Genome-Wide Identification and Expression Analysis of the UGlcAE Gene Family in Tomato

The UGlcAE has the capability of interconverting UDP-d-galacturonic acid and UDP-d-glucuronic acid, and UDP-d-galacturonic acid is an activated precursor for the synthesis of pectins in plants. In this study, we identified nine UGlcAE protein-encoding genes in tomato. The nine UGlcAE genes that were distributed on eight chromosomes in tomato, and the corresponding proteins contained one or two trans-membrane domains. The phylogenetic analysis showed that SlUGlcAE genes could be divided into seven groups, designated UGlcAE1 to UGlcAE6, of which the UGlcAE2 were classified into two groups. Expression profile analysis revealed that the SlUGlcAE genes display diverse expression patterns in various tomato tissues. Selective pressure analysis indicated that all of the amino acid sites of SlUGlcAE proteins are undergoing purifying selection. Fifteen stress-, hormone-, and development-related elements were identified in the upstream regions (0.5 kb) of these SlUGlcAE genes. Furthermore, we investigated the expression patterns of SlUGlcAE genes in response to three hormones (indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA)). We detected firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. Here, we systematically summarize the general characteristics of the SlUGlcAE genes in tomato, which could provide a basis for further function studies of tomato UGlcAE genes.


Introduction
As a major component of the primary cell walls of plants [1], pectins are essential for remodeling cell wall and normal cell-cell adhesion during cellular growth [2][3][4][5]. D-galacturonic acid (GalA) is the constituent of the capsular polysaccharides and lipopolysaccharides of several bacterial species [6]. In plants, GalA residues, which are the precursor of pectin formation, are contained in the backbone of all pectin polymers [7]. UDP-D-galacturonic acid (UDP-GalA), which is the activated nucleotide sugar form of GalA, is required in the synthesis of GalA-containing polymers. UDP is the abbreviation of uridine diphosphate and it is a nucleotide diphosphate that is made up of a pyrophosphate group, a pentose ribose, and a nucleated base uracil. UDP-GalA is synthesized via 4-epimerization of UDP-D-glucuronic acid (UDP-GlcA), which is a nucleotide sugar that is formed by the reputed inositol oxygenation pathway [8] or by the dehydrogenation of UDP-D-glucose (UDP-Glc) in the upstream [9]. Therefore, enzymes that are related to the formation of UDP-GalA and UDP-GlcA are likely to play critical roles in pectin biosynthesis [7,10].

The Identification of UGlcAE Gene Family in Tomato
To identify the UGlcAE genes in the tomato, we searched for sequences that contained the particular domain in the tomato protein database using the hidden Markov model (HMM) model of PF01370, and we found nine potential genes ( Table 1). The open reading frame (ORF) lengths of UGlcAE that were identified in this study ranged from 1221 bp to 1359 bp, encoding peptides varied from 406 to 452 amino acids (aa). All nine UGlcAE genes had a single exon.

Phylogenetic Analysis of the UGlcAE Genes in Tomato and Other Species
To evaluate the classification of the UGlcAE genes in S. lycopersicum, we analyzed the sequence features in 10 different species, including S. lycopersicum, C. sativus, C. annuum, S. tuberosum, A. thaliana, N. tabacum, P. trichocarpa, S. pennelli, Z. mays, and A. lyrata subsp. lyrata, and we constructed a unrooted phylogenetic tree of the UGlcAE genes ( Figure 1) using the N-J methods. The orthologous relationships were evident. Only the tree topology is shown, and the branch lengths do not represent the estimated numbers of amino acid replacements [28]. 1 Figure 1. Phylogenetic analysis of the UGlcAE gene family based amino acids in tomato and other nine species. The unrooted neighbor-joining phylogenetic tree is generated by MEGA 5. The sequence names included three parts: the source numbers from NCBI, the abbreviation of species names, and their respective subfamilies. Red dots highlight the tomato UGlcAE genes.
Combing with the sequence similarity of the mature proteins, the employed UGlcAE genes are distributed into seven groups ( Figure 1). In addition to UGlcAE2 being divided into two clusters, the other five subfamilies are clustered separately.
Interestingly, the UGlcAE4 subfamily is specifically present in A. thaliana and A. lyrata subsp. lyrata, whereas it is absent from other species in this study. This implies that UGlcAE4 may be associated with distinctive functions. It is noteworthy that UGlcAE2 genes were classified into two different groups based on their evolutionary relationship. This finding indicates that UGlcAE2 genes may evolve into new features, which have not been known until today. Moreover, phylogenetic analyses showed that the UGlcAE3 gene in A. thaliana was clustered together with UGlcAE2 genes of A. thaliana and A. lyrata subsp. lyrata, suggesting that there may be some gene fusion among them.

