Ribosomal composition and control of leaf development

Plant Science 179 (2010) 307–315

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Review

Ribosomal composition and control of leaf development Jos H.M. Schippers a,b , Bernd Mueller-Roeber a,b,∗ a b

University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany

a r t i c l e

i n f o

Article history: Received 6 March 2010 Received in revised form 11 June 2010 Accepted 24 June 2010 Available online 1 July 2010 Keywords: Development Translation Ribosome Expansion

a b s t r a c t Protein synthesis in plants occurs at three sub-cellular locations that have their own specific ribosomal compositions: the cytoplasm, mitochondria and plastids. An increased demand for functional and efficient translational machinery is required during development of leaves. The role of specific ribosomal protein (RP-) encoding genes in the regulation of development has been underestimated as housekeeping. However, in Arabidopsis thaliana several RP loss-of-function mutations have been identified that affect cell division or cell expansion and consequently result in deformed leaf size and shape, indicating cell- or development-specific roles of RPs during leaf growth. This view is strengthened by the observation that the expression of many RP genes follows distinct patterns during leaf development. Moreover, translatomics data demonstrate that ribosomal composition is dynamic and organized in a spatio-temporal manner. The regulation of RP gene transcription via different promoter-localized cis-elements allows additional control relevant for leaf growth. We conclude that RPs have a more distinct role in regulating specific processes in leaf development than previously anticipated, and envisage fascinating novel insights in the near future. © 2010 Published by Elsevier Ltd.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Ribosomal protein mutants and developmental defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. Cytoplasmic RPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. RP and RACK1 positions within the cytoplasmic ribosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3. Plastid RPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4. Mitochondrial RPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Expression of ribosomal protein-encoding genes during leaf development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Whole-leaf studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. Leaf cell-specific translation of ribosomal proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Upstream factors controlling ribosomal protein gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1. Telo box, trap40 box, tef box and site II motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2. Inr element, GT1-element, I box and S1-element in plastid RP genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.3. Hormones and ribosomal function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ∗ Corresponding author at: Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany. Fax: +49 331 977 2512. E-mail addresses: [email protected] (J.H.M. Schippers), [email protected] (B. Mueller-Roeber). 0168-9452/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.plantsci.2010.06.012

Protein synthesis involves the binding and translation of mRNA by ribosomal complexes. There are three major protein synthesis locations in the plant cell, each with a distinct composition of its

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ribosomal complex. rRNA molecules form the core of the translational complex while the ribosomal proteins (RPs) are mainly found at the surface [1]. The discovery that the catalytic activity of the ribosome comes from the rRNA and not the RPs led to the idea that the ribosome has evolved as an entire RNA-catalysing enzyme [2]. Later on, apparently more specialized functions evolved that are dependent on the RPs that conjugate with the rRNA molecules. Since RPs are mainly present at the surface of the particle they are in an excellent position for mediating the many interactions of the ribosome with other components, including regulatory proteins of the cell [3]. One typical aspect of multicellular organisms is the demand for the co-ordination of cell proliferation and expansion during development. Leaf size and shape largely vary between plants; such differences arise from genetic and environmental factors. The rapid advances within genome biology have helped to identify dozens of genes that affect leaf size or shape. The regulatory mechanisms that underlie these processes are still insufficiently understood. Leaf development involves a series of characteristic processes such as the initiation of periclinal cell divisions at the shoot apical meristem (SAM), resulting in cellular outgrowth. This group of cells gradually differentiates into a determinate organ with a symmetric architecture, a network of veins, and a regulated distribution of specialized cell types, such as guard cells and trichomes in the epidermal cell layers. The size of leaves is determined by only two physiological processes: cell division leading to an increase in cell number, and cell expansion resulting in enlargement of cells [4]. The basic metabolic and energetic costs of protein synthesis and turnover directly affect the capacity of cellular growth [5]. It was already demonstrated three decades ago [6] that the amount and composition of polysomes change dramatically in growing bean leaves during cell division and the first phases of leaf expansion. The amount of polysomes increases, suggesting an increased demand for protein synthesis. The number of cytoplasmic polysomes drops rapidly after cessation of cell division. However, the number of organellespecific complexes still increases until the leaf has reached its final size. At leaf maturity the total abundance of polysomes drops and a low level is then maintained. Taken together, these data demonstrate that the level of ribosomes during leaf growth substantially fluctuates. Duplicated RP-encoding genes in Brassica napus have undergone functional divergence into highly specialized paralogs and coexpression networks [7]. Translation in plants is most likely also regulated by altering the proteins in the ribosome. This is nicely exemplified by the mere fact that 251 genes in Arabidopsis encode the 81 possible proteins present in the cytoplasmic ribosome [8]. The presence of small multigene families encoding for the various RPs allows for a highly dynamic composition of the ribosome. Most RP mutants are characterized by a decrease in cell number [9]. It can be stated that the total number of cells that arise through cell division contributes to the final size of a leaf. However, in several cases a lower number of cells is compensated by an increase in cell size [10], demonstrating that cell division and expansion are not regulated at the single-cell level alone, but that growth is regulated at the whole-organ level reaching a set final size [11]. The mechanisms behind this compensation effect have been subject of recent studies as discussed below. Ribosomes are generally perceived as housekeeping components within the cell, with a non-selective role in polypeptide synthesis. However, the specific phenotypes of several RP mutations suggest that ribosomal composition may play a fundamental role during development. The big questions here are whether ribosomal composition changes mRNA preference (and thus defines which transcripts are translated) and whether a specific ribosomal composition exists that stimulates leaf growth.

