*2.8. Selective Pressure on UGlcAE Proteins in the Tomato*

**Figure 6.** Assignment of Gene Ontology categories to *UGlcAE* genes in tomato. The lengths of the rectangular columns indicate the number of genes that participated in the corresponding classification. The purple rectangular columns mean gene functions of *UGlcAE* in *Arabidopsis*, the red rectangular columns represent gene functions of *UGlcAE* in tomato. All of the gene functions were classified into three categories, which were further divided into eight minor terms. To examine the evolutionary conservation of the UGlcAE proteins, the selective pressure on the UGlcAE was analyzed with SELECTON. We found that the domain of SlUGlcAE3-like1 protein was undergoing strong purifying selection (Figure 7). Selective pressure analyses of the other SlUGlcAE proteins were also analyzed and the results are shown in Supplementary Material Figure S2. These results confirm that the *SlUGlcAE* genes are undergoing strong purifying selection. The amino acids *2.8. Selective Pressure on UGlcAE Proteins in the Tomato*

that are emphasized in yellow are under positive selection; however, no positive selection site was found in this selection analysis. These results confirm that these gene family members were very conservative in evolution, which imply them playing a pivotal function in pectin biosynthesis. Selection pressure in the promoter regions of *SlUGlcAE* genes indicated that they are also undergoing negative selection (Figure S3). that are emphasized in yellow are under positive selection; however, no positive selection site was found in this selection analysis. These results confirm that these gene family members were very conservative in evolution, which imply them playing a pivotal function in pectin biosynthesis. Selection pressure in the promoter regions of *SlUGlcAE* genes indicated that they are also undergoing negative selection (Figure S3).

results confirm that the *SlUGlcAE* genes are undergoing strong purifying selection. The amino acids

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 9 of 21

To examine the evolutionary conservation of the UGlcAE proteins, the selective pressure on the UGlcAE was analyzed with SELECTON. We found that the domain of SlUGlcAE3-like1 protein was undergoing strong purifying selection (Figure 7). Selective pressure analyses of the other SlUGlcAE

**Figure 7.** Selection pressure analysis of the UGlcAE proteins in tomato. The red shades represent ω < 1 (purifying selection). Amino acid sequence of SlUGlcAE3-like1 is shown, and the sequences of other SlUGlcAE proteins are presented in the Supplementary Materials Figure S2. **Figure 7.** Selection pressure analysis of the UGlcAE proteins in tomato. The red shades represent ω < 1 (purifying selection). Amino acid sequence of SlUGlcAE3-like1 is shown, and the sequences of other SlUGlcAE proteins are presented in the Supplementary Materials Figure S2.

#### *2.9. Cis-Acting Elements Analysis of the UGlcAE Genes in the Tomato 2.9. Cis-Acting Elements Analysis of the UGlcAE Genes in the Tomato*

To explore the *cis*-acting elements of *SlUGlcAE* genes, we analyzed the 0.5 kb upstream sequences of nine *SlUGlcAE* genes using online software Plant CARE and the result was shown in Figure 8 and Supplementary Materials Table S1. The analysis result of 1.5 kb upstream genomic sequences of genes was shown in Supplementary Materials Table S2. To explore the *cis*-acting elements of *SlUGlcAE* genes, we analyzed the 0.5 kb upstream sequences of nine *SlUGlcAE* genes using online software Plant CARE and the result was shown in Figure 8 and Supplementary Materials Table S1. The analysis result of 1.5 kb upstream genomic sequences of genes was shown in Supplementary Materials Table S2.

Kinds, numbers, and locations of *cis*-elements in the upstream of *SlUGlcAE* genes were shown in Figure 8A, and the functional descriptions of these stress-related, hormone-related, and development-related *cis*-elements were exhibited in Figure 8B. As shown in Figure 8A, there are three *cis*-acting elements that are related to hormone, including TGA-element, TATC-box and TCA-element, and 5 stress-related elements including HSE (heat stress-related element), TC-rich repeats (*cis*-acting element involved in defense and stress responsiveness), LTR (*cis*-acting element involved in low-temperature responsiveness), WUN-motif (wound-responsive element), and MBS (MYB Binding Site), and seven elements that are involved in development (Skn-1\_motif, HD-Zip 1, HD-Zip 2, circadian, CAT-box, W box, and ELI-box3). Among them, the 0.5 kb upstream regions of four *SlUGlcAE* genes were found to be the presence of heat stress-related element (HSE), of which had two HSE elements in the 0.5 kb upstream region of *SlUGlcAE6* and 1 HSE elements in the 0.5 kb upstream regions of *SlUGlcAE1*, *SlUGlcAE3,* and *SlUGlcAE5*. Furthermore, defense- and stress-response

