Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins

Violaine Pinon J. Peter Etchells 0 Pascale Rossignol Sarah A. Collier Juana M. Arroyo Robert A. Martienssen Mary E. Byrne ) 0 Present address: University of Manchester , Manchester M13 9PT , UK Leaves are determinate organs that arise from the flanks of the shoot apical meristem as polar structures with distinct adaxial (dorsal) and abaxial (ventral) sides. Opposing regulatory interactions between genes specifying adaxial or abaxial fates function to maintain dorsoventral polarity. One component of this regulatory network is the Myb-domain transcription factor gene ASYMMETRIC LEAVES1 (AS1). The contribution of AS1 to leaf polarity varies across different plant species; however, in Arabidopsis, as1 mutants have only mild defects in leaf polarity, suggesting that alternate pathways exist for leaf patterning. Here, we describe three genes, PIGGYBACK1 (PGY1), PGY2 and PGY3, which alter leaf patterning in the absence of AS1. All three pgy mutants develop dramatic ectopic lamina outgrowths on the adaxial side of the leaf in an as1 mutant background. This leaf-patterning defect is enhanced by mutations in the adaxial HD-ZIPIII gene REVOLUTA (REV), and is suppressed by mutations in abaxial KANADI genes. Thus, PGY genes influence leaf development via genetic interactions with the HD-ZIPIII-KANADI pathway. PGY1, PGY2 and PGY3 encode cytoplasmic large subunit ribosomal proteins, L10a, L9 and L5, respectively. Our results suggest a role for translation in leaf dorsoventral patterning and indicate that ribosomes are regulators of key patterning events in plant development. INTRODUCTION Early in development leaf primordia establish dorsoventral polarity. Outgrowth of the leaf lamina requires juxtaposition of adaxial (dorsal) and abaxial (ventral) domains of the leaf, which are specified by concerted interactions between domain-specific genetic pathways (Barkoulas et al., 2007; Kidner and Timmermans, 2007). Several transcription factor families are involved in establishing adaxial and abaxial fates. One such family is the class III homeodomain-leucine zipper (HD-ZIPIII) genes, which includes PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV) (Byrne, 2006; McConnell et al., 2001; Otsuga et al., 2001; Talbert et al., 1995; Zhong and Ye, 1999). These genes are all expressed throughout incipient primordia and later become localised to the adaxial side of primordia (Emery et al., 2003; McConnell et al., 2001; Otsuga et al., 2001; Prigge et al., 2005; Talbert et al., 1995; Zhong and Ye, 1999). Combined mutations in PHB, PHV and REV reduce adaxial fate, whereas dominant mutations in the HD-ZIPIII genes show replacement of abaxial tissue by adaxial tissue (Emery et al., 2003; McConnell et al., 2001; Ochando et al., 2006; Prigge et al., 2005; Zhong and Ye, 2004). HD-ZIPIII genes have a mutually antagonistic relationship with KANADI genes, which encode GARP putative transcription factors (Eshed et al., 1999; Eshed et al., 2001; Izhaki and Bowman, 2007; Kerstetter et al., 2001). KANADI genes are expressed abaxially in a pattern complementary to HDZIPIII gene expression. Loss of two or more KANADI genes results in polarity defects, and reduced abaxial fate is associated with ectopic outgrowths on the abaxial side of leaves (Eshed et al., 2004; Izhaki and Bowman, 2007). One additional component of the leaf dorsoventral patterning network is the MYB domain transcription factor ASYMMETRIC LEAVES1 (AS1). Loss of function of the AS1 orthologue in Antirrhinum, tobacco, tomato and pea results in adaxial defects, ranging from patches of abaxial cells on the adaxial side of the leaf to leaves that are radial due to complete loss of adaxial fate (Kim et al., 2003; McHale and Koning, 2004; Tattersall et al., 2005; Waites and Hudson, 1995; Waites et al., 1998). By contrast, mutations in AS1 in Arabidopsis have only subtle polarity defects (Byrne et al., 2000; Ori et al., 2000; Xu et al., 2003). One possibility is that AS1 in Arabidopsis contributes to leaf polarity redundantly with other factors (Byrne et al., 2000; Garcia et al., 2006; Huang et al., 2006; Ueno et al., 2007). We have isolated three enhancers of as1, called PIGGYBACK1 (PGY1), PGY2 and PGY3, all of which have a similar phenotype and condition ectopic leaf lamina outgrowths on the adaxial side of the as1 leaf. We refer to this phenotype as a piggyback phenotype, as ectopic outgrowths resemble epiphyllous structures found on the adaxial side of the leaf of the piggyback begonia (Begonia hispida var. cucullifera) (Maier and Sattler, 1977). Here, we describe the as1 pgy phenotype, and demonstrate that AS1 and PGY1 independently promote dorsoventral polarity. AS1 has minor interactions with the HD-ZIPIII-KANADI pathway, whereas genetic interactions position PGY1 as an integral component of this pathway. PGY1, PGY2 and PGY3 genes encode cytoplasmic large subunit ribosomal proteins, L10a, L9 and L5. We propose that leaf-patterning mechanisms involving the HD-ZIPIII-KANADI pathway include ribosome-mediated translational regulation. MATERIALS AND METHODS Plant stocks and growth conditions pgy1-1, pgy1-2, pgy2-1 and pgy3-1 were generated in an as1-1 mutant background, as described previously (Byrne et al., 2002). All pgy alleles segregate as single recessive loci. Mutants were backcrossed to Landsberg erecta (Ler) twice before genetic analysis. pgy2-2 was a GABI-Kat (128H07) T-DNA insertion line (Rosso et al., 2003). rev-6 was obtained from the Arabidopsis Biological Resource Centre (ABRC). kan1-2 and kan2-1 were obtained from John Bowman. All genetic interactions were in a Ler background. Plants were grown either in soil or on Murashige and Skoog media at 22C with a day length of 16 hours. Genetics pgy genes were cloned using Ler Columbia F2 mapping populations. For complementation a 2.1 kb genomic fragment encompassing At2g27530, a 5 kb genomic fragment encompassing At1g33140 and a 3.5 kb genomic fragment encompassing At3g25520 were cloned into the binary vector pMDC123 (Curtis and Grossniklaus, 2003) and transformed into pgy11/pgy1-1 as1/+, pgy2-1/pgy2-1 as1/+ and pgy3-1/pgy3-1 as1/+ plants, respectively, using standard agrobacterium-mediated transformation (Clough and Bent, 1998). For each complementation construct, basta resistant plants with an as1 phenotype were confirmed as as1 pgy homozygotes. as1-1 rev-6 was analysed in the F3 generation of the cross as1-1 rev 6. In the F2 generation of this cross as1-1 rev-6 segregated at 1:15. pgy1-1 rev-6 were obtained from the F3 generation of the cross pgy1-1 rev-6. Progeny from pgy1-1 rev-6/+ individuals segregated 1:3 pgy1-1 rev-6 mutants. as1-1 pgy1-1 rev-6 triple mutants were analysed in the F4 generation of the cross as1-1 pgy1-1 as1-1 rev-6, after selfing as1-1 pgy11 rev-6/+ F3 plants. Segregation of as1-1 pgy1-1 rev-6 in this F4 generation was 1:3. as1-1 kan1-2 and pgy1-1 kan1-2 were obtained from the F3 generation of the respective crosses as1-1 kan1-2 and pgy1-1 kan1-2. as1-1 pgy1-1 (...truncated)