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 (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). These two paralogs are broadly expressed in leaves, flowers and fruits of tomato, potato, and tobacco. Although the eFP browser data (**Figure 4**) shows no expression for FUL1 and FUL2 in tomato leaves, our RT-PCR data (**Figure S1** in **Supplementary Data Sheet S1**) and previous publications (Hileman et al., 2006; Burko et al., 2013) 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 (Massa et al., 2011; Potato Genome Sequencing Consortium et al., 2011; The Tomato Genome Consortium, 2012; Nakasugi et al., 2014) (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). 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 (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**).

In comparison to the euFULI genes, the two euFULII paralogs show more striking differences in spatial expression at the organ level (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**), 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 (**Figure 4**). The online sources and our RT-PCR data also show subtle intra-specific differences in expression between MBP10 and MBP20 in flowers (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). 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 (**Figure S1** in **Supplementary Data Sheet S1**). 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 ways

in 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 (Tanimoto et al., 1999; Kmita et al., 2000; Vogel et al., 2009; Lupiáñez et al., 2015; Puig et al., 2015). To investigate this, we searched for putative TF binding sites in the promoter regions (2 and 5 kb upstream from the transcription start site) of euFUL genes in tomato, potato, and woodland tobacco to compare the differences between the pairs of paralogs (**Table S4** in **Supplementary Data Sheet S1**). Woodland tobacco was used rather than N. benthamiana since relatively longer promoter sequence lengths for euFUL genes were available for this genome assembly (Sierro et al., 2013). 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 (**Table S4** in **Supplementary Data Sheet S1**). 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 (ARF) 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 (STK). ETHYLENE INSENSITIVE 3 (EIN3) 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.

### DISCUSSION

#### Solanaceae euFUL Gene Tree Shows the History of Duplications in This Lineage

In Solanaceae, there has been a major shift to fleshy fruit in the Solanoideae (Knapp, 2002). 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 (Spence et al., 1996; Gu et al., 1998; Liljegren et al., 2000, 2004; Rajani and Sundaresan, 2001; Melzer et al., 2008). 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 (VEG1), an ortholog of FUL in pea (Pisum sativum), is involved in secondary inflorescence meristem identity (Berbel et al., 2012). AGAMOUS-like 79 (AGL79), 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 (Gao et al., 2018). However, the overexpression of an AGL79 ortholog from snapdragon (Antirrhinum majus) in A. thaliana resulted in indehiscent siliques, suggesting a role more similar to A. thaliana FUL (Müller et al., 2001). Evidence suggests that in tomato, one of the AGL79 orthologs, MBP20, plays a role in leaf development (Burko et al., 2013). VERNALIZATION 1 (VRN1) genes, which are FUL-like orthologs in grass species such as wheat (Triticum spp.) and barley (Hordeum vulgare), function in the vernalization response (Preston and Kellogg, 2008). 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 (Smykal et al., 2007). 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 (Bemer et al., 2012; Shima et al., 2014; Wang et al., 2014). To determine if we could identify changes in euFUL sequences or selection that might shed light on this change in function, we analyzed euFUL gene evolution in Solanaceae.

We performed a maximum likelihood phylogenetic analysis (Garli v2.1) (Bazinet et al., 2014) on a data set that consisted of 106 Solanaceae members of the euFUL gene lineage (Litt and Irish, 2003; Shan et al., 2007), which we obtained through amplification and sequencing (37 sequences), generating transcriptome sequence data (29 sequences), or mining databases (40 sequences). As outgroup we used 10 euFUL genes from Convolvulaceae, the sister family to Solanaceae (**Figure 2** and **Supplementary Table S1**) (Stefanoviæ et al., 2003). The resulting tree shows the two major clades of core-eudicot euFUL genes, the euFULI and euFULII lineages (Litt and Irish, 2003; Shan et al., 2007). Within each of these clades there is evidence of a Solanaceae-specific duplication, resulting in two subclades in each lineage. Within each subclade, the order of branches correlates well with the topology of the Solanaceae phylogeny (Olmstead et al., 2008; Särkinen et al., 2013); discrepancies at the genus level are likely due to the short length of some sequences and sequence divergence in some taxa. Each of the subclades includes orthologs from both fleshy- and dry-fruited species, indicating that the subclade duplications preceded the origin of fleshy fruit.

Although duplications in these genes are common (Litt and Irish, 2003; Preston and Kellogg, 2007; Pabón-Mora et al., 2013), we did not find significant evidence of taxon-specific duplications. We did, however, find two genes that did not fall into a specific subclade. A third Streptosolen gene grouped sister to the rest of the euFULI clade (76% identity among the three Streptosolen genes), potentially the result of a taxonspecific duplication followed by sequence divergence. In addition, a Schizanthus gene grouped sister to the euFULII clade (77%

pairwise identity with Schizanthus MBP20). This may also be a divergent genus-specific paralog, but since Schizanthus is one of the earliest diverging genera (Olmstead et al., 2008; Särkinen et al., 2013), it is also possible this gene might be a remaining paralog from the reported whole genome duplication/triplication that occurred early in Solanaceae diversification (Blanc and Wolfe, 2004; Schlueter et al., 2004; Song et al., 2012; The Tomato Genome Consortium, 2012; Albert and Chang, 2014; Bombarely et al., 2016). Examination of sequences showed that these are not likely to be splice isoforms. We also found potential evidence of loss – not every Solanaceae species we studied had a copy of each euFUL gene. We did not, for example, find FUL2 genes in Iochroma, Fabiana, Solandra, Juanulloa, Schizanthus, or Goetzia, even though these all had genes in the FUL1 clade (see **Table S1** in **Supplementary Data Sheet S1** for a complete list). However, although this may represent paralog loss, it is possible we did not recover all gene copies due to PCR primer mismatches, low expression levels, or the absence of transcript in the sampled tissue.

In addition to the major shift to fleshy fruit in the Solanoideae subfamily, fleshy fruits have independently evolved in Cestrum and Duboisia, and there has also been a reversal to a dry fruit in Datura (Knapp, 2002). Our analysis does not include genes from Duboisia, but the euFUL genes from Cestrum and Datura grouped in positions in the tree that were expected based on their phylogenetic position, and did not show any notable differences in sequence from the euFUL genes of their close relatives.

#### euFULI and euFULII Clade Duplicates Have Experienced Different Levels of Purifying Selection

We compared dN/dS ratios between and among Solanaceae euFULI and euFULII lineages, as well as between sequences before and after the transition to fleshy fruit, to investigate if any changes in selection might be correlated with sequence diversification. All ω values from our analyses are closer to 0 than to 1 (**Table 1** and **Table S3** in **Supplementary Data Sheet S1**), which indicates that all euFUL gene clades are under strong purifying selection (Yang, 1997; Yang and Nielsen, 2000; Torgerson et al., 2002; Almeida and Desalle, 2009). Studies suggest that this is the norm for most protein coding genes, and that under such stringent evolutionary constraints, slight differences in evolutionary rates may result in functional diversification (Yu et al., 2017). Our data show a weakening of purifying selection in FUL1 genes relative to FUL2 genes (ω = 0.17 vs. 0.11, p < 0.0005) and in MBP10 genes relative to MBP20 genes (ω = 0.19 vs. 0.15, p < 0.01). Immediately after the euFULI duplication, the FUL1 and FUL2 lineage genes would have been fully redundant, which might have allowed the reduction in purifying selection on the FUL1 genes resulting in potential functional divergence. Similarly, the duplication that resulted in the two euFULII gene clades would have resulted in redundancy in the MBP10 and MBP20 lineages, possibly allowing the more rapid diversification of MBP10 genes.

