Transcription factors (TFs) bind to the cis-acting elements of their target genes and play a key role in gene transcription regulation. The APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily includes the AP2 (APETALA2), ERF (ethylene-responsive factors), and RAV (related to ABI3/VP) gene families and is one of the largest groups of TFs in plants. TFs of the AP2/ERF superfamily contain an AP2 DNA-binding domain (Riechmann and Meyerowitz, 1998; Sakuma et al., 2002). Additionally, the AP2/ERF superfamily is defined by the AP2/ERF domain, which comprises approximately 60–70 amino acids and is involved in DNA-binding (Kumar et al., 2009). The AP2 subfamily members contain two AP2/ERF domains lacking a conserved WLG motif. The ERF subfamily possesses only one AP2/ERF domain, and the RAV subfamily members have a single AP2/ERF domain and a B3 domain (Sakuma et al., 2002). The ERF family is further split up into two subfamilies according to the DNA sequence bound: ERF and CBF/DREB (Sakuma et al., 2002). Proteins encoded by genes from the ERF subfamily bind to the core motif AGCCGCC (Zhou et al., 1997), whereas the CBF/DREB subfamily contains C-repeats recognizing the cis-acting element, A/GCCGAC (Yamaguchi-Shinozaki and Shinozaki, 1994).

The AP2 domain was first described in Arabidopsis and is involved in flower development (Jofuku et al., 1994). AP2/ERF proteins have important functions in the transcriptional regulation of various biological processes related to growth and development, as well as various responses to environmental stimuli (Moose and Sisco, 1996; Liu et al., 2013). Indeed, the combined use of genetic and molecular approaches has shown that the AP2/ERF family participate in the regulation of developmental processes, such as flower development (Elliott et al., 1996), spikelet meristem determinacy (Chuck et al., 1998), leaf epidermal cell identity (Moose and Sisco, 1996), and embryo development (Boutilier et al., 2002). Extensive plant genome sequencing has identified the AP2/ERF gene family in various plants, such as Arabidopsis (Moose and Sisco, 1996), rice, maize (Liu et al., 2013), soybean (Zhang et al., 2008), and foxtail millet (Lata et al., 2014). Although there are data available on the AP2/ERF family in barley (Hordeum vulgare L) (Guo et al., 2016), no investigation has been conducted based on the latest available genomic database.

Barley is the fourth most abundant cereal, after rice, wheat, and corn, in both area and tonnage harvested. It is widely used for feeding animals and beer making (Mayer et al., 2012). The International Barley Sequencing Consortium (IBSC) released the genome sequencing map of barley cultivar Morex, a North American spring six-row malting barley, for the first time in 2012 (Mayer et al., 2012). This map is referred later in the text as V1. In 2017, hierarchical shotgun sequencing of bacterial artificial chromosomes was combined with the use of optical mapping and chromosome-scale scaffolding with chromosome conformation capture sequencing (Hi-C) by the IBSC to create a highly contiguous reference genome sequence for Morex (Mascher et al., 2017). The reference genome assembly for Morex was improved by Monat et al. (2019) with the use of TRITEX, an open-source computational workflow and is referred later in the text as Morex V2. It represents a significant resource for the barley research community. The improved barley genome provides a good opportunity to make better use of barley germplasm resources and carry out the cloning and functional characterization of unknown genes. Specifically, the second version of the reference genome provided the basis for the re-analysis of the HvAP2/ERF gene family in barley.

Starch, the most abundant component of cereal grains, has important biological functions and is a major part of the human diet (Sonnewald and Kossmann, 2013). Starch content and composition are key elements influencing grain yield and quality. They play important roles during endosperm development (James et al., 2003). Starch consists of two types of glucose polymers, namely, amylose and amylopectin (Nakamura et al., 1995). Starch biosynthesis can be broadly divided into three stages: sucrose transport, synthesis of the glucosyl donor, and amylopectin or amylose synthesis (Keeling and Myers, 2010; Bahaji et al., 2014). Starch synthesis in cereals requires several well-characterized enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule bound starch synthase (GBSS), starch synthase (SS), starch branching enzyme (Sun et al., 2003), and starch debranching enzyme (DBE) (James et al., 2003; Hannah and James, 2008; Bahaji et al., 2014). ADP-glucose enters the amyloplast through Brittle1 (BT1, the transporter of ADP-glucose) to be used as a substrate for starch biosynthesis (Bahaji et al., 2014). GBSS is responsible for amylose synthesis and mutation of this enzyme results in lower amylose content (Pérez et al., 2019). Amylopectin synthesis is a complex process involving interaction and feedback between the enzymes SS, SBE, and DBE (Hannah and James, 2008; Jeon et al., 2010).

