*2.11. The Firmness, Pectin Content and the Expression Level of UGlcAE Family Genes in Tomato Fruits at Different Development Stages*

To further explore the change of firmness, pectin contents, and expression levels of *UGlcAE* genes, we investigated the firmness, pectin contents, and the expression levels of *UGlcAE* family genes in fruits at different stages of tomato development (Figure 10). As the fruit matured, the firmness of tomato fruit decreased gradually (Figure 10A). The content of water-soluble pectin (WSP) in the development of tomato fruit showed an increased trend, reached a maximum at the MG stage, and then decreased (Figure 10B). The increase of WSP content in the early may be because of the accumulation of pectin as the fruit grows. The content of WSP gradually decreased after the MG stage, probably due to the gradual degradation of partially WSP by some pectinases. As shown in Figure 10C, nine genes of the UGlcAE family have different expression patterns in the development of tomato fruit. Among them, four genes (*UGlcAE1*, *UGlcAE1-like*, *UGlcAE5*, and *UGlcAE6*) showed relatively high expression levels, and other five genes had lower expression levels. The expression levels of both *UGlcAE1* and *UGlcAE5* first increased, reached the maximum value at the MG stage, and then decreased. This is consistent with the trend of WSP content in tomato fruit development.

*UGlcAE6*) showed relatively high expression levels, and other five genes had lower expression levels. The expression levels of both *UGlcAE1* and *UGlcAE5* first increased, reached the maximum

**Figure 10.** Firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. (**A**) Firmness of tomato fruit in five different stages; (**B**) water-soluble pectin (WSP) contents of fruit in five development stages; and (**C**) Expression level of nine UGlcAE family genes in stages. **Figure 10.** Firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. (**A**) Firmness of tomato fruit in five different stages; (**B**) water-soluble pectin (WSP) contents of fruit in five development stages; and (**C**) Expression level of nine UGlcAE family genes in stages.

#### **3. Discussion 3. Discussion**

UGlcAE is capable of reversibly interconverting UDP-GlcA and UDP-GalA, which plays an important role in pectin synthesis. It bring many new opportunities to study gene families in an evolutionary context with various plant genomes being sequenced [4]. To investigate the phylogenetic relationship of *UGlcAE* gene family members, we searched and collected the amino acid sequences of UGlcAE from 10 plant species. Allof the six subfamilies exist in *Arabidopsis thaliana*. However, in other plant species, the numbers of subfamilies of *UGlcAE* genes vary from three to five. Interestingly, the UGlcAE4 subfamily is specifically present in *Arabidopsis thaliana* and *Arabidopsis lyrata* subsp. *lyrata*, whereas it is absent from the other eight plant species. According to this analysis, we infer that the UGlcAE4 protein may have played specific roles in *Arabidopsis*. UGlcAE is capable of reversibly interconverting UDP-GlcA and UDP-GalA, which plays an important role in pectin synthesis. It bring many new opportunities to study gene families in an evolutionary context with various plant genomes being sequenced [4]. To investigate the phylogenetic relationship of *UGlcAE* gene family members, we searched and collected the amino acid sequences of UGlcAE from 10 plant species. Allof the six subfamilies exist in *Arabidopsis thaliana*. However, in other plant species, the numbers of subfamilies of *UGlcAE* genes vary from three to five. Interestingly, the UGlcAE4 subfamily is specifically present in *Arabidopsis thaliana* and *Arabidopsis lyrata* subsp. *lyrata*, whereas it is absent from the other eight plant species. According to this analysis, we infer that the UGlcAE4 protein may have played specific roles in *Arabidopsis*.