Although studies indicate that the euFULI genes of tomato have novel functions compared to those in dry fruit (Gu et al., 1998; Smykal et al., 2007; Bemer et al., 2012; Shima et al., 2014; Wang et al., 2014), it remains unclear whether the new functions are the result of changes in coding sequences, regulatory regions, or downstream gene targets. Our analysis shows that euFUL genes in both dry- and fleshy-fruited species are evolving at similar rates (**Table S3** in **Supplementary Data Sheet S1**). This suggests conservation of the coding sequences in both fleshy- and dryfruited species despite the central roles in the development of these distinct fruit morphologies.

Sixty-four of the sequences in our analysis were from fleshyfruited species whereas only 42 were from dry-fruited species. Although, we had broad representation across the dry grade, it is possible with additional representation from dry fruited species, more evolutionary patterns would be revealed (Anisimova et al., 2001; Domazet-Loso, 2003; Nielsen et al., 2005).

### FUL1 and MBP10 Proteins Show Amino Acid Changes in Conserved Functional Domains

An analysis of selection across an entire sequence may indicate different types of selection for the whole gene, but this overlooks the fact that key residues may be undergoing rapid evolution that may result in functional changes (Ota and Nei, 1994; Nei et al., 1997; Yang and Bielawski, 2000; Piontkivska et al., 2002; Martinez-Castilla and Alvarez-Buylla, 2003; Jeffares et al., 2006). Other empirical studies have further described functional changes due to a change in a single amino acid residue (Ingram, 1957; Hanzawa et al., 2005; Hichri et al., 2011; Zhao et al., 2012; Fourquin et al., 2013; Dai et al., 2016; Sakuma et al., 2017) specifically associated with changes in polarity (Schröfelbauer et al., 2004; Hoekstra et al., 2006) or conformation (Aseev et al., 2012). Studies in A. thaliana, show that a single amino acid mutation in GLABRA1 (GL1) results in the inhibition of trichome formation (Dai et al., 2016) and a change of a single residue is sufficient to convert the function of TERMINAL FLOWER 1 (TFL1), which inhibits flower formation, to that of the closely related FLOWERING LOCUS T (FT), which promotes flowering (Hanzawa et al., 2005). Three-dimensional modeling has also shown that a single amino acid change in a highly conserved domains may lead to changes in protein–protein interactions (Teng et al., 2009; David et al., 2012; Li et al., 2014). We searched for individual sites in the predicted amino acid sequences that showed evidence of positive selection within the gene groups that, although under purifying selection, were found to have statistically significantly accelerated evolutionary rates (i.e., the FUL1 and MBP10 clades) to determine if any amino acid changes at these sites had the potential to result in a change in protein function.

Our findings show that more residues are rapidly changing in the K domain compared to the M and I domains (**Figure S4** in **Supplementary Data Sheet S1**). The K domain is predicted to have an α-helix structure that facilitates protein–protein interactions (**Figure S5** in **Supplementary Data Sheet S1**) (Yang et al., 2003a,b; Kaufmann et al., 2005; Immink et al., 2010). The α-helix structure depends on conserved hydrophobic residues spaced through the domain (Eisenberg et al., 1982). Therefore,

changes to protein residues that alter charge and/or conformation in this region can lead to changes in such interactions. Most of the rapidly evolving sites did not show an amino acid change specifically associated with the shift to fleshy fruit, but rather showed changes and reversals over the course of gene evolution. Interestingly, in the FUL1 proteins, we found one site in the K domain, corresponding to the 153rd residue in the tomato protein (Slugina et al., 2018), at which 11 out of 15 sequences from dry-fruited species have a negatively charged glutamate (E) residue. In comparison, 100% of the fleshy clade contains a nonpolar residue: valine (V) (13 species) or methionine (1 species). However, since the remaining four FUL1 sequences from dryfruited species have non-polar glutamine (Q) or V at this site, the change from charged to non-polar is not associated with the shift to fleshy fruit. In addition, a PROVEAN analysis predicted the changes at this site to be neutral with regards to function.

Two other sites in the FUL1 K domain show changes that are predicted to have functionally deleterious consequences according to our PROVEAN analysis (Choi, 2012; Choi et al., 2012; Choi and Chan, 2015). These include a charged histidine (H) to a non-polar glutamine/asparagine (Q/N) transition at the 95th residue and a charged lysine (K) to non-polar glutamine/threonine (Q/T) transition at the 157th residue (**Figure S5** in **Supplementary Data Sheet S1**). Polar residues are important for protein–protein interactions of the K domain α-helix (Sheinerman et al., 2000; Curran and Engelman, 2003; Ma et al., 2003; Zhou et al., 2018) and changes might disrupt interactions with other proteins (Liu et al., 2014). However, since these changes are not correlated with the fruit type, it seems unlikely that any alteration to protein function affects fruit morphology. It is also plausible that any negative effect at these sites is masked by the FUL2 paralog, which is likely to be functionally redundant (Bemer et al., 2012; Wang et al., 2014). This is consistent with FUL1 evolving relatively faster (**Table 1** and **Table S3** in **Supplementary Data Sheet S1**), thus enabling divergence compared to FUL2, which appears to be more highly functionally conserved based on stricter sequence conservation.

None of the sites undergoing positive change in the K domain of MBP10 showed a change in charge, suggesting these changes are not likely to affect protein function. We also observed residues in the M domain that are under diversifying selection in both the FUL1 and MBP10 clades. These residues are located not in the α-helix region that directly binds to DNA, but in the β-sheet region of the MADS domain (**Figure S4** in **Supplementary Data Sheet S1**) (Immink et al., 2010). β-sheets are important for protein arrangement in three dimensional space. Therefore, any changes in this region might change protein conformation, influencing DNA binding of the α-helix as well as the ability of the euFUL proteins to form higher order complexes (Pellegrini et al., 1995). However, these shifts were reversible, with no phylogenetic pattern or change in charge, and there was no correlation with the fruit type. Therefore it is unlikely that these shifts have significant functional impact.

A previous report that investigated the evolution of MADSbox genes in A. thaliana also found rapidly evolving sites in the M and K domains of Type II MADS-box proteins, which might have been involved in the functional diversification of this group, but did not report changes in the I domain (Martinez-Castilla and Alvarez-Buylla, 2003). Residues in this domain that are directly involved in forming an α-helix structure are expected to be highly conserved, whereas the remaining residues may not be under such constraints (Yang et al., 2003a,b; Kaufmann et al., 2005). We found residues in the conserved region of the I domain that are undergoing diversifying selection in both FUL1 and MBP10 clades. Of these, one site in FUL1 and three sites in MBP10 had undergone changes in charge but none were predicted to negatively affect the function (**Figures S4, S5** in **Supplementary Data Sheet S1**). In addition, as with the sites in the M and K domains, none of these was correlated with the Solanaceae phylogeny or changes in fruit morphology. It has been reported that higher rates of substitution in lineages that show weakened purifying selection or even diversifying selection may be occurring at residues of minimal functional importance (Jacobsen et al., 2016). This might explain the apparent ease of reversibility and lack of phylogenetic signal among the rapidly changing sites we observed.

## The MBP10 and MBP20 Clades Are the Result of a Tandem Duplication Event

The FUL1 and FUL2 genes of tomato are located on different chromosomes (6 and 3, respectively), which is consistent with the proposed Solanaceae whole genome duplication (Blanc and Wolfe, 2004; Schlueter et al., 2004; Song et al., 2012) or triplication (The Tomato Genome Consortium, 2012; Albert and Chang, 2014; Vanneste et al., 2014; Bombarely et al., 2016) followed by loss of one paralog. The lack of a FUL2 ortholog in our dataset from Goetzia or Schizanthus (**Figure 2**), the two earliest diverging genera that we included in our analyses, raises the possibility that the FUL1/FUL2 clades originated via a duplication that occurred after the diversification of these genera, and not as a result of a whole genome event that preceded the diversification of the family. Whole genome sequences from multiple early diverging lineages will be needed to determine the timing and nature of these early events.

