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Proc Natl Acad Sci U S A. Apr 19, 2005; 102(16): 5779–5784.
Published online Apr 11, 2005. doi:  10.1073/pnas.0501877102
PMCID: PMC556287
Evolution

Heterotopic expression of MPF2 is the key to the evolution of the Chinese lantern of Physalis, a morphological novelty in Solanaceae

Abstract

Morphological novelties arise through changes in development, but the underlying causes of such changes are largely unknown. In the genus Physalis, sepals resume growth after pollination to encapsulate the mature fruit, forming the “Chinese lantern,” a trait also termed inflated-calyx syndrome (ICS). STMADS16, which encodes a MADS-box transcription factor, is expressed only in vegetative tissues in Solanum tuberosum. Its ortholog in Physalis pubescens, MPF2, is expressed in floral tissues. Knockdown of MPF2 function in Physalis by RNA interference (RNAi) reveals that MPF2 function is essential for the development of the ICS. The phenotypes of transgenic S. tuberosum plants that overexpress MPF2 or STMADS16 corroborate these findings: these plants display enlarged sepals. Although heterotopic expression of MPF2 is crucial for ICS, remarkably, fertilization is also required. Although the ICS is less prominent or absent in the knockdown transgenic plants, epidermal cells are larger, suggesting that MPF2 exerts its function by inhibiting cell elongation and promoting cell division. In addition, severely affected Physalis knockdown lines are male sterile. Thus, heterotopic expression of MPF2 in floral tissues is involved in two novel traits: expression of the ICS and control of male fertility. Sequence differences between the promoter regions of the MPF2 and STMADS16 genes perhaps reflect exposure to different selection pressures during evolution, and correlate with the observed differences in their expression patterns. In any case, the effects of heterotopic expression of MPF2 underline the importance of recruitment of preexisting transcription factors in the evolution of novel floral traits.

Keywords: MADS-box gene, morphological novelties, inflated-calyx syndrome

The origin of morphological novelties is a long-standing problem in evolutionary biology. An understanding of this process demands the elucidation of the developmental and genetic mechanisms that produce such structures. In principle, heterochrony [phylogenetic changes in the timing and rates of ontogenetic processes (15)] or heterotopic expression of pre-existing functions (6) offer possible explanations for their appearance. In these scenarios, changes in cis-regulatory elements of genes (7) and/or changes in transacting transcriptional regulators are usually invoked.

MADS-box transcription factors determine floral meristems and floral organ identity (811). These proteins act as (homo- or hetero-) dimers (12, 13) or in higher-order complexes (14, 15). According to the ABC model of flower development the A-function defines sepals, A- and B-functions together define petals, B- plus C-functions control stamen development, and the C-function alone is responsible for carpel formation (9, 10, 16, 17). Most of these functions are mediated by MADS-box proteins. Their modular action is best described in the “floral quartet” hypothesis (18). The composition of oligomers in the functional complex determines the nature of the organ formed. Alterations may lead to homeotic substitutions (19). Thus, variability within such a complex could also provide a basis for the evolution of morphological novelties. For example, ectopic expression of B-function genes in whorl 1 of Nicotiana tabacum results in a homeotic transformation of sepals into petals: the transgenic plants form two whorls of petals (20). Recently, heterotopic expression of B-function genes has been demonstrated in Tulipa, which feature two whorls of petals, their characteristic tepals (6).

Morphological alterations of whorl 1 seem to be characteristic of several genera in the Solanaceae. Although most genera possess small sepals, some have extended tubular calyces. Examples include Brugsmansia, Datura, and Nicotiana, to name just a few. Other genera, like Nicandra, Physalis and Withania, display more spectacular changes, featuring a balloon-like calyx that encapsulates the mature fruit. In Physalis, this structure is often referred to as a “Chinese lantern,” but it has also been more formally termed “inflated-calyx syndrome” (ICS) (21). In this article, we show that recruitment of an existing transcription factor to a novel context by means of heterotopic expression is apparently responsible for this novel morphological trait.

