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JXB Advance Access originally published online on November 16, 2006
Journal of Experimental Botany 2006 57(15):4171-4187; doi:10.1093/jxb/erl194
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense

Madoka Gray-Mitsumune1 *, Martin O'Brien1 *, Charles Bertrand2, Faiza Tebbji1, André Nantel3 and Daniel P. Matton1,{dagger}

1Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2
2Département d'Anatomie-Biologie Cellulaire, Université de Sherbrooke, 3001 12ème Avenue Nord, Sherbrooke, QC, Canada J1H 5N4
3Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montréal, QC, Canada H4P 2R2

{dagger} To whom correspondence should be addressed. E-mail: dp.matton{at}umontreal.ca

Received 7 July 2006; Accepted 8 September 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.

Key words: Carpel, fruit development, MAPKKK, ovule development, Solanaceae


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
The pivotal role played by protein phosphorylation in eukaryotic signal transduction is well illustrated by the wide range of phosphorylation cascades that involve mitogen-activated protein kinases (MAPKs) (Treisman, 1996; Robinson and Cobb, 1997). In vertebrates, MAPKs are typically activated in response to various mitotic agents such as hormones and growth factors (Piwien-Pilipuk et al., 2002). They have been shown to play an important role in the regulation of cell division and differentiation. The activation of MAPKs occurs by phosphorylation of conserved threonine and tyrosine residues in the activation loop between the catalytic subdomains VII and VIII. This phosphorylation step is produced by dual specificity MAPK kinases (MAPKKs). These MAPKKs are themselves activated on serine and/or threonine by serine/threonine MAPKK kinase (MAPKKK) (Nishihama et al., 1995; Madhani and Fink, 1998). In non-plants, the activation of MAPKKKs occurs either through phosphorylation by MAPKKK kinase (MAPKKKK) (Elion, 2000; Sells et al., 1997), by the receiver domain of a two-component histidine kinase module (Posas and Saito, 1997), or commonly by G proteins (Fanger et al., 1997) and G protein-coupled receptors (Sugden and Clerk, 1997). Cell signals are thus transmitted through a chain of phosphorylation events. In plants, direct modulators of MAPKKK are as yet unknown, and complete cascade modules are only just starting to unfold (Asai et al., 2002).

The vast majority of plant developmental mutants described so far have been found to be impaired in the expression of transcription factors, many from the MADS-box family (Jack, 2001). Only a few mutants affecting plant development have been described as encoding protein kinases, although it would be reasonable to assume that post-translational modifications through phosphorylation of a protein involved in a key developmental pathway could be an important regulatory step for its intrinsic activity. Disruption of the signalling cascade would thus also lead to developmental defects. Among the protein kinases known to affect key developmental aspects of plant growth and development, most belong to the receptor-like kinase (RLK) family (Becraft, 2002). Some have also been shown to affect reproductive development. These include the Clavata 1 RLK gene involved in the regulation of meristem size and maintenance (Clark et al., 1993); the BRI1 RLK involved in brassinosteroid perception (Li and Chory, 1997); the Petunia PRK1 RLK involved in pollen and embryo sac development (Lee et al., 1996, 1997); the extra sporogenous cells (EXS) RLK which regulates male germ line cell number, tapetal identity, as well as promoting seed development (Canales et al., 2002); the maize CRINKLY4 (CR4) RLK involved in aleurone cell fate (Becraft and Asuncion-Crabb, 2000); the Arabidopsis CRINKLY4 (ACR4), required for proper embryogenesis (Tanaka et al., 2002) and involved in cell layer organization during ovule integument and sepal margin development (Gifford et al., 2003); the somatic embryogenesis receptor-like kinase (SERK1) involved in somatic embryogenesis (Schmidt et al., 1997), and expressed in developing ovules and embryo (Hecht et al., 2001); and the Strubbelig RLK that affects outer integument formation and organ shape although is does not encode a functional kinase domain (Chevalier et al., 2005). Although downstream kinase modules transducing the initial signal from these RLKs could be expected, like MAPK transducing modules, none has yet been characterized in development, and only a few MAPK family members have been shown to be involved in developmental processes (Hirt, 2000). Recently, a mutation in the Arabidopsis thaliana YODA (YDA) gene, which codes for a MAPKKK, has been shown to cause an early embryonic defect (Bergmann et al., 2004; Lukowitz et al., 2004). In this mutant, the zygote does not elongate properly and the suspensor cells are fused to the embryo. Mutant yda seedlings that can make it through maturity show overproduction and crowding of stomata cells (Bergmann et al., 2004), reminiscent of the receptor kinase two many mouths (tmm) mutant phenotype (Nadeau and Sack, 2002). This hints at the presence of a MAPK module involved in embryo development and stomatal distribution. Most MAPK cascades described so far have been implicated early in stress and disease responses (Asai et al., 2002; Hirt, 2000; Romeis, 2001; Zhang and Klessig, 2001), and hormone perception (Kieber et al., 1993).

In this study, the isolation and functional characterization of a new MAPKKK from the MEKK subfamily in S. chacoense is described. Overexpression of this protein kinase, named ScFRK2, affects ovule identity and suggests an involvement in seed and fruit development.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Plant material
The diploid and self-incompatible wild potato Solanum chacoense Bitt. (2n=2x=24) was grown in a greenhouse with 14–16 h of light per day. The genotypes used were V22 (S alleles S11 and S13) as pollen donor and G4 (S alleles S12 and S14) as female progenitor. Plants were hand pollinated. Transgenic lines were generated in the G4 background.

DNA and RNA gel blot analysis
Total RNA was isolated as described previously (Jones et al., 1985) or with the Plant RNeasy RNA extraction kit from Qiagen (Mississauga, ON, Canada). The RNA concentration was determined by measuring its absorbance at 260 nm. Concentration and RNA quality were verified on an agarose gel with ethidium bromide staining, and RNA concentration adjustment was done if needed. For each tissue tested, 10 µg of total RNA was separated on a formaldehyde/MOPS gel. RNA was then blotted on Hybond N+ membranes (GE Healthcare, Baie d'Urfée, QC, Canada), and were UV cross-linked with a Hoefer UV cross-linker (120 mJ cm–2). To confirm equal loading between RNA samples, a 1 kb fragment of S. chacoense 18S RNA was PCR amplified and used as a control probe. Prehybridization was performed at 45 °C for 3 h in 50% formamide solution (50% deionized formamide, 6x SCC, 5x Denhardt's solution, 0.5% SDS, and 200 µg ml–1 of denatured salmon DNA). Hybridization of the membranes was performed overnight at 45 °C in 50% formamide solution. Genomic DNA isolation was performed with the Plant DNeasy kit from Qiagen. Complete digestion of DNA (10 µg) was carried out overnight with 10 U of restriction enzymes as recommended by the supplier (New England Biolab, Beverly, MA, USA). DNA gel blot analysis was performed as previously described (Sambrook et al., 1989) and DNA was transferred to Hybond N+ membranes prior to cross-linking. Prehybridization was performed for 3 h at 65 °C in 50% phosphate solution [50% of 0.5 M Na2 HPO4 pH 8.0, 1% bovine serum albumin (BSA), 7% SDS, and 1 mM EDTA], while hybridization was performed overnight under the same conditions used for prehybridization. Probes for the RNA and DNA gel blot analysis were synthesized by random labelling using the Strip-EZ DNA labelling kit (Ambion, Austin, TX, USA) in the presence of [{alpha}-32P]dATP (ICN Biochemicals, Irvine, CA, USA). Following hybridization, membranes were washed for 30 min at 25 °C and 30 min at 35 °C in 2x SSC/0.1% SDS; 30 min at 45 °C and 30 min at 55 °C in 1x SSC/0.1% SDS; and for 10 min at 55 °C in 0.1x SSC/0.1% SDS. Prior to control hybridization with the 18S probe, the membranes were stripped as recommended by the manufacturer (Ambion, Austin, TX, USA), and post-hybridization washes were done twice at 60 °C for 30 min in 0.1x SSC/0.1% SDS. Autoradiography was performed at –86 °C on Kodak Biomax MR film (Interscience, Markham, ON, Canada).

