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Keywords:

  • C-function;
  • AGAMOUS;
  • SHATTERPROOF;
  • flower development;
  • dehiscence;
  • VIGS;
  • Nicotiana benthamiana;
  • fruit development

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The C-function, according to the ABC model of floral organ identity, is required for stamen and carpel development and to provide floral meristem determinacy. Members of the AG lineage of the large MADS box gene family specify the C-function in a broadly conserved manner in angiosperms. In core eudicots, two sub-lineages co-exist, euAG and PLE, which have been extensively characterized in Antirrhinum majus and Arabidopsis thaliana, where strong sub-functionalization has led to highly divergent contributions of the respective paralogs to the C-function. Various scenarios have been proposed to reconstruct the evolutionary history of the euAG and PLE lineages in eudicots, but detailed functional analyses of the roles of these genes in additional representative species to validate evolutionary hypotheses are scarce. Here, we report functional characterization of euAG- and PLE-like genes in Nicotiana benthamiana through expression analyses and phenotypic characterization of the defects caused by their specific down-regulation. We show that both paralogs redundantly contribute to the C-function in this species, providing insights on the likely evolution of these gene lineages following divergence of the major groups within the eudicots (rosids and asterids). Moreover, we have demonstrated a conserved role for the PLE-like genes in controlling fruit dehiscence, which strongly supports the ancestral role of PLE-like genes in late fruit development and suggests a common evolutionary origin of late developmental processes in dry (dehiscent) and fleshy (ripening) fruits.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

A major factor for the evolutionary success of the angiosperms, consisting of more of 300 000 extant species, is the carpel. Carpels protect the ovules, provide support for pollination, including incompatibility mechanisms, and, after fertilization, develop into fruits, which in turn protect the developing seeds and ensure seed dispersal. Understanding the evolution and functional diversification of carpels and fruits is therefore a fundamental question in Evolutionary Developmental Biology (Evo-Devo) studies.

Carpel identity in angiosperms is specified by C-function genes, as defined by the ABC model of floral organ identity (Coen and Meyerowitz, 1991). C-function, in addition to specifying carpel identity in the central whorl of the flower, specifies stamen identity in combination with B-function genes in whorl 3, and also controls floral meristem determinacy. Members of the AG lineage of the large MADS box gene family specify the C-function in a broadly conserved manner, as revealed by many functional studies including characterization of mutant or knockdown lines in monocots and basal and core eudicots (Bowman et al., 1989; Bradley et al., 1993; Pnueli et al., 1994; Mena et al., 1996; Davies et al., 1999; Pan et al., 2010; Yellina et al., 2010; Dreni et al., 2011).

In core eudicots, C-class genes were identified in Arabidopsis thaliana and Antirrhinum majus, where they have been thoroughly characterized. In both species, the C-function is mainly carried out by a single AG-like gene, AGAMOUS in Arabidopsis (referred to as AtAG) and PLENA in Antirrhinum (referred to as AmPLE). AtAG and AmPLE encode closely related genes whose expression starts early in the floral sexual organ primordia and remains specific to the carpel and stamens throughout development. In loss-of-function mutants of these genes, the sexual organs are totally absent in the flower and are replaced by several whorls of sepals and petals (Bowman et al., 1989; Bradley et al., 1993).

AtAG and AmPLE belong to the MADS box AG sub-family, although they are not orthologous genes. A major duplication event took place early in the history of the core eudicots that formed the origin of two gene lineages within the AG sub-family: euAG and PLE (Kramer et al., 2004). In Arabidopsis, a member of the rosids, the C-function gene AtAG belongs to the euAG lineage, while in Antirrhinum, a member of the asterids, the C-function gene AmPLE belongs to the PLE lineage. PLE lineage genes in Arabidopsis include two SHATTERPROOF genes (AtSHP), which are the products of a very recent duplication within the Brassicaceae. In contrast with their paralog AtAG, AtSHP genes do not contribute significantly to the C-function, as the only phenotype observed in the double mutant shp1 shp2 is a late defect in fruit dehiscence (Liljegren et al., 2000). Likewise, in Antirrhinum, the euAG lineage is represented by FARINELLI (AmFAR), which provides only a minor contribution to C-function in pollen development (Davies et al., 1999).

These studies suggested an evolutionary scenario whereby a functional switch between the euAG and PLE lineages to specify C-function and sub-functionalization of SHP genes to control fruit dehiscence occurred after divergence of the asterids and the rosids (Causier et al., 2005). This functional switch has been subsequently attributed to changes in both gene regulation and protein activity. The floral regulator gene LEAFY (LFY) is a key activator of AtAG and promotes its very early expression in the center of the flower meristem (Parcy et al., 1998; Busch et al., 1999). Reduced responses to LFY activation in AtSHP and AmFAR have been suggested as an explanation for their minor participation in the C-function (Moyroud et al., 2011). In addition, a single amino acid difference between AmPLE and AmFAR (Q173 insertion in AmFAR) has been identified as a key element explaining the functional differences between the two proteins (Airoldi et al., 2010). All together, these results indicate that the primary C-function has been retained by a different member of the gene pairs in Arabidopsis and Antirrhinum, and maybe more generally in rosids and asterids, and enlighten the plasticity of functional evolution following gene duplication (Causier et al., 2005).

