A FILAMENTOUS FLOWER orthologue plays a key role in leaf patterning in opium poppy

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1 The Plant Journal (2012) 72, doi: /j X x A FILAMENTOUS FLOWER orthologue plays a key role in leaf patterning in opium poppy Nikolaos Vosnakis, Afiqah Maiden, Sofia Kourmpetli, Philip Hands, Donna Sharples and Sinéad Drea* Department of Biology, University of Leicester, University Road, Leciester LE1 7RH, UK Received 2 March 2012; revised 13 July 2012; accepted 17 July 2012; published online 24 September *For correspondence ( sd201@le.ac.uk) These authors contributed equally. SUMMARY The plant-specific YABBY genes were initially defined by their roles in determining abaxial/adaxial cell fate in lateral organs of eudicots, and repressing meristematic genes in differentiating tissues such as leaves. In Arabidopsis thaliana FILAMENTOUS FLOWER (FIL) is also required for inflorescence and floral meristem establishment and flower development in a pathway involving the floral transition and identity genes. Here we describe the characterization of a FIL orthologue from the basal eudicot, Papaver somniferum (the opium poppy), and demonstrate a role for the gene in patterning the highly lobed leaf of the poppy. Silencing of PapsFIL using viral-induced gene silencing resulted in leaves of reduced laminar area, more pronounced margin serration and, in some cases, leaf bifurcation. In contrast, the gene does not appear to affect the development of the flower, and these variations in function are discussed in relation to its taxonomic position as a basal eudicot and its determinate growth habit. Keywords: Papaver somniferum, basal eudicot, leaf development, shoot apical meristem, YABBY, viral-induced gene silencing. INTRODUCTION The YABBY genes are a family of plant-specific transcription factors shown to play a key role in determining leaf polarity in Arabidopsis and Antirrhinum. A Cys2 Cys2 zinc-finger domain at the amino terminus and an HMG box region (also called the YABBY domain) at the C terminus comprise two conserved domains (Sawa et al., 1999b; Bowman, 2000). It was shown that the YABBY gene family in Arabidopsis includes six genes that are categorized in three groups based on sequence analysis. The CRC (CRABS CLAW) group, the INO (INNERNO OUTER) group and the FILAMENTOUS FLOWER (FIL) group, including FIL, YAB3, YAB5 and YAB2 (Bowman, 2000). Expression and functional data analysis from core eudicots Expression and functional data analysis from core eudicots Arabidopsis, Antirrhinum, Solanum lycopersicum (tomato) and Solanum tuberosum (potato), as well as the basal eudicot, Eschscholzia californica, led to the early conclusion that YABBY genes have a highly conserved role in promoting abaxial cell fate, and are expressed in the abaxial domains of lateral organs (Kim et al., 2003; Eshed et al., 2004a; Golz et al., 2004; Navarro et al., 2004; Orashakova et al., 2009). The conclusions of these studies where challenged by the investigation of YABBY gene expression patterns in Oryza sativa (rice, a monocot) that clearly showed that YABBY genes could be expressed in a non-polar pattern. In addition, even when members of the YABBY gene family were expressed ectopically this did not seem to affect the determination of abaxial or adaxial fate (Yamaguchi et al., 2004). More clear evidence supporting the functional diversity of YABBY genes resulted from the study of homologous genes in the monocot Zea mays (maize). It was shown that in Z. mays YABBYs are expressed in an opposite pattern, i.e. in the adaxial side of developing lateral organs (Juarez et al., 2004). Even within the monocots there is variation in the function of YABBY othologues in leaf development, and CRC/DL promotes a flattening of unifacial leaves in Juncus, in contrast to the midrib formation in rice (Yamaguchi et al., 2010). Whereas the YABBY genes are generally implicated in the abaxial/adaxial patterning of lateral organs, at least in the eudicots, in monocots they have also been shown to be involved in organ identity specification (DROOPING LEAF, DL, the orthologue of CRC; Yamaguchi et al., 2004). Another example of a YAB2 orthologue that demonstrates an expression pattern that differs from that 662 The Plant Journal ª 2012 Blackwell Publishing Ltd

2 Role of a FIL orthologue in poppy development 663 found in eudicots came from a study on the basal angiosperm Amborella trichopoda, where the gene was found expressed in the adaxial side (Yamada et al., 2004), although a more recent study reported the abaxial expression of YABBY genes in Cabomba (Yamada et al., 2011). This variation in expression patterns of YABBY genes may indicate that during the evolution of angiosperms the function of these polarity determinants may have diverged significantly. Expression analyses of FIL orthologues in Streptocarpus and Tropaeolum suggest that modulations in gene expression correlate with morphological variations in the form of the phyllomorph basal meristem of Streptocarpus and the peltate leaf development of Tropaeolum (Gleissberg et al., 2005; Tononi