Materials and Methods

Plant Materials. The diploid and interspecific hybrid Solanum tuberosum × Solanum spegazzinii (P40) (22) and the diploid and self-compatible Physalis pubesecens (syn. P. floridana P106) (GenBank, Max Planck Institute for Plant Breeding Research) were grown in the greenhouse. The tetraploid S. tuberosum cv. Désirée (22), Solanum macrocarpon (PI 441915) (23) and the tetraploid Physalis peruviana (P105) (GenBank, Max Planck Institute for Plant Breeding Research) were also used in specific cases.

Isolation of Nucleic Acids and Expression Analysis. Total RNA was isolated by using the total RNA reagent kit (Biomol, Hamburg, Germany). DNA and RNA gel blots, preparation of probes, hybridization, and signal quantification were performed as described by He et al. (24). The filters were exposed to a Storage Phosphor Screen (Molecular Dynamics), and signals were quantified with a Typhoon 8600 PhosphorImager (Amersham Pharmacia).

For RT-PCR analysis, total RNA was treated with DNase I (Roche Diagnostics) to remove genomic DNA contamination. For first-strand cDNA synthesis, 4 μg of total RNA was used with reverse transcriptase (Stratagene) in a 20-μl reaction volume. For PCR of MPF1, the sense primer was 5′-GAG AAG CAC AAG ATG CAT TCA GAA AG-3′, and the antisense primer was 5′-GTA CCT ATA GTT AGC TAT GAC ATC ACT CTC C-3′. The flanking primers used to amplify MPF2 were 5′-GTG CTG AGC TGA TGG AAG AAA AC-3′ and 5′-GAG AAC GAT CGA GTT CAC ATG AAC TCA TGA G-3′. The Actin-specific primers, 5′-ATG GCA GAC GGA GAG GAT ATT CAG-3′ and 5′-GCA CTT CCT GTG GAC AAT AGA AGG-3′, were designed on the basis of the S. tuberosum actin gene sequence (X55751). The three gene-specific primer pairs were used together in the same reaction to monitor the expression of all three target genes simultaneously. PCR (annealing temperature 60°C, extension at 72°C) was performed in 50-μl volumes by using the Taq polymerase (Roche), and the PCR products were separated by electrophoresis on a 1.5% agarose gel.

Isolation of Genes and cDNAs, and Promoter Definition. The Expand High-Fidelity PCR System (Roche) was used for all procedures. cDNAs for STMADS11, STMADS16, and their orthologs were isolated by RACE (rapid amplification of cDNA ends) by using the 5′/3′ Rapid Amplification of cDNA Ends kit (Roche). In all, 210 5′/3′ cDNA clones were isolated from Physalis and sequenced. Only two gene products could be identified, MPF1 and MPF2. Gene-specific primers were derived from these cDNAs and from STMADS11 (25) and STMADS16 cDNA (26) sequences. Genomic loci were obtained with the Expand Long Template PCR System (Roche), by using sequence-specific primers based on the full-length cDNA sequences. The promoter regions were isolated by rapid amplification of gDNA ends (RAGE) according to the Universal Genome Walker kit (Clontech). The final PCR products were cloned into the pGEM-T easy Vector (Promega). Putative transcription initiation sites were deduced from the sequences of the longest cDNA fragments obtained by 5′RACE. The STMADS16-promoter was defined by comparing different alleles from diploid and tetraploid lines of S. tuberosum that showed identical expression patterns. The conserved region from –470 to –1380 was compared with the MPF2-promoter.