However, several functional analyses conducted in the asterid family of Solanaceae provide conflicting results that undermine this hypothesis on C-function evolution, at least regarding the timing of evolutionary events. In petunia, tomato and tobacco, euAG orthologs appear to be significantly involved in stamen and carpel specification, as well as in the control of meristem determinacy (Kempin et al., 1993; Pnueli et al.,1994; Kapoor et al., 2002; Pan et al., 2010). Thus, the reduced contribution of the euAG ortholog to the C-function may be restricted to AmFAR in the Antirrhinum lineage. However, a complete loss of C-function phenotype was not observed in experiments in which euAG orthologs were down-regulated in Solanaceae, strongly suggesting that other genes participate in the C-function in these species, for which PLE-like genes are obvious candidates. Little information is available on the function of PLE lineage genes outside Arabidopsis and Antirrhinum. In tomato (asterids), down-regulation of the PLE-like gene TAGL1 has been shown to affect fruit ripening, but not to cause defects in floral organ specification (Vrebalov et al., 2009; Giménez et al., 2010; Pan et al., 2010). Thus, the role of TAGL1 appears to be restricted to late fruit development, similar to what has been described for the PLE-like SHP genes in Arabidopsis. Likewise, expression studies in various peach varieties (rosids) suggest a role for PLE orthologs in fruit ripening and lignification of pericarp cells (Tani et al., 2007). These studies may indicate that PLE genes may play a reduced early role in floral organ specification and a more prominent role late in fruit development that is not specific to the Arabidopsis lineage but more generally conserved in other eudicots. However, the profound structural differences between fleshy berries (tomato/peach) and dry dehiscent fruits (Arabidopsis), together with the lack of additional examples of mutants in PLE lineage genes, restrict the significance of these speculations.

Therefore, the reported studies on AG lineage genes in core eudicots leave open relevant questions. First, it remains to be studied whether PLE-like genes significantly contribute to C-function in species other than Antirrhinum. In this context, it is currently not clear whether expression studies coupled to regulatory sequence analyses have good predictive value in assessing the relative contribution of AG-like genes to C-function and can be used to formulate evolutionary hypotheses. Finally, a more general question is whether the late role of PLE-like genes in controlling dehiscence pre-dates the divergence of rosids and asterids. This putative ancestral role of PLE lineage genes in late fruit development may indicate a common developmental origin of dehiscence and fruit ripening in dry and fleshy fruits, respectively.

Here, we report a detailed functional analysis of euAG- and PLE-like genes in a solanaceous species, Nicotiana benthamiana, with the aim of unravelling their respective contributions to C-function and therefore better understanding the evolution of these gene lineages following divergence of the rosids and asterids. In addition, as Nicotiana forms dry and dehiscent fruits, this study has allowed us to obtain deeper insights into the function of PLE-like genes in fruit development and possibly fruit evolution.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

To functionally characterize euAG- and PLE-like genes in N. benthamiana, we cloned the corresponding full-length likely orthologs by RT-PCR from cDNA of young flowers using available sequence information for AG-like genes from the close relative Nicotiana tabacum in public databases (NAG1 and NtPLE36, see Experimental procedures). NbAG shows 96% amino acid identity with NAG1, and is more similar to Arabidopsis AtAG (67% identity) or Antirrhinum AmFAR (79% of identity) than to AtSHP or AmPLE. NbSHP shows 67 and 64% identity to AtSHP1 and AtSHP2, respectively, and 76% to AmPLE. The phylogenetic analysis shown in Figure 1(a) clearly groups NbAG in the euAG clade, and NbSHP in the PLE clade.

image

Figure 1.  Sequence analyses for NbAG and NbSHP genes.(a) Phylogenetic tree for protein sequences of AG and PLE homologs: AtAG (X53579), EScaAG1 (DQ088996), EScaAG2 (DQ088997), AmFAR (AJ239057), FBP6 (X68675), OsMADS3 (L37528), OsMADS13 (AF151693), OsMADS58 (AB232157), AmPLE (S53900), pMADS3 (X72912), PPERSHP (DQ777635), AtSHP1 (M55550), AtSHP2 (M55553), TAG1 (L26295), TAGL1 (AY098735), NbAG and NbSHP. Numbers on branches indicate bootstrap values for 10 000 replicates. The gene OsMADS13, the Oryza sativa ortholog of the Arabidopsis class D gene SEEDSTICK, was used as an outgroup in this analysis. Sequences belonging to the euAG and PLE lineages are shaded in light gray and dark gray, respectively. (b) Prediction of LFY occupancy of the largest intron of AG and PLE homologs from Arabidopsis (AtAG, At4g18960; AtSHP1, At3g58780; AtSHP2, At2g42830), Antirrhinum (AmPLE, AY935269; AmFAR, AJ239057) and N. benthamiana (NbAG and NbSHP). The occupancy indicates the relative expected number of bound LFY molecules of each DNA sequence integrating the different binding sites present on the fragment.

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The large second intron of euAG/PLE genes has been shown to be important for their regulation and to contain several conserved motifs, including a LFY binding site that has been reported to be critical to establish early expression of C-function genes during flower development, and two other motifs, an aAGAAT box and a 70 bp element characterized by a CCAATCA repeat (Causier et al., 2009). Because the presence of these conserved motifs has been suggested to have some predictive value regarding the relative contribution of AG-like genes to C-function in different species (Moyroud et al., 2011), we analyzed the sequences of the second intron of NbAG (3897 bp) and NbSHP (2742 bp). We found that the NbAG and NbSHP second introns have the conserved aAGAAT box and the 70 bp element. A consensus binding site for LFY (CCANTGG, as defined by Hong et al., 2003 and Causier et al., 2005) was found in NbAG intron but not in NbSHP, suggesting that NbSHP may have lost early onset of expression and therefore may play a reduced role in C-function (Figure S1). However, we also made use of a bioinformatic tool based on a biophysical model that quantitatively predicts LFY affinity for specific genomic regions (termed ‘predicted occupancy’ or POcc), which out-performs classical searching of consensus binding sites (Moyroud et al., 2011). Interestingly, we found a high level of LFY predicted occupancy in both NbAG and NbSHP second introns, with significantly higher occupancy for the latter, similar to what has been reported for the pair AmFAR/AmPLE from Antirrhinum and in contrast to AtAG/AtSHP in Arabidopsis (Figures 1b and S1).

