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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.


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 HD-ZIPIII 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.


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 22°C with a day length of 16 hours.


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 pgy1-1/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 pgy1-1 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 kan1-2 triple mutants were identified in the F4 generation after selfing as1-1 pgy1-1 kan1-2/+ F3 plants from the cross of kan1-2 × as1-1 pgy1-1. Segregation of as1-1 pgy1-1 kan1-2 in this F4 generation was 1:3. Double and triple mutant combinations of as1-1, pgy1-1 and kan2-1 were generated in an identical manner. as1-1 kan1-2 kan2-1 or kan2-1/+, pgy1-1 kan1-2 kan2-1 or kan2-1/+ and as1-1 pgy1-1 kan1-2 kan2-1 or kan2-1/+ were analysed in the F3 and F4 generations of the respective crosses as1-1 × kan1-2 kan2-1/+, pgy1-1 × kan1-2 kan2-1/+ and as1-1 pgy1-1 × kan1-2 kan2-1/+. In progeny from as1-1 pgy1-1 kan1-2/+ kan2-1/+, plants with a suppressed as1-1 pgy1-1 phenotype segregated 6:10 and plants with a kan double mutant phenotype segregated 1:15. Combinations of pgy mutants in the as1-1 background were generated from the crosses of as1-1 pgy1-1 × as1-1 pgy2-1, and as1-1 pgy1-1 pgy2-1 × as1-1 pgy3-1. The genotype of all combinatorial mutants was confirmed either by sequencing or by CAPS analysis using allele-specific polymorphisms as listed in Table 1.

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Table 1.

Primers used for genotyping and expression studies

Molecular biology

DNA manipulation was carried out using standard methods. RT-PCR and qRT-PCR analysis was carried out using the gene-specific primers listed in Table 1. For RT-PCR, RNA was isolated using Trizol (Invitrogen). cDNA synthesis, following DNase treatment, was performed using a Superscript First-Strand Synthesis System (Invitrogen). For quantitative RT-PCR (qRT-PCR) total RNA was extracted from 10-day-old seedlings using Trizol reagent and purified using an RNeasy Kit (Qiagen). All samples were measured in technical triplicates on three biological replicates. For each pair of primers two controls were used to confirm the absence of genomic DNA in the RNA extract. The qRT-PCR reaction was performed using SYBR Green JumpStart Taq ReadyMix (Sigma) on 10 ng cDNA. A standard curve experiment (Livak and Schmittgen, 2001) showed that all the designed primers bind with the same efficiency to template. PCR reactions were performed using an Opticon real-time PCR instrument (MJ Research). The PCR programme started at 94°C for 2 minutes, followed by 40 cycles of 94°C (15 seconds), 60°C (30 seconds), 72°C (1 minute) and 76°C (1 second). Melting curves were recorded from 65°C to 95°C, reading every 0.5°C. Quantification of gene expression was carried out using the method of Livak and Schmittgen (Livak and Schmittgen, 2001). Results were normalised to that of ACTIN2.

In situ hybridisation

Non-radioactive in situ hybridisations were performed as previously described (Long et al., 1996) using 10-day-old seedlings. For the PGY probes, gene-specific fragments were amplified using gene specific primers (see Table 1) and cloned into the vector pGEM-Teasy (Promega). Antisense and sense probes were transcribed with SP6 or T7 polymerase following linearisation of the clone plasmid.

Fig. 1.

Arabidopsis pgy1, pgy2 and pgy3 leaf phenotypes. (A) Rosettes of pgy mutants. From left to right: wild type, pgy1, pgy2, pgy3. (B) Rosettes of pgy mutants in as1 background. From left to right: as1, as1 pgy1, as1 pgy2, as1 pgy3. (C) Rosette leaves with ectopic outgrowths. From left to right: as1 pgy1, as1 pgy2, as1 pgy3. (D) Rosette of as1 pgy1 pgy2 pgy3 quadruple mutant.

Fig. 2

. Development of ectopic outgrowths in Arabidopsis as1 pgy1. (A-C) Adaxial surface of a late rosette leaf from 12-day-old plants of wild type (A), as1 (B) and as1 pgy1, showing a dome-shaped structure (arrow) of an initiating outgrowth (C). (D,E) as1 pgy1 late rosette leaves from 15-day-old plants, showing ectopic outgrowths forming peg-like structures. (F) as1 pgy1 late rosette leaf from an 18-day-old plant forms a leaf-like structure with trichomes on the adaxial side but not on the abaxial side of an ectopic outgrowth. Scale bars: 100 μm.