Expression Constructs and Plant Transformation. Double-stranded RNA interference (RNAi)-mediated MPF1 and MPF2 knockdown was carried out in P. pubescens. RNAi constructs were assembled by introducing fragments of MPF1 and MPF2 cDNAs encoding I, K, and C domains [sense and antisense sequences, separated by a β-glucuronidase (GUS) intron] into the binary vector pFGC1008 as suggested in the ChromDB (www.chromdb.org/). These MPF1 and MPF2 cDNA fragments share 49% sequence identity. The primer sequences used to generate the MPF1 fragment were 5′-CTA CTA GTG GCG CGC CAG TAT GAT GCA ACT GAT TG-3′ and 5′-CTG GAT CCA TTT AAA TAC GTA TGG ATC GGA GCT C-3′. For MPF2, the primers were 5′-CTA CTA GTG GCG CGC CAG CAT GAA GGA TAT CCT TG-3′ and 5′-CTG GAT CCA TTT AAA TGC TAG CTG AGT GGT AGC-3′. Physalis transformation was performed as described (27).

For overexpression studies, full-length MPF1, MPF2, and STMADS16 cDNAs were cloned into the plant binary vector pBin19, introduced into LBA4404 strain of Agrobacterium tumefaciens and transformed into S. tuberosum cv. Désirée (28). The medium for shoot regeneration and for selection of transformed shoots for the RNAi knockout plants contained 1 μmol·liters–1 α-naphthaleneacetic acid (NAA), 12.5 μmol·liters–1 6-benzylamino-purine (6-BAP) and 15 mg·liters–1 hygromycin. Overexpression lines were selected was on 50 mg·liters–1 kanamycin. MPF2 overexpressing calli regenerated shoots at high frequency, but only a few shoots were obtained from MPF2-RNAi calli. Prolonged cultivation of transgenic calli occasionally resulted in regeneration. However, several of these transgenic plants did not show any phenotypic alterations relative to wild type. Therefore, MPF2 might be essential for vegetative development, and complete knockout of its expression may be lethal.

The genotypes of transgenic lines reported in this study were verified by northern analysis by using HPTII (hygromycin phosphotransferase II) and NPTII (neomycin phosphotransferase II) as a probe in P. pubescens and S. tuberosum, respectively.

Morphological Analyses and Quantification Procedures. For scanning electronic microscopy (SEM), fresh material was frozen in liquid nitrogen, sputter-coated with gold, and examined with a digital scanning microscope (DSM940, Zeiss). Five leaves and the corresponding calyces were taken from the youngest mature fruits on the main branch. Quantification of leaf areas and cell sizes was performed by using the nih image program (imagej, available at http://rsb.info.nih.gov/nih-image). From each line, 20 mature calyces were measured.

Sequence Analysis and Phylogenetic Analysis. DNAs were sequenced at the Automatic DNA Isolation and Sequencing Unit (ADIS) of the Max Planck Institute for Plant Breeding Research (Cologne, Germany). The upstream regions of MPF2, STMADS16, and AGL24 were scanned for potential regulatory sequences in the PLACE database [www.dna.affrc.go.jp/htdocs/PLACE database (29)]. Neighbor-joining trees were generated by paup 4.0 by using full-length protein sequences and SQUA from Antirrhinum majus (30) as the out-group.

Results

The ICS, a Morphological Novelty. Flowers of S. tuberosum and P. pubescens have small sepals that form a fused calyx basally. During flowering, the sepals of P. pubescens are villous, with triangular lobes, and are only ≈10% the size of the petals. In contrast to sepals of S. tuberosum, the calyx of P. pubescens resumes growth after pollination (31) to become large and five-angled at maturity, encasing the fruit (Fig. 1). This condition, the ICS (21), is not unique among the ≈96 genera of the Solanaceae (21, 32). At least 5 solanaceous genera display ICS, including all 75 species of Physalis (21). In all these cases, ICS is formed only after pollination.

Fig. 1.
Stages of flower and fruit development in S. tuberosum and P. pubescens. Sb, Syfl, Smfl, Syfr, Slfr, and Sofr are sepals from buds, young flowers, mature flowers, young fruits, large fruits, and old fruits, respectively. Open columns in the graph represent ...

