Genome-wide identification and evolution of HECT genes in wheat

Introduction

Ubiquitination is a post-translational modification that involves the covalent attachment of ubiquitin to a protein substrate. Ubiquitination is important for cellular homeostatic maintenance and plays essential roles in plant growth, development, and the regulation of abiotic and biotic stresses (Downes et al., 2003; El Refy et al., 2003; Liu & Stone, 2011; Miao & Zentgraf, 2010; Moon, Parry & Estelle, 2004; Rotin & Kumar, 2009; Stone, 2014; Wang & Deng, 2011). At the end of a three-enzyme cascade (E1 ubiquitin activating enzyme; E2 ubiquitin conjugating enzyme; E3 ubiquitin ligase), E3 recruits substrates and promotes or directly catalyzes ubiquitin transfer onto its targets (Huibregtse et al., 1995; Scheffner, Nuber & Huibregtse, 1995; Wang et al., 2006; Wang & Pickart, 2005). E3 generally determines the specificity of the ubiquitination reaction with different substrate recognition domains (Huibregtse et al., 1995; Kim et al., 2007; Scheffner, Nuber & Huibregtse, 1995). According to structural similarities and ubiquitination domains, plant E3 proteins can be classified as three main types (Chen & Hellmann, 2013; Craig et al., 2009; Duplan & Rivas, 2014; Guzman, 2014; Huibregtse et al., 1995; Mach, 2008; Maspero et al., 2013; Qin et al., 2008; Scheffner, Nuber & Huibregtse, 1995; Schwechheimer & Calderon Villalobos, 2004; Stone, 2014; Wang et al., 2006; Wang & Pickart, 2005; Yee & Goring, 2009): HECT (Homologous to E6-AP C-Terminal), RING (Really Interesting New Gene), and U-box.

The HECT-type ubiquitin ligase is an important class of E3s defined by the presence of a C-terminal catalytic HECT domain. The general features of HECT domains are an N-terminal lobe that contains the E2-binding site and a smaller C-terminal lobe that includes the active-site Cys residue that receives ubiquitin from E2 and links itself with the ubiquitin molecule (Downes et al., 2003; Huibregtse et al., 1995). Classification of HECT E3 proteins into different subfamilies is based on the N-terminal domains (Downes et al., 2003; Grau-Bove, Sebe-Pedros & Ruiz-Trillo, 2013; Marin, 2010; Marin, 2013) responsible for recognizing and binding protein substrates (Kamadurai et al., 2013; Kim et al., 2011; Maspero et al., 2011; Maspero et al., 2013; Rotin & Kumar, 2009), while the conserved C-terminal HECT domain catalyzes the transfer of ubiquitin to various substrates. Substrate proteins usually possess recognition motifs that can directly bind to the N-terminal domains, while the special HECT domains are essential to the prediction and evolution of the HECT genes in plants; however, comprehensive research on these genes is limited.

The HECT-type E3 ubiquitin ligases comprise a small class of E3s, and seven genes (UPL1-UPL7) have been identified in Arabidopsis thaliana (Downes et al., 2003). UPL3 is involved in trichome development (Downes et al., 2003; Patra, Pattanaik & Yuan, 2013), genome endoreduplication (El Refy et al., 2003) and seed size (Miller et al., 2019). UPL5 is involved in leaf senescence (Miao & Zentgraf, 2010), and UPL1, UPL3, and UPL5 in plant immunity (Furniss et al., 2018). These seven A. thaliana HECT genes can be classified into five subfamilies or six groups according to the phylogenetic relationships provided in previous studies (Marin, 2013; Meng et al., 2015). However, little research has been conducted on the HECT genes in wheat, which is one of the most important crops produced worldwide (Choulet et al., 2014; International Wheat Genome Sequencing Consortium, 2014; International Wheat Genome Sequencing Consortium et al., 2018). In this research, we conducted a comprehensive genome-wide analysis of the wheat HECT genes to identify HECT genes conserved in wheat, rice, and A. thaliana. Gene exon-intron structure, conserved motif, domain structure, chromosomal distribution, duplication event, and expression profile were also analyzed in detail. Our research data will provide useful information for further functional investigation of the HECT gene family in allohexaploid wheat and their evolution in polyploid plants.