TFs play an important role in the regulation of starch synthesis. Indeed, previous studies identified TFs regulating starch synthesis in rice, maize, wheat, and, to a lesser extent, barley. In rice, TF genes such as NF-YB1, NF-YC12, OsbZIP58, OsbZIP76, and Rice Starch Regulator 1 (RSR1) have been shown to regulate starch synthesis (Fu and Xue, 2010; Wang et al., 2013; Bai et al., 2016; Bello et al., 2019; Niu et al., 2020). ZmNAC36, ZmbZIP91, ZmbZIP22, Opaque 2, Opaque 11, ZmEREB156, ZmNAC128, and ZmNAC130 are central players regulating the expression of starch biosynthesis genes in maize (Zhang et al., 2014, 2016, 2019; Chen et al., 2016; Huang et al., 2016; Feng et al., 2018; Dong et al., 2019). In wheat, TaRSR1, a gene homologous to OsRSR1 and TaNAC019-A1, negatively regulates the expression of many starch synthesis genes, and TubZIP28 and TabZIP28 are transcriptional activators of starch synthesis (Liu et al., 2014, 2016; Song et al., 2020). SUSIBA2, a member of the WRKY TF family, was identified in rice, maize, wheat, and barley (Sun et al., 2003).

The accurate analysis of the AP2/ERF gene family members is important for screening starch synthesis-related genes. In this study, we used the latest published barley genome database for the identification and classification of HvAP2/ERF family members. The data were also confirmed using the Golden Promise genome database. Furthermore, to confirm our results, we compared the latest published database with the previous version. A comprehensive analysis of structural features, phylogenetic relationships, chromosomal location, and expression patterns of the identified AP2/ERF family members was performed. Data regarding the expression of AP2 family members in different barley tissues were retrieved from the database, and the co-expression of these AP2 family members with starch synthesis genes was analyzed. Our results provide new insight into the link between the AP2/ERF family and starch synthesis genes. Particularly, we identified HvAP2-18 as a candidate gene binding to the promoter of AGP-S and SBEI to suppress starch synthesis. The identification of other candidate genes might also provide a better understanding of the AP2 family contribution to starch synthesis in barley.

Starch accumulation occurs in barley endosperm and requires the coordinated regulation of various genes. Many TFs involved in starch production have been identified. The AP2/ERF family of TFs has a crucial role in regulating starch synthesis, as was identified in rice (Fu and Xue, 2010) and wheat. However, no member of the AP2/ERF family has been reported to regulate starch synthesis in barley. The accurate identification and analysis of genes from the AP2/ERF family might provide a reliable basis for the subsequent screening of candidate HvAP2/ERF genes involved in the regulation of starch synthesis. Previous work reported 121 HvAP2/ERF genes in barley (Guo et al., 2016). This study used an earlier version of the barley genome (Beier et al., 2017), which contains some gaps in the physical map and might therefore be lacking some important genes. The updated version (Morex V2) (Monat et al., 2019) of the barley genome has been recently released and is better than the earlier V1 annotation as it contains inclusive and broader information. Thus, the newest V2 version might be helpful for the precise characterization of the HvAP2/ERF family (Monat et al., 2019). In the present study, a total of 185 HvAP2/ERF genes were identified in barley. Hence, we identified 64 more genes than the study using the V1 barley genome, confirming that the Morex V2 dataset contains more gene sequences with better annotation. The HvERF3.1 and HvERF3.2 identified previously matched a single gene, HvERF-16, in Morex V2 and Golden Promise genome databases. Similarly, the previously characterized HvERF4.9 and HvERF4.10 genes corresponded to HvERF27, and the HvERF2.15, HvERF2.16, and HvERF2.17 genes matched the HvERF-58 gene in the Morex V2 genome. These discrepancies might be caused by incorrect sequence splicing or misannotation in the earlier V1 genome. Moreover, multiple sequence alignments revealed that the results of the previous study included repeated genes. Thus, our results provide a more complete understanding of HvAP2/ERF gene family.

