A single gene from Streptosolen grouped sister to the FUL1 and FUL2 clades, while a gene from Schizanthus, one of the earliest diverging genera , grouped as sister to the euFULII clade. To confirm the above were not artifacts, we re-assembled the Streptosolen transcriptome while searching for reads supporting the gene contig, and amplified the Schizanthus sequence using gene-specific primers. The presence of both FUL1 and FUL2 genes in species from across the phylogeny is consistent with the event that produced these two clades being part of a family-wide, whole genome duplication or triplication . However, we did not find a FUL2 ortholog in Schizanthus, using transcriptome data, or Goetzia, using PCR. These two genera are among the earliest diverging in the family , and are the earliest that we sampled. This raises the possibility that the FUL1/FUL2 clades resulted from a duplication that occurred following the diversification of Schizanthus and Goetzia. In addition, although we obtained MBP10 sequences from Nicotiana and most of the genera that diversified subsequently , we did not find members of the MBP10 clade in genera that diverged prior to Brunfelsia. This suggests that the MBP10 and MBP20 subclades were produced by a duplication that occurred later in Solanaceae diversification, after the euFULI duplication and any proposed family-wide whole-genome events.To investigate the nature of the MBP10/MBP20 duplication, we mapped the location of the four euFUL paralogs to the genome of cultivated tomato. FUL1 and FUL2 are located on chromosomes 6 and 3, respectively, hydroponic net pots consistent with their origin from a whole genome multiplication. By contrast, MBP10 and MBP20 are both located on chromosome 2, about 14.3 million base pairs apart .

The location of both euFULII genes on the same chromosome, and the presence of only one ortholog in early diverging species, support the hypothesis that these paralogs may be the result of a tandem gene duplication. Moreover, comparing a 1-million-base-pair region surrounding both MBP10 and MBP20 shows synteny, further supporting a tandem duplication . Annotations indicate that these syntenic zones contain 17 homologous regions. The regions that show homology are located on the opposite sides of MBP10 and MBP20, suggesting an inversion of the tandemly duplicated region. Although we recovered an MBP10-clade member in Brunfelsia australis using transcriptome analysis, we were unable to amplify this gene from leaf or floral tissue of Fabiana or Plowmania, genera that are most closely related to Brunfelsia . In addition, Petunia is also a member of the clade that includes Brunfelsia, and searches of the published Petunia genomes also failed to turn up an MBP10-clade member. However, the Brunfelsia sequence in our analysis, obtained from transcriptome data, falls in the expected place in the phylogeny, and we confirmed the presence of MBP10 transcript in Brunfelsia floribunda floral RNA. This suggests that the MBP10/MBP20 duplication occurred before the divergence of the Brunfelsia/Fabiana/Petunia/Plowmania clade but the MBP10 paralog was lost in Fabiana, Petunia and Plowmania.A long first intron ranging from 1 to 10 kb, with multiple potential TF binding sites, is a general feature of FUL homologs . By contrast, MBP10 has a short first intron of about 80 bp in both cultivated tomato and its closest wild relative, S. pimpinellifolium, and about 110 bp in Nicotiana obtusifolia . The expression of most euFUL genes is strong across nearly all vegetative and reproductive organs ; however, diverse analyses using both quantitative and non-quantitative methods indicate that MBP10 expression is relatively weak in tomato, S. pimpinellifolium, and N. obtusifolia in most organs , however, some studies have suggested moderate expression in leaves . To determine if the short first intron lacks putative TF binding sites, we searched the first intron of MBP10 and MBP20 in tomato .

We found that the first intron of MBP10 contains no putative TF binding sites, while that of MBP20 contains 88 putative TF binding sites for eight different TFs. These TFs belong to five main families : MYB , HSF , Dof , WRKY and MADS-box . A similar situation was observed for Nicotiana obtusifolia, which had 133 putative binding sites in thefirst intron of MBP20 for a similar array of TFs, while MBP10 had only four such sites. In addition, we searched the first intron of AGL79, the euFULII paralog of FUL in A. thaliana, and found 49 putative binding sites, also for similar TFs and TF families. This suggests a loss of regulatory motifs in MBP10.This region corresponds to the K domain . In comparison, the M and the I domains had relatively few sites undergoing diversifying selection. Since these TFs function in complexes with other MADS-domain proteins as well as other proteins, novel interactions made possible by amino acid changes in this region might lead to changes in transcriptional activity. The K domain had 14 sites undergoing diversifying selection in the FUL1 proteins and four of those showed a change in polarity . Of those four, a site that corresponds to the 153rd residue in the tomato protein had negatively charged glutamate in most of the nonSolanoideae species while all Solanoideae species had a nonpolar residue: valine or methionine . All other changes in FUL1 proteins that result in a change in charge appeared to be reversible, and none were correlated with the phylogeny nor with phenotypic changes. We used the PROVEAN tool on all four K-domain sites that showed a change in charge to predict whether these transitions were likely to be deleterious or neutral . Two of these sites, one with a histidine to glutamine/asparagine shift at the 95th residue, and one with a lysine to glutamine/threonine shift at the 157th residue , were predicted to be functionally deleterious while the other two sites, including the 153rd residue with E to V change, blueberry grow pot were predicted to be neutral. There were five rapidly changing sites in the M domain and six sites undergoing positive selection in the I domain of FUL1. None of the sites in the M domain showed a change in polarity.