element (TC-rich repeats) was identified in the 0.5 kb upstream regions of four *SlUGlcAE* genes (*SlUGlcAE1-like*, *SlUGlcAE3-like1*, *SlUGlcAE3-like2*, and *SlUGlcAE5*), and circadian element (circadian) was found in the 0.5 kb upstream regions of three *SlUGlcAE* genes (*SlUGlcAE1-like*, *SlUGlcAE3-like1,* and *SlUGlcAE3-like2*), and endosperm expression-related element (Skn-1\_motif) was discovered in the 0.5 kb upstream regions of two *SlUGlcAE* genes (*SlUGlcAE1* and *SlUGlcAE6*), and other 11 elements are all present in the 0.5 kb upstream regions of only one *SlUGlcAE* gene. Four elements are located in 0.5 kb upstream region of *SlUGlcAE1*, and element numbers are diversified in the 0.5 kb upstream region of other genes, respectively (four in *SlUGlcAE1-like*, three in *SlUGlcAE2-like*, one in *SlUGlcAE3*, two in *SlUGlcAE3-like1*, four in *SlUGlcAE3-like2*, five in *SlUGlcAE5*, four in *SlUGlcAE6*, and zero in *SlUGlcAE6-like*). *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 10 of 21

**Figure 8.** Kinds and numbers of stress-related, hormone-related, and development-related *cis*-elements in the upstream of *SlUGlcAE* genes. (**A**) Various symbols indicate different *cis*-acting elements; and (**B**) Element names and their functional descriptions. **Figure 8.** Kinds and numbers of stress-related, hormone-related, and development-related *cis*-elements in the upstream of *SlUGlcAE* genes. (**A**) Various symbols indicate different *cis*-acting elements; and (**B**) Element names and their functional descriptions.

Kinds, numbers, and locations of *cis*-elements in the upstream of *SlUGlcAE* genes were shown in Figure 8A, and the functional descriptions of these stress-related, hormone-related, and development-related *cis*-elements were exhibited in Figure 8B. As shown in Figure 8A, there are three *cis*-acting elements that are related to hormone, including TGA-element, TATC-box and TCA-element, and 5 stress-related elements including HSE (heat stress-related element), TC-rich repeats (*cis*-acting element involved in defense and stress responsiveness), LTR (*cis*-acting element involved in low-temperature responsiveness), WUN-motif (wound-responsive element), and MBS (MYB Binding Site), and seven elements that are involved in development (Skn-1\_motif, HD-Zip 1, HD-Zip 2, circadian, CAT-box, W box, and ELI-box3). Among them, the 0.5 kb upstream regions of four *SlUGlcAE* genes were found to be the presence of heat stress-related element (HSE), of which had two HSE elements in the 0.5 kb upstream region of *SlUGlcAE6* and 1 HSE elements in the 0.5 kb No of stress-, hormone-, and development-related element was found in the 0.5 kb upstream region of *SlUGlcAE6-like* gene, and the previous spatial expression patterns showed *SlUGlcAE6-like* was lowly expressed at every stages, which are highly consistent. Thus, we infer that *SlUGlcAE6-like* may not be involved in the process of growth and development. In addition, HD-Zip 1 and HD-Zip 2 are located in the same position of *SlUGlcAE1* and *SlUGlcAE1-like*, implying that the two elements might be closely related and complementary to each other. Furthermore, other stress-, hormone-, and development-unrelated *cis*-acting elements have also been identified. For example, core promoter element (TATA-box) and common *cis*-acting element (CAAT-box) are present in the 0.5 kb upstream regions of all nine *UGlcAE* genes. Light responsive *cis*-acting regulatory elements (GATA-motif, chs-CMA1a, G-box) and enhancer (TA-rich region) could also be found.

#### upstream regions of *SlUGlcAE1*, *SlUGlcAE3,* and *SlUGlcAE5*. Furthermore, defense- and *2.10. Expression Patterns of SlUGlcAE Family Genes in Response to IAA, GA and SA*

*SlUGlcAE3-like2*, five in *SlUGlcAE5*, four in *SlUGlcAE6*, and zero in *SlUGlcAE6-like*).

(GATA-motif, chs-CMA1a, G-box) and enhancer (TA-rich region) could also be found.