Structures of the UGlcAE Genes in the Tomato and Arabidopsis Thaliana
Introns, especially UTR introns, in UGlcAE genes may influence the expression level [29]. To analyze the structural characteristics of the UGlcAE genes in tomato and Arabidopsis thaliana, their gene structures were mapped according to the genome sequences and corresponding coding sequences of SlUGlcAE and AtUGlcAE genes ( Figure 2). We found that all of the SlUGlcAE and AtUGlcAE genes do not contain intron in their genomic sequences. In other words, nine UGlcAE genes in the tomato and six UGlcAE genes in Arabidopsis thaliana are single exon structures. distributed into seven groups ( Figure 1). In addition to UGlcAE2 being divided into two clusters, the other five subfamilies are clustered separately.
Interestingly, the UGlcAE4 subfamily is specifically present in A. thaliana and A. lyrata subsp. lyrata, whereas it is absent from other species in this study. This implies that UGlcAE4 may be associated with distinctive functions. It is noteworthy that UGlcAE2 genes were classified into two different groups based on their evolutionary relationship. This finding indicates that UGlcAE2 genes may evolve into new features, which have not been known until today. Moreover, phylogenetic analyses showed that the UGlcAE3 gene in A. thaliana was clustered together with UGlcAE2 genes of A. thaliana and A. lyrata subsp. lyrata, suggesting that there may be some gene fusion among them.

Structures of the UGlcAE Genes in the Tomato and Arabidopsis Thaliana
Introns, especially UTR introns, in UGlcAE genes may influence the expression level [29]. To analyze the structural characteristics of the UGlcAE genes in tomato and Arabidopsis thaliana, their gene structures were mapped according to the genome sequences and corresponding coding sequences of SlUGlcAE and AtUGlcAE genes ( Figure 2). We found that all of the SlUGlcAE and AtUGlcAE genes do not contain intron in their genomic sequences. In other words, nine UGlcAE genes in the tomato and six UGlcAE genes in Arabidopsis thaliana are single exon structures.

Chromosomal Distribution of the UGlcAE Genes in Tomato
To characterize the distribution of UGlcAE genes in the tomato genome, the physical locations of UGlcAE genes on the tomato chromosomes were obtained. According to the genomic sequences of UGlcAE genes, nine SlUGlcAE genes were mapped to eight chromosomes, including chromosome 1, 3, 5, 7, 8, 9, 10, and 12 without regularities of tandem duplication, whose positions were indicated by the black lines in the tomato chromosomes ( Figure 3). Two UGlcAE genes (SlUGlcAE3-like1 and SlUGlcAE6-like) were located on chromosome 5, and the other seven genes (SlUGlcAE1, SlUGlcAE1-like, SlUGlcAE2-like, SlUGlcAE3, SlUGlcAE3-like2, SlUGlcAE5, and SlUGlcAE6) were assigned to different chromosomes, but no gene was mapped to chromosome 2, 4, 6, and 11. There were no tandem duplication events among UGlcAE family members of tomato, suggesting that the functional differentiation may exist among the SlUGlcAE family members. Almost all of the UGlcAE genes in tomato are located near the ends of the chromosome.

Chromosomal Distribution of the UGlcAE Genes in Tomato
To characterize the distribution of UGlcAE genes in the tomato genome, the physical locations of UGlcAE genes on the tomato chromosomes were obtained. According to the genomic sequences of UGlcAE genes, nine SlUGlcAE genes were mapped to eight chromosomes, including chromosome 1, 3,5,7,8,9,10, and 12 without regularities of tandem duplication, whose positions were indicated by the black lines in the tomato chromosomes ( Figure 3). Two UGlcAE genes (SlUGlcAE3-like1 and SlUGlcAE6-like) were located on chromosome 5, and the other seven genes (SlUGlcAE1, SlUGlcAE1-like, SlUGlcAE2-like, SlUGlcAE3, SlUGlcAE3-like2, SlUGlcAE5, and SlUGlcAE6) were assigned to different chromosomes, but no gene was mapped to chromosome 2, 4, 6, and 11. There were no tandem duplication events among UGlcAE family members of tomato, suggesting that the functional differentiation may exist among the SlUGlcAE family members. Almost all of the UGlcAE genes in tomato are located near the ends of the chromosome.