In addition to the role of cis-elements in evolution of C-function genes, a single glutamine insertion in the sequence of the AmFAR protein has been correlated to its possible sub-functionalization in mainly specifying male organ identity. Examination of both NbAG and NbSHP amino acid sequences showed that both deduced proteins lacked this glutamine residue, thus suggesting that functional differences between NbAG and NbSHP proteins produced by this amino acid change should not be expected.

In summary, sequence analyses of NbAG and NbSHP suggested that both factors may share C-function specification, although NbSHP may play a more prominent early role based on predicted LFY occupancy of the second intron.

NbAG and NbSHP are expressed during floral development like typical C-function genes

To elucidate the detailed expression patterns of NbAG and NbSHP, we performed in situ hybridization on young buds of N. benthamiana. As probes, we used the last 500 bp of each coding sequence, a region that shows low sequence similarity between the two genes (64 versus 81% in the MADS box coding region). Both NbAG and NbSHP transcripts began to accumulate at stage 2 of flower development as defined by Mandel et al. (1992), when the sepal primordia arise in the central domain of the flower, which later will give rise to stamen and carpel primordia (Figure 2a,b). At this stage, both transcripts showed similar expression domains, although NbAG expression appeared to occur more externally in the floral meristem than NbSHP expression. At stage 4, when petal primordia were visible, NbAG and NbSHP were detected in the cells that develop into stamens and carpels (Figure 2c,d). At stage 6, NbAG and NbSHP transcripts were uniformly present in the developing stamens and in the apical central zone of the pistil (Figure 2e,f). In the mature flower, expression of both genes was weak in stamens but strong in the placenta (Figure 2g,h). In later stages, expression of both transcripts was also observed in the developing ovules (Figure 2i,j).

image

Figure 2. In situ expression analyses of NbAG and NbSHP in wild-type N. benthamiana floral buds. Sections on the left were probed with NbAG (a, c, e, g, i). Sections on the right were probed with NbSHP (b, d, f, h, j). s, sepal; p, petal; st, stamen; pi, pistil; o, ovule. Developmental stages are as described by Mandel et al. (1992). Control hybridizations with sense probes are shown in Figure S2. (a, b) NbAG and NbSHP expression is restricted to the center of the floral meristem at stage 2 when sepal primordia are clearly visible. (c, d) Expression remains in the center of the floral meristem in stage 4 flowers, when petals begin to form. (e, f) In stage 6 flowers, expression is specific to the developing sexual organs. (g, h) In mature flowers, expression is detected in ovules, placenta, carpel wall and weakly in stamens. (i, j) In later stages, expression is mainly detected in ovules.Scale bars = 100 μm (a-h) and 50 μm (i, j).

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From these experiments, we conclude that expression of these two genes remains specific to the sexual organs from early stages and throughout flower development. NbAG and NbSHP showed nearly identical temporal and spatial expression patterns, similar to those described for Arabidopsis AtAG or Antirrhinum AmPLE genes.

Silencing of NbAG and NbSHP in N. benthamiana using VIGS technology differentially affects flower and fruit development

Expression analyses suggested that both NbAG and NbSHP genes may have similar early and late roles in floral development in N. benthamiana. To investigate the specific contribution of each gene to these roles, we used virus-induced gene silencing (VIGS) to reduce NbAG and NbSHP transcript levels in N. benthamiana. This method transiently down-regulates expression of a specific gene via modified plant viruses, in our case tobacco rattle virus (TRV), and has been shown to efficiently direct the degradation of endogenous mRNAs in Nicotiana as well as other species (Ratcliff et al., 2001; Constantin et al., 2004; Hileman et al., 2005; Wege et al., 2007). In order to specifically silence NbAG and NbSHP genes, we generated TRV constructs carrying the same coding sequence fragments used for the in situ analyses, which showed low sequence similarity to each other and did not present contiguous stretches of more than 20 identical nucleotides.

VIGS of NbAG.  Twelve plants were inoculated with the TRV2-NbAG construct. To evaluate the efficiency and specificity of the VIGS treatment, the levels of NbAG and NbSHP transcripts were measured by quantitative RT-PCR on flowers from five treated plants. In all cases, expression of NbAG was significantly reduced compared to the wild-type (WT), but expression of NbSHP was practically unaffected (Figure S3), proving that the TRV2-NbAG construct was gene-specific.

In the WT flower of N. benthamiana, the first whorl comprises five sepals that are fused for most of their length; in the second whorl, five petals develop that are fused in a tubular corolla; the third whorl comprises five stamens whose long filaments are adnately fused to the second whorl; the fourth whorl is occupied by a two-carpellate gynoecium. The mature pistil comprises a short ovary divided into two locules with central placentation, and a very long style, which ends in a round and flat stigma (Figures 3 and 4).