Histology and microscopy

Scanning electron microscopy (SEM) of leaves was carried out as previously described (Byrne et al., 2003). For SEM analysis of as1 pgy1 rev mutants, tissue was fixed in 3% glutaraldehyde and dehydrated through an ethanol series, before critical point drying using a Watford Polaron and sputter coating with gold (Agar High Resolution Sputter Coater). Samples were viewed using a Gemini Supra 55 VP SEM under high vacuum with a beam accelerating voltage of 3 kV. For analysis of leaf vasculature, tissue was fixed in 3% glutaraldehyde, dehydrated through an ethanol series to 100% ethanol and embedded in JB4 resin (Agar Scientific). Embedded tissue was sectioned at 3 μm and subsequently stained with 0.02% Toluidine Blue. Images were obtained using a Nikon E800 microscope. Leaf 6 of 28-day-old plants was used for vascular sections.


Independent pgy mutants have similar phenotypes

In our screen for modifiers of as1 leaf development we identified a number of mutants, which had similar leaf development defects and formed ectopic lamina outgrowths on the adaxial side of the leaf. Three of these mutants were called pgy1-1, pgy2-1 and pgy3-1. Allelism analysis demonstrated that these three mutants were independent loci.

pgy1, pgy2 and pgy3 single mutants had a subtle phenotype compared with wild type, with formation of slightly pointed leaves and more prominent marginal serrations (Fig. 1A). Often these phenotypes were visible only early in development of rosette leaves. In contrast to single mutants, rosette leaves of as1 pgy1, as1 pgy2 and as1 pgy3 mutants were distinct from as1, and formed narrower, elongate leaves with adaxial ectopic lamina (Fig. 1B,C). Ectopic outgrowths were typically formed in the proximal region of late rosette leaves; however, the penetrance of this phenotype was environmentally conditioned, and in extreme cases ectopic outgrowths were formed on most rosette and cauline leaves. To determine the relationship between PGY genes we generated as1 pgy1 pgy2, as1 pgy2 pgy3 and as1 pgy1 pgy3 triple mutants and the as1 pgy1 pgy2 pgy3 quadruple mutant. Although the frequency of outgrowths was slightly increased in plants with multiple pgy mutations, leaf phenotypes were similar to as1 pgy double mutants, indicating that all three PGY genes contribute to the same developmental pathway (Fig. 1D and see Fig. S1 in the supplementary material). To further understand the developmental defect in these mutants we selected pgy1 for more detailed characterisation.

Initiation and development of ectopic leaf lamina in as1 pgy1 mutants

We analysed the developmental progression of as1 pgy1 outgrowths using SEM. The proximal and adaxial regions of immature late rosette leaves were examined. In wild-type leaves, larger cubical cells of the midvein are evident in the medial region of the leaf, whereas smaller, less regular cells of the lamina are in lateral regions (Fig. 2A). At a similar stage of development, cells in the midvein and lamina regions of as1 leaves could be distinguished. These cells were similar to wild type (Fig. 2B). Early in development of as1 pgy1 mutant leaves, the adaxial surface was characterised by the appearance of dome-shaped structures (Fig. 2C) that developed into radial, peg-like structures (Fig. 2D,E). Outgrowths subsequently became bifacial and leaf-like. One side of fully mature outgrowths was trichome dense and dark green, similar to the adaxial epidermis of wild-type leaves, whereas the opposing side of outgrowths had very few trichomes and was pale green, similar to the abaxial epidermis of wild-type leaves (Fig. 1C and Fig. 2F). These epidermal features of mature outgrowths are consistent with the establishment of dorsoventral polarity. Interestingly, the polarity of outgrowths was always oriented with the adaxial side facing the distal tip of the leaf and the abaxial side facing the proximal base of the leaf and the meristem (Fig. 2F).

Fig. 3.

Vascular patterning defect in Arabidopsis as1 pgy1 leaves. Cross sections through leaf midveins. In wild type (A), as1 (B) and pgy1 (C), xylem is adaxial and phloem is abaxial. In as1 pgy1 mutants (D), xylem is surrounded by phloem. ph, phloem; x, xylem. Scale bars: 50 μm.