et al., 2010). YABBY expression is important for the normal development of lateral organs for an additional reason. Members of the YABBY gene family downregulate the expression of KNOTTED homeobox (KNOX) genes in leaves. KNOX genes are responsible for the preservation of the meristematic activity of the shoot apex (Kumaran et al., 2002). In contrast to the shoot apical meristem, lateral organs show determinate growth. This means that KNOX genes should be repressed in the domains where lateral organs develop. This interaction with KNOX genes seems to be maintained in monocots (Dai et al., 2007). In Antirrhinum, Arabidopsis and rice the function of the FIL orthologues has been studied explicitly using forward (mutagenesis) and reverse (RNAi) approaches. In these species FIL disruption has been shown to have effects in both vegetative and floral tissues (Chen et al., 1999; Sawa et al., 1999a; Golz et al., 2004; Dai et al., 2007; Tanaka et al., 2012). But in Arabidopsis, the effect in vegetative tissues is visible only in combination with a mutation of the closely related and therefore functionally redundant YAB3 gene (Siegfried et al., 1999). In this double mutant there is a reduction in the width of the leaf lamina and in the ectopic activation of meristematic activity, resulting in such phenotypes as bifurcating leaves. Such phenotypes would also imply that the gene could play an important role in the elaboration of leaf lobes in a simple, lobed leaf, as it could with marginal serration in entire simple leaves of Arabidopsis and Antirrhinum. At the functional level, RNAi of a rice orthologue revealed effects on leaves and flowers (Dai et al., 2007), and a mutant in the TONGARI-BOUSHI1 gene (also Os04g ) revealed pleiotropic effects in rice spikelets (Tanaka et al., 2012). Within eudicots, the lobed leaf of Papaver somniferum (the opium poppy) provides a different canvas from the unlobed leaves of Arabidopsis and Antirrhinum, or the compound leaves of tomato or Cardamine hirsuta (Canales et al., 2011), upon which to explore leaf patterning. And these contrast with the parallel-veined, sessile leaves of monocots such as rice and maize. Comparison of pathways regulating leaf development has revealed a significant divergence between dicots and monocots in several aspects (Kidner and Timmermans, 2007). In addition to distinctive leaf features, P. somniferum has a determinate inflorescence, with solitary flowers, in contrast to the prominent indeterminate inflorescences of the other species under study. It is therefore of interest to examine the role of a gene such as FIL that is implicated in both vegetative and inflorescence development. Studying key developmental genes in the opium poppy could provide useful information for their molecular and functional evolution within angiosperms (Kolsch and Gleissberg, 2006). Here we describe the characterisation of an FIL orthologue in the opium poppy and show that its functions are restricted to the vegetative phase generally, and leaf development specifically. RESULTS Leaf development and morphology in Papaver somniferum cv. Persian white Early leaf development in Papaver has been described previously by Gleissberg (1998), and here we provide a brief description of development in the Persian white cultivar of P. somniferum used in this study. The two cotyledons are long and linear, without an obvious petiolar connection (Figure 1a). The first two true leaves are not lobed, are simple and are without trichomes (Figure 1a). Subsequent leaves emerge lobed and with developing trichomes (Figure 1b). The shoot apical meristem (SAM) is well concealed deep at the base of the developing leaves (Figure 1a c), and is not prominently domed, but appears quite flat instead (Figure 1d). The P. somniferum leaf is simple and pinnate but lobed with incised margins. The blade contains several lobes that are subdivided, and along the length of the rachis and the petiole, the lamina is broad (Figure 1e). Leaflet initiation is acropetal, and coincides with the onset of surface growth (Gleissberg, 1998). The transition from vegetative to reproductive phase correlates with the transition from petiolar to amplexicaulous or sessile leaves (Figure 1e), with the upper leaves clasping the stem at their base without a petiole, and with leaves arranged alternately on the stem. Similarly as for Eschscholzia californica (Becker et al., 2005), P. somniferum has a solitary terminal flower (Figure 1f) that emerges from a prominent primary axis. It lacks bracteose leaves, and flowers appear basipetally from a few lateral shoot meristems. An inflorescence meristem stage is difficult to identify, and it seems that there may be a direct transition from vegetative SAM to flower meristem (FM). Isolation and sequence analysis of a full-length FIL orthologue in Papaver somniferum Degenerate primers were used to isolate YABBY gene orthologues from poppy flower tissues using reverse transcription polymerase chain reaction (RT-PCR). By far the