Sequence Alignments and Hydrophilicity Analysis of SlUGlcAE Family
UGlcAE is one of the short-chain dehydrogenase/reductase (SDR) enzyme families, and therefore the amino acid sequences of SlUGlcAE and AtUGlcAE contained two conserved motifs that existed in SDR protein families [30,31]. As shown in Figure 4, the two motifs contain an N-terminal GxxGxxG (x represents any amino acid) sequence to bind to NAD (P) + , and a motif (YxxxK), which play a catalytic role [32].
As indicated in Supplementary Materials Figure S1, all of the SlUGlcAE proteins were trans-membrane proteins. The result was consistent with previous reports [13,14,24]. Among the nine proteins, most of SlUGlcAEs had only one trans-membrane helice with more than 80% probability, except that SlUGlcAE1 and SlUGlcAE5 were more likely to contain two trans-membrane helices.

Sequence Alignments and Hydrophilicity Analysis of SlUGlcAE Family
UGlcAE is one of the short-chain dehydrogenase/reductase (SDR) enzyme families, and therefore the amino acid sequences of SlUGlcAE and AtUGlcAE contained two conserved motifs that existed in SDR protein families [30,31]. As shown in Figure 4, the two motifs contain an N-terminal GxxGxxG (x represents any amino acid) sequence to bind to NAD (P) + , and a motif (YxxxK), which play a catalytic role [32].
As indicated in Supplementary Materials Figure S1, all of the SlUGlcAE proteins were trans-membrane proteins. The result was consistent with previous reports [13,14,24]. Among the nine proteins, most of SlUGlcAEs had only one trans-membrane helice with more than 80% probability, except that SlUGlcAE1 and SlUGlcAE5 were more likely to contain two trans-membrane helices.

Spatiotemporal Expression Patterns Analysis of UGlcAE Genes in Tomato
To gain the expression patterns of UGlcAE genes in different tissues and organs of tomato, and in the developmental stages of the fruit, we performed expression patterns analysis of the SlUGlcAE genes ( Figure 5) with the RNA-Seq database on the website of the functional genomics database of the tomato plant. The expression profiles of the nine tomato UGlcAE genes showed different patterns of temporal-and tissue-specific expression ( Figure 5). The results of the tomato cultivar showed that three genes, including SlUGlcAE1, SlUGlcAE1-like, and SlUGlcAE6, were strongly expressed in the bud, flower, leaf, root, and most fruit ripening stages ( Figure 5A). The same three genes (SlUGlcAE1, SlUGlcAE1-like, SlUGlcAE6) showed a similar expression characteristic in the cultivated tomato, with a lower expression at the breaker stage ( Figure 5A). In addition, SlUGlcAE1 and SlUGlcAE6 had a high expression in leaf and most fruit development stages, and SlUGlcAE1-like and SlUGlcAE3 exhibited a high expression in leaf of currant tomato ( Figure 5B). Specifically, SlUGlcAE6-like exhibited a very low expression level in root, bud, leave, flower, and fruit of the cultivar tomato and wild relative Solanum pimpinellifolium plants ( Figure 5A,B). The black backgrounds indicate the strictly conserved residues, and the gray backgrounds indicate the similar amino acid residues. The GxxGxxG and YxxxK motifs are marked above the sequence alignment in red.

Spatiotemporal Expression Patterns Analysis of UGlcAE Genes in Tomato
To gain the expression patterns of UGlcAE genes in different tissues and organs of tomato, and in the developmental stages of the fruit, we performed expression patterns analysis of the SlUGlcAE genes ( Figure 5) with the RNA-Seq database on the website of the functional genomics database of the tomato plant. The expression profiles of the nine tomato UGlcAE genes showed different patterns of temporal-and tissue-specific expression ( Figure 5). The results of the tomato cultivar showed that three genes, including SlUGlcAE1, SlUGlcAE1-like, and SlUGlcAE6, were strongly expressed in the bud, flower, leaf, root, and most fruit ripening stages ( Figure 5A). The same three genes (SlUGlcAE1, SlUGlcAE1-like, SlUGlcAE6) showed a similar expression characteristic in the cultivated tomato, with a lower expression at the breaker stage ( Figure 5A). In addition, SlUGlcAE1 and SlUGlcAE6 had a high expression in leaf and most fruit development stages, and SlUGlcAE1-like and SlUGlcAE3 exhibited a high expression in leaf of currant tomato ( Figure 5B). Specifically, The black backgrounds indicate the strictly conserved residues, and the gray backgrounds indicate the similar amino acid residues. The GxxGxxG and YxxxK motifs are marked above the sequence alignment in red.