Fig. 4.

rev enhances as1 and as1 pgy1 leaf phenotypes in Arabidopsis. (A) rev, with narrow and elongated leaves. (B) pgy1 rev, with slightly narrower leaves than rev. (C) as1. (D) as1 rev, exhibiting narrower and a more rippled leaf surface compared with as1. (E) as1 pgy1 rev triple mutants have a more severe phenotype than as1 pgy1 double mutants, with formation of very small rosettes accumulating anthocyanin in most leaves. (F) SEM of as1 pgy1 rev rosette, showing radial leaves (arrow). Scale bar: 50μ m in F.

The as1 pgy1 mutant phenotype suggests a defect in adaxial fate

Ectopic outgrowths on leaves are typically associated with dorsoventral polarity defects (Emery et al., 2003; Lin et al., 2003; McConnell and Barton, 1998; Waites and Hudson, 1995). Analysis of the adaxial surface of as1 pgy1 mutants did not reveal epidermal features suggestive of patches of abaxial tissue on the adaxial side of the leaf (Fig. 2). Another marker of leaf-tissue polarity is vascular patterning. In Arabidopsis, leaf vascular bundles develop dorsoventral polarity with xylem tissue in an adaxial position above abaxial phloem (Fig. 3A). We used this anatomical feature to further investigate the leaf defect in as1 pgy1. Transverse sections of leaf midveins of as1 and pgy1 single mutants displayed a vascular pattern similar to wild type (Fig. 3B,C). However, in the as1 pgy1 double mutants, sections of the midvein had a disorganised and sometimes radial vascular pattern, with phloem surrounding xylem (Fig. 3D). Radialisation of the vasculature occurred in regions of the leaf below outgrowths. Both the ectopic outgrowths from the adaxial leaf surface and the radial vascular pattern were consistent with defects in adaxial fate in as1 pgy1 leaves.

rev enhances the as1 pgy1 leaf polarity defect

The dorsoventral polarity defect in as1 pgy1 leaves may occur either through loss of adaxial fate or gain of abaxial fate. HD-ZIPIII family genes PHB, PHV and REV act redundantly in specification of leaf adaxial fate (Emery et al., 2003; Prigge et al., 2005). Loss of REV function results in formation of longer and narrower leaves compared with wild type. rev mutants also exhibit reduced lateral shoot meristem formation, a reduction in floral meristem activity and vascular patterning defects (Otsuga et al., 2001; Talbert et al., 1995; Zhong and Ye, 1999). To determine whether PGY1 interacts with an HD-ZIPIII pathway, we examined genetic interactions between as1, pgy1 and rev. Double mutants, as1 rev and pgy1 rev, and the as1 pgy1 rev triple mutant were generated and leaf development in these mutant combinations was compared with as1 and as1 pgy1.

pgy1 did not dramatically affect rev leaves, although pgy1 rev double mutants had narrower leaves compared with rev (Fig. 4A,B). Rosette leaves of as1 rev mutants were more elongate and narrower compared with as1 single mutants, and the leaf adaxial surface of as1 rev was more rippled than that of as1 (Fig. 4C,D). In these respects as1 rev leaves were similar to those of as1 pgy1. However, as1 rev mutants did not develop ectopic outgrowths. By contrast, as1 pgy1 rev triple mutants had a leaf phenotype more severe than that of as1 pgy1, with formation of radial leaf-like structures (Fig. 4E,F), suggesting a more severe loss of adaxial fate. The as1 pgy1 rev phenotype indicates that all three genes act to regulate leaf adaxial fate. However, REV, PHB and PHV transcript levels were not significantly altered in pgy1 or as1 pgy1 mutants, indicating that PGY1 does not regulate transcription of these genes (Fig. 6K). In contrast to a previous report (Fu et al., 2007), we found transcript levels of these three HD-ZIPIII genes were also not significantly altered in as1 relative to wild type (Fig. 6K).

pgy1 enhances rev inflorescence defects

In addition to leaf defects, rev mutants had a pronounced effect on lateral meristem function, with fewer axillary meristems and abnormal flowers (Fig. 5A,B). Inflorescences of rev single mutants produce wild-type flowers, as well as flowers with a reduced complement of inner whorl organs. Additionally, rev floral meristems may fail to produce a flower, instead only forming a terminal filamentous structure (Otsuga et al., 2001; Talbert et al., 1995). Although the leaf phenotype of rev was only slightly modified by pgy1, the inflorescence and flowers of rev were significantly altered by pgy1. pgy1 rev inflorescences initially formed one or two fertile flowers. Subsequent floral meristems only gave rise to a terminal filamentous organ or did not produce organs (Fig. 5C,D). The pgy1 enhancement of rev was similar to phv and phb/+ enhancement of rev. rev phv, rev phb/+ and rev phv phb/+ mutants initially produce several fertile flowers and subsequent floral meristems mostly form a single radial organ (Prigge et al., 2005). rev phb phv/+ floral meristems also produce filamentous structures as with pgy1 rev mutants (Fig. 5E,F). These interactions further support the suggestion that both PGY1 and REV are together required for organ patterning.