3 664 Nikolaos Vosnakis et al. (a) (b) (f) (c) (d) Figure 1. Development of Papaver somniferum cv. Persian white. (a) SEM of a 10-day-old seedling. (b d) SEM of a 7-day-old seedling, showing the shoot apical meristem (SAM), well protected by emerging leaves. (e) Variation in leaf size and morphology. From left to right, leaves of a single plant from the base to the top. (f) Flowering plant. (g) Persian white flower. Scale bars: (a d) 50 lm; (e g) 1 cm. (e) (g) predominant transcript isolated showed significant BLASTX homology with the FIL/GRAMINIFOLIA (GRAM) YABBY gene products of Arabidopsis and Antirrhinum. Both 5 and 3 rapid amplification of cdna ends (RACE) was used to isolate the full-length sequence of the gene from the opium poppy. As expected, amino acid alignments showed highest sequence similarities in the zinc-finger and YABBY domains (Figure 2b). Phylogenetic analyses confirmed that the gene was an orthologue of the FIL clade YABBY genes, and revealed interesting patterns of FIL gene duplication in the angiosperms (Figure 2a). In eudicots there have been recent duplications in Arabidopsis (FIL and YAB3; Siegfried et al., 1999) and in Eschscholzia (EScaYAB1 and EScaYAB2; Bartholmes et al., 2011), whereas in monocots, where several completed genomes are available for mining, e.g. maize, Sorghum, rice and Brachypodium, a duplication at the base of the Pooideae and a more recent duplication in the maize lineage has occurred, where four orthologues have been identified (Juarez et al., 4004). At the sequence level, these taxonomic groups are discernable, with monocot orthologues having obvious insertions in the region between the conserved zinc-finger and YABBY domains (Figure S1). Expression analysis of PapsFIL throughout the poppy plant In general spatial terms, an RT-PCR survey of the vegetative and floral tissues of the poppy plant showed expression of PapsFIL at readily detectable levels throughout the plant (Figure 3a). Detailed expression analysis was performed on vegetative and reproductive tissues of poppy plants using mrna in situ hybridization. As with AtFIL in Arabidopsis, expression of PapsFIL is tightly restricted to the abaxial cell layers from the early stages of floral primordia emergence (stages P1 P2, as defined in Drea et al., 2007), and is completely absent in the central meristem region (Figure 3f,g). In later stages of flower development, such as P5, it appeared that the expression domain also became localized along the proximo distal axis, towards the distal region of the organ, as the organs extended (Figure 3i,j). By stages P6 and P7 the abaxial expression was detected in only two or three cell layers, primarily in the petal, carpel and sepal (Figure 3h k). The pattern of expression was similar in the shoot, with expression absent from the shoot meristem and restricted to the abaxial domain of the emerging leaf primordia (Figure 3c). As the young leaves differentiate, however, PapsFIL continues to be expressed abaxially, but in cells located mainly in the margins. Functional analysis of PapsFIL in Papaver somniferum using viral-induced gene silencing Fragments of approximately 300 bp, spanning PapsFIL gene regions between the conserved zinc-finger and YA- BBY domains (Y1) and the 3 region downstream of the YABBY domain (Y2; including the 3 untranslated region, 3 -UTR), were amplified and cloned into TRV2 vector for use in viral-induced gene silencing (VIGS) experiments in P. somniferum cv. Persian white. After infiltration with the silencing constructs, plants were monitored for visible phenotypes throughout vegetative and reproductive development. Out of 600 infiltrated plants, leaf samples

4 Role of a FIL orthologue in poppy development 665 (a) (b) Figure 2. Sequence analysis of PapsFIL. (a) Phylogenetic analysis of YABBY proteins from across the angiosperms, with PapsFIL underlined. Accession numbers are listed in Table S1. (b) Multiple protein sequence alignment of PapsFIL and other YABBY genes from various eudicot species. Identical residues are highlighted in black, whereas similar residues are highlighted in grey. Zinc-finger and YABBY domains are indicated by black and grey bars, respectively. were collected from 100 plants showing aberrant phenotypes for further analysis. Samples from wild-type and empty-vector plants were used as controls. Out of the100 individual plants tested, 30 plants were confirmed by RT-PCR to be positive for the TRV2-Y1/Y2 constructs (30%) (Figure 4a). VIGS in plants with either the TRV2-Y1 or the TRV2-Y2 constructs resulted in the same aberrant phenotypes, whereas empty-vector plants resulted in a wild-type phenotype, confirming that the TRV2 vector does not introduce any visible effects to the poppy plants. Infiltrated plants negative for any construct also exhibited the wildtype phenotype, proving that the VIGS procedure did not cause the aberrant phenotypes observed in infiltrated plants positive for TRV2-Y1/Y2 constructs. Wild-type poppy plants grown under the same growth conditions as transformed plants did not exhibit any aberrant phenotypes, eliminating environmental factors as a possible cause for the aberrant phenotypes exhibited by VIGS plants.