Materials & Methods

Results

Phylogenetic analysis of HECT genes in wheat

To understand the evolutionary relationship of the wheat HECT genes, phylogenetic trees were constructed based on the alignment of the full-length protein sequences and HECT domain sequences of 25 wheat, 7 rice, and 7 A. thaliana HECT proteins (Fig. 1 and Fig. S1). According to the classification criteria used for A. thaliana and rice in previous studies (Downes et al., 2003; Grau-Bove, Sebe-Pedros & Ruiz-Trillo, 2013; Marin, 2013; Meng et al., 2015), the wheat HECT genes were categorized into seven groups (Group I, II, III, IV, V, VI and VII), which contained 0, 3, 5, 5, 3, 5 and 4 HECT genes, respectively. Genes in Group III, IV and VI were the most abundant and comprised 60% of the identified genes, while genes in Group I was absent in wheat. Nevertheless, in A. thaliana, Group I included two HECT genes, Group II did not include any HECT genes, and other Groups included one HECT gene, respectively. These seven groups can be further classified into five subfamilies that correspond to those described in a previous study (Marin, 2013).

Gene exon-intron structure and conserved motif and domain architecture of the wheat HECT genes

To investigate the structural characteristics of wheat HECT genes, the exon-intron structures of the wheat HECT genomic sequences, conserved motifs, and the domain architecture of the wheat HECT proteins were compared based on their phylogenetic relationships. Each gene structure was revealed by aligning its coding sequences with the corresponding genomic sequences (Chen et al., 2018; Hu et al., 2015). Most of the wheat HECT genes contained abundant (more than ten) exons and only those in Group III had only three or four exons (Fig. 2A). Closely related HECT genes in the same phylogenetic group had similar exon-intron structures, and those with closer evolutionary relationships were more similar in their number and length of exons and introns. The conserved motifs of wheat HECT proteins in each group were analyzed using MEME software (Bailey et al., 2015). Fifteen conserved motifs (motif1-motif15) were predicted and these motifs were specific to each group (Fig. 2B). The composition of the structural motifs varied among the different HECT groups, while similar motifs were found in the same group. Additionally, the motifs encoding the HECT domain in the C-terminal regions of wheat HECT proteins were relatively conserved, suggesting that the functions of the HECT proteins were intergroup specific. The domain architecture of HECT proteins was obtained using the InterProScan program (Jones et al., 2014) with a three-database annotation (El-Gebali et al., 2019; Letunic & Bork, 2018; Sigrist et al., 2013). In addition to the HECT domain, other domains were found in the N-terminal regions of wheat HECT proteins (Fig. 3). The wheat HECT genes that were derived from the same group generally had similar exon-intron structures (Fig. 2A), motif compositions (Fig. 2B), and domain architectures (Fig. 3).

Chromosomal location and duplication of wheat HECT genes

To decide the chromosomal locations of the wheat HECT genes, the 25 putative wheat HECT genes were located in the 21 chromosomes of the wheat genome database available from Ensembl Plants (Howe et al., 2020; International Wheat Genome Sequencing et al. 2018). The wheat HECT genes were randomly distributed in 17 of 21 chromosomes; chromosome 2D, 3A, 3D, and 7D contained no HECT genes, chromosome 1A contained three HECT genes, chromosome 1B, 1D, 5A, 5B, 5D, and 7B each contained two HECT genes, and the other chromosomes each contained only one HECT gene (Fig. 4). The 25 wheat HECT genes were approximately evenly distributed among the A (9), B (10), and D (6) subgenomes, which was in accordance with the observation that most HECT genes have three homoeologous sequences located across three subgenomes. However, the HECT genes were not randomly distributed among the different chromosomal groups of the three subgenomes. The chromosomal Group II, III, and VII contained two, one, and three sequences, respectively. The remaining 19 sequences were more evenly distributed across chromosomal Group I, IV, V, and VI, and ranged from three to seven genes per group. An interesting finding was that the location of the HECT genes on chromosome 4A was opposite with those of the homoeologous genes on chromosome 4B and chromosome 4D (Fig. 4).

Segmental and tandem duplication are considered two essential factors for gene family expansion in plants (Cannon et al., 2004; Panchy, Lehti-Shiu & Shiu, 2016; Qiao et al., 2019; Zhu et al., 2014). To examine duplication patterns of the wheat HECT genes, we identified tandem and segmental duplication events using MCscanX (Wang et al., 2012) employing default parameters with TBtools (Chen et al., 2018). No tandem duplicated HECT gene pairs were identified in the 25 wheat HECT genes; however, 21 of the 25 wheat HECT genes were involved in segmental duplication. Twenty segmental duplicated HECT gene pairs were identified (Fig. 4 and Table 2), indicating that the segmental duplication events had contributed to HECT gene family expansion. To date the gene duplication time of these segmentally duplicated HECT genes, the Ks and Ka distances, as well as the Ka/Ks ratios were calculated. The Ka/Ks ratios for segmentally duplicated HECT gene pairs ranged from 0.07 to 0.44, with an average value of 0.20 (Table 2), implying that these segmentally duplicated HECT genes were under purifying selection, as indicated by the Ka/Ks ratios were less than 1. The divergence time of duplication events were inferred by Ks (Table 2). Results showed that within six existed phylogenetic groups, the two closest wheat HECT genes were duplicated about 2–12 million years ago (Mya), while the other genes were duplicated about 100–112 Mya.