Fig. 5.

pgy1 enhances rev axillary meristem defects in Arabidopsis. (A) Wild-type flowering plant. (B) rev mutants lack axillary meristems at the base of some cauline leaves (arrows). (C) pgy1 rev plant with reduced stature compared with wild type and rev. Inflorescence forms few or no flowers. (D) SEM of pgy1 rev inflorescence. Most floral meristems either produce filamentous structures or fail to produce any organs. (E) rev phb phv/+ plant with reduced stature and inflorescence with few flowers. (F) SEM of rev phb phv/+ inflorescence. Most floral meristems produce filamentous structures similar to those in pgy1 rev. Scale bars: 200 μm in D,F.

Fig. 6.

kan1 suppresses the as1 pgy1 leaf phenotype in Arabidopsis. (A-C) Rosettes of as1 (A), as1 kan1 (B) and as1 kan1 kan2/+ (C). The rosette leaves of as1 kan1 (B) and as1 kan1 kan2/+ (C) have a surface slightly less rippled than as1 (A). (D-F) Rosettes of as1 pgy1 (D), as1 pgy1 kan1 (E) and as1 pgy1 kan1 kan2/+ (F). The as1 pgy1 phenotype is suppressed by kan1, and to a greater extent in a kan1 kan2/+ background, such that as1 pgy1 kan1 kan2/+ rosette leaves are more similar to as1. (G-J) Plants of kan1 kan2 (G), as1 kan1 kan2 (H), pgy1 kan1 kan2 (I) and as1 pgy1 kan1 kan2 (J). The kan1 kan2 phenotype is not suppressed by as1, pgy1, or by as1 pgy1. (K) Quantitative RT-PCR was used to assay expression levels of PHB, PHV, REV, KAN1 and KAN2 in the following genotypes: wild type, pgy1, as1 and as1 pgy1. Results were normalised to ACTIN2, with the value from wild-type plants arbitrarily set to 1.0. Bars indicate the standard deviation between three biological replicates. ANOVA statistical analysis indicates a significant difference between Ler and as1 pgy1 for KAN1 and KAN2 expression. Scale bars: 1 cm in G-J.

KANADI genes are required for the as1 pgy1 phenotype

KANADI family genes repress HD-ZIPIII genes and specify abaxial fate. kan1 single mutants have only subtle leaf phenotypes, whereas kan1 kan2 double mutants have adaxialised, small and narrow leaves with ectopic radial leaf-like structures forming on the abaxial side of the leaf (Fig. 6G) (Eshed et al., 2001; Eshed et al., 2004; Kerstetter et al., 2001). To further understand the interaction of PGY1 with the HD-ZIPIII-KANADI dorsoventral patterning pathway we generated the as1 kan1 and pgy1 kan1 double mutants and the as1 pgy1 kan1 triple mutant. as1 kan1 leaves were only slightly different from those of as1, being less rolled under at the margins (Fig. 6A,B). The leaf phenotype of pgy1 is subtle and no significant changes were apparent in the pgy1 kan1 double mutants (data not shown). In contrast to as1 kan1, the as1 pgy1 kan1 triple mutant displayed significant suppression of the as1 pgy1 phenotype. In the vegetative rosette, as1 pgy1 kan1 leaves did not have the narrow, elongated shape of as1 pgy1 leaves, and the triple mutant had a phenotype more like that of as1 (Fig. 6D,E), indicating that the suppression of the as1 pgy1 phenotype by kan1 is mainly the effect of an interaction between pgy1 and kan1. As with kan1, the as1 pgy1 phenotype was also suppressed by kan2 (data not shown).

Fig. 7.

pgy1 suppresses the kan1 kan2/+ gynoecium phenotype in Arabidopsis. (A-D) Flowering inflorescences of wild type (A), kan1 kan2/+ (B), as1 kan1 kan2/+ (C) and pgy1 kan1 kan2/+ (D). (E-H) Single siliques of wild type (E), kan1 kan2/+ (F), as1 kan1 kan2/+ (G) and pgy1 kan1 kan2/+ (H). kan1 kan2/+ and as1 kan1 kan2/+ siliques exhibit a similar phenotype, with proliferation of septum and formation of ectopic styles along the abaxial replum, between the two fused carpels (arrows). pgy1 kan1 kan2/+ silique (H) has less ectopic tissue and is more elongated compared with kan1 kan2/+ siliques (F). Scale bars: 1 mm in E-H.