1.1. Ribosomal protein mutants and developmental defects 1.1.1. Cytoplasmic RPs The cytoplasmic ribosome contains 81 RPs encoded by small gene families encompassing a total of 251 genes in Arabidopsis [8]. The presence of multiple copies of individual RPs suggests that functional specialization might have occurred among family members. Still several copies might only act as pseudogenes without any biological function. If they are all housekeeping genes, as it is sometimes taught, it is clear that a mutation in a single RP might cause dysfunction of the whole translational machinery, as with the EMBRYO-DEFECTIVE (EMB) mutants [12]. RPS6B, RPS11A, RPL8A, RPL19A, RPL23C, RPL40B have been identified as EMB mutants. They all belong to families with two or three members, suggesting that there is low functional redundancy or that some of them are weakly active paralogous. Loss of RPS6B is lethal, while partial down-regulation results in an altered leaf pattern [13]. Of the two-gene RPS5 family, RPS5A is mainly expressed in dividing cells, whereas RPS5B is preferentially expressed in differentiating and elongating cells [14], fitting with specification of different family members. Knock-out of RPS5A is embryo lethal, whereas loss of a single allele causes delayed development and altered leaf vascular patterning. A T-DNA insertion in the coding region of RPL23aB has no reported effect on plant morphology [15], whereas suppression of RPL23aA transcript level leads to reduced cell division, retarded growth, morphological abnormalities and altered vascular patterning [16]. The phenotypes of RP mutants show a striking overlap with those of auxin-related mutants [17–19]. The phytohormone auxin is of major importance in plant development, suggesting that translation and auxin signaling might share a common role in regulating leaf growth. The first described RP affecting leaf growth is caused by a T-DNA insertion in the RPS18A gene resulting in the characteristic pointed first leaf (pfl) phenotype [20]. Although the RPS18 family has three members, the other copies cannot compensate for the loss, indicating functional divergence between the three proteins (or slight but important differences in expression patterns). Activation insertion mutants of RPS13B have a similar phenotype and are therefore called pfl2 [21]. A pfl1 pfl2 double mutation does not further enhance the mutant phenotype, demonstrating that both proteins might be needed for the same process. A knock-out of RPL24B leads to a gynoecium development defect in addition to a pointed leaf phenotype [22]. The observed reduction in leaf width of the pfl mutants can either be the result of a lower cell number or a reduction in cell size. In the case of the rps13b mutant fewer but larger cells were present in leaves when compared to wild-type [21]. Conversely, the rpl24b mutant has no change in cell length, which might indicate a defect in cell division. Thus, the change in leaf size is caused both by a defect in cell expansion or cell division depending on the RP gene affected. Two recent studies demonstrate that RPs specifically modulate cell expansion, in line with this conclusion. First, the pointed leaf phenotype of the angusta3 (ang3) mutant is caused by an amino acid change in RPL5B [23]. Interestingly, the total cell number is not affected but leaf epidermal cell size is significantly reduced. Microarray analysis of total RNA of the ang3 mutant revealed no differential expression of genes known to control leaf development suggesting a putative post-transcriptional regulation of cell expansion [23]. The loss of function of individual RP genes often results in detrimental growth defects; however, knock-out of RPS15aE, a gene of a six-member family, results in larger leaves, longer roots, and increased subepidermal palisade cell size [24]. The authors suggested that RPS15aE is a negative growth regulator; however, further in-depth studies are needed to pinpoint the exact mechanism behind the regulation of cell expansion. Taken together, the reported leaf phenotypes for RP mutants may imply that modulation of ribosome composition is a mechanism that can stimulate or repress growth. With this