Table 2:

Estimates of the segmental duplication events in the wheat HECT gene pairs.

Group	Gene 1	Gene 2	Ka	Ks	Ka/Ks	Mya
II	TraesCS5A02G121600	TraesCS5B02G112800	0.01	0.04	0.16	2.93
	TraesCS5A02G121600	TraesCS5D02G118000	0.00	0.03	0.14	2.87
III	TraesCS5A02G262600	TraesCS5B02G261000	0.01	0.08	0.18	6.29
	TraesCS5A02G262600	TraesCS5D02G270200	0.01	0.07	0.18	6.09
	TraesCS5A02G262600	TraesCS7A02G244000	0.55	1.34	0.41	111.99
	TraesCS5B02G261000	TraesCS5D02G270200	0.01	0.06	0.23	4.79
	TraesCS5B02G261000	TraesCS7A02G244000	0.55	1.29	0.42	107.74
	TraesCS5D02G270200	TraesCS7A02G244000	0.55	1.24	0.44	103.15
IV	TraesCS1A02G106100	TraesCS1B02G123400	0.00	0.04	0.07	3.07
	TraesCS1A02G106100	TraesCS1D02G108900	0.00	0.03	0.15	2.44
	TraesCS1B02G123400	TraesCS1D02G108900	0.00	0.03	0.17	2.29
V	TraesCS4A02G285800	TraesCS4B02G027200	0.01	0.03	0.19	2.91
	TraesCS4A02G285800	TraesCS4D02G025000	0.02	0.05	0.30	4.37
	TraesCS4B02G027200	TraesCS4D02G025000	0.01	0.06	0.24	5.04
VI	TraesCS2A02G064700	TraesCS2B02G076900	0.00	0.03	0.15	2.66
	TraesCS6A02G003300	TraesCS6D02G005600	0.01	0.12	0.08	10.01
	TraesCS6B02G000300	TraesCS6D02G005600	0.01	0.13	0.07	11.03
VII	TraesCS1A02G288600	TraesCS1B02G298000	0.01	0.06	0.23	5.41
	TraesCS1A02G288600	TraesCS1D02G287600	0.01	0.05	0.11	4.25
	TraesCS1B02G298000	TraesCS1D02G287600	0.01	0.08	0.17	6.43

DOI: 10.7717/peerj.10457/table-2

Notes:

Ks

synonymous substitution rate

Ka

nonsynonymous substitution rate

Mya

million years ago

Expression profiles of wheat HECT genes

To discover the potential roles of these wheat HECT genes in growth and development, we used public RNA-seq data covering 15 tissues at different growth stages from expVIP (Borrill et al., 2019; Borrill, Ramirez-Gonzalez & Uauy, 2016; Ramírez-González et al., 2018). Based on the wheat RNA-seq data, the 25 wheat HECT genes were detected in all 15 tissues at the gene level (Fig. 5, Table S1, and Table S2). Moreover, the expression of these genes exhibits distinct expression and tissue-specific characteristics. Most HECT genes in Group II, IV, and VI were relatively highly expressed in the roots, stems and spikes, while those in the leaves were expressed at relatively low levels (Fig. 5). Interestingly, in wheat grain tissues, most wheat HECT gene expression in Group II, IV, and VI were high at 2 dpa and 30 dpa and low at 14 dpa. Moreover, genes within each group or in different groups had similar expression patterns in different tissues, such as the high expression of genes in Group II (TraesCS5A02G121600, TraesCS5B02G112800, TraesCS5D02G118000), Group IV (TraesCS1A02G106100, TraesCS1B02G123400, TraesCS1D02G108900), and Group VI (TraesCS6A02G003300, TraesCS6B02G000300, TraesCS6D02G005600, TraesCS2A02G064700, and TraesCS2B02G076900), except for TraesCS3B02G194900 and TraesCS7B02G313300 (Fig. 5). Furthermore, the genes in Group II, IV, and VI were relatively highly expressed in the spikes at different developmental stages and in stems at the one cm spike stage compared to those in other tissues (Fig. 5). According to RNA-seq data of the ten-time point expression time course of wheat senescence in the flag leaf, the expression level of most wheat HECT genes in Group II, IV, and VI gradually increased with the increase of dpa (Fig. 6, Table S3, and Table S4).