*2.10. Expression Patterns of SlUGlcAE Family Genes in Response to IAA, GA and SA*

stress-response element (TC-rich repeats) was identified in the 0.5 kb upstream regions of four *SlUGlcAE* genes (*SlUGlcAE1-like*, *SlUGlcAE3-like1*, *SlUGlcAE3-like2*, and *SlUGlcAE5*), and circadian element (circadian) was found in the 0.5 kb upstream regions of three *SlUGlcAE* genes (*SlUGlcAE1-like*, *SlUGlcAE3-like1,* and *SlUGlcAE3-like2*), and endosperm expression-related element (Skn-1\_motif) was discovered in the 0.5 kb upstream regions of two *SlUGlcAE* genes (*SlUGlcAE1* and *SlUGlcAE6*), and other 11 elements are all present in the 0.5 kb upstream regions of only one *SlUGlcAE* gene. Four elements are located in 0.5 kb upstream region of *SlUGlcAE1*, and element numbers are diversified in the 0.5 kb upstream region of other genes, respectively (four in *SlUGlcAE1-like*, three in *SlUGlcAE2-like*, one in *SlUGlcAE3*, two in *SlUGlcAE3-like1*, four in Plant hormones, such as IAA, GA, and SA are used as endogenous messengers in response to biotic and abiotic stresses in plants [33]. It has been reported that the treatments of plants by exogenous hormones often lead to transient and rapid transcriptional changes in the whole genome [34]. According to *cis*-acting elements analysis of the *SlUGlcAE* genes upstream, three *cis*-acting elements that are related to plant hormones (IAA, GA, and SA) are located in 0.5 kb upstream genomic sequences of *SlUGlcAE1*, *SlUGlcAE2-like*, and *SlUGlcAE5*, respectively (Figure 9A). Thus, we investigated the expression profiles of *SlUGlcAE1*, *SlUGlcAE2-like,* and *SlUGlcAE5* with IAA, GA, and SA treatments (Figure 9B–D).

and development-unrelated *cis*-acting elements have also been identified. For example, core promoter element (TATA-box) and common *cis*-acting element (CAAT-box) are present in the 0.5 kb upstream regions of all nine *UGlcAE* genes. Light responsive *cis*-acting regulatory elements

Plant hormones, such as IAA, GA, and SA are used as endogenous messengers in response to biotic and abiotic stresses in plants [33]. It has been reported that the treatments of plants by

No of stress-, hormone-, and development-related element was found in the 0.5 kb upstream region of *SlUGlcAE6-like* gene, and the previous spatial expression patterns showed *SlUGlcAE6-like* was lowly expressed at every stages, which are highly consistent. Thus, we infer that *SlUGlcAE6-like* 

exogenous hormones often lead to transient and rapid transcriptional changes in the whole genome [34]. According to *cis*-acting elements analysis of the *SlUGlcAE* genes upstream, three *cis*-acting elements that are related to plant hormones (IAA, GA, and SA) are located in 0.5 kb upstream genomic sequences of *SlUGlcAE1*, *SlUGlcAE2-like*, and *SlUGlcAE5*, respectively (Figure 9A). Thus,

**Figure 9.** The detection of expressional level of *SlUGlcAE* genes after plant hormone treatments. (**A**) Hormone-related *cis*-elements prediction in the upstream of three *SlUGlcAE* genes (TGA-element: auxin-responsive element, TATC-box: gibberellin-responsive element, TCA-element: salicylic acid-responsive element); (**B**) The qPCR expression analysis of *SlUGlcAE1* response to plant hormone treatments; (**C**) Responses of *SlUGlcAE2-like* to plant hormones; (**D**) Expression profile analysis of *SlUGlcAE5* under plant hormone treatments; (**E**) Expression of *SlUGlcAE1-like* after hormone treatments; (**F**) Expression level of *SlUGlcAE3* after three hormones treatments; (G) Responses of *SlUGlcAE3-like1* under hormone treatments; (**H**) Expression profile analysis of *SlUGlcAE3-like2* to plant hormones; (**I**) The expression analysis of *SlUGlcAE6* under three hormones treatments; and, (**J**) Expression pattern of *SlUGlcAE6-like* response to plant hormone treatments. The error bars represent the SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, Ns: No significant. **Figure 9.** The detection of expressional level of *SlUGlcAE* genes after plant hormone treatments. (**A**) Hormone-related *cis*-elements prediction in the upstream of three *SlUGlcAE* genes (TGA-element: auxin-responsive element, TATC-box: gibberellin-responsive element, TCA-element: salicylic acid-responsive element); (**B**) The qPCR expression analysis of *SlUGlcAE1* response to plant hormone treatments; (**C**) Responses of *SlUGlcAE2-like* to plant hormones; (**D**) Expression profile analysis of *SlUGlcAE5* under plant hormone treatments; (**E**) Expression of *SlUGlcAE1-like* after hormone treatments; (**F**) Expression level of *SlUGlcAE3* after three hormones treatments; (G) Responses of *SlUGlcAE3-like1* under hormone treatments; (**H**) Expression profile analysis of *SlUGlcAE3-like2* to plant hormones; (**I**) The expression analysis of *SlUGlcAE6* under three hormones treatments; and, (**J**) Expression pattern of *SlUGlcAE6-like* response to plant hormone treatments. The error bars represent the SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, Ns: No significant.

