Identification and expression profiles of the YABBY transcription factors in wheat

The identification of YABBY TFs

The coding sequence, protein sequence, and genome sequence of wheat (IWGSC), rice, and Arabidopsis were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html). To identify wheat YABBY TFs, four steps were performed. First, we compared the Arabidopsis YABBY protein sequences against the wheat genome protein sequences using BLASTP. Second, we used PF04690, a characteristic profile of YABBY TFs from the PFAM database, to run HMMsearch against the wheat protein database with the threshold E< e⁻⁵. Third, we combined the results from the two steps above and manually removed the redundancy and alternative splicing genes. Fourth, the protein sequences of YABBY were submitted to NCBI’s Conserved Domain Database (NCBI CDD) and proteins without YABBY domains were also removed to confirm whether the putative YABBY protein contained the YABBY domain. Finally, putative wheat YABBY TFs were identified.

The physicochemical properties of wheat YABBYs were predicted by using ExPASy (Wilkins et al., 1999), and the amino acid number, theoretical isoelectric point (pI), molecular weight (MW), and grand average of hydropathicity (GRAVY) were predicted using ExPASy’s ProtParam tool (Wilkins et al., 1999). The subcellular location of TaYABBYs was predicted using CELLO v.2.5 (Yu et al., 2006) and Plant-mPLoc (Chou & Shen, 2010).

Phylogenetic relationship, gene structure, and conserved motif analyses

Sequence alignments of YABBY proteins were generated using ClustalW. An unrooted neighbour-joining (NJ) phylogenetic tree was constructed using MEGA7 (Kumar, Stecher & Tamura, 2016) with 1,000 replicates based on aligning the full-length YABBY protein sequence from rice, Arabidopsis, and wheat. To further validate the accuracy of the NJ tree, an unrooted maximum likelihood (ML) tree was built using MEGA7 (Kumar, Stecher & Tamura, 2016) with 1,000 replicates. The gene structure of TaYABBY genes was constructed using the Gene Structure Display Server (GSDS 2.0) (Hu et al., 2015). The conserved motifs of TaYABBYs were predicted using MEME suite (Bailey et al., 2015) with the following parameters: maximum number = 10, motif width = 6–100 amino acids.

Chromosomal location and collinearity analysis

All wheat YABBY genes were mapped onto the wheat chromosomes according to the information obtained from the Ensembl Plants database. The MCScanX program (Wang et al., 2012) was used to predict collinearity relationships between wheat and other species. The chromosomal location and collinearity relationship were visualised by TBtools (Chen et al., 2020).

Plant materials, RNA isolation, cDNA synthesis, and quantitative RT- PCR

The wheat cultivar Chinese Spring (Triticum aestivum) was used in this study. For different tissue expression analyses, the roots, stems, leaves, and inflorescences were collected at the spike formation stage. For different abiotic stresses, 7-day-old seedlings were subjected to salt (200 mM NaCl), drought (20% polyethylene glycol [PEG] 6000), heat (42 °C), and cold (4 °C) for 2h in hydroponic culture to obtain whole plants and collected for RNA isolation. For salt stress at different time points, 7-day-old seedlings were subjected to 200 mM NaCl and collected at 0, 1, 2, 3, 5, 7, 12, and 24 h. Three replicates were performed per treatment, and each replicate included at least 15 plants. After collection, the samples were stored at −80 °C. Total RNA was isolated using the TRIzol reagent (TIANGEN Biotech, Beijing, China) and treated with RNase-free DNase I according the manufacturer’s instruction. The first-strand cDNA synthesis was performed according to the manufacturer’s instructions (TIANGEN Biotech, Beijing, China). Quantitative real-time PCR analysis was performed using Thermo Fish Q3 (Thermo Fisher, Waltham, MA, USA) and all reactions were performed in triplicate. The relative transcript level of a gene was calculated using the 2^−ddct method (Livak & Schmittgen, 2001). Data were normalised to the expression of wheat GAPDH which was assessed in our previous study (Hao et al., 2021). Primers were designed using OLIGO 7 version software, and some primers for the YABBY genes were common to each set because of the highly conserved sequences in the A, B, and D subgenomes. Primers used in this study are listed in Table S1.

Identification of YABBY in wheat

A total of 20 YABBY members were identified in the wheat genome. Among the 21 wheat chromosomes, 20 TaYABBY genes were unevenly distributed in 15 chromosomes according to the annotation of the wheat genome (Table 1). We designated these YABBY genes as TaYABBY1A—TaYABBY7D according to their consecutive chromosomal positions and homology relationships. All were validated using expressed sequence tags (ESTs). Among these 20 TaYABBY TFs, 18 constitute night sets, and every set contains three homoeologous genes in the A, B, and D subgenomes, respectively; two form one set with two homoeologous genes in the A and B subgenome. The deduced length of TaYABBY proteins ranged from 164 amino acids (aa) (TaYABBY2D) to 297 aa (TaYABBY1A and TaYABBY1B) with molecular weights ranging from 17.76 (TaYABBY2D) to 31.44 (TaYABBY1B) kDa. The theoretical pI ranged from 5.62 (TaYABBY2A) to 9.3 (TaYABBY6A and TaYABBY6B), and the GRAVY of each TaYABBY protein was less than zero, indicating that they are hydrophilic proteins. All the 20 YABBY genes are predicted using CELLO v.2.5 (Yu et al., 2006) and Plant-mPLoc (Chou & Shen, 2010) to be located in nuclear.