5 666 Nikolaos Vosnakis et al. (a) (f) (g) (b) (c) (h) (i) (d) (e) (j) (k) Figure 3. Expression analysis of PapsFIL throughout the opium poppy plant. (a) RT-PCR survey for the detection of PapsFIL in different sized floral buds (B0.1 B7), carpels (Ca0.3 and Ca0.7, 0.3 and 0.7 cm long, respectively), and leaves from a vegetative stage plant (LV) and from a reproductive stage plant (LR). Actin was used as a control. (b, d) Transverse sections of a young seedling showing that PapsFIL is not expressed in the SAM (m), but is expressed in the abaxial domain of the leaf primordium (lp). Arrows in (d) highlight the concentration of expression in the marginal abaxial region. (c, e) Longitudinal sections through young seedlings. (f j) mrna in situ hybridization of longitudinal sections of floral buds, indicating the abaxial expression of PapsFIL in sepals (se), petals (pe), stamens (st) and carpels (ca), as well as in leaf primordia (lp). (k) Transverse section of a floral bud. Scale bars: (b e) 25 lm; (f k) 200 lm. PapsFIL affects leaf margin serration and leaf laminar expansion The main effects in leaf development involve the extent and depth of margin incision and the expansion of the leaf blade, and we have classified the phenotypes observed into three classes: (i) increased level of margin serration; (ii) laminar gaps and reduction in blade area; and (iii) lobe-to-leaflet (including bifurcated leaves; Table S2). Of the confirmed VIGS plants, 43.3% showed an increased level of margin serration. Leaves appeared to be severely cut-in and were more serrated compared with wild-type leaves (Figure 5a,b and inset). Figure 5c, and inset, show leaves with laminar gaps, which appeared in 50% of the VIGS-confirmed population: the lamina had not extended, resulting in gaps in the leaf blade. In some cases there was no lamina extension beyond the central vein. Severely asymmetric leaves are shown in Figure 5f,g, in contrast to the symmetrical wildtype leaves (Figure 5d), and appeared in 40% of the population. A few leaves were observed to be bifurcated (Figure 5e). Some plants exhibited only one of the categorized aberrant phenotypes, whereas 26.7% of them exhibited a combination (Table S2). Quantitative RT-PCR revealed that there is a correlation between the level of PapsFIL expression and the resulting phenotype (Figure 4b). Leaf samples from individual VIGS-confirmed plants with a different phenotype severity were tested, and results showed a statistically significant difference in the PapsFIL transcript levels of plants with severe and moderate phenotype, compared with the wild type (P < 0.05). Plants with greater than 50% of leaves affected were designated as severe phenotypes, plants with 30 50% of leaves affected were designated as moderate phenotypes and plants with less than 30% of leaves affected were designated as mild phenotypes. Furthermore, empty-vector plants as well as mild phenotype plants did not show any significant difference compared with the wild type (P > 0.05; Table S3). PapsFIL transcript levels were reduced by approximately 40% in the most severely affected plants, whereas in those with a moderate phenotype the reduction was approximately 15% compared with the wild type.

6 Role of a FIL orthologue in poppy development 667 (a) (b) Figure 4. Expression analysis of PapsFIL in VIGS plants. (a) RT-PCR for the detection of TRV2-Y1 in VIGS plants (Y1 Y4) and in plants carrying an empty TRV2 vector (E1 and E2). Note the difference in the size of the amplified fragment, caused by the insertion of Y1 into TRV2. Wild-type untransformed plants were used as controls (WT1 and WT2). The integrity of cdna was tested using Actin-specific primers. (b) Quantitative RT-PCR results, showing the correlation between the levels of PapsFIL expression in the leaves and the severity of the plant phenotype. PapsFIL expression levels were normalized against Actin and were presented as a percentage of the wild-type PapsFIL expression. Error bars indicate SEM. *Statistically significant difference, compared with the wild type (P < 0.05). Scanning electron microscopy (SEM) was conducted to see the effect of PapsFIL gene silencing at the cellular level. Several leaf samples from VIGS plants with moderate and severe phenotypes, empty vector and wild-type plants were subjected to SEM observation of abaxial and adaxial leaf surfaces to identify any changes in cell shape, organization or size, resulting from PapsFIL silencing. The adaxial epidermal surface of wild-type leaves was composed of tightly packed large pavement cells, whereas cells on the abaxial surface were smaller, with frequent stomata (Figure 5h,i). SEM observations of empty vector VIGS leaf surfaces showed no difference compared with the wildtype, as expected. Also, the adaxial leaf surface of previously confirmed VIGS plants appeared to be similar to that of the wild type (Figure 5m), suggesting that the silencing of PapsFIL has no effect on the shape and organisation of these cells. On the contrary, abaxial cells were found to be constricted, very irregular and in some cases had a flattened appearance (Figure 5l). In the most severe cases, the irregularity of the cell surface made stomata difficult to