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Figure 3.  Phenotypes of N. benthamiana plants inoculated with pTRV2-NbAG, pTRV2-NbSHP or pTRV2-NbAG-NbSHP. (a–d) Top view of N. benthamiana flowers. (a) Wild-type flower at anthesis, showing five petals and five anthers surrounding the stigma. (b) NbAG-VIGS flower showing conversion of stamens into petals and no visible stigma. (c) NbSHP-VIGS flower. Note the absence of a stigma in the center of the five anthers. (d) NbAG-NbSHP-VIGS flower showing additional whorls of petals and no visible sexual organs. (e–i) Longitudinal sections of N. benthamiana flowers. (e) Wild-type flower at anthesis, showing a whorl of sepals, a tubular whorl of petals, stamens whose filaments are adnately fused to the petals, and the central pistil. (f) NbAG-VIGS flower showing homeotic conversion of stamens into petals and the presence of an abnormal fourth whorl. (g) NbSHP-VIGS flower showing a pistil with an unfused and reduced style. (h, i) NbAG-NbSHP-VIGS flowers, in which stamens are replaced by petals. In (h), the fourth whorl is composed of petals. In (i), the flower has developed a new whorl of sepals enclosing petals in the center of the flower. (j–o) Floral organs in the fourth whorl of N. benthamiana flowers. (j) Wild-type pistil comprising an ‘egg-shaped’ ovary, a long style and a flat stigma. (k–m) Central whorl of an NbAG-VIGS flower. (k) Absence of ovary and style differentiation in the highly modified fourth whorl of the NbAG-VIGS flower. (l) Opened fourth whorl of an NbAG-VIGS flower revealing the presence of inner petals. (m) Closer view of the apical part of the fourth whorl of an NbAG-VIGS flower indicating the presence of stigmatic tissue. (n) NbSHP-VIGS N. benthamiana pistils showing fusion defects, reduction of style length and increased number of styles and stigma. (o) Presence of an ectopic floral meristem at the base of the fourth whorl in an NbAG-NbSHP-VIGS flower. Scale bar = 0.5 cm.

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Figure 4.  Sections of N. benthamiana flowers from plants inoculated with pTRV2-NbAG, pTRV2-NbSHP or pTRV2-NbAG-NbSHP. (a–d) Transverse sections of N. benthamiana flowers at 0.5 cm up from the pedicel. (a) Wild-type flower showing an external sepal whorl, a tubular petal whorl with five adnately fused stamens filaments, and the bi-carpellate central ovary. (b) Transverse section of an NbSHP-VIGS flower presenting wild-type first and second whorls, eight stamen filaments fused with the petals, and at least five carpels developing in the ovary. (c) Transverse section of an NbAG-VIGS flower showing a normal first whorl of sepals, an enlarged second whorl of petals surrounding eight stamens converted into free petals, and a fourth whorl lacking carpel tissue differentiation, ovules and placenta. (d) Transverse section of an NbAG-NbSHP-VIGS flower lacking sexual organs and comprising an outer whorl of sepals and numerous whorls of petals. (e, f) Longitudinal sections of the apical part of the pistil. (e) Wild-type stigma and upper style. (f) NbSHP-VIGS plant with unfused style and stigma. (g) Longitudinal section of a wild-type N. benthamiana flower. (h, i) Longitudinal sections of NbAG-VIGS flowers. (h) Conversion of stamens into petals and the presence of some stigmatic tissue in the fourth whorl. (i) The presence of a new floral meristem in the center of the flower. (j) Longitudinal section of an NbSHP-VIGS gynoecium showing additional whorls of carpels developing in its center, and numerous styles. cw, carpel wall; fm, floral meristem; ov, ovary; p, petal; p*, free petal; s, sepal; sg, stigma; st, stamen; sy, style. Scale bars = 500 μm.

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NbAG-VIGS plants showed homeotic transformations specifically restricted to the sexual organs of the flower. This phenotype was observed in approximately 92% of the flowers of all treated plants. In the third whorl, stamens were replaced by petals (Figure 3b,f). The petals developing in the third whorl were free instead of fused into a tubular structure, and in 74% of the flowers there were more than five petals (between 6 and 10) (Figure 4c). In the center of NbAG-VIGS flowers, a short tubular green organ developed that appeared to have mixed sepal/carpel identity (Figure 3f,k). Closer inspection by scanning electron microscopy confirmed the sepal identity of these structures. Instead of the small and rectangular cells typical of the ovary epidermis (Figure 5e), we found jigsaw-shaped cells, and numerous trichomes and stomata, which are typical of the sepal abaxial surface (Figure 5c). However, the organs in the fourth whorl of NbAG-VIGS flowers also retained some carpelloid features such as stigmatic tissue (Figures 3m and 4h).

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Figure 5.  Scanning electron microscope analyses of epidermal cell morphology in flowers of VIGS-treated N. benthamiana plants. (a) Small rectangular cells of a wild-type ovary. (b) Elongated cells of a wild-type style. (c) Jigsaw-shaped cells, trichomes and stomata of a wild-type sepal. (d) Cell types at the surface of an NbAG-VIGS fourth whorl. The cells are irregular and trichomes and stomata are observed. (e, f) Cell types observed in the ovary (e) and style (f) of an NbSHP-VIGS fourth whorl. Note the presence of trichomes and stomata in both cases. Scale bars = 60 μm.