Abaxial defects are enhanced by combining kan mutants, indicating dosage effects between KANADI genes (Eshed et al., 2004; Izhaki and Bowman, 2007). Therefore we tested if a dosage effect would have consequences on as1 and as1 pgy1 mutants. We generated as1 kan1 kan2/+ and as1 pgy1 kan1 kan2/+ combinatorial mutants. Leaves of as1 kan1 kan2/+ were similar to those of as1 kan1, showing only mild suppression of as1 (Fig. 6A-C). By contrast, the suppression of as1 pgy1 was more pronounced in as1 pgy1 kan1 kan2/+ mutants relative to as1 pgy1 kan1, with leaves broader and rounder, and developing only few outgrowths (Fig. 6D-F). These interactions suggest an underlying dosage effect of KANADI function on pgy1. Consistent with these genetic interactions, expression of KAN1 and KAN2 was not significantly altered in as1 or pgy1, whereas both KAN1 and KAN2 were slightly upregulated in as1 pgy1 (Fig. 6K). The suppression of the as1 pgy1 phenotype by kan mutations suggest that ectopic expression of KANADI genes is a cause of the ectopic outgrowths on the adaxial side of as1 pgy1 leaves.

To further test dose effects of kan mutants on as1 pgy1, we generated as1 kan1 kan2 and pgy1 kan1 kan2 triple mutants, as well as the as1 pgy1 kan1 kan2 quadruple mutant. Leaves of as1 kan1 kan2 were more severely affected and smaller in size than those of kan1 kan2, but otherwise had features of kan1 kan2 leaves (Fig. 6H) (Ha et al., 2007). pgy1 kan1 kan2 leaves were indistinguishable from kan1 kan2 leaves (Fig. 6I). In the as1 pgy1 kan1 kan2 quadruple mutant leaves had abaxial outgrowths as in kan1 kan2 (Fig. 6J). These phenotypes indicate that neither AS1 nor PGY1 are required for the severe adaxialised leaf phenotypes resulting from loss of both kan1 and kan2. In the as1 pgy1 kan1 kan2 quadruple mutants, outgrowths were often limited to the abaxial side of the leaves, consistent with KAN requirement for the as1 pgy1 phenotype.

pgy1 suppresses kan gynoecium defects

KANADI genes repress HD-ZIPIII genes and kan mutant phenotypes are in part due to ectopic HD-ZIPIII gene expression (Eshed et al., 2001; Izhaki and Bowman, 2007). Genetic interactions indicate that PGY1 functions together with REV in organ patterning and that pgy1 may enhance rev through effects on other HD-ZIPIII genes. However, pgy1 does not suppress the strong leaf defect of kan1 kan2 mutants. To further explore the relationship between AS1, PGY1 and KANADI genes, we tested whether as1 and pgy1 could suppress other kan mutant phenotypes. The kan1 kan2 double mutant has dramatic effects on gynoecium development, with proliferation of ectopic septum and ovules on the abaxial sides of the carpels (Eshed et al., 2001). As with the kan1 kan2 mutant, the combination of kan1 kan2/+ also displayed gynoecium defects, with ectopic septum and style tissues between the fused carpels, and an increase in apical style and stigma size (Fig. 7F). In as1 kan1 kan2/+ mutants, gynoecia were smaller, but otherwise similar to those of kan1 kan2/+ (Fig. 7G). In pgy1 kan1 kan2/+ mutants, the characteristic silique phenotype of kan1 kan2/+ (Fig. 7B,F) was suppressed by pgy1 (Fig. 7D,H). The siliques were more elongated in pgy1 kan1 kan2/+ compared with kan1 kan2/+, and their morphology was similar to wild type (Fig. 7A,E) except for occasional ectopic style tissue along the abaxial replum (Fig. 7H). pgy1 kan1 kan2/+ are fertile, whereas kan1 kan2/+ gynoecium defects result in greatly reduced fertility. Therefore PGY1, but not AS1, is required for the kan1 kan2/+ gynoecium phenotype.