GO Analysis of the UGlcAE Genes in the Tomato
In order to compare the product functions of UGlcAE genes in tomato and Arabidopsis, we analyzed SlUGlcAE genes and its six orthologous genes in Arabidopsis. As it is shown in the gene ontology (GO) map ( Figure 6), all of the UGlcAE genes of A. thaliana are involved in cellular components, molecular functions, and biological processes. However, the SlUGlcAE genes just have roles in molecular functions and biological processes.

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SlUGlcAE6-like exhibited a very low expression level in root, bud, leave, flower, and fruit of the cultivar tomato and wild relative Solanum pimpinellifolium plants ( Figure 5A,B).

GO Analysis of the UGlcAE Genes in the Tomato
In order to compare the product functions of UGlcAE genes in tomato and Arabidopsis, we analyzed SlUGlcAE genes and its six orthologous genes in Arabidopsis. As it is shown in the gene ontology (GO) map ( Figure 6), all of the UGlcAE genes of A. thaliana are involved in cellular components, molecular functions, and biological processes. However, the SlUGlcAE genes just have roles in molecular functions and biological processes.   tomato cultivar Solanum lycopersicum (mature green stage (MG), breaker stage (B), ten days after breaker stage (B10)); (B) wild relative Solanum pimpinellifolium (immature green stage (IMG), breaker stage (B), five days after breaker stage (B5)). The expression data was gained from pubic RNA-seq data and shown as log2 as calculated by FPKM values (fragments per kilo base of exon model per million) mapped reads. The green boxes represent the lower expression level, whereas the red boxes represent the higher expression level.

GO Analysis of the UGlcAE Genes in the Tomato
In order to compare the product functions of UGlcAE genes in tomato and Arabidopsis, we analyzed SlUGlcAE genes and its six orthologous genes in Arabidopsis. As it is shown in the gene ontology (GO) map ( Figure 6), all of the UGlcAE genes of A. thaliana are involved in cellular components, molecular functions, and biological processes. However, the SlUGlcAE genes just have roles in molecular functions and biological processes.  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.

Selective Pressure on UGlcAE Proteins in the Tomato
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 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).

Selective Pressure on UGlcAE Proteins in the Tomato
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 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).

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.

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.
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 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).
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-, 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.

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

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.

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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.

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.
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

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.
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].

Data Set Collection and Identification of SlUGlcAE Genes
The protein databases of all ten species were retrieved from the National Center for Biotechnology Information (NCBI) FTP site (available online: http://www.ncbi.nlm.nih.gov/Ftp/). The cDNA, CDS, and genome sequence data in tomato were downloaded from the Solanaceae Genomics Network (SGN) (available online: http://solgenomics.net) [37] and Tomato Functional Genomics Database (TFGD) (available online: http://ted.bti.cornell.edu) [38]. Other information and sequences of Arabidopsis thaliana UGlcAEs (AtUGlcAEs) were obtained from the Arabidopsis Information Resource (TAIR; available online: http://www.arabidopsis.org/) [39]. The UGlcAE proteins of tomato (SlUGlcAEs) were predicted depending on the UGlcAE hidden Markov model (HMM) profile from the Pfam database (available online: http://pfam.sanger.ac.uk/) [40], which was used to search the S. lycopersicum UGlcAE proteins sequences by the HMMSEARCH program from HMMER software (available online: http://hmmer.janelia.org) [41]. In the case of the uncompleted protein databases, all of the results were then used as queries in TBLASTN searches against the tomato genomic sequences. To further confirm UGlcAE proteins, the domains of candidate sequences were predicted with the Pfam online server (available online: http://pfam.sanger.ac.uk/) [40] and SMART online server (available online: http://smart.embl-heidelberg.de/) [42]. The tomato genomic sequences were also checked using BLASTP at the NCBI site (available online: http://blast.ncbi.nlm.nih.gov), retaining only those sequences with highly significant matches to annotated UGlcAE proteins. The same procedure was used to search UGlcAE family members in the protein databases of the following nine species: Cucumis sativus, Capsicum annuum, Solanum tuberosum, Arabidopsis thaliana, Nicotiana tabacum, Populus trichocarpa, Solanum pennelli, Zea mays, and Arabidopsis lyrata subsp. lyrata.
The tomato UGlcAE gene subfamilies were named according to the orthologous UGlcAE genes in the A. thaliana genome. The subfamilies of UGlcAE genes in the tomato were distinguished by Arabic numerals, and different members of a subfamily were designated with the numbers.