Sequence analysis and phylogeny
The catalytic kinase domain structure of ScFRK2 was defined following the 12 kinase subdomains assignation described previously (Hanks and Hunter, 1995). Protein secondary structure prediction needed for the ScFRK2 subdomain designation was performed with these four prediction tools: JUFO (http://www.jens-meiler.de/jufo.html), PORTER (http://distill.ucd.ie/porter/), PSIpred (http://bioinf.cs.ucl.ac.uk/psipred/), and SCRATCH (http://www.igb.uci.edu/tools/scratch/). Designations of subdomain boundaries were obtained from a consensus of all prediction tools and conserved amino acids specific to each domain. The phylogenetic analysis was accomplished with the trimmed catalytic kinase domain only. Alignment of the protein sequences was performed using ClustalX employing default parameters (Thompson et al., 1997). The phylogeny was reconstructed in SplitsTree4 (Huson and Bryant, 2006) using the Neighbor–Joining algorithm (Saitou and Nei, 1987) both from uncorrected distances and from distances corrected using a WAG+{Gamma}+F model (Whelan and Goldman, 2001). The phylogenies were validated using the non-parametric bootstrap with 1000 replicates.

Plant transformation
The ScFRK2 cDNA was PCR amplified with Pwo polymerase (Roche Diagnostic, Laval, QC, Canada) with KpnI overhang primers. Primers used were FRK2Kpn1, 5'-GGGGTACCGCGGTCGGCGCAATCTT-3'; and FRK2Kpn2, 5'-GGGGTACCACTTCCATCAGGCTTTG-3'. The PCR product was cleaved with KpnI and inserted in a modified pBin19 transformation vector (Bussière et al., 2003) with a double enhancer cauliflower mosaic virus (CaMV) 35S promoter. Sense and antisense constructs were determined by plasmid digestion with EcoRI, which gives an asymmetric fragmentation according to the orientation of cloned PCR products. Sense and antisense constructs were individually transformed in Agrobacterium tumefaciens LBA4404 by electroporation. Solanum chacoense plants were transformed by the leaf disc method as previously described (Matton et al., 1997).

Tissue fixation and scanning electron microscopy (SEM) observations
Dissected ovaries were fixed in 4% glutaraldehyde for 4 h at room temperature in 0.1 M phosphate buffer (NaHPO4, pH 7.0). After two rinsing steps in 0.1 M phosphate buffer, the tissues were dehydrated in an increasing ethanol series (from 30% to 100%) and critical-point-dried with CO2, coated with gold–palladium, and viewed in a JEOL JSM-35 SEM.

Tissue fixation and optical microscopy observations
Pistils were fixed in FAA for 24 h at 4 °C (50% ethanol, 1.35% formaldehyde, and 5% glacial acetic acid). Samples were then dehydrated in an increasing series of tert-butyl alcohol (from 70% to pure tert-butyl alcohol). Pistils were infiltrated with Paraplast Plus (Tyco Healthcare Group LP, Mansfield, MA, USA) at 60 °C. Thin sections (10 µm) were prepared from embedded samples, and tissue sections were stained in 0.5% Astra Blue and 1% safranin after removal of paraffin. Alternatively, thin sections (10 µm) were prepared from embedded samples and tissue sections were stained in 0.05% toluidine blue O (Fig. 4V–Y). In situ hybridizations were performed as described previously (O'Brien et al., 2005). Microscopic observations were taken on a Leica OrthoPlan microscope and pictures were taken with a Leica DFC320 camera.


Figure 4
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  		Fig. 4 Fruit and ovule development of ScFRK2-OX plants. (A and B) Wild-type (A) and transgenic (B) ovaries. The ovary wall has been removed to expose the ovules. The white arrow in B points to a normal ovule. (C–Q) SEM images from wild-type (C, I, J, L, N, and P) and transgenic (D–H, K, M, O, and Q) plants. (C) Ovules from a wild-type plant. (D and E) Ovules from ScFRK2-OX plants, with D showing a milder phenotype than E. (F) Enlargement of emerging carpelloid structure (CSs). (G) Enlargement of an elongated CS. (H) Papillae on the CS extremity. (I) Papillae on a wild-type stigma. (J) Enlargement of WT stigmatic papillae. (K) Enlargement of papillae on the CS extremity. (L) Style cortex epidermis of a wild-type plant. (M) Epidermis of the stylar section of a CS. (N) Small papillae on a wild-type style 3 d before anthesis. (O) Small papillae from the stylar section of a CS. In N and O, papillar cells are indicated by dark arrows. (P) Enlargement of papillar ornamentations from the style of a wild-type plant. (Q) Enlargement of papillar ornamentations from a CS of a transgenic plant. (R–U) Light microscopy of wild-type (R, T) and transgenic (S, U) plants. (R) Ovary from a wild-type plant (only one locule shown). (S) Ovary from a ScFRK2-OX plant (only one locule shown). (T) Transverse section of a style from a wild-type plant. The arrow indicates the transmitting tract. (U) Transverse section of a CS showing dense cells resembling the transmitting tract (indicated by arrows). (V) Developing ovary from a wild-type plant. The section was taken from a flower bud ~2 mm in length. (W) Enlargement of one of the ovules shown in V. The black arrow indicates the ovule integument and the white arrow indicates the megasporocyte. (X) Developing ovary from a ScFRK2-OX plant. The section was taken from a flower bud ~2 mm in length. The black arrows indicate developing CSs. (Y) Enlargement of the tip of the CSs shown in X. The black arrow indicates the integument. Integuments were often enlarged. The white arrow indicates the megasporocyte. Scale bars, 1 mm (A and B); 100 µm (C, D, F–I, L–O, U, V, and X); 500 µm (E); 50 µm (T); 10 µm (J, K, P, and Q); 250 µm (R and S); 20 µm (W and Y).

 
RNA extraction, probe preparation, cDNA array hybridization, and data analysis
DNA microarrays were printed on UltraGAPSTM Slides (Corning) from 7741 expressed sequence tags (ESTs) corresponding to 6374 unigenes derived from fertilized ovary cDNA libraries covering embryo development from zygote to late torpedo stages in S. chacoense (Germain et al., 2005). This microarray was used to analyse gene expression profiles in ovaries prior to fertilization in wild-type (WT) and ScFRK2 mutant plants. Total RNA was extracted from unfertilized WT and ScFRK2 mutant ovaries using TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. RNA yield and purity were assessed by absorbance determination at both 260 and 280 nm. RNA was only used when the Abs260 nm/Abs280 nm ratio was >1.7. RNA integrity was determined with the RNA 6000 Nano Assay Kit and the Agilent 2100 Bioanalyzer. A 30 µg aliquot of total RNA from WT ovaries was hybridized to microarrays along with the same amount of RNA from ScFRK2 mutant ovaries for 16 h at 42 °C. Each of the four pools of RNA from WT and ScFRK2-OX ovaries was compared with the control RNA in individual microarray hybridizations. Two hybridizations used a Cy3/Cy5 comparison, while the other two used a Cy5/Cy3 ratio. This strategy allowed profiling data to be obtained from four biological replicates whilst simultaneously negating any potential bias that could arise from the choice of labelling dyes. Labelling was performed with cyanine 3-dCTP or cyanine 5-dCTP (1 mM; NEN Life Science, Boston MA, catalogue nos NEL576 and 577). Hybridization and washing were performed as described in the CyScribe Post labeling Kit (Amersham Biosciences) in the CMTTM hybridization chamber (Corning). The DNA microarray slides were scanned with a ScanArray Lite scanner (Perkin Elmer-Cetus, Wellesley, CA; version 2.0) at 10 µm resolution. The resulting 16-bit TIFF files were quantified with QuantArray software (Perkin Elmer-Cetus; versions 2.0 and 3.0). Normalization was performed with Lowess (locally weighted scatter plot smoothing). Statistical analysis and visualization were performed with GeneSpring software (Silicon Genetics, Redwood City, CA, USA) using the available statistical tools (Student's t-test of replicate samples showing a variation different from 1).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
Isolation and sequence analysis of the ScFRK1 and ScFRK2 kinases
Using a negative selection screen targeting only weakly expressed genes expressed during fertilization and early embryogenesis, 16 different protein kinase clones have been isolated from the MAPK family in S. chacoense, a self-incompatible wild potato species close to potato and tomato (Germain et al., 2005). Two of these, named ScFRK1 and ScFRK2, were further investigated for their possible role in fertilization and embryogenesis. Both clones showed strong sequence similarity to the catalytic domain of various MAPKKKs, but lacked a large regulatory domain characteristic of most of these MAPKKKs (Tu et al., 1997; Wu et al., 1999).