Discussion

HECT genes play important roles in A. thaliana and diverse plant growth, developmental and physiological processes (Downes et al., 2003; (El Refy et al., 2003); Furniss et al., 2018; Miao & Zentgraf, 2010; Miller et al., 2019; Patra, Pattanaik & Yuan, 2013), including trichome development (Downes et al., 2003), genome endoreduplication (El Refy et al., 2003), seed size (Miller et al., 2019), leaf senescence (Miao & Zentgraf, 2010), and plant immunity (Furniss et al., 2018). However, this gene family has not been investigated in wheat. In this research, we conducted an extensive analysis of the wheat HECT genes, including their evolution, gene exon-intron structure, conserved motif, domain structure, chromosomal location, duplication event, and expression pattern.

We identified 25 HECT genes in the wheat genome, which is 3.6 times the number present in A. thaliana (Downes et al., 2003). However, a former study discovered 19 soybean HECT genes, which is 2.7 times the number found in A. thaliana (Meng et al., 2015). Our results showed six more HECT genes in wheat than the number previously estimated in the soybean genome. A possible explanation for this difference is that wheat is a hexaploid crop with 21 chromosomes containing three subgenomes (A, B, and D) (International Wheat Genome Sequencing Consortium, 2014; International Wheat Genome Sequencing Consortium et al., 2018), while soybean is a diploid crop with 20 chromosomes derived from an ancient tetraploid that may have had about two times more the number of HECT genes than other normal diploid species (Schmutz et al., 2010).

The phylogenetic analysis of the 25 wheat HECT genes classified them into subfamilies similar to those characterized by previous research (Downes et al., 2003; Grau-Bove, Sebe-Pedros & Ruiz-Trillo, 2013; Marin, 2013; Meng et al., 2015). The classification was according to the corresponding HECT gene sequence homology. Based on the phylogenetic relationships among the HECT genes in wheat, rice, and A. thaliana, the wheat HECT genes were classified into seven groups. Compared with a former report in A. thaliana (Marin, 2013), subfamily IV HECT genes were absent in wheat. Wheat subfamily V (Group I, UPL1/2 and Group II, UPL8 in this study) contained three genes, subfamily VI (Group III, UPL5) contained five genes, subfamily III (Group IV, UPL6) contained five genes, Subfamily II (Group V, UPL7) contained three genes, and subfamily I (Group VI, UPL3 and Group VII, UPL4) contained nine genes. The HECT gene Group I was not observed in the wheat genome. With the exception of Group I and II, other wheat Groups own HECT genes orthologous with A. thaliana. This is basically consistent with the results of a previous HECT gene investigation in plants (Marin, 2013), suggesting that A. thaliana HECT gene Group II (UPL8 in this study) was lost, while the wheat HECT gene Group I (UPL1/UPL2) was not observed in our analysis. Gene members of each phylogenetic Group often possess identical gene exon-intron structures, conserved motifs, and domain architectures, indicating that they probably recognize, bind, and might interact with same or similar substrate protein.

Segmental duplication events, tandem duplication events, as well as transposition events are three main evolutionary mechanisms of duplication events that expand the members of gene family (Cannon et al., 2004; Panchy, Lehti-Shiu & Shiu, 2016; Qiao et al., 2019; Zhu et al., 2014). Segmental duplications frequently occur in higher plants, because they are diploidized polyploids that have maintained various duplicated chromosomal blocks in the existing genomes (Cannon et al., 2004; Qiao et al., 2019). In this present research, we discovered that 21 of the 25 wheat HECT genes were located in chromosomes across the three subgenomes (A, B, D), indicating that segmental duplication obviously contributed to expanding the wheat HECT gene family. A previous study has shown that the allohexaploid wheat subgenomes A, B, and D were originally derived from three diploid (2x; 2n = 14) species and underwent three hybridization events (International Wheat Genome Sequencing 2014). The A and B subgenomes diverged from a common ancestor ∼7 million years ago and the first hybridization occurred ∼5.5 million years ago between A and B subgenomes, leading to the D subgenome through homoploid hybrid speciation. The second hybridization between the A and B subgenomes gave rise to the AABB genome <0.8 million years ago via polyploidization. Wheat originated <0.4 million years ago by allopolyploidization from a third hybridization. By estimating the approximate dates of the segmentally duplicated pairs of wheat HECT genes, we infer that the paralogous genes in wheat HECT groups originated from a relatively recent duplication event during the shaping of the three subgenomes (A, B, D) that occurred before the second hybridization event in wheat evolution history, except for TraesCS7A02G244000 in Group III, which originated from a relatively ancient duplication event before the appearance of the common ancestor of the A and B subgenomes. Thus, segmental duplication events were the primary driving forces for HECT gene evolution during the speciation and evolution of allohexaploid wheat.