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Fig. 1. RP protein position in the 40S and 60S eukaryotic ribosome model [27]. Left, 40S model (Protein Databank accession code 3JYV). RP proteins with known plant Arabidopsis mutants are RPS5 (yellow), RPS11 (firebrick red), RPS13 (cyan), RPS15 (magenta), RPS18 (green), and RACK1 (RED). The 18S rRNA is given in orange while a tRNA loaded into the 40S subunit is shown in blue. Landmarks for the 40S subunit: h, head; bk, beak; sh, shoulder; pt, platform; rf, release factor site; if, initiation factor sit. Right, modeling of the 60S subunit (Protein Databank accession codes 3JYW and 3JYX). Shown are CP, central protuberance; RPL5 (red) and the 5S rRNA (blue) on top; RPL8 (hotpink); RPL28 (green); RPL10 (marine blue); RPL9 (magenta); RPL23 (limegreen); RPL24 (purpleblue) and RPL19 (yellow).

in mind, it is even more fascinating that studies on the compensation syndrome – decreased cell number but same final leaf size through increased cell expansion – have revealed a role for several ribosomal proteins. Radiation induced mutants for RPL5A (oli5) and RPL5B (oli7) have a decreased cell number [25]. The decrease in cell number of oli7 leaves was reported to be due to a reduced number of palisade cells. Crossing the oli mutants into the angustifolia3 mutant (AN3 encodes a transcription co-activator) resulted in a 2.5-fold increase of cell size compared to wild-type suggesting that ribosomal proteins contribute to setting final organ size [25]. RPL5A was also identified in second-site enhancer screens for the known leaf development-related transcription factor mutant asymmetric leaves1 (as1) and called asymmetric leaves1/2 enhancer6 (ae6) [26] or PIGGYBACK3 (PGY) [27]. Next to that, EMS mutants in RPL28A (ae5), RPL10aB (PGY1) and RPL9C (PGY2) have a similar enhancement in leaf growth of the as1 phenotype. Taken together, these elegant studies have started to reveal new insights into the mechanisms that control leaf size with RPs as fundamental regulators. 1.1.2. RP and RACK1 positions within the cytoplasmic ribosome A first comprehensive computer-modeled structure of the eukaryotic cytoplasmic ribosome has recently been published [28]. RPs and RACKs (see Section 1.3.3 Hormones and ribosomal function) can potentially be employed for fine-tuning translation efficiency and specificity of the ribosome core. If true it would be logical to assume that the ribosome proteins that cause the leaf developmental defects encode for those ribosome modules that can be easily exchanged. Of the small subunit proteins affected in EMB mutants, RPS11 is deeply buried within the rRNA, whereas proteins affected in the viable mutants (RPS5, RPS15, RPS18 and RPS13) are almost all located at the head of the 40S subunit (Fig. 1). In humans the RPS19 subunit in the head of the small ribosome has a crucial function in ribosome biogenesis [29]. In yeast the r-proteins rpS5, rpS18, and rpS19 are likely required for late nuclear pre-40S maturation [30]. Interestingly, in yeast the rps11 and rps13 subunits are involved in early steps of rRNA processing. As is evident from the modeled structure RACK1 protein is located at the side of the head. The head of the 40S subunit contains one tRNA loading site which is surrounded by the RPs that affect leaf development when mutated. Proteins of the large 60S subunit affected in embryo lethal mutants (RPL8, RPL19, RPL23, RPL40) locate to one side of the

ribosome, whereas proteins affected in the other mutants (RPL5, RPL9, RPL10, RPL28) surround the tRNA loading/release site (Fig. 1). RPL19 is known to be essential for ribosome assembly and thus mutating its gene leads to embryo lethality [28]. During protein synthesis, the ribosome alternates between a ratcheted and a non-ratcheted conformation, catalyzed by elongation factor eEF2, facilitating the translocation of tRNA molecules [31,32]. RPL9 and RPL10 facilitate the interaction with elongation factors thereby regulating translation [31]. The loss of RPL24 activity in mice causes activation of a p53-dependent checkpoint mechanism that stimulates a survival response upon ribosomal protein deficiency [33]. The RPL5 protein is required for stabilizing the 5S-rRNA and sits along the mRNA channel binding the start codon [34]. Therefore RPL5 appears to have a role in translation initiation. Interestingly, RP proteins that affect leaf development when mutated are mainly found around tRNA loading and release sites both in the 40S and 60S subunits. As already mentioned above, RP mutants show similarity to auxin mutants. Interestingly, auxin-related transcripts appear to be under strict translation control (see Section 1.3.3), suggesting a direct link between the RP proteins and auxin mediated development. 1.1.3. Plastid RPs Proplastids reside in meristematic cells of the shoot apex. There plastids start to differentiate and divide once a leaf primordium is differentiated and plastids give rise to functional chloroplasts [35]. The number of chloroplasts largely correlates with the final size of the mesophyll cell, although light also has a profound effect [36,37]. The leaf initial is heterotrophic, depending on other plant organs for carbon supply [38]. The leaf eventually, becomes a net exporter of photosynthate, undergoing the so-called sink-to-source transition [39]. At this point, the leaf has accumulated enough functional chloroplasts that sustain its own energy demands. Chloroplast development is tightly linked with that of the leaf; a maximum number of chloroplasts are reached at the start of leaf maturity whereas the breakdown of chlorophyll is the first visible sign of the last step of leaf development, i.e. senescence [40]. Translation within the chloroplast occurs on the plastid ribosomes (PR) that consist of 30S and 50S subunits. Fifteen of the 24 genes encoding for the 30S subunit are encoded by the chloroplast genome and nine by the nuclear genome [41]. In contrast, 24 of the