The ScFRK1 kinase clone (accession no. AY427828) is 1539 bp long [excluding the poly(A) tail] and is likely to represent the full-length ScFRK1 mRNA since the size of this cDNA corresponds to the size of the mRNA as determined from RNA gel blot analyses (~1.5 kb, data not shown). It codes for an open reading frame of 320 amino acids with an estimated mol. wt. of 36.2 kDa. A stop codon is found upstream and in-frame with the first AUG initiation codon, indicating that the coding region is complete. The longest ScFRK2 cDNA isolated (accession no. AY427829) consists of 1115 bp [excluding the poly(A) tail] and codes for a predicted 304 amino acid protein with an estimated mol. wt. of 34.3 kDa. A stop codon is also found upstream and in-frame with the first AUG initiation codon, indicating that the coding region is also complete. Both proteins are composed of a catalytic domain that constitutes the major part of the protein (Fig. 1A). The kinase domain starts after the first 10 (for ScFRK1) and five (for ScFRK2) amino acids, and stops 42 (for ScFRK1) and 47 (for ScFRK2) amino acids before the end of the protein, leaving ~50 amino acids for putative regulatory domains.


Figure 1
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  		Fig. 1 Sequence analyses of ScFRK1 and ScFRK2. (A) Sequence alignment of the deduced protein sequences from S. chacoense ScFRK1 and ScFRK2, and with the three most similar MAPKKKs from A. thaliana. Kinase subdomains are identified with thick black lines and were defined as described by Hanks and Hunter (1995), where {alpha}-helices and ß-sheets were deduced by four different prediction tools (see Materials and methods). (B) Phylogenetic tree obtained with the Neighbor–Joining method from uncorrected distances. Bootstrap support values from 1000 replicates are indicated beside the branches. All A. thaliana sequences were directly obtained from the TAIR website (http://www.arabidopsis.org/). The phylogenic analysis was performed using the catalytic kinase domain. Tree branch length is proportional to the number of substitutions, and the scale bar represents the expected number of substitution per site.

 
The ScFRK2 deduced protein showed the strongest amino acid sequence identity with ScFRK1 (45% identity; 60% overall similarity), and with a predicted protein kinase from A. thaliana (At5g67080, renamed MAPKKK19), with 36% identity (51% overall similarity) (Fig. 1A). The ScFRK1 deduced protein showed the greatest sequence identity (39%; 50% similarity) with the At3g50310 protein sequence (renamed MAPKKK20), a putative protein kinase from A. thaliana. Since initial BLAST comparisons retrieved mostly protein kinases defined as MAPKKKs, and since ScFRK1, ScFRK2, and their most similar A. thaliana putative orthologues were considerably smaller than most typical MAPKKKs, lacking a large N- or C-terminal regulatory domain, a phylogenetic analysis was performed with representatives from the MAPK (as the outgroup), MAPKK, MAPKKK, and MAPKKKK families, in order to determine to which family of MAPKs ScFRK1 and ScFRK2 belonged. The phylogenetic analysis was achieved after aligning trimmed kinase domains based on previous sequence alignments and secondary structure predictions (Hanks and Quinn, 1991). Protein kinases from A. thaliana previously classified into these various MAPK families were used for the analysis. Two members of the MAPK family were chosen (outgroup), as well as 10 MAPKKs, 80 MAPKKKs (48 from the RAF subfamily, 21 from the MEKK subfamily, and 11 from the ZIK subfamily), and six MAPKKKKs. To simplify the display, and since the ScFRK1 and ScFRK2 kinases were closest to the MEKK, a phylogenetic tree obtained with only the MEKK subfamily from the MAPKKK family is shown in Fig. 1B. Only the tree obtained from uncorrected distances is shown, but the use of corrected distances resulted in a tree with an identical topology (data not shown). This analysis clearly showed that both ScFRK1 and ScFRK2 are members of the MEKK subfamily of the MAPKKKs.

The genomic organization of ScFRK2 was assayed by PCR amplification on genomic DNA and by restriction enzyme digestion of genomic DNA followed by DNA gel blot analyses. PCR amplification was achieved with primers that amplified the complete ScFRK2 cDNA clone, including all available 5' and 3' untranslated region sequences. Genomic DNA and plasmid PCR amplifications were run side by side on an agarose gel, and no differences in the length of the PCR products could be detected (data not shown), indicating the absence of an intron in the region defined by the primers used. In A. thaliana, At5g67080 (MAPKKK19), At3g50310 (MAPKKK20), and At4g36950 (MAPKKK21) are also intronless. DNA gel blot analyses of genomic DNA restriction fragments revealed a simple multibanding pattern for ScFRK2 hybridization (Fig. 2A). The EcoRI and NcoI digestions showed three bands, while the BamHI and XbaI digestions showed two bands. Since EcoRI and BamHI cleave the ScFRK2 cDNA once, and NcoI and XbaI do not cleave the ScFRK2 cDNA, these results suggest that, in S. chacoense, the ScFRK2 gene is present possibly in two to three copies if one of the BamHI fragments contains two copies of ScFRK2 in tandem.


Figure 2
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  		Fig. 2 Transcript profile of ScFRK2. (A) DNA gel blot analysis of the ScFRK2 gene. Genomic DNA (10 µg) isolated from S. chacoense leaves was digested with BamHI, EcoRI, NcoI, or XbaI restriction enzymes, transferred onto membranes and probed with the complete 1.1 kb ScFRK2 cDNA. Estimated molecular weights of the fragments obtained appear on the left of each DNA blot. (B) RNA gel blot analysis of the ScFRK2 gene. All tissues were collected from greenhouse-grown plants. Pollen tubes were germinated in vitro. Fertilized ovules were dissected from ovaries 2–6 d after pollination (DAP). Pistils at 2–4 DAP were separated into styles and ovaries. A 10 mg aliquot of total RNA isolated from S. chacoense tissues was blotted and probed using the full-length ScFRK2 cDNA (upper panel). Membranes were stripped and re-probed using a partial 18S rRNA to ensure equal loading of each RNA sample (lower panel). (C) In situ localization of ScFRK2 transcripts in mature ovaries. I and II, unfertilized mature ovary sections. III and IV, ovary sections 2 DAP (~12 h post-fertilization). V and VI, magnifications of III and IV, respectively. VII and VIII, magnifications of ovary sections 2 DAP showing staining in the zygote. I, III, V, and VII, control sense probe. II, IV, VI, and VIII, antisense sense probe. Digoxigenin labelling is visible as red to purple staining. All hybridizations used 10 µm thick sections and an equal amount of either ScFRK2 sense or antisense probe. Scale bars, 250 µm (I–IV); 125 µm (V and VI); 20 µm (VII and VIII). In, integument; ov, ovule; ow, ovary wall (pericarp); p, placenta; zy, zygote.