It is mentionable that the members of the UGlcAE2 subfamily were not classified into the same cluster. This indicates that there are great differences in the sequences of different members within the UGlcAE2 subfamily. We further speculate that the UGlcAE2 subfamily may be dividing new functions. Moreover, some members of the UGlcAE2 subfamily grouped with UGlcAE3 in *Arabidopsis*. It indicates that there are similarities in the sequences of these UGlcAE2 members and It is mentionable that the members of the UGlcAE2 subfamily were not classified into the same cluster. This indicates that there are great differences in the sequences of different members within the UGlcAE2 subfamily. We further speculate that the UGlcAE2 subfamily may be dividing new functions. Moreover, some members of the UGlcAE2 subfamily grouped with UGlcAE3 in *Arabidopsis*. It indicates that there are similarities in the sequences of these UGlcAE2 members and AtUGlcAE3. Therefore, it is likely that the members of the UGlcAE2 subfamily and AtUGlcAE3 might have similar functions or undergo gene fusion.

Similar to the previous studies of the UGlcAE in *Arabidopsis* [12,13], two branches of the phylogenetic tree are trustworthily occupied by UGlcAE1 and UGlcAE6, respectively, meanwhile, UGlcAE2, UGlcAE3, UGlcAE4, and UGlcAE5 are located together in one branch of the phylogenetic tree. This result implies a more ancient role of UGlcAE1 and UGlcAE6, concurrently, the other UGlcAEs might have evolved later [12,13].

In all ten plant species, only *Arabidopsis* contains all of the six *UGlcAE* subfamilies and every subfamily has at least one member. This is comprehensive and regular, which is very congruent with its identity of the model plant.

The expression patterns of the nine genes differed in the different tissues and development stages of tomato. However, it is still possible to find a certain rule from Figure 5. As mentioned by Mølhøj in 2004 [13], the heatmap representation of all the expression patterns reveals that *UGlcAE1* and *UGlcAE6* subfamilies (except *UGlcAE6-like*) were strongly expressed in cultivar tomato, whereas *UGlcAE2*, *UGlcAE3*, and *UGlcAE5* subfamilies were lowly expressed isoforms. However, *UGlcAE6-like* showed considerably lower expression levels in tomato. This is consistent with the result of *cis*-element analysis of *UGlcAE* gene families in Figure 8, which is no significant (stress-, hormoneand development-related) *cis*-acting elements being found within the range of 0.5 kb in front of the *UGlcAE6-like* gene coding region.

The expression trends of *UGlcAE1* and *UGlcAE5* in tomato fruit development were consistent with those of WSP content, indicating that *UGlcAE1* and *UGlcAE5* may be more closely related to the formation of WSP during the fruit ripening when compared to other members of the *UGlcAE* gene family. The expression level of *UGlcAE5* was high in Figure 10C and low in Figure 5A, which indicate that *UGlcAE5* may be easily affected by some factors in the environment and cause its expression level to be unstable. In addition, other results of Figure 10C (high expression level of the three genes (*UGlcAE1*, *UGlcAE1-like*, and *UGlcAE6*) and low expression level of the five genes (*UGlcAE2-like*, *UGlcAE3*, *UGlcAE3-like1*, *UGlcAE3-like2* and *UGlcAE6-like*)) were basically consistent with the results of Figure 5A. This may suggest that the expression of these eight genes is relatively stable during tomato fruit development.

After three hormones treatments, the expression of *UGlcAE1* was more susceptible to IAA, and the expression of *UGlcAE5* was more susceptible to SA. These results suggest that the WSP content of tomato may be more susceptible to IAA and SA in fruit development. *UGlcAE6-like* exhibited the very low expressions in Figures 5, 9J and 10C, indicating that *UGlcAE6-like* is less likely to affect WSP content during the tomato fruit ripening.

Pectin degradation is a major effect on fruit softening [35]. The identifications of the family genes help to understand more about these genes and can better investigate the mechanisms of pectin production and degradation. An in-depth understanding of specific gene expression during ripening and maturation of tomato fruits [36] will enable the precise manipulation of expression of new associate genes to more precisely control the mechanisms of cell wall modification and softening. This is still an outstanding question so far [35].