Genes Involved in ICS Formation. Two key observations guided our search for the molecular basis of the ICS. First, the Tunicate phenotype in Zea mays, where the kernels are enclosed by glumes (21, 33, 34), apparently results from ectopic expression of the vegetative-specific MADS-box gene ZMM19, a STMADS11-like gene (35), in mutant inflorescences (21). Second, overexpression of another STMADS11-like gene, STMADS16 from S. tuberosum, in N. tabacum results in the formation of leaf-like sepals (25). Therefore, a STMADS11-like gene, such as an ortholog of STMADS16, seemed likely to be involved in ICS formation in Physalis.

We isolated cDNAs for STMADS11 family members from P. pubescens, P. peruviana, S. macrocarpon, and S. tuberosum. Molecular phylogeny revealed that these genes fall into the STMADS16 and STMADS11 subclades within the STMADS11-superclade (Fig. 7, which is published as supporting information on the PNAS web site). Overexpression of members of the subclades ZMM19 (21), SVP (21), and AGL24 (21, 36) in Arabidopsis thaliana generates enlarged, leaf-like sepals and thus suggests that they have common functions. Overexpression of STMADS16 does not give rise to this trait in Arabidopsis (data not shown), but, if the gene is overexpressed in the solanaceous N. tabacum, foliose sepals are formed (25). This result supports the idea that a STMADS16 ortholog might be involved in ICS formation in Physalis. The Physalis orthologs of STMADS11 and STMADS16 were designated MPF1 and MPF2, respectively, and the gene pair STMADS11/MPF1 was used as a control throughout the study. Both orthologous pairs are present as single-copy genes, as revealed by Southern analysis (refs. 25 and 26 and Fig. 8, which is published as supporting information on the PNAS web site).

Heterotopic Expression of MPF2 Is Essential for ICS Formation. STMADS11 and MPF1 are expressed in leaves and in sepals of Solanum and Physalis species (Fig. 2). STMADS16, however, is expressed only in vegetative tissues in S. tuberosum (ref. 25; verified by RT-PCR in the present study; data not shown), although its ortholog MPF2 is expressed highly in leaves and at an ≈5-fold lower level in sepals (Fig. 2 A and C) and other floral organs of Physalis (data not shown). Similar expression patterns for STMADS16-orthologous genes are also observed in P. peruviana and S. macrocarpon (Fig. 2 B and D). The observed expression patterns correlate with the presence or absence of enlarged sepals: S. tuberosum does not express STMADS16 in floral tissues and displays neither ICS nor leaf-like sepals, whereas species that express orthologs of STMADS16 in floral organs, like P. pubescens and P. peruviana, show the ICS, or have foliose sepals, like S. macrocarpon (ref. 21 and Fig. 2). If restriction of the STMADS16 expression pattern to vegetative tissues represents the ancestral state, then heterotopic expression of MPF2 in floral organs, and hence ICS formation, seems to be the derived state. We tested this hypothesis by analyzing the consequences of down-regulation of MPF2 in transgenic Physalis plants using the RNAi technology, and by overexpressing MPF2 and STMADS16 in transgenic S. tuberosum.

Fig. 2.
Analysis of gene expression. The Northern blot studies were performed with total RNA derived from S. tuberosum Désirée(A), S. macrocarpon (B), P. pubescens (C), and P. peruviana (D). The graphs show relative mRNA levels: all values are ...

MPF2 Function Is Required for ICS. RNA interference has been shown to be an efficient tool for gene silencing, affecting gene expression, mRNA degradation, or mRNA translation (37). In six of the eight 35S::MPF2-RNAi transgenic Physalis plants, leaf size is reduced and formation of the “Chinese lantern” structure is impaired relative to wild type. Indeed, in the most severely affected line, Ko45, no ICS is formed (Fig. 3 A and B). Except in the case of Ko27, the reduction in the sizes of leaves and calyces correlates well with the extent to which MPF2 RNA levels are reduced. In Ko27, the translation of MPF2 RNA might also have been affected (Fig. 3 AC). Down-regulation of MPF2 by RNAi expression is specific, because transcripts of MPF1, the closest relative of MPF2, are not affected in the transgenic plants (Fig. 3C).