The AP2/ERF superfamily is a large and significant TF group and plays a role in various processes related to plant growth and development. It has been shown to be involved at different growth stages including seed germination, flowering and ripening as well as in response to various environmental stresses (Schmidt et al., 2013). With the advancement of second-generation sequencing technology, a lot of reports have identified AP2/ERF family members in various plant species (Nakano et al., 2006; Zhu et al., 2014; Zhao et al., 2019). The conserved motifs of AP2/ERF TFs have a specific role in the proper function of these genes (Sakuma et al., 2002). A total of 50 conserved motifs located outside the AP2 domain were detected in Arabidopsis (Nakano et al., 2006). Here, we analyzed 10 motifs in HvAP2/ERF proteins, and motif 1 (partial AP2/ERF domain) was observed in all genes (Figures 2, 3). All the AP2/ERF subfamilies contain the WLG domain except the AP2 subfamily, which has been confirmed by our study. Additionally, in the ERF subfamily, there was no WLG domain from HvERF-38 to Hv-ERF54, but we still classified them into the ERF subfamily according to the annotation information (Supplementary Figure 2). Furthermore, we verified this classification via phylogenetic analysis. The genes HvERF-38 to HvERF-54 were part of the C1 group of the ERF subfamily because of their high homology (Figure 1) and the identical motif structures (Figure 3), confirming that they belong to the ERF subfamily.

Expression profiling has further confirmed that the AP2/ERF family has specific roles in various processes ranging from seed germination to fruit ripening and from response to environmental stress to response to pathogen attack (Klucher et al., 1996). However, there are relatively few studies on the transcriptional regulation of AP2/ERF genes during grain development. Transcriptomic expression analysis indicated that HvAP2/ERF genes were active in different barley tissues and showed tissue-specific differential expression. Most of the HvAP2/ERF genes of the first, second, and 16th groups were highly expressed in early grain developmental stages (5 DPA), but their expression levels were significantly decreased at 15 DPA. In the AP2 subfamily, 11 genes were highly expressed during endosperm development, with expression levels higher at 5 DPA compared with that at the 15 DPA. The other three subfamilies, namely, ERF, RAV, and DREB, contained 28, 1, and 26 genes, respectively, with higher expression levels during endosperm development. Although the relevant TFs involved in starch synthesis have not been identified in the study of the barley HvAP2/ERF family, it is possible to identify some family members involved in seed development and starch synthesis through the analysis of transcriptome data.

Previous work found that AP2/ERF TFs can regulate the expression of genes involved in starch synthesis. For example, RSR1 was identified using gene co-expression analysis in rice (Fu and Xue, 2010) and wheat (Kang et al., 2013). Our results indicated that 12 HvAP2/ERF genes had an expression pattern similar to that of the starch synthesis genes (Figure 6). Among the starch synthesis enzymes, AGPase is responsible for the first key step of starch synthesis and is the rate-limiting enzyme (Ballicora et al., 2004). AGPase is a heterotetramer composed of two large subunits (AGPL) and two small subunits (AGP-S) in higher plants. AGPL and AGP-S have complementary roles in AGPase function (Qu et al., 2018). In wheat, TubZIP28 and TabZIP28 regulate starch synthesis by binding to the promoter of cytosolic AGPase and enhancing its transcription and activity (Song et al., 2020). TaNAC-019-A1 regulates the expression of multiple starch synthase genes such as AGP-S, SBE1, and SBE2a, thus affecting starch synthesis in grains (Liu et al., 2020). In our study, LUC and Y1H analyses showed that HvAP2-18 could bind to the promoter of HvAGP-S and SBE1. Additionally, HvAP2-18 is homologous to the rice RSR1 gene. The RSR1 gene negatively regulates starch synthesis in endosperm. However, the mechanisms triggered by RSR1 to regulate starch synthesis in barley have not been studied in detail. We provide qRT-PCR data suggesting that the expression pattern of HvAP2-18 was different to that of genes involved in starch synthesis. It was further observed by LUC and Y1H that HvAP2-18 binds to the promoter region of starch synthesis genes. Additionally, transcriptome data and qPCR analyses showed that most AP2 subfamily members, highly expressed at the grain development stage, tended to be highly expressed at the initial stage, whereas the expression was low later on. This pattern of expression suggests a possible negative regulatory role. The candidate gene HvAP2-18 might therefore be the first transcriptional regulator of starch synthesis identified in barley and as such, could be a valuable target for further study.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

JD carried out analysis of gene members, qRT-PCR and wrote this article. HK and YL downloaded sequences and did the yeast one-hybrid assay. WH, CG and NL analyzed the expression data. HT contributed to the material planting. QJ conceived and designed the experiments. QX, YZZ, PQ, YJ, MD, JM, JW, GC, XL, YW, and YLZ partially participated in its design and revised the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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