 
Fertilization induces ScFRK2 mRNA accumulation in ovules
The ScFRK2 expression pattern was determined by RNA gel blot analysis (Fig. 2B) with various vegetative (stems and leaves), generative (petals), and reproductive tissues (stamens, pollen, pollen tubes, fertilized ovules, styles, and ovaries). Prior to fertilization, the strongest ScFRK2 mRNA accumulation is observed in stamens and styles, although a basal level of mRNA can be detected in most tissues examined, including roots or tubers (Fig. 2B, and data not shown). Fertilization had a dramatic effect on ScFRK2 accumulation only in ovaries, as can be seen 2 d after pollination (DAP), and corresponding to ~12 h after fertilization (Fig. 2B). Intriguingly, hybridization with the ScFRK2 probe detected two transcripts, although not consistently in all experiments (for example, see Fig. 3A). The smaller transcript is estimated at 1.1 kb and corresponds to the expected size for the ScFRK2 cDNA (1.11 kb). The larger 1.6 kb ScFRK2 transcript is slightly less abundant in all tissues tested except stamens. Since previous results suggest that the gene has no introns, the present finding suggests that the longer transcript is most probably not a splice variant. Nonetheless, since ScFRK2 is part of a small gene family, this prompted an attempt to isolate a cDNA corresponding to the longer 1.6 kb transcript. One million phages from a cDNA library made from 2 DAP pistils were screened using the full-length ScFRK2 cDNA as a probe. All eight clones that were retrieved corresponded to the 1.1 kb transcript (data not shown). Thus the ScFRK2 mRNA abundance can be estimated at 0.0008% of total mRNAs in pistil tissues. This is reflected in the exposure time of the hybridization membranes, with 4 d exposure with the ScFRK2 probe versus 5 min for the control 18S rRNA probe. A 5' rapid amplifcation of cDNA ends (RACE) PCR was also attempted, but again no product longer than the original ScFRK2 cDNA could be isolated. Since it is unlikely that the larger ScFRK2 hybridizing transcript corresponds to an unspliced ScFRK2 pre-mRNA from the above-mentioned results, the higher molecular weight band may correspond to a cross-hybridizing member of the ScFRK2 family (Fig. 2A). Alternatively, it cannot be excluded that there is a possibility of an alternative transcription termination site that would generate a longer transcript, although again this species should have been found in the library re-screening.


Figure 3
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  		Fig. 3 Analyses of ScFRK2 transgenic plants carrying sense constructs. (A) RNA gel blot analyses of ScFRK2 mRNA (upper panel) and 18S rRNA (lower panel) of ovary tissues derived from wild-type plants (WT), overexpression lines S2 and S17, and co-suppression line S20. (B) Comparison of fruit slices. Scale bar, 1 cm. (C) Growth in fruit size after pollination.

 
In order to determine the spatial expression pattern of the ScFRK2 gene, in situ RNA hybridizations were performed using ovaries taken before and after fertilization (Fig. 2C). Before fertilization, a weak ScFRK2 mRNA signal was detected only in mature ovules (Fig. 2CII). After fertilization, much stronger ScFRK2 mRNA signals were detected in ovules and, also, diffuse signals were detected in the placenta and ovary wall (Fig. 2CIV and VI). Although preservation and observation of the zygote is difficult in these paraffin-embedded sections, staining can occasionally be observed in the embryo sac where the zygote would be located (e.g. see magnification in Fig. 2CVIII). The absence of or very weak sense probe hybridization signals confirmed the specificity of the detection pattern obtained (Fig. 2CI, III, V, and VII).

ScFRK2 overexpression lines have defects in ovule development
Transgenic plants were generated carrying the sense or antisense construct of ScFRK2. The ScFRK2 cDNA was placed downstream of a double enhancer 35S promoter in a modified pBin19 vector in a sense or antisense orientation (Bussière et al., 2003). Kanamycin-resistant plants were grown to maturity in the greenhouse, and the ScFRK2 expression level was monitored by RNA gel blot analyses of stamen, leaf, and ovary tissues. Identical results were obtained for the three tissues tested, indicating that the CaMV 35S promoter used was equally active in ovaries, stamens, and leaves. Among 13 transgenic lines containing the sense construct, one showed complete co-suppression for ScFRK2 (line S20 in Fig. 3A), while 12 others showed various levels of ScFRK2 mRNA overexpression (ScFRK2-OX lines) (e.g. lines S2 and S17 in Fig. 3A). Plants containing the antisense construct showed reduced ScFRK2 mRNA levels compared with the WT (data not shown), but none showed mRNA down-regulation as severe as the co-suppression line S20 (Fig. 3A). No obvious developmental defects were detected either in co-suppression line S20 or in the antisense lines. Overall plant growth and development appeared unaffected in all ScFRK2-OX transgenic lines. However, severe defects in seed and fruit development were exhibited. Half of the ScFRK2-OX lines (OX lines S2, S17, S5, S7, S9, and S11) produced smaller than normal fruits containing fewer seeds (Fig. 3B, C; Table I). Two OX lines, S2 and S17, were selected for detailed phenotypic observation because they exhibited the highest levels of ScFRK2 mRNA. Development of the fruits started similarly in WT and transgenic lines, but significant differences in fruit size were detected from 10 DAP in OX lines (Fig. 3C). In WT S. chacoense plants, fruits develop to maturity in ~21 DAP. Thereafter, mature embryos start to desiccate in order to reach seed maturity at ~40 DAP. At 21 DAP, fruit diameters from ScFRK2 transgenic lines reached only 60% of those of the normal WT fruits, weighed only 36% of the weight of normal WT fruits, and contained only 6.4% of the total number of seeds normally produced in WT fruits. The reduced seed set could explain the small fruit size observed. Furthermore, mature seeds obtained from OX lines bore embryos that were retarded in their development, most of them still being at the torpedo stage compared with mature embryos in WT plants (Table 1). These embryos would eventually proceed to a mature stage and plants could be regenerated from these when hand dissected and placed on a sterile solid MS medium (data not shown).


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  		Table 1 Comparison of fruit size, weight, ovule number, and embryo developmental stage between wild-type and mutant plants

 
Further analyses of the ScFRK2-S2 and ScFRK2-S17 mutants revealed that overexpression of ScFRK2 led to the transformation of ovules into carpelloid structures (CSs). In hand-dissected ovaries from the ScFRK2-OX lines, the uppermost ovules in the ovary developed as filiform structures (Fig. 4B). A normal size ovule in this transgenic background is also visible, indicated with a white arrow in Fig. 4B. By comparison, a WT ovary containing normal ovules is shown in Fig. 4A. Ovaries with a severe phenotype showed small bumps on their surface instead of a perfectly round and smooth surface as seen in control ovaries (data not shown). Inside these ovaries, the filiform structures filled most of the locular space, leaving a few empty pockets. The transformed ovules took on a twisted spaghetti-like appearance due to the physical constraint of the available growth space in the ovary (Fig. 4B). To identify the nature of these transformed ovules, their morphology was analysed by SEM. WT ovaries showed well distributed ovules in the two locules (Fig. 4C). In contrast, mutant ovaries showed variable numbers of normal sized ovules and ovules with abnormal growth. The severity of the defect was also influenced by environmental factors, such as day length or temperature, with more abnormal ovules observed in warmer periods during summer in the greenhouse (data not shown). The length of these modified ovules varied from small outgrowths to long and thin structure (Fig. 4D–G). Figure 4D shows modified ovules with a mild phenotype, while Fig. 4E shows a more severe phenotype where filiform structures are longer and become twisted (Fig. 4G). These structures developed from the placenta as for normal ovules (Fig. 4F) and had an intrusive growth that disrupted ovule organization in their surroundings. Cells on the filiform structure were elongated without being wider than normal ovule cells (Fig. 4F). The tip of the filiform structure had cell projections (Fig. 4H) similar to stigmatic papillae found on WT pistils (Fig. 4I). As a comparison, a magnification of stigmatic papillae of a WT style is shown in Fig. 4J. The papillae on the filiform structure (Fig. 4K) resemble normal stigmatic papillae (Fig. 4J), except that no mucilage could be observed on their surface. Considering that the CS most probably corresponds to immature styles, this is not unexpected. Cell morphology was also compared between WT stylar and CS cells (Fig. 4L, N, and P, and M, O, and Q, respectively). Cells from CS (Fig. 4M) showed rectangular cells that were more similar to stylar cells (Fig. 4L) than to ovule cells. Moreover, CS shared the same smaller triangular cells found on stylar cells (data not shown), but lacked guard cells and stomata that mature styles show along their length (compare Fig. 4L and M). Another feature of S. chacoense style is the presence of small papillae-like protrusions that develops as the style matures (O'Brien et al., 2002a). Along their length, style (Fig. 4N) and CS (Fig. 4O) shared similar protruding papillae-like cells (Fig. 4N, O; black arrows). When magnified, these papillae-like cells showed similar cuticular ornamentations in both transgenic (Fig. 4Q) and WT cells (Fig. 4P).