PGY genes encode ribosomal proteins

PGY1, PGY2 and PGY3 were identified by positional cloning, and all three genes were found to encode cytoplasmic large subunit ribosomal proteins (Fig. 8A). pgy1-1 had a base pair change resulting in a premature stop codon in At2g27530. A second independent allele of pgy1, designated pgy1-2, also had a base pair change resulting in a premature stop codon in At2g27530. The as1 pgy1-2 mutant phenotype was the same as as1 pgy1-1. This gene encodes ribosomal protein L10a, which is a member of the L1p/L10e family, involved in binding and release of deacylated tRNA from the E site of the ribosome (Nikulin et al., 2003; Yusupov et al., 2001). A point mutation in pgy2-1 resulted in a splicing defect in At1g33140, whereas an insertion mutant allele, pgy2-2, failed to accumulate significant levels of transcript (see Fig. S2A in the supplementary material). As with as1 pgy2-1 mutants, as1 pgy2-2 mutants formed adaxial ectopic outgrowths. PGY2 encodes ribosomal protein L9, which is a member of the L6p/L9e family. This ribosomal protein is located at the base of the stalk protuberance, in a region important for translation-initiation-factor binding (Ban et al., 1999). A point mutation in pgy3-1 resulted in an amino acid change in the protein encoded by At3g25520, which encodes ribosomal protein L5. This ribosomal protein is a member of the L18p/L5e family. L18p/L5e binds the 5S RNA and is involved in anchoring of peptidyl-tRNA during translation (Meskauskas and Dinman, 2001).

Fig. 8.

Arabidopsis PGY genes encode ribosomal proteins. (A) Schematic representation of PGY1/RPL10aB, PGY2/RPL9C, PGY3/RPL5A genes showing sites of point mutations in pgy1-1, pgy1-2, pgy2-1, pgy3-1 alleles and site of T-DNA insertion in pgy2-2. (B-D) mRNA expression patterns of PGY1 (B), PGY2 (C) and PGY3 (D) in transverse sections of 10-day-old vegetative apices. All three genes are expressed in the meristem and throughout leaf primordia. In older leaves expression is higher in the vasculature and at the margins of the leaf, in cytoplasmically dense cells. Scale bars: 50 μm.

The ribosomal proteins encoded by all three PGY genes are members of small gene families and have one or two closely related members in Arabidopsis (see Table S1 in the supplementary material) (Barakat et al., 2001). The relatively subtle phenotype of pgy mutants may be due to redundancy between ribosomal proteins in Arabidopsis,or PGY genes may not have a role in global translation due to tissue-specific expression. To test for possible differential expression between the PGY ribosomal protein family members, we analysed expression in shoot tissues using RT-PCR. All of these ribosomal protein genes were ubiquitously expressed in the shoot (see Fig. S2B in the supplementary material). To investigate PGY function in leaf development further, we analysed the mRNA expression pattern of PGY1, PGY2 and PGY3 using in situ hybridisation. All three PGY genes were expressed throughout the vegetative meristem and in young organ primordia. In older organ primordia, expression was most prominent in adaxial cells and in the margins of developing leaves (Fig. 8B-D and see Fig. S3 in the supplementary material). These high levels of expression are coincident with small cytoplasmically dense cells. PGY genes, therefore, do not appear to regulate organ patterning through tissue-specific expression.


PGY1, PGY2 and PGY3 genes encode ribosomal proteins. A role for these genes in leaf patterning is revealed by leaf development phenotypes in an as1 background. Genetic interactions demonstrated that PGY1 and AS1 act independently in promoting adaxial fate or repressing abaxial fate, with PGY1 being an integral component of the HD-ZIPIII-KANADI pathway.

Development of ectopic leaves

Establishment of dorsoventral polarity in incipient leaf primordia requires specification of both adaxial and abaxial domains. In a model in which leaf outgrowth is promoted by juxtaposition of these two domains it might be expected that an ectopic patch of one fate within the field of the other would form a continuous boundary, and lamina outgrowth at this boundary would surround the circumference of the ectopic patch. This appears to be the case in Antirrhinum, where mutations in the AS1 orthologue PHANTASTICA (PHAN) result in lamina surrounding ectopic patches of abaxial tissue (Waites and Hudson, 1995). Likewise, in tobacco plants with reduced PHAN function, adaxial ectopic lamina outgrowth is contiguous along a boundary of tissue with reduced adaxial fate (McHale and Koning, 2004). Ectopic lamina outgrowths in maize dorsoventral polarity mutants are also most often formed as a continuum along an adaxial-abaxial boundary (Evans, 2007; Juarez et al., 2004a; Juarez et al., 2004b; Schichnes et al., 1997; Timmermans et al., 1998). As in these examples, outgrowths in as1 pgy1 mutants could result from small patches of abaxial cells on the adaxial side of the leaf. However, in as1 pgy1 mutants there was no clear evidence of abaxial cell types on the adaxial epidermis of the leaf. One possibility is that ectopic abaxial patches in subepidermal layers, such as in the vasculature, recapitulate an adaxial-abaxial boundary for ectopic outgrowth.