We did not recover an MBP10 ortholog from any of the genera that diverged prior to Brunfelsia (**Figure 2** and **Figure S2** in **Supplementary Data Sheet S1**). Our investigation revealed that in tomato, both MBP10 and MBP20 are located on chromosome 2, about 14.3 million base pairs apart. The 1 million base-pair region surrounding each gene shows synteny, but the order of the homologous regions is reversed (**Figure 3**). Together, this suggests that the MBP10/MBP20 clades are the result of a tandem duplication accompanied by an inversion (Purugganan et al., 1995; Vision et al., 2000; Achaz et al., 2001; Prince and Pickett, 2002). Supporting this, a previous report that investigated genomic duplication events in tomato also found evidence for large-scale intra-chromosomal duplications in chromosome 2 (Song et al., 2012). Although the authors suggest this event was concurrent with a whole genome duplication at the origin of the family, they give a large window, 36–82 million years ago (MYA), for the timing of this event. The stem age of the family is predicted to be approximately 49 MYA (Särkinen et al., 2013), indicating that this duplication might have happened later in Solanaceae diversification. Our data suggest that this duplication event is

independent of the reported whole genome events, occurring prior to the diversification of the Brunfelsia clade but after the event that produced the FUL1 and FUL2 clades (**Figure 2** and **Figure S2** in **Supplementary Data Sheet S1**).

The expected topology for the euFULII clade, based on a duplication prior to the divergence of the Brunfelsia clade, would be a paraphyletic grade of pre-duplication euFULII genes, from species that diversified prior to Brunfelsia, and nested MBP10 and MBP20 clades that would include post-duplication genes from all species that diversified subsequent to the duplication. However, in our tree, the pre-duplication genes do not form such a basal grade (**Figure 2**). Rather, they form a clade with the post-duplication MBP20 genes. The results of our PAML analyses indicate that the MBP20-clade genes show less sequence divergence than MBP10 genes; this higher degree of similarity among pre-duplication sequences and post-duplication MBP20 genes may underlie their grouping into one clade (Pegueroles et al., 2013).

Our results indicate that the euFULII duplication occurred prior to the origin of the clade containing Brunfelsia. We would therefore expect to find both an MBP10 and an MBP20 in all species of that clade. However, we did not find an MBP10 ortholog in members of this clade other than Brunfelsia. MBP10 appears to have been lost from the genome of Petunia, based on analyses of multiple fully sequenced genomes (Bombarely et al., 2016), and potentially from Plowmania and Fabiana. We were able to recover MBP10 orthologs from Nicotiana and most other later-diverging genera. However, our analysis includes fewer species from the dry grade of the Solanaceae phylogeny than the fleshy-fruited Solanoideae clade (17 out of 45) and even fewer species that diverged prior to Brunfelsia (7). In the MBP10 clade in particular, our analysis includes 13 orthologs from species in the fleshy-fruited clade but just four from the dryfruited species, and our analysis only includes sequence data from four genera that diverged prior to the origin of the Brunfelsia clade (Streptosolen, Cestrum, Goetzia, Schizanthus) (**Figures 1**, **2**). Thus there may be genera that originated prior to Brunfelsia that contain MBP10 that our sampling did not include. Floral and fruit transcriptomes, which provided MBP10 orthologs from later diverging species, yielded no MBP10 sequences from Cestrum and Schizanthus; nonetheless, whole genome sequences of early diverging species are needed to determine the timing of the MBP10/MBP20 duplication.

#### euFULII Expression Divergence May Be Associated With Cis-Regulatory Re-coupling

Our analysis of Solanaceae euFUL homologs show that FUL1 and FUL2 are broadly expressed in leaves, flowers, and fruit (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). This overall similarity in expression may indicate a conservation of cis-regulatory elements in gene copies following duplication (Haberer et al., 2004). Supporting this, our investigation into the number of putative TF binding sites in the promoter region of euFULI homologs did not reveal statistically significant differences (**Table S4** in **Supplementary Data Sheet S1**). In tomato fruit development, FUL1 expression increases with time, whereas FUL2 expression reaches a maximum at early stages and then decreases over later stages (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). This variation in expression associated with the developmental stages might be due to changes in cis-elements as a result of the accumulation of random mutations over time (Force et al., 1999; Haberer et al., 2004).

Our analysis did find differences in the number and location of predicted binding sites for specific TFs or families, for instance for ARF, STK, and EIN3 TFs, which may account for the types of differences in expression seen between euFUL paralogs. The 5 kb region upstream of the FUL1 transcription start site in tomato contains three putative ARF binding sites but the corresponding region of FUL2 in tomato contains no such motifs (**Table S4** in **Supplementary Data Sheet S1**). ARF TFs, important in tomato fruit development, are activated in response to auxin and may upregulate or repress downstream genes (de Jong et al., 2010, 2015; Liu et al., 2018); the absence of binding sites from the FUL2 promoter is the type of factor that might underlie differences in expression observed between FUL1 and FUL2. Predicted STK binding sites are only found in the promoters of potato FUL1, tomato FUL2 and woodland tobacco MBP10. STK and STKlike proteins appear to function in storage protein synthesis, glucose reception, and vegetative and reproductive development (Zourelidou et al., 2002; Curaba et al., 2003; Bömer et al., 2011; Chung et al., 2016; Nietzsche et al., 2017). Meanwhile, the 2 kb upstream region of FUL2 contains a putative site for EIN3. This protein is involved in the development of tomato in response to ripening-associated ethylene production (Tieman et al., 2001). No such motifs are found in the corresponding region of FUL1. In contrast, the 2–5 kb region in FUL2 contains four putative sites for EIN3 while the corresponding region in FUL1 contains three such sites (**Table S4** in **Supplementary Data Sheet S1**).

Whereas the euFULI members largely overlap in spatial expression with some variation associated with developmental stages, the euFULII homologs show less consistent spatial expression patterns. Only MBP20 is expressed in tomato roots and potato fruit while only MBP10 is expressed in potato tubers (**Figure 4**). However, these "on" or "off " expression patterns cannot be explained by the presence or absence of any putative TF binding sites (**Table S4** in **Supplementary Data Sheet S1**). These two paralogs, which appear to be the result of a tandem duplication and inversion, are located approximately 14.3 Mbp apart (**Figure 3**) on chromosome 2. Although gene clusters resulting from tandem duplications are often coexpressed, this is not the case when there are large physical distances between the genes (Lercher et al., 2003). An investigation into the expression of human transgenes in mice also found changes in expression as a consequence of an inversion, possibly through disrupting enhancer activity or changes to chromatin structure (Tanimoto et al., 1999; Vogel et al., 2009; Puig et al., 2015). Chromosomal rearrangements

such as inversions may also result in novel connections between coding regions and other promoters or long distance regulatory motifs while disrupting the original regulatory mechanisms (Kmita et al., 2000; Lupiáñez et al., 2015). This sort of re-coupling of one of the two paralogs might lead to the types of contrasting expression patterns observed for MBP10 and MBP20. However, the expression patterns are not consistent across species (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**) and this might be due to additional changes following the inversion (Cosner et al., 1997; Lupski, 1998; Haberle et al., 2008; Chiang et al., 2012). An in-depth analysis of the entire loci and their genomic environment for all paralogs in multiple species would be necessary to determine if the tandem duplication and inversion are associated with changes in proximity to heterochromatin, additional rearrangements, or other phenomena that might have altered gene expression.