To analyse the cell types present in the transformed ovules in more detail, thin sections of fixed ovaries from ScFRK2-OX plants were observed. The WT ovary contained ovules uniform in size and evenly arranged, extending from the placenta (Fig. 4R, only one locule shown). In contrast, ovaries in ScFRK2-OX plants contained irregularly shaped ovules as well as CSs similarly extending from the placenta (Fig. 4S, only one locule shown). The locule organization was disrupted due to an excessive growth of the long filiform CS (Fig. 4S). These CSs showed a clearly different cellular organization from WT ovules. Transverse sections through the CS revealed strands of thin, long, and more compacted cells (arrows in Fig. 4S, U), reminiscent of the transmitting tissue cells found in WT style (arrow in Fig. 4T). CSs contained vessels throughout almost the entire length of the filiform (Fig. 4S, U). This is also similar to the transmitting tract of the WT style. On the other hand, WT ovules contained vessels only in the funiculus (data not shown). An attempt was made to identify CS further using molecular markers for mature style-transmitting tissue (S-RNase, HT gametophytic self-incompatibility modifier) (O'Brien et al., 2002b). However, the signal intensities of the marker transcripts were about the same between WT and mutant ovaries (data not shown). This may be because these CSs are more similar to immature styles and the expression of these genes had not yet initiated. At this stage, CS did not contain any structures resembling an embryo sac. In the section shown in Fig. 4S, most ovules, including those that are normal sizes, appeared abnormal, and only a few would have led to seed formation, which explains the low seed yield in mature fruits (Table 1). At 7 DAP, the filiform CS had already started to wither and turn brown, and none could be observed at fruit maturity (data not shown).

The ontogeny of CS in developing pistils was investigated further. Ovule primordia of ScFRK2-OX plants appeared identical to those of WT plants (data not shown). The first ovule abnormalities were detected when integument growth became apparent. Instead of forming a single layer integument characteristic of Solanum species (Fig. 4W, black arrow), the stalk of ovule primordium continued to grow and expanded excessively, forming enlarged ovules (Fig. 4X, Y, black arrows). At this stage, the enlarged ovule still retained a structure resembling the nucellus containing a megasporocyte at the tip (Fig. 4Y, white arrow). Some megasporocytes were undergoing meiosis, which appeared relatively normal (data not shown), suggesting that the conversion to carpelloids initiates from abnormal growth of the integument and stalk. Since no structures that resembled the megasporocyte or megagametophyte were visible in mature CS (Fig. 4S, U), the megagametophyte may either have degenerated or have been engulfed by the outgrowth of its surrounding tissues.

Expression of the ScFRK2 gene in developing ovules
Since ScFRK2 overexpression interfered with ovule development before fertilization (see above), the ScFRK2 expression pattern was investigated further in developing ovules. RNA gel blot analyses using flower buds 1, 2, 3, 4, or 12 mm in length failed to detect any ScFRK2 mRNA signals (data not shown), possibly because the cells expressing ScFRK2 comprised only a small portion of the flower buds. However, RNA in situ hybridization revealed cell-specific accumulation of ScFRK2 mRNA in early ovule development (Fig. 5). ScFRK2 mRNA signals were detected in the ovule primordia of young pistils (Fig. 5B). Particularly intense signals were detected from the megasporocyte (Fig. 5D). Signals in the megasporocyte weakened as the ovule developed, and stronger signals were instead detected from the ovule integument (Fig. 5C). Intense signals were detected near the growing end of the ovule integument (Fig. 5C, E). Only diffuse signals were detected in the ovule integument after the integument reached full growth, and little or no signal was detected from megagametophytes (Fig. 2CII).


Figure 5
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  		Fig. 5 In situ RNA hybridization analyses in developing ovules. (A–E) ScFRK2 expression in wild-type plants. (A) Sense probe. (B–E) Antisense probe. (A and B) A developing ovary showing ovule primordia arising from the placenta. Sections were taken from a flower bud ~1.5 mm in length. (C) Ovary at a later stage showing a developing ovule with growing integuments. The section was taken from a flower bud ~2 mm in length. As ovules mature, the signal in the megasporocyte became weaker while the signal in the integument became stronger, particularly at the growing tip. (D) Enlargement of B showing an intense ScFRK2 mRNA signal in the megasporocyte. (E) Enlargement of C. (F–I) Comparison of ScFRK2 expression between wild-type and ScFRK2-OX plants. In situ RNA hybridization was performed simultaneously using identical conditions to allow direct comparison. Sections were taken from ovaries at a similar stage to those shown in C. (F) Sense probe. (G–I) Antisense probe. (F and G) Wild-type plant. (H) ScFRK2-OX plant. Arrows show developing CSs. Although signals are almost absent from the stalk, intense signals remain strong at the tip. (I) Enlargement of H, showing the megasporocyte at the tip of the developing CS. The signal in the megasporocyte remains strong. (J–N) ScFBP11 expression in developing ovules. Tissue sections were taken from ovaries at about the same stage as those in C–I. In situ RNA hybridization was performed simultaneously using identical conditions to allow direct comparison. (J) Sense probe. (K–N) Antisense probe. (J and K) Wild-type plant. (K) Diffuse signals were detected from the ovule integument while weak signals were detected from the megasporocyte. (L) Ovary from a ScFRK2-OX plant. Strong signals were detected in ovules that appear normal and also at the tip of the developing CS. Very weak signals were detected in the stalk of the CS. (M) Enlargement of K, showing the absence of signal in the megasporocyte. (N) Enlargement of L, showing intense signals in the megasporocyte of the CS. cs, carpelloid; in, integument; mmc, megasporocyte; op, ovule primodium; ov, ovule; ow, ovary wall; p, placenta. Scale bars, 100 µm (A–C, F–H, J–L); 20 µm (D, E, I, M, and N).

 
As expected, ScFRK2 mRNA signals were stronger in ScFRK2-OX plants than in WT plants (compare Fig. 5G and H). Surprisingly, transgenic plants retained a spatial expression pattern of ScFRK2 that somewhat resembled the WT, i.e. strong expression in gametophytes. Ovule-specific signals in the transgenic plant are shown in Fig. 5H. However, there were distinct differences in the cell specificity of ScFRK2 distribution. Developing ovules of transgenic plants retained high levels of ScFRK2 mRNA in megasporocytes (Fig. 5H, I), whereas little or no signal was detected in the megasporocytes of WT ovules (Fig. 5C, E, G). Much weaker signals were detected in the stalk of developing CS, while intense signals were always associated with the megasporocyte and its surrounding tissues at the tip (Fig. 5H).