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31 proteins of the 50S subunit are encoded by the nuclear genome [42]. In addition, seven plastid-specific, ribosome associated proteins have been identified that function as regulators of chloroplast translation [43]. The rate of protein synthesis within chloroplasts is highly light dependent, whereas the mRNA levels remain relatively unchanged throughout light/dark transitions [44]. The formation of the large Rubicso protein RbcL is fully dependent on and tightly regulated by the plastid ribosome complex [45]. Therefore, any disturbance of the plastid ribosome might have profound effects on the global energy status of the plant. This is especially evident from experiments with spectinomycin, a specific inhibitor of plastid translation [46]. Treatment of plants with this chemical results in severe leaf developmental defects. Non-photosynthetic tissues like petals are also deformed upon spectinomycin treatment, demonstrating that plastids may play a specific role in organ development in addition to photosynthesis. This is supported by the observation that embryolethal knock-out lines have been identified for the PRPL6, PRPL13 and PRPL31 genes [47]. Finally, the non-photosynthetic, parasitic flowering plant Epifagus virginiana has a tiny plastid genome that encodes for nine proteins including two RPs [48]. Therefore plastids appear to have an essential non-photosynthetic function [49], which might well be related to development. The first Arabidopsis report on a knock-out of a PRP gene involves the loss of the PRPL11 subunit, which causes a dramatic decrease in size and a pale green coloration of the leaves [50]. The authors suggested that PRLP11 is required for maintaining the stability of the plastid ribosome. In contrast, the leaf longevity mutant oresara4 is caused by a T-DNA insertion in the promoter of PRPS17 causing a 90-fold reduction in transcript level [51]. Leaves of the ore4-1 mutant have a reduced photosynthetic rate, which might facilitate the observed longevity. Although ore4-1 leaves have the same maturation time as those of the wild-type they have less fresh weight and are smaller. The knock-out of PRPS21 is characterized by hypersensitivity to glucose (GLUCOSEHYPERSENSITIVE1, GHS1) causing leaf developmental arrest [52]. Prps21 mutant plants grown in soil possess a very similar phenotype as the prpl11 knockout although they have lower levels of chloroplast proteins. Taken together, mutational evidence so far demonstrates a role for PRPs in development, however, it needs to be confirmed whether or not this is independent from their role in photosynthesis. 1.1.4. Mitochondrial RPs Plant morphogenesis relies on cell division and cell expansion, two processes that put a high demand on energy and metabolic resources. The number of mitochondria and the steady-state levels of several nuclear-encoded mRNAs for mitochondrial proteins increase during leaf development [53,54]. Mitochondrial respiration changes during the three phases of leaf development and is modulated in proliferating cells during stress [55]. Therefore, translational control within the mitochondria might modulate leaf development through changes in the level of mitochondrialencoded proteins [56]. The 49S large ribosome subunit is encoded by 42 genes, of which three are encoded by the mitochondrial genome, whereas the 29S small subunit is encoded by 24 genes of which four are located on the mitochondrial genome [56,57]. The first reported leaf developmental defects resulting from a mutation in mitochondrial ribosomal protein (MRP)-encoding genes of Arabidopsis is due to a rearrangement in the mtDNA sequences of MRPS3 and MRPL16 causing a reduced level of expression [58]. The observed phenotype was designated maternal distorted leaf (MDL), which is characterized by an irregular leaf shape, rough leaf surface and a dark green appearance. A second RP that affects leaf growth is MRPL11 [59]. A T-DNA insertion in the promoter of MRPL11 results in a 90% reduction in transcript abundance, with a decrease in leaf size and a darker-green leaf