Phylogenetic Analysis
A phylogenetic tree of UGlcAE was constructed by analyzing full-length proteins from S. lycopersicum, C. sativus, C. annuum, S. tuberosum, A. thaliana, N. tabacum, P. trichocarpa, S. pennelli, Z. mays, and A. lyrata subsp. lyrata in the MEGA5 software (Center for Evolutionary Medicine and Informatics, Arizona State University, Tempe, AZ, USA) using the Neighbor-Joining method [43]. Bootstrap analysis was employed using 1000 replicates.

Selective Pressure Analysis on UGlcAE Proteins in the Tomato
The ratio of non-synonymous to synonymous substitutions (dN/dS; termed ω) at each codon site of each protein was identified, according to an empirical Bayesian method using the Server for the identification of site-specific positive selection and purifying selection (SELECTON version 2.4, Tel Aviv University, Tel Aviv, Israel [44,45]. Selection pressure analysis can be used to identify purifying or positive selection of specific areas in a sequence, and the sites that ω values significantly >1 or <1 suggest positive (Darwinian) or purifying, respectively [46]. The selection pressure acting on the coding sequences of the SlUGlcAE genes was recognized with the M8 model (extra category ωs ≥ 1, beta distribution, and positive selection allowed). In order to ensure the accuracy of the results, a likelihood ratio test was used to test the significance of the ω values [47], which compares two nested models: a null model that assumes no positive selection (M8a) and an alternative model that assumes positive selection (M8). Non-nested models, including M8a (extra category ωs set to 1) and MEC (positive selection allowing model), were also used in the pressure analysis.

Gene Structure Analysis
To analyze gene structure, the exon, and intron structures of SlUGlcAE and AtUGlcAE genes were generated using the Gene Structure Display Server 2.0 (available online: http://gsds.cbi.pku.edu. cn) [48] by aligning the CDS sequences with the corresponding genomic DNA sequences from the SGN (available online: http://solgenomics.net/) [37].

Analysis of Expression Profile of UGlcAE Genes in Tomato Various Tissues
The expression profile was obtained through analyzing microarray data. The microarray data were downloaded from the Tomato Functional Genomics Database (available online: http://ted. bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi) [38], including the UGlcAE genes expression in 10 tissues (bud, flower, leaf, root, 1 cm fruit, 2 cm fruit, 3 cm fruit, mature green stage (MG) fruit, breaker stage (B) fruit, and ten days after breaker stage (B10) fruit) of the tomato cultivar (Solanum lycopersicum), and four tissues (immature green stage (IMG) fruit, breaker stage (B) fruit, five days after breaker stage (B5) fruit, and leaf) of the wild species (Solanum pimpinellifolium). Only genes with an at least five units average expression signal at one time point and the similar trend in different biological replicates were considered to be expressed at the time point. The expression patterns of the SlUGlcAE genes were estimated by intensity values and were visualized using MultiExperiment Viewer (Broad Institute of MIT and Harvard University, Boston, MA, USA [51].

Sequence Alignments and Prediction of Transmembrane Domains of SlUGlcAE Family
All nine SlUGlcAE and six AtUGlcAE protein sequences were aligned using the (version 5.0.6, North Carolina State University, Raleigh, NC, USA), and then the results were output by genedoc program. Next, the hydrophilicity of the SlUGlcAE protein sequences was predicted by the trans-membrane Hidden Markov model algorithm (available online: http://www.cbs.dtu.dk/ services/TMHMM/) [52].

The Analysis of SlUGlcAE Family Protein Domains
The domains of UGlcAE family proteins in tomato were analyzed by the Pfam (available online: http://pfam.xfam.org/search) [40].

