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Boundaries are composed of distinctive sets of cells that are formed between the shoot apical meristem (SAM) and lateral organs or between lateral organs during embryogenesis and post-embryonic development. Boundaries separate lateral organ primordia from the SAM and adjacent organ primordia, and promote SAM and organ primordium formation (Aida & Tasaka, 2006a,b; Rast & Simon, 2008). Boundary cells have specific morphological and cytological characteristics. Cells at the meristem and organ boundaries display a saddle-shaped surface and are elongated along the boundaries (Kwiatkowska, 2004, 2006; Reddy et al., 2004). Cell proliferation analysis reveals that boundaries between inflorescence and floral meristems, and between floral organs, consist of nondividing cells.
CUP-SHAPED COTYLEDON (CUC)/NO APICAL MERISTEM (NAM), a small group of plant-specific NAC transcription factors, plays important roles in the regulation of boundary development (Aida & Tasaka, 2006a; Rast & Simon, 2008). The CUC/NAM gene family includes CUP-SHAPED COTYLEDON1, 2 and 3 (CUC1, 2 and 3) in Arabidopsis thaliana, NO APICAL MERISTEM (NAM) in Petunia, GOBLET (GOB) in tomato (Solanum lycopersicum) and CUPULIFORMIS (CUP) in Antirrhinum majus (Souer et al., 1996; Aida et al., 1997; Takada et al., 2001; Vroemen et al., 2003; Weir et al., 2004; Berger et al., 2009). CUC/NAM genes are expressed in all boundaries between organ primordia and meristems from early embryogenesis to floral developmental stages. Mutations in CUC1/CUC2 of A. thaliana and in NAM of Petunia lead to fusion of cotyledons and some floral organs, as well as severe defects of the primary apical meristem (Souer et al., 1996; Aida et al., 1997; Takada et al., 2001). Double mutants cuc3/cuc2 or cuc3/cuc1 in A. thaliana and the co-suppression plants of NAM and its homolog genes NAM HOMOLOG-1 (NH-1) and NAM HOMOLOG-3 (NH-3) in Petunia exhibit lateral organ fusion during vegetative development (Souer et al., 1998; Hibara et al., 2006). Mutations in the CUP gene in snapdragon (Antirrhinum majus), however, strongly affect all lateral organ boundaries during embryonic, vegetative and reproductive development (Weir et al., 2004). These studies not only demonstrate that CUC/NAM genes play a pivotal role in organ separation and primary apical meristem formation but also show that different degrees of functional redundancy exist amongst the CUC/NAM family members.
Plant leaf primordia initiate from the flanks of the SAM and go through primary and secondary morphogenesis to form various leaf patterns, such as simple or compound leaves. Of particular importance for leaf patterning is the region at the leaf margins, the marginal blastozone or leaf marginal meristems, which maintains a morphogenetic activity and is responsible for the initiation of secondary structures, such as leaflets (Blein et al., 2010; Efroni et al., 2010). In compound-leafed species, both class I homeodomain KNOTTED1-like genes (KNOXI), which were initially identified for their roles in the maintenance of shoot meristem identity (Long & Barton, 1998), and the floral meristem identity gene LEAFY (LFY) and its orthologs, UNIFOLIATA (UNI ) in pea (Pisum sativum) and SINGLE LEAFLET1 (SGL1) in Medicago truncatula, are species-specific positive regulators in compound leaf development (Hofer et al., 1997; Champagne et al., 2007; Wang et al., 2008; Blein et al., 2010). Recent studies revealed that CUC/NAM genes also share a conserved function in compound leaf development and leaf margin formation (Nikovics et al., 2006; Blein et al., 2008, 2010; Bilsborough et al., 2011; Hasson et al., 2011). Reduced expression of NAM/CUC leads to the suppression of marginal outgrowth and thus formation of reduced and/or fused leaflets during compound leaf development in a diverse compound-leafed species (Blein et al., 2008). GOB, a NAM ortholog in tomato, is essential for proper specification of lateral organ boundaries at the apical meristem and proper specification of leaflet boundaries in developing compound leaves (Berger et al., 2009). Furthermore, it has been reported that the ectopic expression of CUC1 in the margins of developing leaves is sufficient to change their architecture from simple to compound in A. thaliana (Hasson et al., 2011). It is thus proposed that NAM/CUC genes have a common role in promoting leaflet formation and separation.
Previous studies suggest that CUC/NAM genes prevent organ fusion through repression of boundary cell growth and that the possible cytological function of these genes is to regulate cell division or orientation as well as cell expansion (Aida & Tasaka, 2006a,b; Rast & Simon, 2008). CUP in snapdragon directly interacts with a TCP-domain transcription factor, which has previously been shown to regulate organ outgrowth (Weir et al., 2004). Over the last decade, several regulators of CUC genes have been identified in A. thaliana, including SHOOTMERISTEMLESS (STM), PINFORMED1 (PIN1), MONOPTEROS (MP), and microRNA164 (Aida et al., 1999, 2002; Mallory et al., 2004; Aida & Tasaka, 2006a; Larue et al., 2009). STM plays a major role in SAM initiation and is also implicated in cotyledon separation. Activation of STM expression in the embryo apical end requires CUC1 and CUC2, whereas at later stages of A. thaliana embryogenesis STM is required for proper expression patterns of CUC1 and CUC2 (Aida et al., 1999; Hibara et al., 2003). Further studies showed that STM directly binds to the promoter of CUC1 and thus up-regulates CUC1 expression (Spinelli et al., 2011). PIN1 and MP repress CUC1 expression in cotyledons and promote CUC2 expression in cotyledon boundaries (Aida et al., 2002; Furutani et al., 2004). During leaf margin development, CUC2 promotes the generation of PIN1-dependent auxin accumulation while auxin represses CUC2 expression. This feedback loop regulates the activity of the conserved auxin efflux module in leaf margins to form stable serration patterns (Bilsborough et al., 2011). Both CUC1/2 and GOB are post-transcriptionally regulated by microRNA164 for fine-tuning of organ boundaries both temporally and spatially, especially in leaf margin development (Laufs et al., 2004; Mallory et al., 2004; Nikovics et al., 2006; Larue et al., 2009). These genes are negative or reciprocal regulators of CUC/NAM. More recently, it was reported that CUC1 directly activates the expression of LIGHT-DEPENDENT SHORT HYPOCOTYLS (LSH) (LSH3 and LSH4) in shoot organ boundaries (Takeda et al., 2011). Despite these observations, how CUC/NAM regulates downstream gene(s) to affect lateral organ development remains elusive.
In this study, two insertion mutant alleles of MtNAM, one null allele with a retrotransposon Tnt1 insertion, and one weak allele with a native Medicago Endogenous Retrotansposon 1 (MERE1) insertion, were characterized in detail in M. truncatula. The mtnam mutants display unique simplified floral organ phenotypes in addition to the common fused-cotyledon and leaflet phenotypes shared with other cuc/nam mutants. Microarray and real-time quantitative PCR analyses revealed that mutations in MtNAM down-regulate the expression of floral homeotic genes. MtNAM is expressed at boundaries between lateral organs/organ primordia and meristems. These results indicate that MtNAM plays an essential role in controlling floral organ formation and lateral organ separation in M. truncatula.