Genome-wide identification and expression analysis of U-box gene family in wild emmer wheat (Triticum turgidum L. ssp. dicoccoides)

Highlights

• The U-box gene family was firstly identified in wild emmer wheat and other 3 triticum species.

• Segmental duplication and polyploidization mainly contributed to the expansion of U-box family in wild emmer wheat.

• Expression patterns and co-expression network of the TdPUBs were comprehensively investigated using RNA-seq samples and the stress-responsive candidates were obtained.

• Genetic variation analysis of U-box genes based on the public resequencing data showed that significant genetic bottleneck has occurred during evolution process of tetraploid wheat.

Abstract

In this study, 82 U-box genes were identified in wild emmer wheat (TdPUBs) through a genome-search method. Phylogenetic analysis classified them into seven groups and the genes belonging to the same group shared the similar exon–intron structure, motif organization and cis-element compositions. Synteny analysis of the U-box genes between different species revealed that segmental duplication and polyploidization mainly contributed to the expansion of TdPUBs. Furthermore, the genetic variations of U-box were investigated in wild emmer, domesticated emmer and durum wheat. Results showed that significant genetic bottleneck has occurred during domestication process of tetraploid emmer wheat. Meanwhile, 12 TdPUBs were co-located with known domestication related QTLs. Finally, the tissue-specific and stress-responsive TdPUB genes were identified through RNA-seq analysis. Combined with qPCR validation of 19 salt-responsive TdPUBs, the candidates involving in salt response were obtained. It lays the foundation to better understand the regulatory roles of U-box family in emmer wheat and beyond.

Introduction

Ubiquitination is a conserved post-translational modification of proteins to regulate many cellular processes in vertebrates and higher plants, including cell division, cell death, hormone responses, biotic and abiotic stress responses (Zeng et al., 2008). The major effect of ubiquitination is to add the ubiquitin molecule with the size of 76-amino acid (AA) on the lysine residues of the target protein. In eukaryotic, the ubiquitin proteasome system (UPS) degrades the aberrant and short-lived proteins and then controls the protein loads of the cell, which contains in-concert catalytic activities of three types of enzymes, namely a large number of ubiquitin ligases (E3), together with a few ubiquitin-activating enzymes (E1) and ubiquitin-conjugating enzymes (E2) (Yan et al., 2003). In the process of ubiquitination, E1 is the activator to activate the ubiquitin molecule to form as a thioester bond through an ATP-dependent reaction at cysteine residue on E1, and then the ubiquitin is transferred to ubiquitin-conjugating enzyme E2. Furthermore, E2 functions as the transporter to transfer the activated ubiquitin to E3 to form the ubiquitin thioester, which finally mediates the ubiquitination of target proteins (Pickart, 2001, Finley and Chau, 1991). Among them, E3 ubiquitin ligases are the largest family and the different E3 ligases can regulate the system to emerge different functions. According to their structure, E3 ligases can be classified into HECT-type, RING-type and U-box-type (Callis, 2014).

U-box family is one group of E3 ubiquitin ligase proteins with U-box motif comprising of ~70 AA, which has a tertiary structure resembling of RING domain but loses the characteristic zinc-chelating cysteine and histidine residues to chelate the zinc (Ohi et al., 2003). Extensive studies have reported that U-box genes played the vital roles in regulating diverse developmental processes and stress signaling in plants (Azevedo et al., 2001). Up to date U box family has been widely identified in many plant species, including 64 members belonging to 7 groups in Arabidopsis (Wiborg et al., 2008), 77 in rice and 67 barley (Zeng et al., 2008, Ryu et al., 2019) as well as 91, 99 and 101 in banana, Brassica oleracea.L and B. rapa. L, respectively (Hu et al., 2018, Hu et al., 2019, Wang et al., 2015). Meanwhile, some plant U-box genes have been functionally validated. Wang et al found that mutation of AtPUB4 in Arabidopsis could result in male sterility due to sporophytic defect, suggesting AtPUB4 was a vital regulator in sporophyte development (Wang et al., 2013). AtPUB18 and AtPUB19 were found to coordinately function as regulatory components in development and stress response in Arabidopsis (Bergler, 2011). Furthermore; NtACRE276 was confirmed to have the E3 ligase activity and involved in cell death and defense signaling, and its ortholog in Arabidopsis (AtPUB17) and canola (BnARC1) showed similar biological function (Yang et al., 2006). In wheat, overexpression and RNAi-mediated knockdown of TaPUB1 have revealed that TaPUB1 could induce the expression of target genes to improve the antioxidant capacity under stress condition (Wang et al., 2020).

Wheat is one of the most important staple crops all over the world, which occupied around 17% of global cultivated lands and provided the 30% of global calorie consume (Shewry, 2009). However, its yield is dramatically reduced by abiotic stress, such as drought and high salinity, in particular under the challenge of climate change. As the ancestor donor of A and B genome of bread wheat, wild emmer wheat was considered as the indispensable invaluable genetic resource for wheat breeding due to its high adaptability to abiotic stress (Xie and Nevo, 2008, Nevo et al., 1993). It was found that wild emmer wheat had the extreme tolerant genotypes through comparison of the salt tolerance between wild emmer and durum wheat accessions (Feng et al., 2018). Thus, identification and mining of the gene associated with stress tolerance in wild emmer wheat holds the promise for underlying the mechanism underlying stress response and also breeding for elite varieties in emmer and bread wheat. Some studies have been carried out to identify the genes related to stress tolerance in wild emmer wheat. Chen et al. reported that TdCBL6 encoded a calcineurin B-like protein and overexpression of TdCBL6 improved the salt tolerance, which could regulate both Na + and K + uptake/translocation to keep ion homeostasis under salt stress conditions (Chen et al., 2015). An autophagy-related gene, TdAtg8, was found to be a positive regulator in osmotic and drought stress response (Kuzuoglu-Ozturk et al., 2012). However, few ubiquitin pathway related genes have been identified in wild emmer wheat at present.

The completion of its reference genome provides the opportunity to investigate the genomic organization and evolution dynamics of gene family in wild emmer wheat at the genomic level (Avni et al., 2017). At present, the detailed information of U-box gene family, especially in its roles in stress tolerance, has not been well studied in wild emmer wheat. Here, we performed an in-silico genome-wide search to identify U-box family in wild emmer (TdPUBs) using the updated reference genome information. Then, the phylogenetic relationship, chromosome localization, conserved domain of the identified TdPUBs were analyzed. Furthermore, the expression patterns of these TdPUBs in different tissues and under stress conditions were systematically investigated, and then 19 salt-responsive candidates were selected to validate their expression through qRT-PCR analysis. Finally, genetic variations of these TdPUBs were investigated based on resequencing data to reveal the selection effect during emmer wheat evolution. This was the first study to identify U-Box family in wild emmer, which not only provided the important candidate genes for further functional study, but also contribute to the evolution of this family in wheat and beyond.