Fig. 3.
MPF2 is essential for ICS formation. (A) Leaf size and ICS formation in the two MPF2-RNAi transgenic Physalis lines Ko34 and Ko45 compared with wild type. Shown are leaf sizes (Left) and calyx sizes (Right) during development. (B) Quantification of phenotypic ...

Interestingly, in the lines Ko34 and Ko45, in which ICS is drastically reduced or absent, a small amount of MPF2 RNA is still detectable by multiple rounds of RT-PCR in the flowers, although Northern analysis failed to reveal gene expression (Fig. 3 C and D). This finding indicates that plant survival might depend on a residual level of MPF2 expression (Fig. 3D), perhaps explaining the difficulties we encountered in obtaining transgenic knockout lines (see Materials and Methods).

SEM reveals that epidermal cell size in the leaves and sepals of the transgenic RNAi line Ko45 is increased by 10–30% compared with wild type (Fig. 9, which is published as supporting information on the PNAS web site), despite the overall reduction in the size of these organs (Fig. 3 A and B). This finding suggests that MPF2 negatively controls cell elongation in Physalis leaves and in sepals. Because cells become larger and organs smaller after down-regulation of MPF2 function, it seems that MPF2 positively controls cell proliferation in the wild type. This assumption is supported by observations made on transgenic potato plants, which overexpress MPF2 and STMADS16, as shown below.

MPF2 Inhibits Cell Expansion and Promotes Cell Division. 35S::MPF2 or 35S::STMADS16 transgenic S. tuberosum plants form enlarged leaf-like calyces (Fig. 4A). SEM observations demonstrate two striking alterations in both 35S::MPF2 and 35S::STMADS16 transgenic sepals: the density of stomata is dramatically increased (Fig. 4Bc) as on the surface of the wild-type leaf (data not shown), and cell size is decreased by at least 50% (Fig. 4Bc). Given that the organ is larger and the cell size is smaller, MPF2 seems to promote cell division and to inhibit cell expansion in sepals of the transgenic potato plants. These observations support the results of our MPF2-RNAi analysis in Physalis and corroborate the key role of MPF2 in ICS formation.

Fig. 4.
Effects of overexpression of MPF2 in transgenic lines of S. tuberosum. (A) Phenotypes of wild-type and transgenic S. tuberosum plants. Flowers and fruits from wild-type and transgenic plants (genotypes indicated above the panels) are shown. (Far Right ...

In contrast to MPF2, the related Physalis gene MPF1, which belongs to the STMADS11 rather than to the STMADS16 subclade (Fig. 7), does not affect ICS formation when inhibited using RNAi in Physalis (data not shown). Furthermore, when MPF1 is overexpressed in potato (Fig. 4A), calyx size, epidermal cell size, and cell number are not affected (Fig. 4Bb). These observations thus support the specificity of MPF2 function in ICS formation.

MPF2 Is Required for Male Fertility. Although MPF1 RNAi transgenic Physalis plants are perfectly fertile, severely affected transgenic RNAi MPF2 knockout lines, like Ko45, are almost entirely sterile. The wild-type stigma shown in Fig. 5 is loaded with pollen, and the developing berry displays good seed set (Fig. 5 AC). In contrast, the transgenic line Ko45 shows very little pollen on the stigma and only occasional fertilization of ovules (Fig. 5 EG). In F2 plants grown from the few seeds obtained by selfing, the traits small leaves, small ICS, and reduced male fertility cosegregated with the transgene. However, upon fertilization with wild-type pollen, berries developed normally, although they remained tightly enwrapped by the calyx (Fig. 5H), in marked contrast to the balloon-like ICS formed in wild type Physalis (Fig. 5D). Thus, female fertility is not affected in the RNAi lines, but MPF2, in addition to its role in ICS, has apparently been integrated into the male fertility program during the evolution of Physalis.