The expression of ScFBP11, a Solanum orthologue of FBP7 and FBP11, is altered in ScFRK2-OX plants
In Petunia, down-regulation of the class D MADS-box genes FBP7 and FBP11 changes the fate of the ovule primordium to a carpel primordium and spaghetti-like structures in positions normally occupied by ovules (Angenent et al., 1995). These abnormal structures morphologically resemble style and stigma tissues, like the ones obtained in ScFRK2-OX lines. To determine if ScFRK2 overexpression affected the expression of the class D MADS-box gene, an EST corresponding to a putative FBP7/11 orthologue from the S. chacoense ovary library was isolated and termed ScFBP11. ScFBP11 shares 87% amino acid sequence identity with both FBP7 and 11, and 86% with TAGL11, a tomato FBP7/11 orthologue (Busi et al., 2003). A phylogenetic analysis confirmed the close association of ScFBP11 with Petunia FBP7 and FBP11 as well as with other class D MADS-box genes (data not shown). In situ RNA hybridization was performed using a partial ScFBP11 cDNA sequence to determine the spatial expression pattern of ScFBP11. In WT plants, diffuse ScFBP11 mRNA signals were detected in the integument of the developing ovule, and weak or no signals were detected from megasporocytes (Fig. 5K). ScFBP11 appeared to be upregulated in the developing ovule of ScFRK2-OX transgenic plants. Ovule signals were much stronger in transgenic plants than in WT plants (compare Fig. 5L and K). More importantly, transgenic ovules retained ScFBP11 signals in megasporocytes (Fig. 5N), in striking contrast to WT ovules (compare Fig. 5M and N). Developing CSs retained intense signals in megasporocytes at the tip, while signals were mostly absent from the stalk (Fig. 5L).

cDNA microarray analyses
To determine whether the overexpression of ScFRK2 showed any alterations in the expression of other genes, the mutant plants were compared with WT S. chacoense plants using DNA microarrays from a 7.7 K array made from ovule-derived ESTs (Germain et al., 2005). ANOVA (analysis of variance) testing was used, along with a Benjamini and Hochberg multiple testing correction algorithm, to select 389 ESTs that showed a statistically significant difference in transcript abundance between the WT and the ScFRK2-overexpressing ovules. A Welch t-test (P <0.05) was initially used to compare the profiles from the ScFRK2 versus control and the control versus control comparisons. They were then further restricted with a 1.5-fold variation (1.5 cut-off up or down). A relatively even split was observed between up-regulated (160) and downregulated (213) transcripts, and this result is presented in Supplementary Table S1 at JXB online. The observed changes in transcript abundance are relatively modest compared with what has been observed in other experiments (time-course analysis following fertilization in WT plants; data not shown). Since the phenotype in ScFRK2-OX lines is only observable in a fraction of the samples (see Fig. 4, showing ovules at different stages of transformation into CS structures in the same ovary), it is possible that the changes in transcript abundance are somewhat ‘diluted’ by contributions from unaffected tissue. Furthermore, for the microarray experiment, whole ovaries were used instead of isolated ovules, increasing the dilution effect. Nevertheless, statistically significant results were obtained for 373 ESTs. To determine whether the differentially expressed genes were involved in similar biological processes, these were categorized according to their gene ontology (GO) classification. Apart from a large class of hypothetical/unknown proteins (23.7%), the largest functional groups observed were implicated in metabolism (17.5%), proteins with binding function, including RNA, DNA, protein, and ion binding (9.8%), development and organogenesis (8.5%), protein synthesis (7%), defence response-related proteins (6.9%), protein fate (8.5%), signal transduction (3.3%), transcription (5.1%), and transport (4.3%). Interestingly, of the selected genes, some have previously been shown to be involved in flower or fruit development. In the ScFRK2-OX downregulated category, among the genes involved in transcriptional regulation, homologues of LEUNIG and EIL2 transcription factors have been found. LEUNIG has been shown to regulate AGAMOUS negatively during flower development (Conner and Liu, 2000), while EIL2 (ETHYLENE INSENSITIVE3-like) is involved in ethylene perception, flower abscission, and fruit ripening (Tieman et al., 2001). Among the genes involved in signal transduction in the down-regulated category, three RLKs from S. chacoense, ORK3, 12, and 23, and an ethylene receptor homologue have been found. ORK3 and 12 transcript levels had been shown to increase after fertilization in the ovary, while transcript levels for ORK23 were induced at a distance in the ovary by pollination alone (Germain et al., 2005). In the ScFRK2-OX up-regulated category, two RLKs and one serine/threonine kinase with possible involvement in developmental processes were retrieved. The ORK6 RLK from S. chacoense was previously shown to be strongly induced (17-fold) following fertilization in ovaries (Germain et al., 2005), while the second EST had significant sequence identity with putative orthologues of the phytosulphokine (PSK) receptor. PSKs are small peptide ligands that induce plant cells to dedifferentiate and re-enter the cell cycle at nanomolar concentrations (Matsubayashi et al., 2002). Two PSK genes in maize, ZmPSK1 and ZmPSK3, were also detected in egg and central cells of the female gametophyte, while ZmPSK1 mRNA was present in synergids, indicating that the PSK peptide probably plays a role during gametogenesis and fertilization (Lorbiecke et al., 2005). The other protein kinase was most similar to the APK1a kinase, a kinase very similar to the APK2a kinase, whose gene was originally isolated as having cis-regulatory elements bound by AGAMOUS through an in vivo binding assay (Ito et al., 1997).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
ScFRK2 belong to the MEKK family of MAPKKKs
Using the basic local alignment search tool on publicly available databases, both ScFRK1 and ScFRK2 were found to be most similar to the catalytic domain of numerous MAPKKKs, as well as to three uncharacterized protein kinases from A. thaliana (MAPKKK19, 20, and 21). When compared with most MAPKKK family members, these protein kinases (ScFRK1 and -2, and MAPKKK19, 20, and 21) lacked a typical regulatory domain. A phylogenetic analysis confirmed that these five MAPKKKs belonged to the same subgroup inside the MEKK family of MAPKKKs. Although kinase activity could be obtained from the ScFRK1 and ScFRK2 kinases after in vitro translation (data not shown), complementation in a yeast mutant background deficient for the Ste11 MAPKKK could not be achieved with either kinase (data not shown). This suggests that they may not be involved in a typical MAPK cascade, and/or that the lack of a large N-terminal regulatory domain, like in Ste11, might hamper proper function in yeast and make complementation ineffective.

Developmental defects caused by overexpression of ScFRK2
Overexpression of the ScFRK2 gene leads to developmental defects in ovules, and delayed seed and fruit development. Although ectopic expression can sometime lead to pleiotropic effects on plant development, phenotypic abnormalities were found only in developing ovule and pollen (O'Brien et al., 2007) where ScFRK2 was most strongly expressed during normal development. Ectopic expression of ScFRK2 in vegetative tissues did not cause any visible changes in vegetative development. This suggests that the ScFRK2 kinase acts only on the cells that contain its cognate signalling pathway partners and targets. Specific effects of ScFRK2 overexpression may be explained partly by the ovule-specific accumulation of ScFRK2 mRNAs in transgenic plants (Fig. 5H). Although one would expect the activity of the CaMV 35S promoter to be more ubiquitous, some tissue preferences have been reported previously (Wilkinson et al., 1997). Alternatively, the coding region of the ScFRK2 gene might itself contain regulatory elements curbing the expression pattern obtained in ovules under the CaMV 35S promoter. In addition, steady-state ScFRK2 mRNA levels may be controlled post-transcriptionally by affecting mRNA processing and stability. Also, there may be a positive feedback mechanism that promotes up-regulation of endogenous ScFRK2 transcription.