An alternative, but not exclusive, possibility is that as1 pgy1 outgrowths are the result of establishment of a new proximodistal axis of growth. Two of the earliest signatures of leaf primordia initiation are downregulation of class I KNOX genes and formation of a localised auxin maxima in the peripheral region of the meristem (Heisler et al., 2005; Jackson et al., 1994; Reinhardt et al., 2000; Reinhardt et al., 2003). In addition, auxin has been implicated in the formation of ectopic outgrowths on the hypocotyls of kan1 kan2 kan4 mutants (Izhaki and Bowman, 2007). Ectopic outgrowths in as1 pgy1 mutants may, likewise, initiate at sites of a localised auxin maximum, possibly established through interactions between patterns of ectopic KNOX expression, conferred by as1, and regulators of auxin distribution.

There is no morphological evidence that outgrowths are associated with a structured meristem. In addition, the KNOX genes BP (also known as KNAT1 - TAIR) and KNAT2 are not required for as1 pgy1 outgrowths (M.E.B., unpublished), although the role of KNOX genes in the piggyback phenotype may be masked by redundancy. Interestingly, all bifacial outgrowths in as1 pgy1 mutants were oriented with the adaxial side towards the distal tip of the leaf and the abaxial side towards the proximal base of the leaf. This invariant polarity, in the absence of a meristem, suggests sensitivity to additional patterning cues.

PGY1 is a component of the HD-ZIPIII-KANADI pathway

Genetic interactions indicate that AS1 interacts to a minor extent with the HD-ZIPIII-KANADI gene pathway. The as1 leaf phenotype was only moderately affected by loss of REV, KAN1 and KAN2 genes. Potentially other members of these gene families are downstream of AS1 and more dramatic polarity effects are masked by misexpression of the YABBY gene FILAMENTOUS FLOWER (FIL) in as1 (Garcia et al., 2006; Li et al., 2005).

In comparison, PGY1 has reciprocal interactions with both HD-ZIPIII and KANADI family genes. PGY1 may have common downstream targets with REV, or may regulate other HD-ZIPIII genes. Aspects of as1 pgy1 leaf phenotypes resemble rev phv and rev phv phb/+ mutant leaves. These combinatorial mutants exhibit a rippled leaf surface and develop ectopic leaf lamina on the adaxial side of the leaf (this study) (Prigge et al., 2005). Likewise, the similarity of the pgy1 rev inflorescence phenotype to the enhanced rev phenotype from loss of phv or phb/+ opens the possibility that downstream targets of PGY1 are HD-ZIPIII genes.

Loss of KANADI function suppresses the leaf phenotype of as1 pgy1, and this suppression is specific to pgy1. Interactions with KANADI genes are also evident in the flower, and pgy1 can rescue the carpel patterning defects of kan1 kan2/+ mutants. However, neither as1 pgy1 nor pgy1 can rescue the kan1 kan2 phenotype. Therefore, PGY1 is not strictly necessary for kan1 kan2 phenotype, and pgy1 loss-of-function is not sufficient to compensate the loss of both KAN1 and KAN2. Together these interactions suggest an antagonistic relationship between PGY1 and KANADI genes, possibly through opposing interactions with a common downstream target. Potential common downstream targets are HD-ZIPIII genes, which are negatively regulated by KANADI genes and, as noted above, may be positively regulated by PGY1.

Potential role of translation in development

Most ribosomal proteins in animals are encoded by a single copy gene, and mutations in these genes have gross effects on development. For example, mutations in ribosomal protein genes in Drosophila result in a semi-dominant Minute phenotype characterised by slow growth and reduced size of heterozygotes, and homozygous lethality (Lambertsson, 1998; Marygold et al., 2007). However, Drosophila Minute mutants also display various developmental defects, as does the mouse Minute mutant belly spot and tail (Bst; also known as Rpl24 - Mouse Genome Informatics), and ribosomal protein mutants in zebrafish (Amsterdam et al., 2004; Oliver et al., 2004; Uechi et al., 2006).