Collinearity analysis

Among the 20 TaYABBY genes, three segmental duplication events were constructed by four TaYABBY genes (Table S2). In addition, 0, 0, 7, 8, 12, and 8 orthologous were found between wheat and Arabidopsis, Brassica napus, Brachypodium, rice, maize, and sorghum respectively (Table S3 and Fig. 1). These results indicate that TaYABBY genes in monocot plants are closely related.

Phylogenetic tree of wheat YABBYs

To better understand the phylogenetic relationship of YABBY genes among rice, wheat, and its ancestor species, an NJ tree was constructed. Consistent with previous reports, these YABBY proteins can be classified into five subgroups, including 5 CRC, 5 INO, 9 YAB2, 14 FIL, and 1 YAB5 member (Fig. 2). To further evaluate the accuracy of the NJ tree, we created a tree topology using the maximum likelihood (ML) method. This tree topology was the same as that of the NJ tree in Fig. S1, indicating that the tree is suitable for further analysis. In clade YAB1, no genes of wheat or its ancestor species were included. Clade FIL is a large group, which contains night wheat YABBY members.

An unrooted NJ phylogenetic tree was also constructed in wheat YABBYs; 9, 5, 3, and 3 TaYABBYs were classified into the FIL, YAB2, INO, and CRC groups (Fig. 2A).

Structural characteristic analysis of TaYABBY TFs

Two highly conserved domains were identified in all TaYABBYs, including a C2–C2 zinc finger domain at the N-terminus and a YABBY domain at the C-terminus (Fig. 3). Within the C2–C2 domain (C-X2-C-X20-C-X1-HC), the cysteine (C) and histidine (H) residues directly involved in Zn²⁺ binding are conserved. At the C-terminus, 28 conserved amino acids were 100% conserved inside the YABBY domain, including five alanine (A), three proline (P), three serine (S), three isoleucine (I), and other amino acid residues. The YABBY domain, like the HMG-box domain, has been confirmed to be associated with DNA binding (Sawa et al., 1999). In plants, the YABBY domain of Arabidopsis CRC is able to bind to the promoter regions of KCS7 and KCS15, two genes involved in the synthesis of very long chain fatty acids (Han, Yin & Xue, 2012). The YABBY domain of rice OsYABBY1 specifically binds to a GA-responsive element in the promoter of 2GA3ox2 (Dai et al., 2007), and the FIL has been confirmed to bind non-specifically to DNA via its YABBY domain (Kanaya, Nakajima & Okada, 2002). These results indicate that YABBY domain is the main structural domain that performs the function, and that the main amino acids play dominant roles.

Members of the same group shared similar conserved motifs and structures. As shown in Fig. 4B, motifs were conserved within the same group; motifs 1, 2, 3, and 4 were found in all TaYABBYs, while motif 5 was only identified in FIL and INO members. Among them, motifs 1 and 4 constitute the YABBY domain, while motifs 2 and 3 form the C2–C2 domain at the N-terminus. All TaYABBY genes contained six or seven exons, and the phylogenesis-related genes had similar gene structures (Fig. 4C).

Expression pattern of wheat YABBY genes

We used quantitative reverse transcription PCR (qRT-PCR) to analyse the expression patterns of TaYABBY genes in different tissues. The results showed that many TaYABBY genes were highly expressed in leaf tissues (Fig. 5), including TaYABBY1A/D, 4A, 4B, 4D, 5A, 5B, 5D, 6A/B, and 7A/B/D. Some homoeologous genes had similar expression patterns; for example, TaYABBY4A, TaYABBY4B, and TaYABBY4D are highly expressed in leaf tissues; TaYABBY7A, TaYABBY7B, and TaYABBY7D are mainly expressed in leaf tissues.

We also analysed the expression patterns under different abiotic stress conditions. As shown in Fig. 6, the expression of all of them was induced by abiotic stresses; 13 and 10 were upregulated by salt and PEG treatments, respectively, while 10 and 10 were down-regulated by heat and cold treatments, respectively. Because all TaYABBY genes were upregulated by salt, we also analysed the expression patterns of TaYABBY genes under salt stress at different time points. As shown in Fig. 7, the expression levels of all genes peaked at 2 h, and then began to decline, indicating that the expression of the YABBY gene can be induced in a short period of time by salt stress. These results indicate that TaYABBY genes are involved in plant responses to abiotic stresses.