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In addition to homeotic transformation of stamens and carpels, NbAG-VIGS treatment caused indeterminacy of the floral meristem. NbAG-VIGS flowers developed additional whorls of petals and sepals inside the sepalloid gynoecium (Figure 3l). These supernumerary organs were evident in histological sections of the flower (Figure 4c,h,i). Moreover, the prolonged activity of the floral meristem in the NbAG-VIGS plants occasionally produced an ectopic floral meristem in the center of the flower (Figure 4i).

Our results indicated that NbAG plays a major role in C-function, being required to both specify stamen and carpel identity and to control floral determinacy. However, the presence of carpel tissue in the fourth whorl of all the NbAG-VIGS flowers analyzed also suggested that additional factors, most likely NbSHP, may participate in carpel identity specification, or, alternatively, that the residual NbAG activity in the VIGS plants may be sufficient to partly direct carpel development.

VIGS of NbSHP.  To functionally characterize NbSHP and to specifically evaluate its possible contribution to C-function and to fruit development, we performed an equivalent experiment with a specific TRV-NbSHP construct. Quantification of NbAG and NbSHP transcripts in treated plants revealed that the expression of NbSHP was strongly reduced in NbSHP-VIGS flowers but expression of NbAG was not significantly altered or even increased (Figure S3).

NbSHP-VIGS plants showed phenotypic alterations restricted to flower development. Stamen development was mostly unaffected, although occasionally mild transformations of anthers into petaloid tissue were observed (Figure S4). In contrast, gynoecium development was severely affected in 94% of the NbSHP-VIGS flowers. Incomplete carpel fusion led to a complete split of style and stigma. Moreover, the style was much shorter than in WT plants (Figures 3c,g,n and 4f). Close inspection of the epidermal cells of the NbSHP-VIGS modified ovary revealed that the cell morphology was intermediate between typical ovary and sepal cells, with development of stomata and trichomes, which are characteristics of sepal tissue (Figure 5e). Stomata and trichomes were also observed on the style epidermis of the NbSHP-VIGS gynoecium (Figure 5f). These observations indicate that a partial homeotic conversion occurred in the fourth whorl of NbSHP-VIGS flowers.

In addition, most of the NbSHP-VIGS flowers displayed mild to strong defects in floral meristem determinacy. In 43% of the flowers, the gynoecium was formed by more than two carpels and developed internal whorls of carpeloid organs (Figures 3n and 4b,j). Supernumerary organs were also found in the third whorl of 36% of the flowers, where up to 10 stamens developed instead of the typical five formed in WT flowers (Figure 4b).

Although the vast majority of the NbSHP-VIGS gynoecia were severely affected and therefore sterile, fruits were able to develop on a small proportion of NbSHP-VIGS flowers several weeks after inoculation, allowing us to evaluate the possible role of NbSHP in fruit development. These fruits were anatomically almost identical to WT fruits and did not show any sign of homeotic transformations, as revealed by close inspection of ovary cell morphology and ovary wall organization (Figure S5). The WT fruit of N. benthamiana is a dry capsule, which opens at maturity to allow seed dispersal along four dehiscence zones at the top of the fruit that correspond to the two fused carpel margins and the central carpel midveins (Figure 6a). In contrast, all the NbSHP-VIGS fruits that formed remained indehiscent even weeks after maturity, a phenotype similar to that caused by loss of function of AtSHP genes in Arabidopsis (Figure 6b). It is known that lignification of dehiscence zones plays a crucial role in fruit shattering, and that dehiscence zone lignification is greatly reduced in Arabidopsis shp1 shp2 mutants (Liljegren et al., 2000). For this reason, we studied the lignification pattern in NbSHP-VIGS mature fruits compared to WT. In the WT fruit, four strongly lignified narrow stripes were observed at the top of the fruit, which corresponded to the dehiscence zones (Figure 6c). By contrast, no lignification was detected along the fused carpel margins or the midveins of the carpels in the indehiscent NbSHP-VIGS fruits (Figure 6d).

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Figure 6.  Fruit dehiscence and lignification altered in N. benthamiana plants inoculated with pTRV2-NbSHP. (a) Open mature capsule of an N. benthamiana wild-type plant. (b) Closed mature capsule of an NbSHP-VIGS plant. (c) Lignification pattern of an N. benthaniana wild-type fruit. (d) Absence of lignin in an N. benthamiana NbSHP-VIGS fruit.

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VIGS of NbAG1 and NbSHP.  In addition to its role in fruit dehiscence, the described phenotypes of NbSHP-VIGS plants suggested that this gene plays a major role in the control of meristem determinacy, while also participating in specification of carpel identity, and, to a lesser degree, stamen identity. In this sense, NbSHP appeared to be responsible for the presumptive residual C-function in NbAG-VIGS flowers. Thus, in order to determine whether down-regulation of NbSHP modified the NbAG-VIGS phenotype, we silenced both genes simultaneously by inoculating 12 plants with the TRV2-NbAG-NbSHP vector. Quantitative RT-PCR in the flowers of five treated plants confirmed that both genes were strongly down-regulated (Figure S3).