By contrast to animals, all ribosomal protein genes in Arabidopsis are represented by small gene families (Barakat et al., 2001). Mutations in an S5 ribosomal protein gene, arabidopsis minute-like1 (aml1), are semi-dominant. As with Drosophila Minute mutants, aml1 heterozygotes are smaller in size than wild type, and homozygotes are embryo lethal (Weijers et al., 2001). Unlike aml1, mutations in several ribosomal protein genes are recessive and result in subtle developmental defects. Mutations in POINTED FIRST LEAF (PFL) and POINTED FIRST LEAF2 (PFL2), which encode an S18 ribosomal protein and an S13 ribosomal protein, respectively, have some reduced growth and changes to the shape of early formed leaves (Ito et al., 2000; Van Lijsebettens et al., 1994). Mutations in SHORT VALVE1 (STV1), which encodes an L24 ribosomal protein, are reduced in stature but, in addition, display carpel-tissue-patterning defects (Nishimura et al., 2005). This phenotype mimics the floral phenotype of AUXIN RESPONSE FACTOR gene mutants, ettin (ett) and monopteros (mp), and it has been proposed that STV1 modulates ETT and MP expression via translation of short upstream open-reading-frames (uORFs) encoded in the 5′ UTR of ETT and MP transcripts (Nishimura et al., 2005).

Mutations in PGY1, PGY2 and PGY3 and combined mutations in these genes have relatively subtle effects on development, suggesting that select targets are more sensitive to loss of these ribosomal proteins rather than a global effect on protein synthesis. The nature of these three ribosomal proteins does not immediately suggest a mechanism for the specificity of the phenotype. One possibility is that PGY proteins have a function independent from the ribosome, as reported for some other ribosomal proteins (Wool, 1996). However, PGY genes and related family members are expressed throughout actively dividing tissues, as would be anticipated for ribosomal proteins involved in global protein synthesis (this study) (Schmid et al., 2005). An alternate possibility is that not all ribosomes are equivalent. The presence of different isoforms and post-translational modifications of ribosomal proteins result in ribosome heterogeneity, and this may generate functionally distinct ribosomes with target-transcript specificity (Carroll et al., 2007; Giavalisco et al., 2005; Komili et al., 2007). This appears to be the case in Saccharomyces cerevisiae, where mutations in ribosomal protein paralogues have different phenotypic consequences (Komili et al., 2007). PGY target specificity may be conferred by interaction with other proteins or protein complexes. An interacting complex may be the small RNA silencing complex, RISC, which interacts with large subunit ribosomal proteins (Chendrimada et al., 2007; Ishizuka et al., 2002). Or specificity may be inherent in target transcripts, either in non-coding UTR sequences, as with STV1 targets or, for example, may be mediated through the binding of small RNAs such as mircoRNAs or trans-acting siRNAs (ta-siRNAs). In this respect, it is noteworthy that several leaf dorsoventral polarity gene families are targeted by small RNAs. The adaxial HD-ZIPIII genes are targets of microRNAs mir165 and mir166, whereas the AUXIN RESPONSE FACTOR genes ETT and ARF4, which are required for abaxial fate, are targets of TAS3-derived ta-siRNAs (Allen et al., 2005; Rhoades et al., 2002; Tang et al., 2003). These proposed models for PGY function are not necessarily mutually exclusive, and it seems likely that global effects on translation are, to some extent, masked by redundancy between family members in Arabidopsis. Although the mechanism is still to be defined, PGY mutants provide new insights into regulatory networks controlling leaf patterning and begin to address the role of the ribosome as a regulator of development.

Supplementary material

Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/135/7/1315/DC1


We thank colleagues at JIC for valuable discussion and comments on the manuscript. We also thank Desmond Bradley and Sinead Drea for advice on in situ hybridisation, Adrian Turner for advice on qRT-PCR, Andrew Davis for photography, Kim Findley for SEM training and the JIC Horticultural Services staff for plant care. We thank John Bowman for providing seed of kan1 and kan2 mutants. V.P. is supported by a Marie Curie Early Stage Training Programme Studentship (JICISBEST). This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) to M.E.B. and by the Royal Society as a Wolfson Merit Award to M.E.B.


  • * These authors contributed equally to this work

  • Present address: University of Manchester, Manchester M13 9PT, UK

    • Accepted January 3, 2008.


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