase domain and the presence of HEAT repeats [14]. Investigations of the plant-TOR pathway have been hampered by the natural resistance of plants to rapamycin. Identification of a potential FRB domain in the AtTOR protein raises the question of the basis of this resistance, since plants also possess orthologues of FKBP12 proteins. A yeast three-hybrid analysis was therefore undertaken to determine if the AtTOR FRB domain was capable of interacting with plant orthologues of Saccharomyces cerevisiae FKBP12 in the presence of rapamycin. This revealed that none of the plant FKBPs tested (AtFKBP12, AtFKBP15-1, AtFKBP152 and AtFKBP62) could interact with the AtTOR FRB domain in the presence of rapamycin (B. Menand and C. Robaglia, unpublished work). However, a control with yeast FKBP12 (ScFKBP12), revealed that complex formation could occur [14]. This showed that the FRB domain of Arabidopsis thaliana TOR was still functional for a ternary-complex formation, despite the fact that none of the plant FKBPs could engage in such a complex and suggests that this domain C 2004 Biochemical Society is involved in a TOR function conserved in plants, yeast and animals. Mutants of AtTOR were readily identified in T-DNA insertion libraries and two mutants with insertions interrupting the gene near the FRB domain and upstream of the kinase domain were analysed in depth [14]. The siliques of those plants contained one quarter aborted seeds suggesting that the mutation was lethal at the homozygous stage, indeed the observation of embryos showed that development was arrested at the globular stage. Interestingly, those arrested embryos still occasionally contained cells undertaking division. This shows that AtTOR inactivation was not detrimental for the cell-division machinery. During the early stages of Arabidopsis embryo development, there is a small increase in the total embryo volume and the size of cells decreases during divisions. The developmental arrest of a tor mutant embryo at the globular stage correlates with the change from divisions without growth to divisions coupled with growth [15]. Therefore AtTOR is probably required for premitotic cytoplasmic growth during cell proliferation. This is supported by the absence of segregation distortion in the transmission of the tor mutation showing that correct divisions of the tor gametophytes can occur, leading to gamete formation. Again, these divisions are the result of cleavages within a pre-existing cell mass without net enlargement of the cytoplasm (the alternance of generations where both haploid and diploid forms can divide is specific to plants). In-frame fusion with the GUS reporter further reveals that the synthesis of the AtTOR–GUS fusion protein occurs in embryo, endosperm, primary meristems and primordia but not in differentiated organs such as fully expanded leaf cells and root tissue (Figure 3, lower panel). During the formation of secondary roots AtTOR expression occurs very early, as soon as the first periclinal divisions of the lateral root primordia and well before the meristem structure was clearly established [14]. Remarkably, the precursors of the stomatal guard cells, which return to a cycle of cell division after differentiation and with a small increase in cytosol mass, are not expressing the AtTOR-GUS marker, whereas they express the CycB-GUS marker, further providing an indication that AtTOR is essential for cytoplasmic Post-Transcriptional Regulation of Plant Gene Expression Figure 3 Post-transcriptional regulation of AtTOR Upper panel: reverse transcriptase–PCR analysis of AtTOR expression in plant tissues. Lower panel: GUS staining of an heterozygous TOR/tor-1 plant, the tor-1 allele is fused with the GUS gene at amino acid 1555 of AtTOR. Total RNA was extracted from the indicated tissues and 1 µg was reverse transcribed using oligo(dT). After a 10× dilution, PCR analysis was performed using AtTOR or actin 8 specific primers for the indicated number of cycles. PCR products were separated by agarose gel electrophoresis, blotted on to nylon and hybridized with AtTOR or actin 8 32 P-labelled probes. growth. The above observations and their interpretation are summarized in Figure 2. Post-transcriptional regulation of AtTOR gene expression As stated above, the production of the TOR protein, as attested by the in-frame GUS fusion is limited to proliferating cells and tissues. However, reverse transcriptase– PCR experiments designed to detect the AtTOR mRNA reveal that it is expressed at nearly equal levels in all plant tissues including differentiated cells at the tip of the leaves and fully expanded leaves where TOR-GUS protein is undetectable (Figure 3). This suggests that AtTOR is regulated post-transcriptionally by translational repression in differentiated cells. As translational regulation is often linked to particular features of untranslated mRNA sequences, the AtTOR 5 -leader sequence was obtained by 5 -RACE (where RACE stands for rapid amplification of cDNA ends) analysis. Visual inspection of the resulting 230 bp sequence reveals the presence of a short microORF (open reading frame) having the potential to encode for a single amino acid, AUGUGUUGA. MicroORFs were found to positively or negatively regulate translation of downstream genes in many cases [16]. An indication of their function is their conservation since sequence constraints are generally not more rigid in untranslated regions than in coding sequences. Indeed, sequencing the DNA upstream of C 2004 Biochemical Society 583 584 Biochemical Society Transactions (2004) Volume 32, part 4 the AtTOR coding region in 12 Arabidopsis ecotypes chosen from a collection maximizing genetic diversity [17] shows that the AtTOR leader region, including the uORF, is highly conserved, thus supporting the maintenance of a functional regulatory role (results not shown). Concluding remarks The lethal phenotype of knockout AtTOR mutants and the insensitivity of plants to rapamycin currently limits the study of the plant TOR pathway. Innovative methods to conditionally inactivate AtTOR or the isolation of hypomorphic mutants are necessary. Post-transcriptional gene silencing through the expression of double-stranded RNA is a way to achieve this goal, since a range of phenotypes can in principle be obtained [18]. Another strategy would be to generate tissue sectors devoid of AtTOR by complementation of the available mutants with the AtTOR gene bordered by target sites for an inducible recombinase such as FLP [19]. Plants harbour orthologues of many genes linked to TOR in other organisms such as those coding for ribosomal S6 kinase, PDK, eIF4E and TAP 42, but lack others such as genes coding for PKB and eIF4E-BP [20]. Therefore the precise architecture of the plant TOR pathway remains to be determined. Since plants and animals independently acquire multicellularity, it is probable that signals acting upstream of TOR to orchestrate growth and morphogenesis are different [21]. An important area of future research will be the exploration of the integration of the plant TOR at the level of the organism. C 2004 Biochemical Society References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Hafen, E. and Stocker, H. (2003) PLoS Biol. 1, 319–322 Cosgrove, D.J. 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