Fig. 5.
MPF2 is a component of male fertility. (A) Wild-type flower. (B) Stigma covered with pollen. (C) Unripe berry cut open to reveal the developing seeds. (D) Berry inside ICS (front portion removed). (E) Flower from transgenic line Ko45. (F) No pollen on ...

Heterotopic Expression of MPF2 Correlates with Changes in the Promoter Sequence. Recruitment of a STMADS16-like function for the establishment of the ICS in Physalis during evolution might have occurred in response to mutations in a transacting regulator of MPF2 expression or to changes in cis-regulatory elements of the MPF2 gene itself. A comparison of the molecular structure of the STMADS16 and MPF2 genes revealed that the organization of the transcription units, and the respective coding sequences, are highly conserved. This finding is in marked contrast to the poor conservation seen in their promoter regions (Fig. 6): although the coding sequences are 86% identical, the promoter regions show only 42% sequence identity. Intriguingly, four CArG-boxes [MADS-box protein-binding motifs (38)] are present in the STMADS16 upstream region but absent in the corresponding part of MPF2 (Fig. 6). This finding suggests that the promoter regions and the coding sequences of these genes have been subjected to different selective pressures during evolution. This finding is in accord with the functional similarity between the two genes in leaves as contrasted by the differences in their expression in the flowers of S. tuberosum and P. pubescens.

Fig. 6.
Gene structures of STMADS16 and MPF2 (A) and comparison of their promoters (B). (A) The solid boxes indicate exons, and the 5′ and 3′ UTRs are depicted as open boxes. M, MADS-box (DNA-binding domain); I, intervening domain; K, keratin-like ...

Unlike STMADS16 and MPF2, the orthologous gene pair STMADS11 and MPF1 exhibit similar expression patterns (Fig. 2), suggesting that their promoters are structurally related. Indeed, both promoters contain CArG-boxes and share 60% DNA sequence identity (C.H. and H.S., unpublished data).

Thus, the sequence divergence between the MPF2 and STMADS16 promoters could be responsible for the heterotopic expression of MPF2 in Physalis, although this assumption needs to be verified functionally.

Discussion

Gene duplication and subsequent alterations in cis-regulatory elements are often postulated to account for the evolution of new morphological features in related genera or species (7). The ICS of Physalis seems to be an unusual case in that it does not involve gene duplication. Here, it seems that changes in promoter structure alone were sufficient to ensure heterotopic expression of MPF2. MPF2 encodes a MADS-box transcription factor that, like other MADS-box proteins in other tissues (25, 3941), influences sepal size by controlling cell division and cell elongation.

Heterotopic MPF2 expression is essential for ICS formation, but a signal from the developing fruit is also required for the Chinese lantern to form, because MPF2 function in controlling cell division and elongation in sepals depends on successful fertilization in Physalis. The precise nature of this signal is not clear; possibly, hormones can take on this role, because developing fruits produce cytokinins and gibberellins, which are known to promote either cell division or cell elongation (4244). MPF2 RNA is present in the sepals before pollination, and therefore the control event is likely to be posttranslational; either the MPF2 RNA cannot be immediately translated and the protein is synthesized only in response to the “fertilization signal,” or the protein might be initially inactive and have to be activated by modification(s). Biochemical analyses of the MPF2 protein and of plant hormones before and after fertilization are required to substantiate these assumptions.

Intriguingly, in addition to controlling cell division and elongation in sepals and thus leading to ICS formation, MPF2 also plays a role in male fertility. How MPF2 became an integral part of the male fertility program during the evolution of Physalis and how it performs this function is not clear. Protein–protein interaction studies might reveal whether or not MPF2 replaces a component in the “floral quartets” (18). The transition from the fertility system in the progenitor, which still needs to be identified, to the fertility system of Physalis is the next challenge in understanding the sequence of events in the evolution of the morphological novelty termed the ICS.