coloration. Interestingly, the mutant has a large reduction in the abundance of mitochondrial protein complexes but has a normal photosynthetic rate. Furthermore, a mild RNAi-dependent silencing of MRPS10 results in serrated and distorted leaves [60]. Taken together, the leaf developmental alterations caused by defects in mitochondrial translation indicate a role for the mitochondrial RPs during growth. This is underlined by several specific mutants such as in the gene MRPL14-2 (HUELLENLOS), which is essential for ovule development [61,62]. The loss of MRPL14-2 can be compensated for by MRPL14-1 (HUELLENLOS-PARALOG), demonstrating that this small gene family is developmentally regulated. Furthermore, MRPL21 and MRPS11 are required during female gametophyte development [63] and are also known as NUCLEAR FUSION DEFECTIVE 1 and 3, respectively. Thus, specific subunits appear to be necessary during different phases of development emphasizing the specialization among RPs. Altering the ribosome composition might be a regulatory mechanism to support specific stages of development. 1.2. Expression of ribosomal protein-encoding genes during leaf development 1.2.1. Whole-leaf studies RPs are commonly considered to be housekeeping genes and therefore regularly used as reference genes in quantitative (q)RT-PCR expression analyses. However, as the above-mentioned development defects caused by different RP mutations already indicate, RPs appear to be regulated in a tissue- and developmentdependent way. To visualize the expressional behavior of RP-encoding genes during leaf development we made use of a highquality dataset on leaf growth at ten time-points [4]. Physiological analysis of the first leaf pair of Arabidopsis shows that all cells in young leaves are undergoing proliferation. This phase continues until day 11, after which cell division rates decline and cell expansion begins to determine final leaf area. Leaf expansion stops at around day 20 and is followed by a maturation stage [4]. We identified 73 published RP-encoding nuclear genes for the three different ribosome complexes that exist in plant cells (Table S1). Cluster analysis suggests the RPs for the three compartments are clearly differentially regulated during leaf growth (Fig. 2A). Interestingly, cytoplasmic RPs are mainly found in clusters 1 and 6, which are related to differential gene expression during cell expansion. The expression of cytoplasmic RPs is down regulated after proliferation but increases at the end of the expansion phase (Fig. 2B). In contrast, chloroplast RPs fall into gene clusters 2 and 5, which show high expression during cell proliferation and cell expansion. A drastic decrease in their expression is observed at the onset of maturation (Fig. 2B). The expression of mitochondrial RPs is distributed over the different clusters and is highly variable during leaf development. This maybe related to the changes in leaf metabolism during development. Taken together, the expression level of RP-encoding genes during leaf growth is dependent upon the developmental phase of the leaf. However, it has to be kept in mind that translation efficiency is mainly considered to be a post-transcriptionally regulated process. 1.2.2. Leaf cell-specific translation of ribosomal proteins It appears from the analysis above that expression of RP genes during leaf development is highly regulated. To further differentiate the three ribosomal complexes and demonstrate specialization among the RP-encoding genes, we made use of recently published cell-specific expression data [64]. A cell-specific ribosome-associated mRNA isolation technique was developed by expressing a FLAG-epitope-tagged ribosomal protein L18 (FLAGRPL18) in transgenic Arabidopsis plants under the control of cell-specific promoters [65]. This exciting new technique greatly

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Fig. 2. Expression of RP encoding genes during leaf development. (A) Quality threshold (QT) clustering of genes differentially expressed during leaf growth resulted in 16 clusters that share a similar expression pattern [4]. The distribution of plastid, cytoplasmic and mitochondrial RPs within these clusters show that the different RP types correlate with different leaf growth stages. (B) Relative expression level of 73 RP genes during leaf growth (data based on Beemster et al. [4]). All experiments were performed on the first leaf pair of Arabidopsis. Color code as in (A). DAS, days after sowing.

advances research capabilities in the field of translatomics, which is expected to correlate better with proteome data than transcriptomics. Current procedures for mRNA extraction from organs not only perturb cell-specific gene expression patterns but also largely discount the fact that the functionality of individual mRNAs is posttranscriptionally regulated via localization, translation, storage and degradation [66]. Since mRNA species loaded onto polysomes are actively translated, it is expected that changes in their abundance directly affect protein levels. It has to be noted that still a minor subpopulation of these loaded mRNAs are stalled in initiation or elongation, which might result in a difference between final protein level and loaded mRNA abundance [67]. The dataset covers expression levels from six different leaf tissues that are measurable due to expressing the tagged RPL18 under control of six tissue-specific promoters; epidermis (CER5), trichomes (GL2), photosynthetic tissue (RBCS), guard cells (KAT1),

phloem companion cells (SUC2), and shoot bundle sheath cells (Sultr2;2). Since ribosomes are present in all living leaf cells one might expect that the expression level of each subunit depends on the need for protein synthesis within the different tissues. With this technique only nuclear expressed genes can be measured. Of the 148 RP genes encoding the cytoplasmic large subunit we found 106 were expressed. The absolute expression value of each gene was taken; subsequently the tissue with the highest signal value was given a score of 100% and the relative signal level within the other five tissues was calculated based on the difference with this cell type. A dot-plot of the relative signal level shows variability in mRNA loading for the different RPs (Fig. 3A). About 50% of all RPL-encoding mRNAs have the highest expression level within guard cells, whereas lower levels are observed in companion cells and the epidermis (Fig. 3B and Table S1). Individual RPL-encoding mRNAs can for example have a translation activity of 100% in the

Fig. 3. Leaf cell-specific translational activities of mRNAs encoding RPs. (A) Relative translational activity of different RP transcripts in six leaf tissues (RbcS, photosynthetic cells; SUC2, phloem companion cells; Sultr2;2, shoot bundle sheath; GL2, trichome; CER5, epidermis; KAT1, guard cells). Relative activity is estimated based on the highest level, set to 100%. (B) Cell-specific eFP viewer. Expression level of representative RP genes from four different classes, as visualized by the eFP browser (http://efp.ucr.edu/).