Almost all (95%) of the NbAG-NbSHP-VIGS flowers completely lacked stamens and carpels, and showed a greatly enhanced floral indeterminacy compared to specific down-regulation of the individual genes. Sepals and petals developed normally, although occasionally some supernumerary organs were observed (Figure S4). In the third whorl, stamens were converted into petals (Figure 3d). In 90% of the flowers, a new tubular corolla of petals developed in the fourth whorl, which enclosed numerous petals, reiterating the pattern in the second and third whorls (Figures 3h and 4d). However, approximately 5% of the flowers showed a different phenotype: instead of the fourth whorl, a new flower developed composed of sepals and petals (Figure 3i), similar to what has been described for Arabidopsis ag mutants (Bowman et al., 1989). In very few flowers, an ectopic flower emerging from the axil of a third whorl organ was also observed (Figure 3o), suggesting that NbAG and NbSHP may also play a minor role in floral meristem specification.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

In this study, we present a detailed characterization of the role of AG-like genes from euAG and PLE lineages in flower and fruit development in the solanaceous species N. benthamiana. Our studies provide additional data to address several open questions regarding the evolution of AG-like genes in eudicots. First, we have assessed the specific contribution of a member of the PLE lineage in asterids to the C-function. Second, our results help to understand the temporal and quantitative contributions of AG-like factors to the different aspects of C-function. Finally, we have demonstrated the conserved role of PLE-like genes in the regulation of fruit dehiscence in distantly related species.

The C-function in N. benthamiana

Previous work in other solanaceous species such as petunia or tomato suggested that, in contrast to the situation in Arabidopsis or Antirrhinum, both paralogs from the PLE and euAG lineages may be significantly required to contribute to the C-function (Kapoor et al., 2002; Vrebalov et al., 2009; Pan et al., 2010). However, functional data, especially for PLE-like genes, were only partial, and were therefore insufficient to fully confirm this hypothesis. In this work, we demonstrate that both NbAG (euAG lineage) and NbSHP (PLE lineage) jointly perform the C-function in the solanaceous species N. benthamiana.

Simultaneous down-regulation of both NbAG and NbSHP genes caused typical loss of C-function phenotypes, with complete homeotic transformation of stamens into petals, lack of carpels and loss of floral determinacy, with formation of numerous concentric whorls of petals or new flowers arising from the center of the floral meristem. The specific contribution of NbAG and NbSHP to these phenotypes was also assessed: NbAG but not NbSHP appeared to contribute significantly to stamen identity; both NbAG and NbSHP were required to confer carpel identity, although specific down-regulation of NbAG had a stronger effect; and NbAG and NbSHP similarly controlled floral meristem determination.

In Arabidopsis and Antirrhinum, in which functional studies are extensive and allelic series are available, it has been shown that the different components of the C-function (stamen identity, carpel identity and determinacy) can be genetically separated (Mizukami and Ma, 1992; Davies et al., 1999; Causier et al., 2009). The phenotypes of weak mutant alleles of AtAG or AmPLE genes support a threshold model in which stamen identity, carpel identity and floral meristem determinacy each require different levels of C-function activity. In addition, temporal requirements can be deduced from the phenotypes of heterochronic alleles of AmPLE, which indicate that an early onset of expression is needed to specify stamen identity, while correct carpel formation and meristem termination may still be achieved if C-function activation is only attained at later stages of floral development (Causier et al., 2009). In this work, we show that NbAG and NbSHP exhibit highly similar temporal and spatial patterns of expression, in partial agreement with the presence of previously described regulatory elements in the second intron of both genes that predict early onset of expression for both, although predicting also a higher level of expression for NbSHP (Causier et al., 2009; Moyroud et al., 2011). As NbSHP down-regulation has little or no effect on stamen formation and only causes mild loss of carpel identity, it may be proposed that the different quantitative contribution of both genes to the C-function may rely on differences in protein activity, compatible with the threshold model. In fact, this hypothesis is indirectly supported by the phenotypes of petunia plants over-expressing pMADS3 (the Petunia hybrida euAG-like gene, which is 91% identical in deduced amino acid sequence to NbAG) or FBP6 (Petunia hybrida PLE-like gene, 90% identical to NbSHP). While pMADS3 over-expression induced homeotic transformations of sepals and petals, FBP6 over-expression did not (Kater et al., 1998), therefore indicating that the two proteins have different biochemical potential. Our results indicate that, in N. benthamiana, NbAG and NbSHP are redundantly required for C-function, but NbAG shows higher activity, probably reflecting differences in protein function. Thus, NbAG alone provides enough C-activity to specify stamen identity but NbSHP does not in the absence of NbAG; the higher requirements of C-function for carpel specification require both NbAG and NbSHP, but absence of the less active NbSHP factor only causes mild defects; and finally, only simultaneous expression of both NbAG and NbSHP provides sufficient C-activity to promote floral meristem termination.

These conclusions, which were not anticipated from expression or in silico analyses, highlight the importance of functional data to address evolutionary questions and to validate predictions based on bioinformatic analyses of regulatory sequences to enable formulation of evolutionary hypotheses.

The evolution of C-function

The redundancy of euAG and PLE lineage genes in Nicotiana adds valuable information to clarify the evolution of C-function in eudicots. Expression patterns, protein activities and relative contributions to the C-function for the euAG/PLE paralogs in Arabidopsis, Antirrhinum and Nicotiana are different in each species.

Together with similar evidence in monocots or basal dicots, in which AG-like genes have independently diversified but also redundantly provide the C-funcion (Zahn et al., 2006; Yellina et al., 2010; Dreni et al., 2011), these results strongly support an scenario where sub-functionalization of AG-like genes to perform the various aspects of C-function occurred multiple times in evolution, involving both changes in expression patterns and protein activity.