# MBP10 Shows Signs of Pseudogenization

The first intron of some MADS-box genes contains cis-elements important for the regulation of expression (Gazzani et al., 2003; Michaels et al., 2003; Schauer et al., 2009). Studies have found that deletions in the first intron of a FUL-like gene in Aegilops tauschii alters expression and results in the loss of the vernalization requirement (Fu et al., 2005; Takumi et al., 2011). Consistent with this, the first introns of angiosperm euFUL orthologs are generally in the range of 1–10 kb (**Table 2**) (Takumi et al., 2011). In contrast, tomato MBP10 has a short first intron of 80 bp. We compared the putative TF binding sites in the first introns of MBP10 and MBP20 in tomato to characterize potential loss of such sites, which might suggest reduced gene regulation. The first intron of MBP10 is predicted to have no TF binding sites, while the first intron of MBP20 is predicted to contain 88 TF binding sites (**Figure S3** in **Supplementary Data Sheet S1**). These included binding sites for MYB, HSF, Dof, WRKY, and MADS-box TFs. Specific TFs predicted to bind to these sites include MYB2 and C1 (MYB), which play roles in anthocyanin accumulation and lignin biosynthesis, PBF (Dof), which plays a role in endosperm storage protein accumulation, and SPF1 (WRKY), thought to function in fruit ripening (Bovy et al., 2002; Fei et al., 2004; Hwang et al., 2004; Lee et al., 2010; Xu et al., 2014; Jun et al., 2015). A similar pattern was found in analysis of the first intron of MBP10 in Nicotiana obtusifolia, which is 110 bp (**Table 2**). This analysis found three putative TF binding sites for MYB2 and one for PBF. By contrast, the first intron of N. obtusifolia MBP20 is predicted to have 133 TF binding sites and include a repertoire similar to those found for tomato MBP20. To determine whether the difference in TF binding site number between the paralogs represented a gain of sites in the MBP20 genes or a loss in the MBP10 genes, we also searched for TF binding sites in the first intron of AGL79, the single euFULII ortholog in A. thaliana (Gao et al., 2018). We found that it contains 49 predicted TF binding sites for five different TFs in four families: MYB (MYB2, GAMYB), HSF (HSF1), WRKY (SPF1), and GT-box (GT-1). Although this number is substantially smaller than the number of sites predicted in the first introns of the Solanaceae MBP20 genes, the results suggest that there has been a loss of TF binding sites in MBP10.

 Therefore, the absence or extremely weak expression of MBP10 in fruits of all species, and its weak expression in most organs of tomato and potato is notable (**Figure 4** and **Figure S1** in **Supplementary Data Sheet S1**). This relatively weak expression may at least in part be due to the loss of TF binding sites in the first intron and suggests a potentially reduced role in regulating fruit-related developmental processes. Importantly, the loss of putative TF binding sites and low expression, combined with the faster evolutionary rate, suggest MBP10 might be in the process of becoming a pseudogene. Further support for this hypothesis comes from an examination of the MBP10 sequences, which suggests that at least two of the sequences in our study (from N. sylvestris and Dunalia spinosa) show a frameshift that would result in an premature stop codon.

## CONCLUSION

Our results suggest that there was a weakening in purifying selection following the euFUL gene duplications in Solanaceae, resulting in coding sequence diversification in FUL1 and MBP10 clades relative to FUL2 and MBP20. Expression of the euFULI genes is broad, while the euFULII genes have contrasting patterns at the organ level, potentially resulting from cis-regulatory changes associated with the inversion event. We also found evidence to suggest that the MBP10 clade is becoming a pseudogene. Although at least some clades of Solanaceae euFUL genes took on new functions associated with the development of fleshy fruit we did not find any amino acid shifts that were correlated with the change in fruit type. It is also possible that the novel functions are a consequence of downstream changes, perhaps as the result of changes in binding partners or targets. Therefore, the mechanism underlying the shift in euFUL function from dry to fleshy fruit in Solanaceae awaits additional analyses.

#### AUTHOR CONTRIBUTIONS

AL designed and supervised research, and assisted in writing the paper. DM contributed to the design of the study, generated Cestrum diurnum, C. nocturnum, and Schizanthus grahamii transcriptome libraries, retrieved sequences from PCR-based methods and database mining, analyzed the data, and wrote the paper. CE assisted with PAML analysis, contributed suggestions for analyses, and made suggestions

on the paper. AR generated Dunalia spinosa, Fabiana viscosa, Grabowskia glauca, and Salpiglossis sinuata transcriptome libraries, contributed suggestions for analyses, contributed in recording the associated protocols, and commented on the paper. JM retrieved sequences from PCR-based methods. MS generated the Nicotiana obtusifolia transcriptome libraries and additional sequences using PCR-based methods. NP-M generated Brunfelsia australis and Streptosolen jamesonii transcriptome libraries, contributed suggestions for analyses, and made suggestions on this paper.

#### FUNDING

This research was funded by the National Science Foundation (IOS 1456109). DM was partly supported by UCR Graduate Assistance in Areas of National Need (GAANN) and Graduate Research Mentoring Program (GRMP) fellowships. NP-M acknowledges grants by COLCIENCIAS<PHONE_NUMBER>12), the Convocatoria Programaticas 2017–2018, and the Estrategia de Sostenibilidad 2018–2019, in the

#### REFERENCES

Universidad de Antioquia given to the group Evo-Devo en Plantas.

#### ACKNOWLEDGMENTS

We are grateful for the help with transcriptome library compilation and analyses by Glenn Hicks, John Weger, Holly Eckelhoefer, and Clay Clark at the UCR IIGB Core Facility, the advice on the gene tree generation by John Heraty, feedback by Elizabeth McCarthy, Jacob Landis, and Patricia Springer and Jaimie Van Norman lab members, and greenhouse and lab support by Arman Baghaei, Alan Le, and Victor Herrera.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00043/ full#supplementary-material

of nucleosome occupancy in target site selection. Genome Res. 16, 1517–1528. doi: 10.1101/gr.5655606

factor (Vif). Proc. Natl. Acad. Sci. U.S.A. 101, 3927–3932. doi: 10.1073/pnas.<PHONE_NUMBER>

regulate ethylene responses throughout plant development. Plant J. 26, 47–58. doi: 10.1046/j.1365-313x.2001.01006.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Maheepala, Emerling, Rajewski, Macon, Strahl, Pabón-Mora and Litt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


*A.2. The Experiments of the Values in Table 2*

Figure A1 illustrates the examples using different settings for the constant value *C*. It is noteworthy that *C* in Table 2 aims to correct the difference from the " *ρ* ≈ *ρ*" in Equation (4). According to the assumption in Section 2.2.1, this difference is small but sensitive to rock property. The goal of *C* is to optimize the visual comfort mentioned in Section 3.1. Therefore, *C* is affected by two settings, range and scale.

The range of the pixel value is between 0 and 256. (i) In Table 2, the range of *C* is set from 0 to 50. Thus, the maximum adjustment is less than 20% of the pixel value. Figure A1b shows a synthetic example using Table 2, and the white pixels in Figure A1a highlight the rocks. Figure A1a,b depict that Table 2 is a visually comfortable setting. Figure A1c,d respectively increase the range of *C* by three times and five times, and the embedded rock samples are very unreal to the background. Figure A1e reduces the range of *C* to about half of Figure A1b. The inserted rock is too bright compared to the background. Therefore, the range setting of *C* in Table 2 is in a reasonable range. (ii) *C* in Table 2 is divided into 11 scales according to the corresponding conditions (*imgmean*). A higher img\_mean corresponds to a higher *C*. Figure A1f doubles the scale-setting to 21 scales, but there is no significant change compared to Figure A1b. Therefore, the classification method in Table 2 is also reasonable.