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bundle sheath, as in the case of RPL10aB/PGY1, whereas in other tissues this is between 70 and 76% (Table S1). Moreover, its paralogue RPL10aC has the highest translation activity in guard cells and only 37% activity in epidermal cells, suggesting a subtle specialization between these RP family members. The mRNA species of 79 genes encoding for the small subunit have, as with the large subunit, the highest translational activity in the guard cells, while the average activity within the other tissues is ca. 80%. The polysome loading of mRNA is coined here as translational activity, since the loaded mRNA is located in an active translation complex. There is also considerable variation in translation activity between different tissues. For instance the activity of RPS20A is 100% in photosynthetic tissue and guard cells, 75% in trichomes and bundle sheath and about 48% in companion cells and the epidermis. The highest level of chloroplast translational activity is within photosynthetic tissue and guard cells, whereas the translation within the epidermis is as low as 25%. The translational activity for each subunit is quite similar within each tissue, suggesting a common regulation of the expression of genes encoding PRPs. The mitochondrial RPs have the highest expression level in companion cells, which fits well with previous studies showing an increased number of mitochondria in companion cells [68]. However, the expression levels in other leaf tissues have a high degree of variability, which might be related to specific metabolic functions within the different cell types. The previously mentioned MRPL11 gene [59] has the highest translation activity within companion cells. The decrease in leaf size might therefore be caused by a defect in the sucrose loading that depends on energy generated by mitochondria [69]. The visualization of cell-specific expression data for four representative RP genes of different classes was performed by using the eFP browser (Fig. 3B). The schematic representations summarize the plotted data. The development of FLAG tagged ribosomes has set a new resolution to research at the translational level and is a valuable tool to study both mRNA loading onto ribosomes as well as polysomal protein composition during leaf growth. 1.3. Upstream factors controlling ribosomal protein gene expression The tissue and development-specific expression of genes is regulated through cis-acting elements within their promoters. Several studies have focused on identifying the regulatory sequences for RP genes. A couple of interesting elements are enriched in promoters of RP genes, which we will discuss here briefly. 1.3.1. Telo box, trap40 box, tef box and site II motif The telo box (AAACCCTAA) was first identified in the promoter of the elongation factor EF1A gene [70] and is identical to the (AAACCCT)n repeat motif of plant telomers [71]. This motif is present within the first 150 bp from the initiation codon of most promoters of genes encoding cytoplasmic RPs [72]. The presence of the telo box in the EF1A and RP promoters has a striking similarity with the distribution of the rpg box in yeast which is bound by the DNA-binding factor repressor/activator protein 1 (RAP1) [73]. RAP1 appears to modulate chromatin structure [74] by promoting heterochromatin formation. The telo box in Arabidopsis is recognized by the DNA-binding protein PUR␣1 which has high similarity to PUR␣ from humans [72]. It was speculated that RAP1 and PUR␣ are involved in similar mechanisms to control chromatin opening and recruitment of transcription factors. One interesting observation is the overrepresentation of the telo motif in promoters of auxin upregulated genes during axillary bud outgrowth in Arabidopsis [75]. The telo box itself is not sufficient for the activation of expression but acts in synergy with several other cis-acting elements [76]. The first element that acts in concert with the telo box is the well-characterized translation–elongation factor box (tef box;