Interestingly, we observed that reduction of NbSHP mRNA levels in NbSHP-VIGS plants frequently produces a concomitant increase in NbAG mRNA levels. Similar effects were previously reported for other species (Davies et al., 1999; Kapoor et al., 2002; Vrebalov et al., 2009; Giménez et al., 2010), suggesting that the ancestral C-function was auto-regulated and that this regulation was maintained following duplication. Thus, auto-regulation of C-function genes may provide a compensation mechanism that facilitates independent sub-functionalization of duplicated C-function genes.

An ancestral role for PLE genes in late carpel development

In addition to their sexual organ identity and floral meristem maintenance phenotype, NbSHP-VIGS flowers show defects in late pistil development, with unfused carpels in the style and stigma regions. In Arabidopsis, a recent study uncovered a similar late role for AtSHP genes in the development of the apical regions of the pistil, although it was only observed in certain mutant backgrounds (Colombo et al., 2010). Thus, as PLE-like genes appear to share a common role in style and stigma development in both Arabidopsis and Nicotiana, it is possible to speculate that this function is ancestral and was already present in the PLE lineage before separation of the asterids and the rosids. However, it is difficult to assess whether this role is specific to the PLE lineage, or whether euAG-like genes also participate in this function. Carpel development in NbAG-VIGS plants is strongly affected, and only patches of stigmatic tissue develop. Likewise, ag mutants do not form any carpels in Arabidopsis, and late roles of AtAG in pistil development will therefore go unnoticed. The fact that AtAG and AtSHP genes are expressed in the apical regions of the developing pistil, together with the absence of evident style defects in shp1 shp2 mutants, may also support an alternative scenario whereby AtAG, AtSHP1 and AtSHP2 redundantly regulate this function.

An ancestral role for PLE lineage genes in fruit development

In addition to the various floral phenotypes discussed above, we show that down-regulation of the NbSHP gene leads to development of indehiscent fruits with strongly reduced lignification at the presumptive dehiscence zone. Remarkably, these fruits phenocopy the shp1 shp2 double mutants in Arabidopsis (Liljegren et al., 2000). The fruit-shattering process is well documented in Arabidopsis (for review, see Ferrándiz, 2002; Ferrándiz et al., 2010). The AtSHP genes are expressed in the valve margins and later in the fruit dehiscence zones, where they are required for differentiation of the dehiscence zone in the Arabidopsis silique and lignification of the adjacent cells. AtSHP expression is restricted to the valve margins by the negative regulation exerted by FRUITFULL (AtFUL), another member of the MADS box family from the AP1/FUL clade that is expressed in the valves. In Arabidopsis, AtFUL over-expression phenocopies the shp1 shp2 mutant, resulting in indehiscent fruits with reduced lignin (Ferrándiz et al., 2000). The FUL ortholog of N. tabacum, NtFUL, has been studied previously. When NtFUL is over-expressed in the related species Nicotiana sylvestris, indehiscent fruits are also formed (Smykal et al., 2007). Thus, in both Arabidopsis and Nicotiana, down-regulation of PLE orthologs (AtSHP in Arabidopsis, NbSHP in N. benthamiana) or over-expression of FUL orthologs (AtFUL in Arabidopsis, NtFUL in N. sylvestris) produce nearly identical effects in fruit dehiscence and lignification. It is very unlikely that this regulatory network directing dehiscence zone formation has been established twice during eudicot evolution, and it may therefore be proposed that this network was already present in the common ancestor of asterids and rosids.

Recent studies of the PLE/SHP ortholog TAGL1 in tomato, another solanaceous species with fleshy fruits, have shown that TAGL1 is required for fruit ripening (Itkin et al., 2009; Vrebalov et al., 2009; Giménez et al., 2010; Pan et al., 2010). Also, expression analyses of FUL and PLE/SHP orthologs in peach (a rosid species with fleshy fruits) suggest a conserved function of these genes in controlling peach fruit lignification (Tani et al., 2007). A wealth of studies mapping fleshy-to-dry or dry-to-fleshy transformations into phylogenetic trees suggest that these have occurred multiple times within most main taxa of angiosperms (Pabon-Mora and Litt, 2011). The likely role of the FUL–SHP network in fleshy fruit development across rosids and asterids, together with the conserved role of this same gene network in dehiscence zone formation in distantly related species such as Arabidopsis and Nicotiana, allows us to draw two main conclusions. First, it strongly supports the idea of an ancestral sub-functionalization of PLE-like genes to direct late fruit development that pre-dates the divergence of rosids and asterids. Second, it suggests a common evolutionary origin of dehiscence in dry fruits and ripening in fleshy fruits that places the genetic switch that allows transformation of dry capsules into fleshy fruits and vice versa downstream of the FUL–SHP genetic network.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Plant materials and growth conditions

N. benthamiana plants were grown in the greenhouse at 22°C (day) and 18°C (night) with a 16 h light/8 h dark photoperiod, in soil irrigated with Hoagland No. 1 solution supplemented with oligo elements (Hewitt, 1966).

Cloning and sequence analyses

Full-length coding sequences of N. benthamiana AG and PLE genes were isolated by RT-PCR on cDNA of young flowers of N. benthamiana using available sequence information from the close relative N. tabacum and the more distant tomato (Solanum lycopersicum). From the sequence of NAG1 (N. tabacum AG ortholog, accession number L23925), the primers NbAGFor and NbAGRev were designed and used to isolate the gene NbAG (deposited in Genbank under accession number JQ699177). From the sequence of TAGL1 (tomato PLE ortholog, accession number AY098735) and the partial sequence of NtPLE36 (N. tabacum PLE ortholog, accession number U63163), the primers NbSHPFor5′ATG and NbSHPRev were designed and used to amplify a partial sequence of NbSHP. The 3′ end of NbSHP was then isolated by RT-PCR using primers NbSHPFor2 and RT [sequence present in the oligo (dT) primer used for retrotranscription]. The NbSHP sequence has been deposited in Genbank under accession number JQ699178.