**Figure A1.** The visualized results of the experiments for the constant *C* in Table 2. (**a**) refers to the synthetic annotation, while (**b**–**f**) corresponds to the synthetic images through different settings of *C*. (**b**) applies the same setting as Table 2. (**c**) keeps the grade setting but increases the range of the *C*, the maximum *C* is set to 250. (**d**) refers to the results of only increasing maximum *C* to 150. (**e**) refers to the results of decreasing maximum *C* to 20. (**f**) keeps the maximum *C* as the same as (**b**), while the grade setting applies 21 grades.

**Figure A2.** Some examples from the synthetic dataset. "Synthetic image" and "annotation" refer to the synthetic images and corresponding annotations, respectively. Annotations use white and black pixels to represent the rock and background pixels.


*Review* **Agricultural Sustainability: A Review of Concepts and Methods**

#### **Maria G. Lampridi 1,2,\*, Claus G. Sørensen 3 and Dionysis Bochtis 1**

Received: 21 August 2019; Accepted: 18 September 2019; Published: 19 September 2019

**Abstract:** This paper presents a methodological framework for the systematic literature review of agricultural sustainability studies. The framework synthesizes all the available literature review criteria and introduces a two-level analysis facilitating systematization, data mining, and methodology analysis. The framework was implemented for the systematic literature review of 38 crop agricultural sustainability assessment studies at farm-level for the last decade. The investigation of the methodologies used is of particular importance since there are no standards or norms for the sustainability assessment of farming practices. The chronological analysis revealed that the scientific community's interest in agricultural sustainability is increasing in the last three years. The most used methods include indicator-based tools, frameworks, and indexes, followed by multicriteria methods. In the reviewed studies, stakeholder participation is proved crucial in the determination of the level of sustainability. It should also be mentioned that combinational use of methodologies is often observed, thus a clear distinction of methodologies is not always possible.

**Keywords:** agricultural sustainability; sustainability assessment; review


**3. Results and discussion**

#### **3.1. FTIR spectrum of pure epoxy**

FTIR spectrum of pure epoxy is shown in **Figure 5**. In the below figure, peaks corresponding to the presence of functional groups in the epoxy system and are listed in **Table 1**.

The epoxy resin contains several polar groups which can interact with the surface –OH groups on the nanoparticles through hydrogen bonding rather easily, similar to the bonding between the –OH groups and H2 O molecules in the atmosphere. An uncured DGEBA epoxy resin is polar in nature and contains two epoxide groups at both ends.

#### **3.2. Hydrogen bond formation in polymer and nanoparticle**

In the fabrication process of the epoxy nanocomposites, the filler (oMMT) is first mixed with DGEBA based epoxy subsequently the addition of hardner (DETDA) into the epoxy and oMMT nanoparticle mix to initiate the curing process. When DETDA hardner is added to the epoxy and oMMT particle mix, the epoxide group open up and form hydrogen bond with the free –OH groups on the nanoparticle surface in addition to reacting with amine groups of the hardner as shown in **Figure 6**.

**Figure 5.** FTIR spectra of pure epoxy.

The electrical breakdown voltage was measured at 300.15 K as per ASTM D-149 as shown in

Electrical breakdown voltage of nanocomposite insulator was documented. The dielectric

FTIR spectrum of pure epoxy is shown in **Figure 5**. In the below figure, peaks corresponding

The epoxy resin contains several polar groups which can interact with the surface –OH groups on the nanoparticles through hydrogen bonding rather easily, similar to the bonding between

In the fabrication process of the epoxy nanocomposites, the filler (oMMT) is first mixed with DGEBA based epoxy subsequently the addition of hardner (DETDA) into the epoxy and oMMT nanoparticle mix to initiate the curing process. When DETDA hardner is added to the epoxy and oMMT particle mix, the epoxide group open up and form hydrogen bond with the free –OH groups on the nanoparticle surface in addition to reacting with amine groups

O molecules in the atmosphere. An uncured DGEBA epoxy resin is

to the presence of functional groups in the epoxy system and are listed in **Table 1**.

polar in nature and contains two epoxide groups at both ends.

**3.2. Hydrogen bond formation in polymer and nanoparticle**

*t* KV/mm (2)

*V*

**Figure 4**.

146 Optimum Composite Structures

breakdown strength (DES) was computed using,

**Figure 4.** Insulation breakdown test method by needle-plate electrode geometry.

here, V = Electrical breakdown voltage in kilo volts,

*E* = \_\_

t = thickness of nanocomposite sample in m.

**3. Results and discussion**

**3.1. FTIR spectrum of pure epoxy**

of the hardner as shown in **Figure 6**.

the –OH groups and H2

**Table 1.** FTIR peaks corresponding to functional groups in pure epoxy.

**Figure 6.** Hydrogen bonding between –OH groups on the nanoparticle surface and epoxide groups in epoxy during curing.

Since, there is abundantly free –OH groups are available on the nanoparticle surface, the hydrogen bonded epoxy segments will be more in number nearer to the nanoparticle surface. Development of the hydrogen bonds at interface region of epoxy and nanofiller is due to strongly bonded first nanolayer and tightly bounded second nanolayer of nanofiller and epoxy segments. This is in accordance with multi core model [1].

H2

O molecule is highly prone to hydrogen bonding with free hydroxyl groups present on the OMMT particle surfaces as shown in **Figure 7**. Hence, there will be a tendency for all the –OH

erties of the epoxy based nanocomposites [12]. Although, it is very difficult to fully remove the

the nanoparticles. One of the important processes which has been adopted in the present work is to adequately dry the OMMT particles before they are adding into the polymer matrix.

With reference to the **Figure 7**, the width of the peak, intensity of the peak and –OH infrared peak positions are influenced by the existence of hydrogen bonding in the oMMT nanofiller and epoxy matrix. However, it is well known that for –OH bands i)width of peak, and ii) intensity of the peak increases as the incorporation of oMMT particles in epoxy resin increases which is considered as sign of adding up of hydrogen bonds in the nanofilled filled composites.

**)**

of the epoxy nanocomposites are shown in the **Figure 8**. It is noted from

value of pure epoxy resin as 442.15 K. It is also noted from the plot

O molecules will tend to influence the thermal and electrical prop-

O molecules

O molecules are

by 278.15 K as

O molecules, the experi-

O layer on the surface of

O layer on the surface of the OMMT

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149

groups present in the oMMT nanoparticle surface to form hydrogen bonds with H2

particles. When such oMMT particles having high concentration of surface H2

that inclusion of 2 wt.% oMMT, into epoxy results in increased value of Tg

formation of hydrogen bonds between the surface –OH groups and H2

ments carried out have to be controlled to reduce the formation of a H2

in the atmosphere which may result in the formation of a H2

**Figure 7.** FTIR spectra of epoxy-oMMT nanocomposites.

**3.4. Analysis of the glass transition temperature (Tg**

introduced into epoxy, the H2

The variations in Tg

the figure, about the Tg

compared to pure epoxy.

#### **3.3. FTIR spectra of epoxy-oMMT nanocomposites**

The FTIR spectrum of epoxy-oMMT nanocomposites is shown in **Figure 7**. The FTIR spectra clearly show that the incorporation of the filler particles leads to formation of hydrogen bonds. The interaction mechanism between nanofiller particles and base epoxy is the formation of hydrogen bond with free surface hydroxyl (–OH) groups present on the nanofiller particle outer surface and the base polymer epoxy. One of the major understand with the hydrogen bonding is that they will not exhibit any such new peaks in FTIR spectra.