ArGGRYANNNNNGTaa) [77]. The tef box and telo box cooperate in the activation of genes during cell cycling [72]. Furthermore, the tef box is overrepresented in the promoters of genes encoding for RPs [78]. The positioning of the telo and tef boxes within the promoters of the different RPs is highly conserved [72]. The second synergistic element is the transcription of ribosomal-associated protein 40 (trap40) box (GGGGGTAGAATAG) originally identified in the RPSaA promoter [79]. In contrast to the telo box, the tef box is sufficient for inducing expression of cell cycle-regulated genes [78]. Although the tef and trap40 boxes are known for more than a decade, the identities of the proteins recognizing them have remained elusive [80]. The third interacting element was first found in studies with a rice promoter of the gene encoding PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA), which contains the so-called site II motif [81]. The site II consensus motif of sites IIa and IIb (T/GGTCCCAT) occurs in promoter regions of auxin-regulated genes. Interestingly, the site II motif is present in 153 promoters of genes encoding for RPs in association with a telo box [76]. Moreover, promoter topology is highly conserved, with the telo box residing between 60 and 150 bp upstream of the translation start codon and the site II motif is preferentially located upstream of it (between 120 and 180 bp). The site II motifs are recognized and bound by TEOSINTE-BRANCHED CYCLOIDEA PCNA FACTOR (TCP) 20 [76], a bHLH-like transcription factor with a critical role in leaf growth [82,83]. Moreover, a detailed study of the TCP20 binding site (GCCCR) shows that a clustering of these cis-elements is preferred for enabling high expression of RP encoding genes in cycling cells [84]. This clustering of motifs occurs in about half of all RP promoters suggesting that only a subset of all RPs functions during cell cycle progression [84]. Currently it is assumed that organ growth rates, and shape can be regulated by balancing the level of TCP proteins, which can both act as repressors and activators of gene expression. There is the direct link between the activation of expression via site II motifs and the telo box, indicated by the fact that TCP20 interacts with the telo box-binding protein PUR-␣1 [76]. 1.3.2. Inr element, GT1-element, I box and S1-element in plastid RP genes The lack of a TATA box is a characteristic feature of promoters of plastid genes encoded by the nucleus [85]. A pyrimidine-rich Initiator (Inr) element (TTTTCATCTTC) is found instead of the TATA box within the transcription start site that is essential for lightresponsive transcription of these genes. Plastid targeted ribosomal proteins have two light responsive motifs within their promoters; the GT-1 element (GGTTAA) and the I box (GATAAA) [86]. The GT-1 element was first reported as a binding site for the GT-1 transcription factor in the promoter of the pea rbcs-3A gene [87]. GT members belong to the family of trihelix transcription factors [88] of which a few bind to the RbcS promoter in Arabidopsis [89]. The I-box was initially found to be a GATA-like box that is present in most promoter sequences of RbcS encoding genes [90,91]. Thus far a single DNA-binding protein was identified that binds to the I-box in plants, LeMYBI [92], a MYB-like transcriptional activator. The observation that all PRP encoding genes have three cis-elements in common fits well with the observed similar expression behavior for these genes (Fig. 3). Taken together the above-mentioned motifs have a fundamental role in controlling the expression of RP genes during cell proliferation and/or photosynthesis. However, information on other DNA binding sites controlling RP expression is limited. 1.3.3. Hormones and ribosomal function The RPs decorating the rRNA cores appear to become extensively altered upon ABA treatment [93]. Five genes encoding RPs are induced within 6 h of ABA treatment, while 16 genes are

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down regulated. This observation suggests that composition of the protein synthesis machinery is adjusted to restrict or regulate protein synthesis during stress. Gibberellin, auxin, ethylene, cytokinin, brassinosteroid, and jasmonate also affect the expression of subsets of RP encoding genes [94]. Since phytohormones are key signaling molecules in regulating plant development and growth, it is very likely that they also act at the site of protein synthesis. An example of how phytohormone signaling is perceived by the ribosome is suggested by studies on the family of Receptor for Activated C Kinase 1 (RACK1) proteins. RACK1 harbors seven tryptophan–aspartic acid–domain (WD40) repeats. RACK1 is involved in auxin [95], cytokinin [96], salicylic acid [97] and ABA signaling [98]. RACK1 is important in cell cycle control as well as cell movement and growth in other eukaryotes, which quickly led to the idea that RACK1 is a protein hub for multiple signal transduction pathways. RACK1 is associated with the ribosome, both in mammals and in plants [99,100]. RACK1 recruits activated protein kinase C to the ribosome, which might result in a stimulation of translation by the phosphorylation of initiation factors or of mRNAassociated proteins in mammals [101]. It would be very attractive to uncover RACK1 distribution on ribosomes during leaf growth. The action of phytohormones might be mediated by RACK1 modulation of the translational machinery that directs translation of specific mRNA species. The translation efficiency of a given mRNA is generally dependent on the sequence of the 5 -untranslated region (UTR) [102]. mRNA species with an optimal initiation codon (5 (A/G)NNAUGG-3 ) containing an A-rich sequence from −10 to −1 are more likely to be translated [102]. In general, the consensus initiation sequence of most mRNA species in Arabidopsis overlaps with the optimal sequence, indicating that they are likely to be efficiently translated [103]. Still, 20–30% of all mRNAs contain one or more small upstream ORFs (uORFs) 5 of the main ORF (mORF) [102,103]. Moreover, mRNAs encoding regulatory proteins are more often associated with uORFs. Currently, several mutants with defects in uORF-processing are known. These include specific RPs and components of the eukaryotic initiation factor 3 (eIF3) complex RPL24 is encoded by two genes in Arabidopsis [8], a knock-out of one causes similar phenotypic defects as two auxin-related mutants, ettin (ett) and monopteros (mp) [22]. Half of all auxin-response factors (ARFs) have one or more uORF. Taken together these observations suggest that auxin-mediated signaling is highly regulated at the level of the ribosome. RP mutants with auxin-mutant like phenotypes might be caused due to lower translational efficiency of the ribosomes lacking specific subunits. RPs and RACKs can potentially be employed for fine-tuning translation efficiency and specificity of the ribosome core. This should allow for rapid responses to growth stimuli or stresses without prior transcription of new mRNA species. Since different sets of RPs are affected at the expression level by various phytohormones, it is very likely that specialization of RPs to specific hormone signaling pathways has developed during evolution. 1.4. Outlook The big questions are whether ribosomal composition changes mRNA preference (and thus defines which transcripts are translated) and whether a specific ribosome set-up exists that stimulates leaf growth or specific cellular functions. The current knowledge about RPs and the expression patterns of their underlying genes is still limited and insufficient to accurately define the mechanisms by which they modulate growth. The work summarized above clearly demonstrates that the ribosome ensemble change during leaf growth. There is evidence for specific ribosome constitutions that can stimulate or even suppress growth. Unravelling the cellular and developmental peculiarities of ribosome make-up and mRNA loading preference will be interesting research challenge for the coming years.