The expression of *SlUGlcAE1* in response to IAA was increased at 1 h, decreased at 3 h, and increased thereafter, including 6 and 12 h, and returned to background level until 24 h (Figure 9B). On the whole, the expressional level of *SlUGlcAE1* gene was up-regulated after IAA treatment, although accompanying an unclear reason of being down-regulated only at 3 h. The expressional level of *SlUGlcAE1* gene was not significant different when comparing to the control in response to GA and SA throughout the treated process (Figure 9B). Combining to the result analysis of *cis*-acting elements in *SlUGlcAE1* gene upstream, we found that *SlUGlcAE1* expressional regulation was consistent with TGA *cis*-acting elements existing in the upstream of this gene, which is an auxin-responsive element, while GA or SA responsive element is non-existent (Figure 9A). This indicated that the expression of *SlUGlcAE1* gene could be regulated by IAA, but it could not be regulated by GA and SA (Figure 9B). The *SlUGlcAE2-like* gene under IAA treatment was down-regulated at 6 and 24 h, and no obvious difference was found at other time points (Figure 9C). The change of *SlUGlcAE2-like* expression pattern was unobvious under SA treatment. *SlUGlcAE2-like* showed increasingly strong down-regulation in expression at 6, 12 and 24 h, while similar expression level to the control within the first 3 h under GA treatment (Figure 9C). These results indicated that the expression of *SlUGlcAE2-like* gene could be regulated by GA, and be regulated by IAA irregularly, but not be regulated by SA (Figure 9C). This is aligned with the previous *cis*-acting elements analysis result. That is the fact that there are a gibberellin-responsive element (TATC-box) within 0.5 kb genomic sequences of *SlUGlcAE2-like* gene upstream (Figure 9A) and another gibberellin-responsive element (GARE-motif) within 1.5 kb genomic sequences outside 0.5 kb genomic sequences of *SlUGlcAE2-like* gene upstream (Table S2). There is an auxin-responsive element (AuxRR-core) within 1.5 kb genomic sequences outside 0.5 kb genomic sequences of *SlUGlcAE2-like* gene upstream (Table S2). *SlUGlcAE5* after IAA treatment showed a similar expression level to the control at different time points (Figure 9D). The expression level of *SlUGlcAE5* after GA treatment was no significant change in the first 12 h, but it reduced at 24 h with unknown reasons). *SlUGlcAE5* was up-regulated at 1 h, not affected at 3 h, and up-regulated continuously at later time points, including 6, 12, and 24 h after SA treatment (Figure 9D). The results suggested the expression of *SlUGlcAE5* gene could indeed be regulated by SA. It keeps consistent with the result of *cis*-acting elements analysis, which has a salicylic acid-responsive element (TCA element) in 0.5 kb genomic sequences of *SlUGlcAE5* gene upstream (Figure 9A). Taken together, these data suggest that although the result of *cis*-acting elements analysis may contain a few false positives, the prediction of these three hormone response sites in our study is still relatively reliable.

In addition, we also examined the expression level of the other six *UGlcAE* genes after three hormones treatments. The results were shown in Figure 9E–J. Both *SlUGlcAE1-like* and *SlUGlcAE3* were down-regulated by SA (Figure 9E,F). *SlUGlcAE3-like2* was up-regulated by the three hormones within 12 h of hormone treatment. However, it was down-regulated by three hormones at 24 h (Figure 9H). *SlUGlcAE3-like1*, *SlUGlcAE6*, and *SlUGlcAE6-like* were affected by different hormones at different times after hormones treatments (Figure 9G,I,J).