The band at 3444 cm−1 correspond to the stretching vibration of hydroxyl (–OH) groups attached to the nanoparticle surface. The –OH groups may be attached on the surface of nanofiller particles as free –OH groups, or as –OH groups attached to absorbed H2 O molecules. When the band appears at 1638 cm−1, this corresponds to the bending vibrations of absorbed H2 O molecules. The presence of the FTIR peaks at the above mentioned wavelengths reports the presence of hydroxyl (-OH) groups on the nanofiller surfaces. This result obtained through FTIR measurements in the present work have been well supported by several researchers [9–11].

The reason for the presence of H2 O molecules on the surfaces of oMMT particles are affinity of H2 O molecules present in atmosphere to get bonded to the surface OH groups on the oMMT particles through hydrogen bonding. H2 O is a polar particle or molecule and O2 attached to the

**Figure 7.** FTIR spectra of epoxy-oMMT nanocomposites.

Since, there is abundantly free –OH groups are available on the nanoparticle surface, the hydrogen bonded epoxy segments will be more in number nearer to the nanoparticle surface. Development of the hydrogen bonds at interface region of epoxy and nanofiller is due to strongly bonded first nanolayer and tightly bounded second nanolayer of nanofiller and

**Figure 6.** Hydrogen bonding between –OH groups on the nanoparticle surface and epoxide groups in epoxy during curing.

The FTIR spectrum of epoxy-oMMT nanocomposites is shown in **Figure 7**. The FTIR spectra clearly show that the incorporation of the filler particles leads to formation of hydrogen bonds. The interaction mechanism between nanofiller particles and base epoxy is the formation of hydrogen bond with free surface hydroxyl (–OH) groups present on the nanofiller particle outer surface and the base polymer epoxy. One of the major understand with the hydrogen

The band at 3444 cm−1 correspond to the stretching vibration of hydroxyl (–OH) groups attached to the nanoparticle surface. The –OH groups may be attached on the surface of nanofiller par-

ecules. The presence of the FTIR peaks at the above mentioned wavelengths reports the presence of hydroxyl (-OH) groups on the nanofiller surfaces. This result obtained through FTIR measurements in the present work have been well supported by several researchers [9–11].

O molecules present in atmosphere to get bonded to the surface OH groups on the oMMT

O molecules on the surfaces of oMMT particles are affinity of

O is a polar particle or molecule and O2

band appears at 1638 cm−1, this corresponds to the bending vibrations of absorbed H2

O molecules. When the

O mol-

attached to the

epoxy segments. This is in accordance with multi core model [1].

bonding is that they will not exhibit any such new peaks in FTIR spectra.

ticles as free –OH groups, or as –OH groups attached to absorbed H2

**3.3. FTIR spectra of epoxy-oMMT nanocomposites**

148 Optimum Composite Structures

The reason for the presence of H2

particles through hydrogen bonding. H2

H2

H2 O molecule is highly prone to hydrogen bonding with free hydroxyl groups present on the OMMT particle surfaces as shown in **Figure 7**. Hence, there will be a tendency for all the –OH groups present in the oMMT nanoparticle surface to form hydrogen bonds with H2 O molecules in the atmosphere which may result in the formation of a H2 O layer on the surface of the OMMT particles. When such oMMT particles having high concentration of surface H2 O molecules are introduced into epoxy, the H2 O molecules will tend to influence the thermal and electrical properties of the epoxy based nanocomposites [12]. Although, it is very difficult to fully remove the formation of hydrogen bonds between the surface –OH groups and H2 O molecules, the experiments carried out have to be controlled to reduce the formation of a H2 O layer on the surface of the nanoparticles. One of the important processes which has been adopted in the present work is to adequately dry the OMMT particles before they are adding into the polymer matrix.

With reference to the **Figure 7**, the width of the peak, intensity of the peak and –OH infrared peak positions are influenced by the existence of hydrogen bonding in the oMMT nanofiller and epoxy matrix. However, it is well known that for –OH bands i)width of peak, and ii) intensity of the peak increases as the incorporation of oMMT particles in epoxy resin increases which is considered as sign of adding up of hydrogen bonds in the nanofilled filled composites.

#### **3.4. Analysis of the glass transition temperature (Tg )**

The variations in Tg of the epoxy nanocomposites are shown in the **Figure 8**. It is noted from the figure, about the Tg value of pure epoxy resin as 442.15 K. It is also noted from the plot that inclusion of 2 wt.% oMMT, into epoxy results in increased value of Tg by 278.15 K as compared to pure epoxy.

nanolayer near to the nanoparticle surface and this interfacial polymer nanolayer influences

or remain constant. In the present work, the results obtained for 2 wt.% nanocomposites, may be due to few cross links but for higher loading, the nanoparticle-polymer interaction

with the published literatures [16, 17]. The cross linking density reduces due to etherification mechanism and curing agent preferably tends to attach to the surfaces and very thin layer of hardner or curing agent will surround the nanofiller particles. This thin layer of curing agent will keep the curing agent around the nanofiller particles from reacting with the epoxy resin. This 'curing agent concentration' mechanism will also cause decrease in cross-link density

1.75 ns, that is size or area of the free volume and free volume content increases from 7.17 to

may be because of addition of nanofiller in polymer matrix creates additional free volume. It further suggests that, the filler will not occupy the pre-existing free volume cavities due to

versus filler content (wt.%) of epoxy-oMMT nanocomposites.

and 2.28 to 2.45% respectively. However, at 7 wt.% oMMT loading, both τ3

) with 2, 5 and 7 wt.% of oMMT in epoxy nanocomposites is shown in

and 2.14% respectively. The modifications has taken place

tion, repulsive interaction or the interaction is neutral and as such Tg

decreases at 5 and 7 wt.% of filler loading.

The variation of free volume content or fractional free volume (Fv

The addition of oMMT content in epoxy resin (2-5 wt.%), causes τ3

found to be repulsive and therefore Tg

**3.5. Characterization of free volume content**

decrease slightly to 1.74 ns, 7.34 nm3

. These nanoparticle and polymer interactions can be developed as attractive interac-

can increase, decrease,

), free volume size (Vf

to increase from 1.72 to

, Vf

) and

151

and Fv

decreases and the present results are in good concord

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the Tg

and thereby Tg

longest life time (τ3

**Figure 9**.

7.45 nm3

**Figure 9.** Plot of τ3

, Vf and Fv

**Figure 8.** DSC plots of epoxy-oMMT nanocomposite.

With the incorporation of 5 and 7 wt.% of nanofiller Tg value decreases. The interfacial interaction of polymer nanocomposites and following in the formation of nanolayer in nanocomposites are concluded in the literature followed. The formation of nanolayer and its influence on Tg is also been reported. The higher value of in Tg [13, 14] when nanofiller has been added is reported in some cases whereas, some others report a smaller value of Tg due to addition of nanoparticles into base polymer matrix [15], but the majority of the proposed theories are uncertain till today. The influence of oMMT nanofiller on the curing reaction and glass transition values of the epoxy nanocomposites is investigated by DSC in order to understand the molecular mobility in the nanocomposites. When the nanofiller concentration is increased from 5 to 7 wt.%, the values of Tg in epoxy nanocomposites is observed to decrease. Similar to the results obtained in the case of epoxy filled with oMMT nanofillers in the present work, reduced value of Tg have been reported for nanoalumina filled PMMA composites [16]. The increase in Tg with the addition of 2 wt.% of nanofiller is due to few cross links that developed between the polymer matrix and nanoparticle. However, the reduction in Tg values at 5 and 7 wt.% of nanofiller loading may be due to the many reasons such as changes in molecular weight, tacticity, cross-linking density and the presence of residues from incomplete reactions. The other possible reasons could be the size of the nanofillers which are certainly larger than the free volume hole sizes (discussed in Section 2.4) in the matrix and therefore the possible slide between the chains can result in increased free volume.