Nevertheless, there were distinctive differences in the ScFRK2 mRNA accumulation pattern in developing ovules between WT and transgenic plants. A schematic representation of ScFRK2 mRNA distribution is shown in Fig. 6. In WT plants, ScFRK2 mRNA signals in megasporocytes weakened after integument initiation while maintaining mRNA levels in integuments (Fig. 5C, E, and G). In contrast, megasporocytes of transgenic ovules, including those that appeared of normal size, maintained high levels of ScFRK2 mRNAs (Fig. 5H, I). The abnormal elevation of ScFRK2 in megasporocytes may hinder ovule maturation processes, possibly by preventing megagametogenesis and integument differentiation. This may be the reason why most ovules, including normal sized ones, failed to mature in ScFRK2-OX plants (Fig. 4S). There were few ScFRK2 mRNA signals in the stalk of developing CSs (Fig. 5H, I). It is unlikely that the absence of ScFRK2 is the cause of loss of ovule identity because ScFRK2 down-regulation either by antisense or by co-suppression does not cause ovule abnormality (see above). Instead, the down-regulation may have been caused by the loss of ovule identity itself. The abnormal elevation of ScFRK2 in megasporocytes may have triggered the loss of identity in the integument, which in turn down-regulates ScFRK2 expression in the CS. This scenario is also consistent with the persistence of ScFRK2 mRNA in the nucellus-like structures at the tip of the CS. Once the programme for CS formation is initiated, the CS may have taken over the entire ovule to eliminate any structures reminiscent of megasporocytes or megagametophytes.


Figure 6
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  		Fig. 6 Schematic representation of ScFRK2 mRNA distribution during wild-type and ScFRK2-OX ovule development. ScFRK2 in situ RNA hybridization signal distribution is represented by the grey scale, where black is strongest, and decreasing grey intensity corresponds to lower expression levels. Wild-type. ScFRK2 mRNA accumulates in the megasporocyte during ovule primordium formation. As ovules mature, ScFRK2 mRNA levels decrease in megasporocyte and increase near the growing tip of the ovule's integument. When ovules reach maturity, ScFRK2 mRNA is only detected in the integument. ScFRK2-OX. In general, ScFRK2 mRNA accumulates more than the wild type but the accumulation pattern during ovule primordium formation is identical to that of the wild type (i.e. accumulation mostly found in megasporocyte). ScFRK2 mRNA remains abundant in megasporocyte during ovule/CS formation. Also, ScFRK2 mRNA accumulates at the tip of overgrowing integument, whereas no or little ScFRK2 mRNA is detected in the overgrowing funiculus (stalk of the CS).

 
The ScFRK2 expression in pistils prior to fertilization, taken together with a strong increase in expression observed in ovaries immediately after fertilization, suggests that this protein kinase has both pre- and post-fertilization roles. Unfortunately, the role of ScFRK2 in post-fertilization ovule development could not be assessed thoroughly in this study since overexpression leads to the homeotic conversion of ovules into CSs that did not give rise to seeds. The few seeds that were obtained exhibited delayed development (Table 1). Although this is consistent with the ScFRK2 function in post-fertilization ovule development, it is difficult to distinguish a direct effect on embryo development from an indirect effect due to pre-fertilization ovule deficiency. To study the role of ScFRK2 in post-fertilization ovule development, it will be necessary to direct ScFRK2 expression only after fertilization using either a tissue-specific promoter or an inducible promoter.

Down-regulation of ScFRK2 via antisense inhibition or co-suppression should have been more informative; however, no obvious defects were observed in these transgenic plants (data not shown). One explanation for this is functional redundancy shared with other related genes. The presence of such gene(s) was suggested by the DNA gel blot analysis (Fig. 2A). It will be interesting to see the effects of dominant-negative mutations, which may overcome the functional redundancy problem. In this regard, it should be noted that the effect of ScFRK2 overexpression may not always result in the enhancement of the normal ScFRK2 function. Proteins involved in MAPK cascades often function in protein complexes where MAPKKKs, MAPKKs, and MAPKs are anchored together and phosphorylate its targets within the complex (Murphy and Blenis, 2006). Overexpression of one component may not always enhance the function of its complex as a whole. In an extreme situation, the presence of excess proteins that do not participate in its cognate complex may actually hinder the complex function by competing for the same upstream targets. It is not known whether ScFRK2 overexpression enhances or inhibits its downstream functions. Neither is it known whether ScFRK2 overexpression results in accumulation of active ScFRK2 kinase. It will be necessary to identify downstream target proteins of ScFRK2. ScFRK2 function can then be monitored through the phosphorylation status of the target proteins.

Cells in multicellular organisms are equipped with an intricate network of signalling pathways that may interact to maintain a delicate balance of cell fate determination. This is why, unlike mutations in transcription factors, mutations in signalling pathways are difficult to interpret. Interplay of different MAPK cascades is illustrated by interactions between SIPK and WIPK, two closely related MAPKs in tobacco. Both SIPK and WIPK are phosphorylated after exposure to ozone, which triggers production of reactive oxygen species scavengers (Samuel and Ellis, 2002). Stable overexpression of SIPK does not result in accumulation of phosphor-SIPK. SIPK is phosphorylated only after the exposure to ozone and, even then, the amount of phosphor-SIPK is no more than in the WT. An unexpected side-effect of SIPK overexpression was instead found in the phosphorylation status of WIPK. SIPK overexpression inhibits accumulation of phosphor-WIPK and, as a result, cells become hypersensitive to ozone stress. Inhibition of SIPK via RNA interference (RNAi) also results in hypersensitivity to ozone. In this case, phosphor-WIPK accumulation is stimulated while phosphor-SIPK accumulation is inhibited (Samuel and Ellis, 2002). Does the FRK2 pathway interact with other pathway(s) in a similar manner? Future biochemical analyses should reveal the web of FRK2 signalling pathways on ovule fate determination.

Gene mutations that result in loss of ovule identity
Overexpression of ScFRK2 resulted in homeotic conversion of ovules into CSs (Fig. 4). Several genes coding for transcription factors have been identified as regulators of ovule development (Schneitz et al., 1997; Gasser et al., 1998), and misexpression of some of these genes leads to reiteration of carpel development instead of ovules to form CSs and thus cause the loss of ovule identity. In many cases, the loss of ovule identity can be linked to either the misexpression of a class C MADS-box gene or downregulation of class C and/or D MADS-box genes. In Arabidopsis, a mutation in APETALA2 (AP2), a negative regulator of the class C MADS-box gene AGAMOUS (AG), causes the conversion of ovule to CSs within the gynoecium although at a low frequency (Modrusan et al., 1994). BELL1 (BEL1) is also postulated as a negative regulator of AG (Ray et al., 1994). The bel1 mutation likewise causes the loss of ovule identity, albeit with different timing to the ap2 mutant (Modrusan et al., 1994). In the bel1 mutant, the ovule integument continues to grow and differentiate into structures with carpel-like morphology (Modrusan et al., 1994). In the ap2 mutant, homeotic conversion occurs at an earlier stage than in bel1 mutants, as the CS forms directly from an ovule primordium projection (Modrusan et al., 1994). AG is misexpressed in both ap2 and bel1 mutants, and this may have caused the loss of ovule identity (Drews et al., 1991; Ray et al., 1994). Consistent with this idea, ectopic expression of AG orthologues results in homeotic conversion of ovule to CS in Arabidopsis (Ray et al., 1994) and tobacco (Mandel et al., 1992). Interestingly, the frequency of transformation in the bel1 mutant decreases with decreasing temperatures, as well as with reducing day length (Modrusan et al., 1994). Ovule to CS conversion in the ScFRK2-OX lines was also influenced by temperature and day length (data not shown).