Heterotopic expression of MPF2 is thus a prerequisite for two novel functions of MPF2 in Physalis: calyx cell proliferation/cell expansion and male fertility. The need to maintain fertility thus provides a strong selective force for the maintenance of the ICS. Specific functions in calyx formation and male fertility may or may not prove to be separable for MPF2.

Recruitment of a transcription factor into a new context can be achieved by changes in transacting regulators or by mutations that affect cis-elements in the regulatory sequences of the transcription factor itself (5, 45). Although the STMADS16 promoter contains two N10-like CArG-boxes, the promoter of MPF2 in Physalis has no such motifs (Fig. 6). The presence or absence of CArG-boxes suggests a mechanism of regulation. Expression of MADS-box genes is often subject to autoregulation (13, 46), or is under the control of other MADS-box proteins (36), suggesting that STMADS16 might be under the negative control of another MADS-box protein that prevents its expression in floral organs of S. tuberosum. SQUA-like proteins (30) are candidates for such a regulatory role, because loss of their function in, for instance, the Arabidopsis ap1 mutant (47), or in the lemads-mc mutant of Solanum lycopersicum (tomato) (48), a close relative of potato and Physalis, results in enlarged and leaf-like sepals. Furthermore, AGL24, which codes for an STMADS16 ortholog (Fig. 7), is ectopically expressed in Arabidopsis ap1 mutants (36). This finding is consistent with the presence of two N10-like CArG-boxes in the AGL24 upstream region (data not shown) and supports the idea that AP1 represses its expression in floral tissues (36). SQUA homodimers have been shown to bind efficiently to N10-like CArG-boxes, whereas heterodimers of SQUA with other MADS-box proteins do not (49). SQUA-like proteins such as LeMADS-MC (48) and AP1 (47), therefore, seem to restrict the expression of STMADS16-like genes to vegetative tissues. Lemads-mc and ap1 mutants are characterized by large, foliose sepals, possibly due to ectopic expression of the STMADS16-like gene (36, 48). Accordingly, the absence of N10-like CArG-boxes in the promoter of MPF2 of P. pubescens seems to be a prerequisite for the expression of this gene in floral tissues.

The differential expression patterns of STMADS16 and MPF2 correlate with differences in calyx structure of S. tuberosum and P. pubescens. Changes in the expression of MPF2 during evolution might have been effected by accumulating mutations in the promoter regions or by a recombination event that led to a promoter exchange. Preliminary experiments suggest that the sequences of MPF2 (P. pubescens) and MPP3 (P. peruviana) promoters both diverge from that of the STMADS16 promoter of S. tuberosum at the same site. For this reason, we favor a recombination event that fused the coding region of an STMADS16-like precursor gene to a novel promoter sequence as the cause of the change in the tissue-specific expression of MPF2. This assumption needs to be further substantiated. Regardless of the precise reason in this particular case, recruitment of an existing transcription factor into a different functional context might be a common phenomenon in the evolution of morphological novelties (3, 50).

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Drs. C. Gebhardt, T. Münster, and S. Zachgo (Max Planck Institute for Plant Breeding Research) and S. Y. Chen (Genetics Institute, Chinese Academy of Sciences, Beijing) for providing plant materials and clones, and J. Hu for help in the phylogenetic reconstruction. We thank Dr. Zs. Schwarz-Sommer for comments and help in preparing the manuscript. The comments by Drs. S. Davis, T. Münster, A. Yephemov, and S. Zachgo on the manuscript are appreciated. The technical assistance of H. Henselewski and R.-D. Hirtz is gratefully acknowledged. C.H. was supported by a postdoctoral fellowship from the Max-Planck Society.

Notes

Abbreviations: ICS, inflated-calyx syndrome; RNAi, RNA interference; SEM, scanning electronic microscopy.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY643725–AY643734 (cDNA sequence data), AY643735 (MPF2), and AY643736 (STMADS16)].

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