Sun and co-authors [17] reported that the Tg depression is associated with the improved polymer chain dynamics because of the additional free volume at the resin-nanofiller interface. Becker and co-authors [18] remarked, interfacial interactions between polymer chains and positively or negatively charged nanofiller surface leads to the development of a polymer nanolayer near to the nanoparticle surface and this interfacial polymer nanolayer influences the Tg . These nanoparticle and polymer interactions can be developed as attractive interaction, repulsive interaction or the interaction is neutral and as such Tg can increase, decrease, or remain constant. In the present work, the results obtained for 2 wt.% nanocomposites, may be due to few cross links but for higher loading, the nanoparticle-polymer interaction found to be repulsive and therefore Tg decreases and the present results are in good concord with the published literatures [16, 17]. The cross linking density reduces due to etherification mechanism and curing agent preferably tends to attach to the surfaces and very thin layer of hardner or curing agent will surround the nanofiller particles. This thin layer of curing agent will keep the curing agent around the nanofiller particles from reacting with the epoxy resin. This 'curing agent concentration' mechanism will also cause decrease in cross-link density and thereby Tg decreases at 5 and 7 wt.% of filler loading.

#### **3.5. Characterization of free volume content**

With the incorporation of 5 and 7 wt.% of nanofiller Tg

**Figure 8.** DSC plots of epoxy-oMMT nanocomposite.

is also been reported. The higher value of in Tg

from 5 to 7 wt.%, the values of Tg

Sun and co-authors [17] reported that the Tg

reduced value of Tg

150 Optimum Composite Structures

increase in Tg

Tg

tion of polymer nanocomposites and following in the formation of nanolayer in nanocomposites are concluded in the literature followed. The formation of nanolayer and its influence on

of nanoparticles into base polymer matrix [15], but the majority of the proposed theories are uncertain till today. The influence of oMMT nanofiller on the curing reaction and glass transition values of the epoxy nanocomposites is investigated by DSC in order to understand the molecular mobility in the nanocomposites. When the nanofiller concentration is increased

to the results obtained in the case of epoxy filled with oMMT nanofillers in the present work,

7 wt.% of nanofiller loading may be due to the many reasons such as changes in molecular weight, tacticity, cross-linking density and the presence of residues from incomplete reactions. The other possible reasons could be the size of the nanofillers which are certainly larger than the free volume hole sizes (discussed in Section 2.4) in the matrix and therefore the

mer chain dynamics because of the additional free volume at the resin-nanofiller interface. Becker and co-authors [18] remarked, interfacial interactions between polymer chains and positively or negatively charged nanofiller surface leads to the development of a polymer

is reported in some cases whereas, some others report a smaller value of Tg

between the polymer matrix and nanoparticle. However, the reduction in Tg

possible slide between the chains can result in increased free volume.

value decreases. The interfacial interac-

[13, 14] when nanofiller has been added

in epoxy nanocomposites is observed to decrease. Similar

depression is associated with the improved poly-

have been reported for nanoalumina filled PMMA composites [16]. The

with the addition of 2 wt.% of nanofiller is due to few cross links that developed

due to addition

values at 5 and

The variation of free volume content or fractional free volume (Fv ), free volume size (Vf ) and longest life time (τ3 ) with 2, 5 and 7 wt.% of oMMT in epoxy nanocomposites is shown in **Figure 9**.

The addition of oMMT content in epoxy resin (2-5 wt.%), causes τ3 to increase from 1.72 to 1.75 ns, that is size or area of the free volume and free volume content increases from 7.17 to 7.45 nm3 and 2.28 to 2.45% respectively. However, at 7 wt.% oMMT loading, both τ3 , Vf and Fv decrease slightly to 1.74 ns, 7.34 nm3 and 2.14% respectively. The modifications has taken place may be because of addition of nanofiller in polymer matrix creates additional free volume. It further suggests that, the filler will not occupy the pre-existing free volume cavities due to

**Figure 9.** Plot of τ3 , Vf and Fv versus filler content (wt.%) of epoxy-oMMT nanocomposites.

bigger size of the fillers but creates additional free volume probably at the interface. The results also indicate that the layers of oMMT will result in favorable interaction with the epoxy resins and thereby the segmental motion is hindered. Hence, the significant increase in Fv percentage is not observed. The decrease in Tg is justified by the decrease in free volume content.

Because of this cause, charge carriers are accelerated over shorter distances and have reduced mobility and kinetic energy. This process is considered as a scattering mechanism. The energy of charge carriers is distributed more evenly in the polymer and thus causes less damage in the material and prolongs the lifetime and service of the epoxy. This scattering process decreases the electric field at the electrodes and increase the voltage required for charge injection.

These fillers can also act as barriers for the penetration of the charge carriers throughout the depth of the sample. In case of 5 wt.% oMMT, the number of nanoparticles is much more and the inter particle distance is also less than 100 nm. This inter particle distance was justified by morphological studies. Hence, there is a possibility of overlapping of the loose polymer regions in the nanocomposites leading to the reduction in the loose polymer regions. This polymer layer along with a large number of fillers can also obstruct the discharge path, thereby dielectric strength increases. The increase in dielectric strength values is well supported by reduction in dielectric constant values and increase in free volume content at 2 and

When oMMT filler content is increased to 7 wt.%, there is a reduction in the dielectric strength occurs due to the overlapping of the tightly bound polymer regions over the interface, since the inter particle distances are comparable to the filler diameter. The interphase region close to the nanoparticle is found to be conductive [11]. As the conductive interphase regions tends to overlap, the pure polymer region is reduced, leading to an easier conducting path for the charge transfer and thereby a reduction in the dielectric strength is observed. With the addi-

free charge carriers are available, but reduction in free volume leads to an easier conduction path for the charge carriers and thereby DES decreases. The reduction in DES at 7 wt.% of oMMT filled nanocomposite is well supported by increase in dielectric constant values and free volume content of the nanocomposites. This has been well supported by many authors. Many researchers [19] have reported that layered silicate nanofillers modify the trapping property of both isotactic and syndiotactic PP. Roy and co-authors [20] have reported that

**i.** Existence of hydrogen bond between nanofillers and epoxy polymer chains in the nano-

**ii.** The interaction takes place between epoxy and nanoparticle in small region around the oMMT nanofiller surface called as "interface region" and the nature of interaction is

**iii.** The interaction between the oMMT and epoxy chains has a direct effect on dielectric strength characteristics of the nanocomposite and accordingly, a three core interface

found to be depends on the chemical bond of the oMMT and epoxy.

model has been used to elucidate the characteristics of the interface region.

has taken place, and hence more

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153

, and free volume measurements.

5 wt.% oMMT filled epoxy-oMMT.

tion of 7 wt.% of oMMT into epoxy matrix, reduction in Tg

composites has been established through FTIR, Tg

deep trap sites have been identified in SiO2

lated current (TSC) measurements.

The following remarks are drawn:

**4. Conclusions**

#### **3.6. Effect of interface on dielectric strength (DES)**

The dielectric strength (DES) is the vital properties of dielectric insulators. With reference to the discussion in Section 2.4, the properties of epoxy nanocomposites are mainly explained by interfacial interaction of polymer and of nanofillers. This interfacial area is responsible for the interaction of the electric field between the base epoxy and nanofiller. The DES of the nanocomposites depends largely on nanofiller content and even a very less quantity of nanofiller can cause improvement. When nanofiller particles are incorporated into the epoxy matrix, there is a change in morphology of the epoxy due to the interfacial interaction of epoxy with the oMMT nanofiller.