To better understand the roles of the HECT genes during the life cycle of wheat, we performed an expression analysis of public RNA-seq data (Borrill, Ramirez-Gonzalez & Uauy, 2016; Choulet et al., 2014; Ramírez-González et al., 2018) in 15 tissues at different developmental stages. Analysis of the expression patterns of these wheat genes in 15 tissues showed that most wheat HECT genes in Group II, IV, and VI were relatively highly expressed in the roots, stems, and spikes. In particular, the genes in Groups II, IV, and VI were relatively highly expressed in the spikes at different developmental stages and in stems at the 1-cm spike stage compared to other tissues. Therefore, the expression of these genes may be closely related to wheat spike growth and development, suggesting that the HECT genes in highly expressed spikes may be involved in the regulation or degradation of proteins via ubiquitination during spike development stage. Previous studies have revealed that A. thaliana AT4G38600/UPL3 plays a specific role during trichome development (Downes et al., 2003; Patra, Pattanaik & Yuan, 2013) and seed size (Miller et al., 2019) and that AT4G12570/UPL5 is an important transcription factor that positively regulates leaf senescence by the ubiquitination and degradation of AT4G23810/WRKY5 3 (Miao & Zentgraf, 2010). In our investigation, the wheat genes orthologous to A. thaliana AT4G38600/UPL3 included five paralogous genes in Group VI. Except for grain at the 14 dpa stage, these five genes were all relatively highly expressed in wheat, particularly in spikes. A reasonable explanation is that the relatively low expression of UPL3 at 14 dpa stage may be related to the size of wheat seeds and is an adaptive regulation mechanism during seed formation. This is consistent with a recent study of UPL3 in Brassica napus (Miller et al., 2019). Miller et al. determined a mechanism in which the proteasomal degradation of LEC2, a transcription factor controlling seed maturation, is mediated by UPL3 and reduced UPL3 expression would increase LEC2 protein levels and seed size. The wheat genes orthologous to A. thaliana AT4G12570/UPL5 were five paralogous genes in Group III, which were expressed in different wheat tissues but showed distinct features. At different developmental stages, the expression levels of TraesCS5D02G270200, TraesCS5A02G262600, and TraesCS5B02G261000 in roots, stems, and spikes were relatively unchanged, but gradually increased in leaves and decreased in grain. The three wheat genes TraesCS1A02G106100, TraesCS1B02G123400, and TraesCS1D02G108900 are orthologous to A. thaliana AT3G17205/UPL6 in Group IV, and the genes in Group II (UPL8 absent in A. thaliana) showed similar expression patterns to those in Group VI (UPL3). RNA-seq data of wheat leaf senescence (Borrill et al., 2019) indicated that HECT genes in Group II, IV and VI (UPL8, UPL6, and UPL3) might also play crucial roles in plant leaf senescence. The differential expression of paralogous HECT genes in or among groups in wheat suggests that they might have the same or similar functions as their orthologous genes in A. thaliana and Brassica napus; nevertheless, they might have evolved functional differences.

A former research discovered that AT4G38600/UPL3 mediated UPS-dependent proteolysis of the two transcription factors AT5G41315/GL3 and AT1G63650/EGL3, which interact with the ARM domains of UPL3 and function as positive regulators during A. thaliana trichome development (Patra, Pattanaik & Yuan, 2013). The evolutionarily closely related Group VII (UPL4) and VI (UPL3) belong to the same subfamily I defined in previous studies (Grau-Bove, Sebe-Pedros & Ruiz-Trillo, 2013; Marin, 2013) and genes in Group VII contain no ARM domains (Fig. 3) and thus, are differentially expressed at relatively low levels (Fig. 5) compared to those genes in Group VI. More functional explorations of these genes could improve our understanding of the roles of HECT genes in wheat and other plants during growth and development.

Conclusions

Herein, 25 identified wheat HECT genes were classified into six phylogenetic groups and distributed evenly in 17 of 21 chromosomes of the three subgenomes. Twenty-one hypothesized segmentally duplicated genes indicated that segmental duplication was significantly associated with the expansion of these HECT genes. The expression analysis revealed that most wheat HECT genes in Group II, IV, and VI (UPL8, UPL6, and UPL3) were highly expressed in roots, stems, and spikes at different developmental stages and gradually increased with the increase of dpa, suggesting that these genes may be involved in wheat growth, development and leaf senescence. This study provides useful information for further biological functional analysis of the HECT gene family in allohexaploid wheat.

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