Only one site in the I domain showed a change in polarity, but this site was predicted to be neutral functionally. MBP10 proteins had 20 sites undergoing diversifying selection in the K domain, only 1 such site in the M domain and 3 in the I domain .Of these, only three sites in the I domain showed a change in charge, all of which were also predicted not to have a negative effect on function.We compared euFUL expression data for the cultivated and wild tomato species, potato and Nicotiana benthamiana to identify any patterns that might be the result of changes in the regulatory regions following the duplications of these genes. Not all data from online sources were comparable across species, as different studies included different organs and developmental stages in their analyses, limiting cross-species comparisons. The analysis shows similar spatial expression patterns for FUL1 and FUL2 . These two paralogs are broadly expressed in leaves, flowers and fruits of tomato, potato, and tobacco. Although the eFP browser data shows no expression for FUL1 and FUL2 in tomato leaves, our RT-PCR data and previous publications show expression of all four euFUL homologs in these organs. Both euFULI genes are expressed relatively weakly in the roots of tomato, potato, and tobacco . Although spatial domains of expression are similar for the euFULI genes, they differ in temporal expression over the course of fruit developmental stages in tomato. Although both FUL1 and FUL2 are expressed in the fruits of all species, in tomato FUL2 is highly expressed during the early stages of fruit development and then tapers off, whereas FUL1 expression increases with time . In comparison to the euFULI genes, the two euFULII paralogs show more striking differences in spatial expression at the organ level , and also between species. In all species for which expression is reported, MBP10, alone among the euFUL genes in Solanaceae, is not expressed in fruits, or is expressed at barely detectable levels. In tomato, MBP20 is expressed strongly in roots while MBP10 is not. By contrast, in potato tubers, MBP10 expression is high and MBP20 is not expressed . The online sources and our RT-PCR data also show subtle intra-specific differences in expression between MBP10 and MBP20 in flowers . In addition, our RT-PCR data show that MBP10 is expressed relatively weakly in petals and stamens in tomato while MBP20 is expressed throughout the flower . However, these differences seem to be a matter of expression intensity in comparison to the more striking contrasts seen in roots, tubers, and fruits. The types of differences in expression between FUL1 and FUL2 versus MBP10 and MBP20 might be due to differences in the regulatory environment as a result of the different waysin which these duplicates arose. A tandem duplication and inversion may have disrupted regulatory regions in ways that would not be associated with a whole genome duplication or triplication . To investigate this, we searched for putative TF binding sites in the promoter regions of euFUL genes in tomato, potato, and woodland tobacco to compare the differences between the pairs of paralogs . Woodland tobacco was used rather than N. benthamiana since relatively longer promoter sequence lengths for euFUL genes were available for this genome assembly . Despite this, the maximum available promoter length for NsMBP10 was about 3.3 kb. We found that the differences in types and numbers of predicted TF binding sites between FUL1 and FUL2 were comparable to the differences between MBP10 and MBP20 . Nonetheless we did find some differences that may underlie observed differences in expression between paralogs. Some of these differences were presence/absence of binding sites for a particular TF, and some were in the number and distribution of sites. Putative binding sites for AUXIN RESPONSE FACTORS were absent from the tomato FUL2 promoter while they were present in the promoters of all other euFUL genes in all species examined. Only FUL2 in tomato, FUL1 in potato, and MBP10 in woodland tobacco contained binding sites for STOREKEEPER .

ETHYLENE INSENSITIVE 3 has three sites in tomato FUL1 and five in tomato FUL2, but the distribution of the sites differs. In FUL1, there are no sites within 2 kb of the coding sequence, and three within 5 kb, whereas in FUL2 there is one site in the 2 kb region and four in the full 5 kb region. In woodland tobacco, there are three EIN3 sites in FUL1, all of which are within the 2 kb region, and only one in FUL2, which is located between 2 and 5 kb. These types of differences may underlie observed differences in expression.In Solanaceae, there has been a major shift to fleshy fruit in the Solanoideae . However, we do not know the molecular basis of this economically and ecologically important evolutionary event. FUL negatively regulates lignification in the dehiscence zone in the dry silique of A. thaliana, and functions in cauline leaf development, the transition to flowering and determinacy . Studies of FUL ortholog function across the angiosperms have shown that it is labile, and orthologs have acquired diverse roles over evolutionary time. VEGETATIVE 1 , an ortholog of FUL in pea , is involved in secondary inflorescence meristem identity . AGAMOUS-like 79 , the A. thaliana euFULII paralog of FUL, is mainly expressed in the root and has functions in lateral root development and may also play a role function in leaf shape, leaf number, branching, and time to flowering . However, the over expression of an AGL79 ortholog from snapdragon in A. thaliana resulted in indehiscent siliques, suggesting a role more similar to A. thaliana FUL . Evidence suggests that in tomato, one of the AGL79 orthologs, MBP20, plays a role in leaf development . VERNALIZATION 1 genes, which are FUL-like orthologs in grass species such as wheat and barley , function in the vernalization response . Evidence to date, therefore, suggests that euFUL function is labile, and has changed substantially in different plant lineages during the course of angiosperm evolution. Thus it is not surprising to find a change in function of euFUL orthologs in Solanaceae. There is evidence to suggest that Solanaceae euFUL orthologs play a role similar to that of A. thaliana FUL in the development of dry dehiscent fruits . However, studies suggest that in the fleshy fruit of Solanoideae, FUL orthologs play roles in pigmentation as well as ethylene response, cell wall modification, glutamic acid degradation, volatile production, and pericarp and cuticle thickness .