Involvement of the class D MADS-box genes in ovule identity determination was first identified in Petunia, a Solanaceae species like S. chacoense. Down-regulation of the class D MADS-box genes FBP7 and FBP11 via co-suppression changes the fate of the ovule primordia to the carpel primordia. Hence, CSs develop on the placenta (Angenent et al., 1995). FBP7 and FBP11 work redundantly because neither an FBP7 nor an FBP11 single knockout mutant causes a similar phenotype (Vandenbussche et al., 2003). Normally, the expression of FBP7 and 11 mRNAs is found in carpels. However, ectopic expression of the FBP11 gene causes ectopic ovule formation in the sepal, suggesting that FBP11 is sufficient for ovule induction (Colombo et al., 1995). The Arabidopsis genome contains only one gene, SEEDSTICK (STK), that belongs to the class D MADS-box gene clade (Kramer et al., 2004). STK alone does not determine ovule identity but, instead, acts redundantly with three closely related class C MADS-box genes, AG, SHATTERPROOF1 (SHP1) and 2 (SHP2) (Pinyopich et al., 2003). In the shp1 shp2 stk triple mutant, ovules are converted into carpel- or leaf-like structures with style-like characteristics (Pinyopich et al., 2003). Similar to FBP11 in Petunia, ectopic expression of STK, SHP1, or SHP2 in Arabidopsis causes conversion of sepals to carpelloid organs with ectopic ovules (Favaro et al., 2003). Interplay of class C and D MADS-box genes has further been demonstrated by protein interaction assays. Yeast two-hybrid and three-hybrid analyses have shown that when the class E MADS-box transcription factor SEPALLATA 3 (SEP3) is present, AG forms a complex with either STK, SHP1, or SHP2, and this complex (AG–SEP3–STK, AG–SEP3–SHP1, or AG–SEP3–SHP2) may be sufficient for promoting ovule identity (Favaro et al., 2003). Similarly, FBP11 forms a higher order complex with FBP2 (class E) and FBP6 (class C) transcription factors (Tonaco et al., 2006).

Possible interactions between ScFRK2 and FBP7/11 and/or AG pathways
The ScFRK2 expression pattern in S. chacoense ovules was very similar to those of FBP7 and FBP11 in Petunia and ScFBP11 in S. chacoense. Before fertilization, ScFRK2 mRNAs are detected in ovule primordia in the young ovary (Fig. 5B) and ovule integuments of the mature ovary (Fig. 5C). Similarly, in Petunia, FBP11 mRNA is detected in ovule primordia of the young ovary and the integuments of mature ovules (Angenent et al., 1995). A similar distribution of ScFBP11 mRNA was observed in S. chacoense ovules (Fig. 5K). ScFRK2 mRNA levels in ovules increased greatly after fertilization (2 DAP) and decreased back to pre-fertilization levels by 4 DAP (Fig. 2B, C). Petunia FBP7 and FBP11 mRNA levels in ovules also increase after fertilization (Colombo et al., 1997). However, unlike ScFRK2, the levels of FBP7 and FBP11 remain high for several days and decrease after 7 DAP (Colombo et al., 1997).

ScFRK2-overexpressing plants exhibit strikingly similar ovule defects to FBP7/11 co-suppressed plants. In the ovary, ovules were transformed into filiform CSs, with the uppermost ovules being more frequently transformed. An identical situation was also found in the Petunia FBP7/11 co-suppression lines (Angenent et al., 1995). The filiform structures exhibit characteristics similar to the style, such as structures resembling transmitting tracts and cell projections at the extremity resembling stigmatic papillae (Fig. 4K). These were also characteristics found in CSs of FBP7/11 co-suppressed plants (Angenent et al., 1995). The only difference is that the CSs found in FBP7/11 co-suppressed plants were devoid of any structures resembling the nucellus (Angenent et al., 1995), whereas nucellus-like structures were found in the ScFRK2-overepressing ovules at the beginning of abnormal growth (Fig. 4Y). This suggests that ScFRK2 may be acting on ovule development at a later stage than FBP7/11.

FBP11 accumulation following fertilization has been associated with its role in seed development. Endosperm development is altered in FBP7/11 co-suppression lines (Colombo et al., 1997). Between the time of pollination, and up to 9 DAP, seed development is unaffected. However, later on, the endothelium of the ovule starts to degenerate. By 18 DAP, the endothelium completely degenerates, which leads to a disturbance of endosperm development and delayed embryo development (Colombo et al., 1997). ScFRK2-overexpressing lines also exhibited a delay in embryo development. WT fruits 21 DAP contained a large proportion of mature embryos, the remaining embryos being at the walking-stick stage. In ScFRK2-OX lines, embryo development was delayed, and at 21 DAP, most embryos are still at the late torpedo stage (Table 1). Although only a few ovules develop to mature seeds (around six per fruit compared with >100 seeds in WT plants), this delay has been consistently and repeatedly observed. These embryos would normally correspond to the ones observed 16–17 DAP in a WT plant. A small fruit phenotype was also observed (Fig. 3B, C). Mutations in STK, an Arabidopsis orthologue of FBP7/11, also cause defects in fruit (silique) development (Pinyopich et al., 2003).

Phenotype similarities between ScFRK2-OX plants and FBP7/11 co-suppressed plants suggested that ScFRK2 may be interfering with the function of class D MADS-box genes. Contrary to expectations, ScFRK2 overexpression resulted in up-regulation of ScFBP11 (Fig. 5L). If ScFBP11 is up-regulated in ScFRK2-OX plants, how do they phenocopy FBP7/11 down-regulated plants and AG up-regulated plants? There are at least two possible explanations for this apparent discrepancy. First, upregulation of ScFBP11 may cause ectopic expression of an AG orthologue, which subsequently may cause ovule to CS conversion in transgenic plants. In Arabidopsis, ectopic expression of the class D gene STK causes ectopic accumulation of AG transcripts (Favaro et al., 2003). Unfortunately, no information is available regarding the expression levels of AG orthologues, because AG orthologues have not been cloned from S. chacoense. Secondly, ectopically expressed ScFBP11 may take on AG function due to functional redundancy between class C and D MADS-box genes. Functional redundancy between class C and D genes has been reported in Arabidopsis in which loss of ovule identity occurs only when both C and D gene functions are lost (Pinyopich et al., 2003). Furthermore, 35S::STK partially restores AG function in ag1 mutants (Favaro et al., 2003). In either case, the fact that ScFBP11 expression was altered by ScFRK2 overexpression suggests that there are at least some interactions, direct or indirect, between the ScFRK2 and ScFBP11 pathways.

To identify further target genes of the ScFRK2 pathway, global changes in gene expression levels were examined in transgenic plants using a microarray analysis. Genes differentially regulated between transgenic and wild-type ovaries were identified (see supplementary Table 1 at JXB online). Particularly interesting is the down-regulation of LEUNIG (LUG) in ScFRK2-OX ovaries. The lug mutation was initially identified as an enhancer of ap2 mutant defects (Liu and Meyerowitz, 1995). Like ap2 and bel1 mutation, lug mutation causes ectopic expression of AG, suggesting that LUG is a negative regulator of AG (Liu and Meyerowitz, 1995). The lug mutation alone causes abnormal gynoecium development where two carpel valves remain open (Liu et al., 2000). LUG mRNA accumulation in ovules suggests that LUG is also involved in ovule development (Conner and Liu, 2000). LUG works redundantly with AINTEGUMENTA (ANT), another negative regulator of AG, to control ovule development. Double mutants of lug and ant cause complete abolishment of ovules as well as other marginal tissues of the gynoecium (Liu et al., 2000). Down-regulation of a LUG orthologue predicts up-regulation of an AG orthologue in the transgenic plants and is consistent with the ovule phenotype. However, further identification and expression analyses of AG orthologues will be necessary to confirm this point. Finally, it will be interesting to know if any of the differentially expressed genes are regulated by class C or D MADS-box genes.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available at JXB online.


   Acknowledgements
 
We thank Dr Malcolm Whiteway (Biotechnology Research Institute, NRC, Montréal, Canada) for providing the ste11 mutant yeast strain and Gabriel Téodorescu for plant care and maintenance. We also thank Simon Joly for his help and advice regarding the phylogenetic analysis. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Canada) and by le Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT, Québec). MO is the recipient of PhD fellowships from NSERC and from FQRNT. DPM holds a Canada Research Chair in Functional Genomics and Plant Signal Transduction.


   Footnotes
 
* These authors contributed equally to this work. Back

GenBank accession numbers: ScFRK1, AY427828; ScFRK2, AY427829; ScFBP11, DN980993.


   Abbreviations
 
CaMV, cauliflower mosaic virus; CS, carpelloid structure; DAP, days after pollination; EST, expressed sequence tag; FRK, fertilization-related kinase; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; RLK, receptor-like kinase; SEM, scanning electron microscopy; WT, wild type.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
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