Leaves, the main photosynthetic organs of flowering plants, show tremendous morphological variation. Leaf shape may be classified as simple, when the leaf blade is entire, or dissected (also referred to as compound), when the blade is sub-divided into distinct leaflets. Both simple and dissected leaves initiate as entire structures at the flanks of the pluripotent shoot apical meristem (SAM). However, in dissected leaves, successive generation of lateral growth axes after leaf initiation results in the formation of leaflets. Leaflets form at the flanks of a central stalk called a rachis, and their development requires re-deployment of many of the genetic pathways that drive leaf initiation at the SAM (Hay and Tsiantis, 2006; Barkoulas et al., 2008; Blein et al., 2008; Jasinski et al., 2008; Berger et al., 2009; Koenig et al., 2009; Shani et al., 2010). Genetic analyses in tomato, Cardamine hirsuta and dissected-leaved legumes have highlighted two processes with a key role in both leaf initiation and leaflet development: growth polarization and control of the timing of tissue differentiation.
Growth polarization requires the auxin efflux carrier PINFORMED 1 (PIN1) (Galweiler et al., 1998; Benkova et al., 2003). In the SAM, PIN1 activity facilitates the formation of auxin activity maxima, which underpin leaf initiation (Reinhardt et al., 2003; Heisler et al., 2005). Class I KNOX proteins (KNOXI) maintain the activity of the SAM by preventing tissue differentiation (Long et al., 1996; Vollbrecht et al., 2000). In compound leaves, PIN1 promotes a polar flow of auxin towards sites of initiating leaflets, and establishes local auxin maxima that promote leaflet outgrowth (Barkoulas et al., 2008; Koenig et al., 2009; Scarpella et al., 2010). KNOX1 proteins are also required for leaflet formation by delaying tissue differentiation, which allows cells to respond to signals that promote growth polarization, such as local auxin maxima (Hay and Tsiantis, 2006, 2010; Barkoulas et al., 2008; Shani et al., 2009). Leaflets form within a morphogenetic window defined by the antagonistic activity of the transcriptional regulators KNOX and TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR). In contrast to KNOX1 proteins, class II TCP proteins act in the leaf to accelerate differentiation (Efroni et al., 2008; Shani et al., 2008). Negative regulation of TCP activity is required to maintain the morphogenetic competence of compound leaves to develop leaflets and TCP over-expression prevents leaflet formation (Ori et al., 2007). KNOXI proteins are excluded from most simple-leaved seed plants, indicating that the species-specific regulation of these genes probably contributed to the evolutionary diversification of leaf form (Hay and Tsiantis, 2006; Efroni et al., 2010; Piazza et al., 2010). KNOX and TCP proteins act at least in part by influencing the activity of the hormones cytokinin and gibberellin (Hay et al., 2002; Jasinski et al., 2005; Shani et al., 2010; Fleishon et al., 2011; Yanai et al., 2011). KNOXI-mediated regulation of these two hormones is required for both SAM function and dissected-leaf development, thus defining a common genetic module underlying shoot and dissected-leaf development (Hay et al., 2002; Shani et al., 2010). Another class of shoot regulators that are required for leaflet formation are CUC (CUP-SHAPED COTYLEDONS) proteins, which de-limit developing leaflets along the leaf, thus recapitulating their earlier developmental role in organ separation at the SAM during leaf initiation (Blein et al., 2008; Berger et al., 2009). The activities of CUC and PIN1 proteins are also required for serration development at the margin of simple leaves (Hay et al., 2006; Nikovics et al., 2006; Bilsborough et al., 2011); therefore, current evidence suggests that flexible deployment of a common suite of shoot regulators (PIN1, KNOX1, TCP, CUC) underlies the development of various types of shoot outgrowths (Efroni et al., 2010).
Here, we report the isolation and characterization of the C. hirsuta mutant sil3, which we selected for study because it shows a dramatic reduction in leaflet but not leaf number, and, as such, may provide new insight into dissected-leaf development. We present evidence that SIL3 is required for leaflet initiation, leaf growth, regulation of auxin homeostasis and KNOX gene expression. We show that SIL3 encodes the ATP binding cassette (ABC-type ATPase ABCE2 or RNase L inhibitor 2 (RLI2). In eukaryotes and archaea, the highly conserved protein ABCE1 plays important roles in ribosome-driven protein biosynthesis, coupling translation termination to ribosome recycling, and eventually re-initiation (Andersen and Leevers, 2007; Barthelme et al., 2007, 2011; Verrier et al., 2008; Khoshnevis et al., 2010; Pisarev et al., 2010). Our data reveal C. hirsuta RLI2 as a novel regulator of leaflet development, and offer a route to explore the recently discovered but poorly understood inputs of ribosomal activity into leaf development.