Hormone Treatments
S. lycopersicum plants were grown at 25 ± 2 • C with a 12 h light/dark photoperiod. The humidity was maintained at approximately 60% to 70%, and the photosynthetic photon flux density was controlled at about 120 µmol photons/m 2 /s. When the seedlings were six weeks old, the plants were treated with IAA (100 µM), GA (100 µM) and SA (100 µM), respectively [54]. Plant leaves were collected at 0, 1, 3, 6, 12, and 24 h after treatments, immediately frozen in liquid nitrogen, and then stored at −80 • C until use.

Plant Materials
Other S. lycopersicum seedlings were grown in the same conditions with the above mentioned seeds (see Section 4.11). Different fruits were harvested in the following five stages: immature green stage (IMG), mature green stage (MG), breaker stage (B), four days after breaker stage (B + 4), and seven days after breaker stage (B + 7). All of the plant samples were retrieved at the same time each day, and then frozen in liquid nitrogen and stored at −80 • C.

Real-Time PCR
Total RNA was extracted from the leaves using the Total RNA Kit (BioTeke Corporation, Beijing, China), following the manufacturer's instructions. Integrity of the RNA was verified by agarose gel electrophoresis. Synthesis of the cDNA was performed from the total RNA samples using the PrimeScript™ RT Reagent Kit, according to the protocol with gDNA Eraser (TaKaRa, Dalian, China). All of the primer sequences are shown in Table 2. EF1α gene was used as the internal control under abiotic stress [55], and the SlCAC gene was selected as an internal standard during tomato development [56] to quantitate the expression of SlUGlcAE genes. Real-time PCR was performed using CFX96 Touch™ real-time PCR system (Bio-Rad, Hercules, CA, USA) with a SYBR Premix Ex Taq™ II Kit (Bio-Rad). The reactions were carried out in the following conditions: denaturation at 94 • C for 4 min, 40 cycles of 5 s at 95 • C, 30 s at 60 • C, 15 s at 95 • C, 20 s at 60 • C, and 15 s at 95 • C. Three biological duplications were used. The 2 −∆∆Ct method was used to visualize and analyze the real-time PCR data [57,58].

Fruit Firmness Measurement and Determination of Water-Soluble Pectin Content
As described by Wu and Abbott [59], the fruit firmness was quantified using a Firmness tester (GY-2). Fifteen unbroken tomatoes were taken from each group. The equator of the fruit was placed under a flat probe, and the maximum value was read after pressing down. Each fruit was measured at least three times. Test parameters: Probe pressure rate 1 mm/s, Pressing distance 3 mm.
The content of pectin in tomato fruit was detected by the water-soluble pectin content kit of Suzhou Keming Biotechnology Co., Ltd. (Suzhou, China The principle of determination is to use the acid solution to extract water-soluble pectin, and to determine the content of pectin by carbazole colorimetry. Pectin is hydrolyzed to galacturonic acid, which condenses with carbazole reagent in sulfuric acid solution. The resulting material has a maximum absorption peak at 530 nm.

Conclusions
The Solanaceae genus is one of the most morphologically various plant families, with more than 3000 described species being distributed worldwide [60]. Apart from being of economic value, tomato is also a model crop for fleshy fruit development [35,61]. In addition, tomato is still the first horticultural crop for which its genome has been sequenced [62]. In this study, we identified nine SlUGlcAE genes and analyzed the spatiotemporal expression patterns, the phylogenetic relationships, the selective pressure, the cis-acting elements, and so on. We also focused on the response patterns of nine SlUGlcAE genes to IAA, GA, and SA, according to the results of cis-acting elements analysis. Moreover, the firmness decreased gradually, and WSP showed an increased trend, reached a maximum at the MG stage, and then decreased in the development of tomato fruit. All of the results above have allowed for us to identify tomato orthologs that are related to known UGlcAE genes in Arabidopsis for in-depth studies. It would also accelerate for executing functional studies based genomics to elucidate their elaborate roles in tomato fruit development, and to be helpful for revealing the roles of other members in the Solanaceae genus.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/ 1583/s1. Author Contributions: X.Z., X.D. conceived and designed the study; X.D. collected data, processed and analyzed the data, created the figures, wrote the manuscript and completed the submission; J.L. and Y.P. helped design the experiments, provided the research facility and helped write the manuscript; Y.Z., L.N. and Y.W. provided daily care to the plant and helped collect plant leaves at different times.