distinguish. Transverse sections of these leaves, stained with toluidine blue, clearly highlighted the collapsed cells of the abaxial side, confirming the SEM findings (Figure 5o). VIGS plants of moderate phenotype had moderately irregular abaxial sides, whereas those with a severe phenotype had highly irregular abaxial cells, suggesting that the resulting phenotype is caused by this disorganisation of the abaxial leaf side. Moreover, SEM observations of the leaf edges of severely serrated VIGS plants revealed a complete lack of cell organisation in some of these areas, and the appearance of cauliflower-like structures (Figure 5n) that could explain the arrest of lamina expansion in those leaves, resulting in asymmetrical and highly serrated phenotypes. Given the role of the FIL/GRAM genes in Arabidopsis, Antirrhinum and other species (Chen et al., 1999; Golz et al., 2004; Dai et al., 2007), it was unexpected that no aberrant phenotype was detected in the flowers of all 600 infiltrated plants. Previous studies illustrate the efficiency of the same VIGS procedure in silencing flower-expressed genes in the same species and cultivar of opium poppy (Hileman et al., 2005; Drea et al., 2007; Hands et al., 2011; Pabon-Mora et al., 2012). However, to eliminate the possibility that the lack of a phenotype in floral tissues was caused by the absence of VIGS penetration as far as the flower, we also tested the flowers of plants with defective leaves for the presence of the TRV2-Y1/Y2 fragment. In the majority of those flower samples, TRV2-Y1/Y2 was detected, as shown in Figure S2. Investigation of PapsFIL functional mechanism in Arabidopsis To explore the functional orthology of PapsFIL with AtFIL, the entire PapsFIL coding region was expressed under the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into wild-type Arabidopsis (Ler-0 ecotype). A summary of the analyses of transgenic lines is available in Table S4. The overexpression of PapsFIL produced seedlings with severe defects in SAM development. Most transgenic seedlings did not develop far beyond the cotyledon phase (Figure 6b,i,j). Cotyledons appeared to be larger than the wild type, and in many cases curled downwards. In cases where true leaves developed, these were usually of an irregular shape, curling inwards, and in some extreme cases were clavate (Figure 6b). Table S4 shows details of the different seedling phenotypes exhibited by CaMV35S::PapsFIL seedlings 1 month after germination. Detailed examination of the transgenic seedlings showed that the SAM was much larger than the wild type (Figure 6c), whereas SEM revealed a high degree of cell disorganisation and lack of differentiation (Figure 6k m). RT-PCR on whole seedlings confirmed the expression of the PapsFIL gene at high levels, and also showed that SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS) expression levels were higher than in the wild type (Figure 7a). Two different wild-type samples were used as controls: one seedling of the same age (3 weeks old) and one at the same developmental stage (two true leaves; 10 days old).

7 668 Nikolaos Vosnakis et al. (a) (b) (c) (d) (f) (e) (g) (h) (i) (j) (k) (l) (m) (n) (o) Figure 5. The effect of PapsFIL silencing on Papaver somniferum leaves. (a) Fully developed wild-type leaf. (b) Deeply serrated leaf from a VIGS plant, with another example as an inset (class 1). (c) Leaf with undeveloped lamina, with another example as an inset (class 2). (d) Young wild-type leaf. (e) Bifurcated leaf from a VIGS plant (class 3). (f, g) Highly asymmetric leaves from VIGS plants (class 3). (h) Abaxial side of a wild-type leaf. (i) Adaxial side of a wild-type leaf. (j) Leaf edge of a wild-type leaf. (k) Transverse section of a wild-type leaf stained with toluidine blue. (l) Abaxial leaf side of a VIGS plant showing the irregular shape of cells. Arrows indicate stomata. (m) Adaxial leaf side of a VIGS plant that does not show any abnormalities compared with the wild type. (n) Leaf edge of a severely serrated and asymmetrical VIGS plant showing high levels of disorganization and the formation of cauliflower-like structures. (o) Transverse leaf section of a VIGS plant stained with toluidine blue. Arrows indicate collapsed cells on the abaxial side. Scale bars: (a g) 1 cm; (h o) 20 lm. The few plants that managed to switch to the reproductive stage (nine plants out of the 86) showed a range of severity in leaf phenotypes as well as in the length of the inflorescence stem (Figure 6d,e,f). However, they all produced flowers that were similar to the wild type and set seed as normal (Figure 6g). The RT-PCR survey of separate rosette leaf and inflorescence samples of these plants showed that PapsFIL was expressed in abundance in both tissues (Figure 7b), suggesting that the heterologous expression of PapsFIL in Arabidopsis has no effect on flower development. In addition, there was no STM or WUS ectopic expression detected in the leaves of these plants, suggesting that the previously detected elevated transcript levels in the seedling samples were probably the result of enlarged meristems. mrna in situ hybridization confirmed the higher STM levels in the SAMs of the transgenic seedlings, compared with the wild type, and the domain of STM expression correlated with the larger meristem area (Figure 7c). SEM observations of the abaxial and adaxial surfaces of leaves from flowering plants did not show any obvious differences compared with the wild type leaves, except for cellular disorganization (Figure S3). DISCUSSION The YABBY genes have been designated as primarily vegetative or reproductive in terms of function and/or expression patterns in Arabidopsis (Bowman, 2000). FIL, YAB2, YAB3 and YAB5 are expressed in both vegetative and reproductive tissues, whereas INO and CRC expression is restricted to the flowers. Depending on the species in question, these categorizations can overlap, with CRC and FIL orthologues having both vegetative and reproductive roles in monocots