For the phylogenetic tree, the deduced amino acid sequences were aligned using the clustal w tool in macvector 12.0 software (MacVector Inc., http://www.macvector.com/) and further refined by hand. Pairwise Poisson genetic distances were estimated from the alignment, and a neighbor-joining tree was estimated using distance matrices from 10 000 bootstrap replicates and rooted to OsMADS13, an Oryza sativa ortholog of the Arabidopsis class D gene SEEDSTICK.

The second introns were obtained by PCR using the primers NbAGintronFor and NbAGintronRev for NbAG (accession number for intron sequence JQ699179), and NbSHPintronF and NbSHPintronR for NbSHP (accession number for intron sequence JQ699180). To predict the probability of the presence of LFY binding sites in the intron sequences, we used the Morpheus webpage facility (http://biodev.cea.fr/morpheus/Default.aspx) with the LFY matrix. The score program permits localization of the best transcription factor binding sites in DNA fragments. The occupancy program calculates the expected number of bound transcription factor molecules of a DNA fragment using a biophysical model that integrates the various binding sites present on the fragment.

Primer sequences are given in Table S1.

In situ hybridization

RNA in situ hybridization with digoxigenin-labeled probes was performed on 8 μm paraffin sections of N. benthamiana buds as described by Ferrándiz et al. (2000). The RNA antisense and sense probes were generated from a 504 bp fragment of the NbAG cDNA (positions 244–747) and a 510 bp fragment of the NbSHP cDNA (positions 244–754). Both fragments were cloned into the pGemT-Easy vector (Promega, http://www.promega.com), and sense and antisense probes were synthesized using SP6 or T7 polymerases.

Virus-induced gene silencing in N. benthamiana

The same regions of NbAG and NbSHP coding sequence used for in situ hybridization were used for the VIGS experiments. In the case of the single gene constructions, a XbaI restriction site was added to the 5′ end of the PCR fragment and a BamHI restriction site was added to the 3′ end. The amplicon was digested by XbaI and BamHI and cloned into a similarly digested pTRV2 vector (Liu et al, 2002). For the double gene construction, the fragment of NbAG coding sequence was introduced into the pTRV2-NbSHP vector using the EcoRI restriction site. The three resulting plasmids (pTRV2-NbAG, pTRV2-NbSHP and pTRV2-NbAG-NbSHP) were confirmed by digestion and sequencing, before being introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterium inoculation of N. bentahamiana leaves was performed as described by Dinesh-Kumar et al. (2003).

Quantitative RT-PCR

Total RNA was extracted from flowers at anthesis using an RNeasy Plant Mini kit (Qiagen, http://www.qiagen.com). Four micrograms of total RNA were used for cDNA synthesis performed using a First-Strand cDNA Synthesis kit (Invitrogen, http://www.invitrogen.com) and the quantitative PCR master mix was prepared using iQTM SYBR Green Supermix (Bio-Rad, http://www.Bio-rad.com). The primers used to amplify NbAG (qNbAGFor and qNbAGRev) and NbSHP (qNbSHPFor and qNbSHPRev) generated products of 81 bp and did not show any cross-amplification. Results were normalized to expression of Elongation Factor 1 (EF1) mRNA (accession number AY206004), amplified by qNbEF1for and qNbEF1rev. The efficiency of amplification of NbAG, NbSHP and the EF1 reference gene was similar. The PCR reactions were run and analyzed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, http://www.appliedbiosystems.com). Primer sequences are given in Table S1.

Scanning electron microscopy (SEM) and histology

VIGS-treated plants were analyzed by cryoSEM on fresh tissue under a JEOL JSM 5410 microscope equiped with a CRIOSEM instrument CT 15000-C (Oxford Instruments, http://www.oxford-instruments.com). Young buds were collected for histological analyses, fixed in FAA (3,7% formaldehyde, 5% acetic acid, 50% ethanol) under vacuum and embedded into paraffin. Sections 10 μm thick were stained in 0.2% toluidine blue solution, and observed under a Nikon Eclipse E-600 microscope (http://www.nikoninstruments.com). For lignin observation, fruits were fixed in FAA, stained for 5 min in 2.5% phloroglucinol, and soaked for 30 sec in 50% HCl.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank Rafael Martínez-Pardo and Eugenio Grau for technical support, Amy Litt (New York Botanical Gradens, Bronx, NY, USA) for providing VIGS plasmids, Eugenio Minguet for help with the LFY matrix and Morpheus tool, and Francisco Madueño, Monica Colombo and Barbara Ambrose (NYBG) for critical reading of the manuscript. The work was funded by grant BIO2009-09920 from the Spanish Ministerio de Ciencia e Innovación to C.F.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Figure S1. Analyses of the NbAG and NbSHP second intron.

Figure S2. Sense controls for in situ hybridization.

Figure S3. Expression level of NbAG and NbSHP by real-time PCR analysis in TRV2-NbAG, TRV2-NbSHP or TRV2-NbAG-NbSHP flowers.

Figure S4. Additional phenotypes of NbSHP-VIGS flowers.

Figure S5. Morphology of indehiscent NbSHP-VIGS fruits.

Table S1. Primers used in this work.

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