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Growth of a leaf largely depends on the expansion of epidermal cells and the building of a venation network. In contrast, expansion of a mesophyll cell will first push away ‘air’ and less so other cells in its surrounding. Therefore it would be highly interesting to employ a cell-specific complementation strategy to test reversion of developmental defects caused by RP mutations. For example, complementation of the pfl mutant (RPS18A) could be analyzed by reintroducing the wild-type open reading frame under the control of an epidermal or vascular tissue-specific promoter. A tissue layer-specific complementation approach for ANGUSTIFOLIA has demonstrated that leaf length and width depend on different cell layers [104]. Conversely, one could test whether the introduction of a PRP into photosynthetic tissue (mesophyll cells) is sufficient for restoring leaf size. None of the overexpression lines for RPs have been tested for an effect on leaf growth, although the loss-of-function of RPL15aE demonstrates that it is a negative growth regulator. Proteomic approaches aimed at characterizing the dynamics of ribosome composition may reveal developmental stage-dependent protein occupancies. Next to that the ribosome-associated RACK proteins participate in cell signaling via phosphorylation underscoring the complexity of regulating translation. With the tremendous amount of techniques available we foresee that specialized functions for specific RPs can be pinpointed in the coming years. Perhaps controlling protein synthesis turns out to be a hotspot for genetic engineering to increase plant tolerances as well as yield. Acknowledgements Support by the Bundesministerium fuer Bildung und Forschung (BMBF), Germany, for funding of the GoFORSYS centre (Grant No. 0313924) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.plantsci.2010.06.012. References [1] N. Ban, P. Nissen, J. Hansen, P.B. Moore, T.A. Steitz, The complete atomic structure of the large ribosomal subunit at 2.4 A◦ resolution, Science 289 (2000) 905–920. [2] P. Nissen, J. Hansen, N. Ban, P.B. Moore, T.A. Steitz, The structural basis of ribosome activity in peptide bond synthesis, Science 289 (2000) 920–930. [3] D.E. Brodersen, P. Nissen, The social life of ribosomal proteins, FEBS J. 272 (2005) 2098–2108. [4] G.T. Beemster, et al., Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis, Plant Physiol. 138 (2005) 734–743. [5] M. Piques, et al., Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis, Mol. Syst. Biol. 5 (2009) 314. [6] S.C. Makrides, J. Goldthwaite, Biochemical changes during bean leaf growth, maturity, and senescence, J. Exp. Bot. 32 (1981) 725–735. [7] C.A. Whittle, J.E. Krochko, Transcript profiling provides evidence of functional divergence and expression networks among ribosomal protein gene paralogs in Brassica napus, Plant Cell 21 (2009) 2203–2219. [8] A. Barakat, et al., The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome, Plant Physiol. 127 (2001) 398–415. [9] M.E. Byrne, A role for the ribosome in development, Trends Plant Sci. 14 (2009) 512–519. [10] A. Hemerly, et al., Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development, EMBO J. 14 (1995) 3925–3936. [11] C.J. Potter, T. Xu, Mechanisms of size control, Curr. Opin. Genet. Dev. 11 (2001) 279–286. [12] D. Meinke, R. Muralla, C. Sweeney, A. Dickerman, Identifying essential genes in Arabidopsis thaliana, Trends Plant Sci. 13 (2008) 483–491. [13] T. Morimoto, Y. Suzuki, I. Yamaguchi, Effects of partial suppression of ribosomal protein S6 on organ formation in Arabidopsis thaliana, Biosci. Biotechnol. Biochem. 66 (2002) 2437–2443. [14] D. Weijers, et al., An Arabidopsis minute-like phenotype caused by a semidominant mutation in a RIBOSOMAL PROTEIN S5 gene, Development 128 (2001) 4289–4299.

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