8 Role of a FIL orthologue in poppy development 669 Figure 6. Heterologous expression of PapsFIL in Arabidopsis thaliana. (a) Wild-type seedling, 10 days old. (b) Transgenic seedling showing enlarged SAM and abnormal leaf development. (c) Detail of enlarged SAM in a seedling expressing 35S::PapsFIL, showing the initiation of leaf-like structures from two lateral meristems. (d) Wild-type A. thaliana (Ler-0) flowering plant. (e, f) Arabidopsis plants overexpressing PapsFIL at the flowering stage. (g) Flower of transgenic plant, showing normal number of floral parts. (h) Wild-type A. thaliana seedling (10 days old). (i, j) Arabidopsis seedlings overexpressing PapsFIL (3 weeks old). (k m) SAM of Arabidopsis 35S::Paps- FIL seedlings. Scale bars: (a c) 50 lm; (d f) 1 cm; (g, k m) 100 lm; (h j) 500 lm. (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (Yamaguchi et al., 2004, 2010; Dai et al., 2007), compared with reproductive phenotypes (as single mutants) in Arabidopsis (Alvarez and Smyth, 1999; Bowman and Smyth, 1999; Sawa et al., 1999a; Chen et al., 1999; Lugassi et al., 2010). A recent survey of YABBY genes in E. californica showed that orthologues from all vegetative clades were expressed in both vegetative and reproductive tissues, as in Arabidopsis (Bartholomes et al., 2011). Here, using VIGS, we show that an FIL orthologue in P. somniferum, a basal eudicot, has an effect on vegetative development. PapsFIL plays a role in several aspects of leaf patterning Elaboration of the dicot leaf involves growth in three axes: the adaxial abaxial; the proximal distal; and the lateral axes (reviewed in Szakonyi et al., 2010). The genetic basis of leaf form is an intensively studied topic in developmental biology, and has centred around the involvement of KNOX genes, polarity genes (HD-ZIPIII, KANADI and YABBY families), ARP-MYB genes, complementary small RNAs, boundary genes of the CUC family and the auxin hormone (reviewed in Blein et al., 2010; Efroni et al., 2010; Kidner and Timmermans, 2010; Scarpella et al., 2011). The phenotypes observed in poppy plants with reduced PapsFIL expression categorized as lobe leaflet formation is reminiscent of defects in KNOX gene regulation, as found in Arabidopsis fil yab3 double mutants (Kumaran et al., 2002), although we did not detect ectopic expression of the two class-1 KNOX genes PapsSTM1 and PapsSTM2 in our study.

9 670 Nikolaos Vosnakis et al. (a) (b) (c) Figure 7. Molecular and histological analysis of transgenic Arabidopsis plants. (a) RT-PCR survey for the expression of PapsFIL, STM and WUS in 35S::PapsFIL transgenic seedlings (T 1 T 3 ), compared with wild-type seedlings at two developmental stages: 3 weeks old (WT3w, same age as the transgenic seedlings) and 10 days old (WT10d, same developmental stage as the transgenic seedlings, with two true leaves). Actin was used as a control. (b) RT-PCR survey of leaf and inflorescence samples from wild-type (WT) and 35S::PapsFIL transgenic plants (A, B and C), showing that PapsFIL is expressed in the flowers, even though there is no observed phenotype. Actin was used as the control ( ve, negative control, water). (c) Longitudinal section of a transgenic seedling hybridized with an STM probe. Inset shows the expression pattern of the same probe in wild-type seedling shoot apical meristem (SAM). lp, leaf primordium; m, meristem. Scale bars: 100 lm. It should be noted that our analyses on VIGS plants were on mature leaves, and so it could be that we missed the earlier expression of various candidate genes. The margin serration effects could implicate the CUC genes and auxin (Blein et al., 2008; Sarojam et al., 2010). Sarojam et al. (2010) proposed that YABBY activity might be involved with auxin-mediated effects in leaf margins based on previous observations of margin effects in Antirrhinum leaves (Golz et al., 2004). Ben- Gera et al. (2012) have shown that auxin maxima coincide with initiating leaflets and lobes in the compound leaves of tomato, whereas the microapplication of auxin caused ectopic leaflet outgrowths. This effect in leaf margins is very clear in the incised, lobed leaves of P. somniferum. Blein et al. (2008) found that overexpression of KNOTTED1 in the compound leaves of Cardamine hirsute led to an increase in CUC gene expression, and as the silencing of CUC genes had led to reduced lobing and leaflet fusion, one possibility is that the phenotypes observed in Papaver by silencing PapsFIL might be caused