The **Figure 10** shows the variation of Dielectric strength, free volume with respect to nanofiller loading. With the addition of 2 wt.% oMMT into epoxy matrix, it is observed that Tg increases due to increase in cross linking density and hence less free charge carriers are available leading to slightly higher value of DES than that of pure epoxy. A further increase in the filler loading up to 5 wt.% of oMMT shows an increase in the DES above that of 2 wt.% of nanocomposite. In this case the Tg decreases, and hence more free charge carriers are available.

Here the effect of third interface layer also called loose layer comes into scenario. The loose polymer layers contain more traps or free volumes as discussed in Section 2.4. These charge carriers are easily and more frequently trapped in trap sites rather than in the base epoxy.

**Figure 10.** Plot of DES, Fv with respect to filler content (wt.%) of epoxy-oMMT nanocomposites.

Because of this cause, charge carriers are accelerated over shorter distances and have reduced mobility and kinetic energy. This process is considered as a scattering mechanism. The energy of charge carriers is distributed more evenly in the polymer and thus causes less damage in the material and prolongs the lifetime and service of the epoxy. This scattering process decreases the electric field at the electrodes and increase the voltage required for charge injection.

These fillers can also act as barriers for the penetration of the charge carriers throughout the depth of the sample. In case of 5 wt.% oMMT, the number of nanoparticles is much more and the inter particle distance is also less than 100 nm. This inter particle distance was justified by morphological studies. Hence, there is a possibility of overlapping of the loose polymer regions in the nanocomposites leading to the reduction in the loose polymer regions. This polymer layer along with a large number of fillers can also obstruct the discharge path, thereby dielectric strength increases. The increase in dielectric strength values is well supported by reduction in dielectric constant values and increase in free volume content at 2 and 5 wt.% oMMT filled epoxy-oMMT.

When oMMT filler content is increased to 7 wt.%, there is a reduction in the dielectric strength occurs due to the overlapping of the tightly bound polymer regions over the interface, since the inter particle distances are comparable to the filler diameter. The interphase region close to the nanoparticle is found to be conductive [11]. As the conductive interphase regions tends to overlap, the pure polymer region is reduced, leading to an easier conducting path for the charge transfer and thereby a reduction in the dielectric strength is observed. With the addition of 7 wt.% of oMMT into epoxy matrix, reduction in Tg has taken place, and hence more free charge carriers are available, but reduction in free volume leads to an easier conduction path for the charge carriers and thereby DES decreases. The reduction in DES at 7 wt.% of oMMT filled nanocomposite is well supported by increase in dielectric constant values and free volume content of the nanocomposites. This has been well supported by many authors. Many researchers [19] have reported that layered silicate nanofillers modify the trapping property of both isotactic and syndiotactic PP. Roy and co-authors [20] have reported that deep trap sites have been identified in SiO2 -XLPE nanocomposites through thermally stimulated current (TSC) measurements.


**2.9 Thermal imidization of a polyamic acid (PAA)**

The PI films can be cured of the PAA solution after applying thermal imidization. After casting the PAA solution onto the substrate, thermal steps with temperature from 100 to 350°C were used to evaporate the solvents. According to the literature, various thermal steps and different temperature ranges have been utilized to achieve 100% imidization from PAA to PI. Two main thermal imidization ways are as follows:

1.Baking films with slowly increasing in temperature up to 350°C according to the flexibility and Tg of the PI film.

### **Figure 8.**

*Film thickness variation at different spin coater speeds (a) PAA solution high molecular weight and (b) PAA solution slight less molecular weight.*

*Synthesis Process Optimization of Polyimide Nanocomposite Multilayer Films, Their Dielectric… DOI: http://dx.doi.org/10.5772/intechopen.91206*

2.Start baking PAA with the temperature 100°C for 1 h, then baking at 200°C for 1 hour, then baking at 300°C and keeping for 1 h and slowly cool down to room temperature.

Several arduous factors are involved in the above simple-looking thermal imidization process to predispose the degree of imidization of PI films. During imidization, the remaining solvent at the later stages determines the stability of PI films. In early stages, the imidization process is faster, and several dynamic changing in physical properties happen due to the basicity of the amide solvent to accept protons and the amicable conformation of the amic acid to increase the mobility of the reacting functional groups [6]. At later stages, the rate of imidization slows down due to the decyclohydration of the open amic acid group into the closed imide ring, which decreases the chain mobility, and the Tg approaches the reaction temperature [6].

### **2.10 Determination of the degree of imidization using FTIR**

When one monomer reacts with other monomer, it forms a carbon chain of the polymer. In our case, a single unit of two monomers such as diamine (ODA) and dianhydride (PMDA) reacts with each other to form a single unit of polyamic acid (PAA) solution. In this reaction, an oxygen atom of diamine reacts with the hydrogen atom of dianhydride, and the hydrogen atom of dianhydride reacts with the carbon atom of diamine to give us a repeated unit of PAA. After obtaining PAA, thermal imidization was applied to cure PI and PI/ SiO2 films.

Fourier transform infrared spectroscopy (FTIR) is a promising tool to determine the degree of imidization despite a minor error in measurement due to its sensitivity to chemical change. The bond composition of PI atomic structure was studied by FTIR spectroscopy to analyze the chemical bonds present in PI. The bands most frequently utilized are imide absorption bands near 1780 cm−1 (C═O asymmetrical stretching), 1380 cm−1 (C▬N stretching), and 725 cm−1 (C═O bending). This study describes the chemical characterization of half cured and fully cured PI and PI/SiO2 films. The range of wavelength used was from 650 to 4000 cm−1 . The PI subatomic structure is composed of a molecular chain containing the functional groups such as aromatic rings, amide rings, and some noncyclic ether rings. These functional groups have discrete chemical bonds such as C▬N▬C, C═O, benzene (C6Hn), ether link (C▬O▬C), and ▬OCH2▬CH2 deformation. The functional groups of PI polymer chains provide the level of linkage with subatoms as well as the linkage with silicon oxide nanoparticles. Imide group contains imide carbonyl in-phase and out-of-phase stretching, the C▬N▬C axial, transverse, and out-of-phase stretching. The absorbance peaks at 1371, 1112, and 721 cm−1 indicate the transverse and axial stretching of C▬N▬C bond, and the reasons of 1781 and 1720 cm−1 peaks are in-phase and out-of-phase stretching of C═O in imide ring. Aromatic ring is divided into tangential, radial skeletal, and outof-plane vibrations. The peak at 1590 cm−1 relates to tangential C▬C vibrations. The peak at 1280 cm−1 is tangential phenyl ring vibrations. The other 1480, 1168, 1087, 1011, 843, and 788 cm−1 peaks describe about the tangential, radial skeletal, and out-of-plane bending vibrations of C6Hn in aromatic ring. The peaks at 1234 and 1454 cm−1 relate to the bond of ether link (C▬O▬C), and 1416 cm−1 peak corresponds to ▬OCH2▬CH2 deformation. **Figure 9(a)** and **(b)** shows the FTIR spectra of changes in the bonds of half cured and fully cured PI films at different temperatures and time to determine the degree of imidization to obtain imide ring.

**Figure 9.** *(a) Half cured PI film at different temperature and time and (b) zoom version of main function group peaks.*

In fully cured PI films, as shown from **Figure 9**, the bonds are grouped into amide rings, aromatic rings, and noncyclical stretching based on atom vibrations [18]. The intensity of the absorption band at 3463 cm−1 corresponding to the stretch vibration of ▬OH bond.