by a downstream effect through derepression of KNOX gene activity in the leaves. Further work is required to demonstrate the involvement of CUC genes or altered auxin distribution in poppy. This downstream effect is likely to be indirect, requiring other leaf-specific determinants, as when PapsFIL is ectopically expressed in Arabidopsis it led to an increase in STM expression. The overexpression of PapsFIL in Arabidopsis produced leaf development phenotypes that differ from previous AtFIL overexpression analyses (Sawa et al., 1999b; Siegfried et al.,1999). Specifically, we did not detect expression of KNOX1 genes in Papaver VIGS leaves, and we did not see direct evidence of actual abaxialization of adaxial epidermis cells. The presence of PapsFIL in the SAM (when driven by 35S) resulted in larger SAMs and expanded STM expression domains, showing that the central meristem domain is at least sensitive to YABBY activity, as shown in Arabidopsis (Siegfried et al., 1999; Goldshmidt et al., 2008), even though PapsFIL is not expressed in this central domain. Based on the highly abaxially-restricted expression pattern of PapsFIL and the exclusion of expression in the central meristem, a non-autonomous effect is also possible in P. somniferum. Differential effects on gene expression in the context of the SAM or the leaf have been seen with the GOBLET gene (CUC2 orthologue) in tomato, the expression of which is reduced in the SAM in response to auxin, but is unaffected in the leaf (Ben-Gera et al., 2012). Whereas KNOX, ARP and HD-ZIPIII genes have been identified and examined in species beyond the angiosperms (Harrison et al., 2005; Floyd and Bowman, 2006), YABBY genes are absent from completed genomes of bryophytes and lycophytes (Floyd and Bowman, 2007). It has been suggested that the YABBY genes are the key novel regulators in the evolution of spermatophyte leaves that allow for the difference between the meristematic domains of the SAM and the blastozones of the leaves, and the possible derivation of the latter from the former (Floyd and Bowman, 2010). Silencing or heterologous overexpression of PapsFIL does not affect flower development In Arabidopsis and Antirrhinum, LEAFY (LFY) and FLORI- CAULA (FLO; Coen et al., 1990; Weigel et al., 1992) act with APETALA 1 (AP1) and SQUAMOSA (Irish and Sussex,1990; Huijser et al., 1992) to promote the conversion of vegetative and inflorescence meristems to a floral state. As genetic studies have revealed that the basis of the fil floral phenotypes in Arabidopsis is through AP1, LFY and the ABC genes

10 Role of a FIL orthologue in poppy development 671 (Sawa et al., 1999a), it could also be suggested that the absence of the AP1 lineage outside of the core eudicots (Litt and Irish, 2003; Pabon-Mora et al., 2012) might affect the function of genes such as FIL. Arabidopsis, Antirrhinum and rice all have distinct inflorescence stages where an inflorescence meristem is easily recognizable. This is in contrast with species that have terminal inflorescences, such as P. somniferum and E. californica (Becker et al., 2005). In the latter case the expression of EScaFLO was found to be stable throughout the transition from vegetative to reproductive phases (Becker et al., 2005), unlike the modulation in expression levels of LFY in Arabidopsis as it moves to the floral initiation stage (Blazquez et al., 1997). As with the abaxial expression of the EScaCRC orthologue in E. californica (Orashakova et al., 2009), PapsFIL is also expressed in a distinctly polarized abaxial pattern in P. somniferum. This suggests that the modulation in expression of YABBY genes from abaxial in core eudicots to adaxial or central domains is particular to the monocots. Across the angiosperms the expression patterns of YABBY genes can vary in terms of being polar or non-polar, and the phenotypic effects can vary in terms of whether they affect vegetative and/or reproductive development, the function of YABBY genes is central to fundamental developmental processes. There is the possibility that another FIL paralogue in opium poppy may exist that plays a role in flower development, although we did not identify another paralogue in opium poppy in our analyses. The FIL and YAB3 paralogues in Arabidopsis have distinct and redundant roles in flower and vegetative development, respectively (Chen et al., 1999; Sawa et al., 1999a,b; Siegfried et al., 1999). Recently, two closely related FIL paralogues have been identified in Eschscholzia, and both genes are expressed extensively throughout the plant in both vegetative and floral tissues (Bartholmes et al., 2011). It will be interesting to explore their functional conservation or divergence. Our results suggest that despite their very similar expression patterns, PapsFIL and AtFIL are functionally divergent. EXPERIMENTAL PROCEDURES Isolation of PapsFIL We used RT-PCR reactions with the dcrc-znf and dcrc-yb degenerate primers (Lee et al., 2005) to isolate YABBY genes from carpel and flower cdna. Total RNA was extracted using Trizol reagent (Invitrogen, and converted to cdna using Superscript III (Invitrogen), following the manufacturer s instructions. Isolated sequences were cloned into pcr4- TOPO sequencing vector (Invitrogen) and sequenced to identify FIL-like clones. Sequence and phylogenetic analyses Translated sequences of FIL orthologues were aligned using CLU- STALX (Thompson et al., 1997), and alignments were refined by hand using BIOEDIT (Hall, 1999). The sequence for PapsFIL was deposited in GenBank with accession number JQ Expression analyses using RT-PCR Total RNA was extracted from P. somniferum and Arabidopsis leaf and flower tissues using the Trizol reagent (Invitrogen), and approximately 300 ng were used in 10-ll cdna synthesis reactions using SuperscriptÔ III Reverse Transcriptase (Invitrogen). For cdna synthesis the poly(t) primer used was 5 -GACTCGAGTCGACATCGA(T) Primers for testing the expression of PapsFIL were: RT-YABF1, 5 -AC- CGTTTCTACAAGGACGAG-3 ; RT-YABR1, 5 -CTGCAGTGAAGAACC- CATC-3 ; and RT-YABR2, 5 -CATAGTGATTGGTGTGCTG-3. Actin primers were: ACT1, 5 -ATGGATCCTCCAATCCAGAC-3 ; and ACT2, 5 - TATTGTGTTGGACTCTGGTG-3. Primers used for PapsSTM1 were: STM1 F, 5 -TAAGCAGCATGACAACACCAG-3 ; and STM1 R, 5 -GAG- GATCTATGAAGTTCtCACTAGC-3. Primers for PapsSTM2 were: STM2 F, 5 -TACCTAAGGACGCAAGGCAAC-3 ; and STM2 R, 5 -AT- GATGGTGCACATGAGTAGTCG-3. Expression analyses using qrt-pcr Quantification of gene expression level by real-time PCR was performed using 1 ll of 1 : 10 dilution of cdna template in 20 ll total reaction volume containing SYBR Ò Green JumpStartÔ Taq Ready- MixÔ (Sigma-Aldrich, and 10 lm of each PapsFIL-specific primer (RTYABF1 and RTYABR1) or Actin (PapsAct qf and PapsAct qr; Allen et al., 2008). Actin was used as the reference gene. Each qpcr reaction was performed in triplicate. The PCR reaction was performed in a PTC-200 Peltier thermal cycler (MJ Research) as follows: the samples were initially denatured at 95 C for 3 min followed by 40 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s and with an extension at 72 C for 30 s. The PapsFIL expression level was normalized using Actin as a reference. Expression analyses using in situ hybridization Hybridizations were carried out as previously described by Drea et al. (2005, 2007), with minor modifications. Gene regions derived from the central and C-terminal/3 -UTR of PapsFIL were used to generate digoxigenin-labelled RNA probes. PCR fragments amplified with YAB1F/YAB1R and YAB2F/YAB2R were cleaned using a Qiagen PCR purification kit and used in an in vitro transcription reaction with DIG-UTP and T7 RNA polymerase (Roche, Primers for the Arabidopsis STMprobe: 5 -TGTCAGAAGGTTGGAGCACCA-3 and 5 -GAATTGTAATACGAC TCACTATAGGGTTTGTTGCTCCGAAGGGTAA-3. Virus-induced gene silencing Two regions of the PapsFIL gene sequence were introduced into the TRV2 vector and transformed into Agrobacterium strain GV3101 and used to infiltrate poppy seedlings at the P3 P5 stage, as previously described (Hileman et al., 2005). The resulting individual plants were assayed for the presence of the viral vector using RT-PCR as well as for any visible phenotype. Two constructs incorporating regions of PapsFIL between the conserved zinc-finger and YABBY domains (YAB1) and the 3 -UTR (YAB2) were cloned into the TRV2 vector cut with XbaI and BamHI, and were assembled as follows: YAB1FXba, 5 -GCTCTAGATGTGGCAGTTTGTACAA GAC-3 ; Y AB1RBam, 5 -CGGGATCCATGGAAGCCTGTGCTTCTTC -3 ; YAB2FX ba, 5 -GCTCTAGAATGGAGAGGATGTTCCATTC-3 ; and YAB2RBam, 5 -CGGGATCCGCAGATTTGCTGATGAGTTC-3.

11 672 Nikolaos Vosnakis et al. Scanning electron microscopy Plant material was fixed overnight in FAA (formaldehyde 3.7%, acetic acid 5% and ethanol 50%), and then transferred to 70% ethanol. Samples were dehydrated in an ethanol series (80, 90, 100% ethanol) before critical-point drying in a Bal-Tec 030 Critical Point Drier. Samples were coated in gold using a Polaron SC7640 Sputter Coater and analysed on a Hitachi S3000H scanning electron microscope equipped with digital image capture. Overexpression in Arabidopsis thaliana The coding region of PapsFIL was amplified with primers 35sFILf, 5 - GGGGTACCATGTCATCCTCTTCTGCCTT-3, and 35sFILr, 5 -CGGGA TCCCTAGCTAGATACATAGTGAT-3, and cloned between KpnI and BamHI under the CaMV35S promoter in the pcambia1300-derived vector (described in Yi et al., 2010), and transformed using the floral-dip method (Clough and Bent, 1998) into the A. thaliana Landsberg erecta (Ler-0) ecotype. The primers used for Arabidopsis gene expression analysis were the same as described in Kumaran et al. (2002). ACKNOWLEDGEMENTS The authors would like to thank Stefan Hyman (EM Unit, University of Leicester) for his help with SEM. This work was supported by a Leverhulme Trust Research Grant to SD. NV and AM were supported by MSc Molecular Genetics funds from the University of Leicester. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Full amino acid alignment of all FIL/GRAM orthologues used to generate the phylogeny. Figure S2. Detection of TRV2 in the flower tissues of VIGS plants. Figure S3. SEM observations of Arabidopsis leaves. Table S1. Accession numbers for all sequences used in the alignment in Figure S1 and the phylogenetic tree in Figure 2. Table S2. Abberant phenotypes of Papaver somniferum VIGSconfirmed plants. Table S3. Details of qpcr analysis on VIGS plants. Table S4. 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