In the present study, for the first time, thirteen hevein genes were identified and characterized from barley. Also in this study, gene structure analysis, protein structure analysis, physicochemical properties analysis, conserved motifs and domains analysis, signal peptide and subcellular localization prediction, phylogenetic analysis, promoter analysis, expression analysis, and antimicrobial activity prediction were performed. The hevein family, which takes its name from the rubber tree or Hevea brasiliensis, was first identified from the latex of this tree. Heveins belong to a large group of plant pathogenicity-related proteins (PR) (Andreev et al. 2012). Hevein genes are present in all plants and belong to a larger family of chitinase proteins that are involved in plant defense processes (Beintema 1994). So far, several hevein gene families have been identified from rubber, mulberry, soybean, wheat and other plants (Berthelot et al. 2016a; Berthelot et al. 2016b; Slavokhotova et al. 2017; Saeed et al. 2016; Porto et al. 2012; Van Holle and Van Damme 2015; Andreev et al. 2012). Most of barley hevein genes were intronless, while some had only one intron in their structure. This gene structure and intron-exon pattern has also been observed in other plants. Both intronless and single intron hevein genes have been observed in rubber tree (Berthelot et al. 2016a; Berthelot et al. 2016b). In a study, three intronless heveins were identified from wheat plant (Andreev et al. 2012). In similar study, an intronless hevein gene was identified in tomato plant (KOUZAI and SAITO 2013). All identified members of mulberry hevein gene family were intronless genes (Saeed et al. 2016). Various hypotheses have been suggested for the existence of intronless genes, including inheritance from ancient prokaryotes, gene duplications, retroposition, and other mechanisms (Yan et al. 2016). The intronless genes can effectively express and this may be a reason for the importance of the functional role of these genes in maintaining cell processes (Zou et al. 2011; Gentles and Karlin 1999).
Search for conserved functional domains showed that the ChtBD1 domain has repeated one to four times in some barley heveins, while this domain was associated with another domain called Lyz-like in some other. The MSA results showed that ChtBD1 domain is highly conserved in barley heveins. The MSA also revealed eight conserved cysteine amino acids that formed four intramolecular disulfide bonds in all barley heveins. Sixteen intramolecular disulfide bonds were observed in barley heveins with four ChtBD1 domains. Hevein domain, which is a small chitin binding protein, is present in all plants and was first identified in latex of rubber tree. This domain consists of 30 to 40 amino acids and eight cysteine amino acids is present in its structure (Van Parijs et al. 1991; Damme et al. 1998).
The secondary and 3D structures of the ChtBD1 domain are stabilized by disulfide bonds (Martins et al. 1996; Xiang et al. 2004). Heveins display antifungal and antibacterial activity which is mainly related to their ChtBD1 functional domain. The antifungal activity of heveins is greatly decreased by the loss of the ChtBD1 domain (Iseli et al. 1993). The cationic and amphipathic properties of ChtBD1 domain enable it to interact with negatively charged membranes of fungi and bacteria (Martins et al. 1996; Xiang et al. 2004; Lipkin et al. 2005). So far, heveins have been expressed in various plants, including tobacco, tomato, and Arabidopsis to improve resistance to fungal pathogens (Choon Koo et al. 2002; R Shukurov et al. 2012; Khaliluev et al. 2011; Shukurov et al. 2010).
The search for signal peptide showed extracellular signal peptides in all barley heveins. The signal peptide regions were highly variable in the barley heveins. The pathogens invade plants through extracellular space and therefore, the presence of heveins and other antimicrobial proteins in the extracellular space is essential to combat pathogens (Drikvand et al. 2019). Previous studies have reported N-terminal signal peptides in heveins that lead mature peptides to the extracellular space (Peumans and Van Damme 1995; Damme et al. 1998; Van Holle and Van Damme 2015; Lee et al. 1991; Slavokhotova et al. 2017).
The analysis of physicochemical properties of barley heveins revealed thermal stability, acidic and alkaline properties, hydrophilic properties, and positive and negative net charge for these proteins. The thermal stability of a protein is depending on the parameters including, aliphatic index, amino acid composition, and instability index. The aliphatic index is the relative volume of the side chains of the amino acids valine, alanine, isoleucine, and leucine, and is directly correlated with the thermal stability of a protein (Ikai 1980). The high content of alanine amino acid in the structure of barley heveins increases their aliphatic index and thermal stability. The instability index predicts the stability of a protein based on its amino acid composition. instability index value less than 40 indicates protein stability (Guruprasad et al. 1990). The calculated instability indices for barley heveins (except HORVU2Hr1G126720 and HORVU3Hr1G064470) indicated their proper protein stability. Since heveins are extracellular proteins, protein stability is vital for their activity. Positive charge allows heveins to interact with negatively charged membranes of pathogens. On the other hand, negatively charged heveins interact with pathogen membranes through other pathways or at specific pH conditions (Yeaman and Yount 2003). The grand average of hydropathy (GRAVY) is a measure of protein solubility. The proteins with positive GRAVY tend to be hydrophobic, while proteins with negative GRAVY are hydrophilic. The GRAVY indicates thermal stability of proteins (Kyte and Doolittle 1982; Moghadam et al. 2016). The calculated GRAVY for barley heveins (except HORVU0Hr1G016310 and HORVU2Hr1G126720) indicates the hydrophilicity of these proteins. The calculated GRAVY values are consistent with the calculated aliphatic and instability indices and show the thermal stability of barley heveins.
The search for conserved protein motifs revealed three motifs in the Lyz-like domain of barley heveins, while there was no motif within the ChtBD1 domain. This may be due to the short length of the ChtBD1 domain in the structure of the heveins. The phylogenetic analysis divided barley heveins into two groups. In the phylogenetic tree, heveins with conserved protein motifs classified together in one group, and heveins without conserved protein motifs were divided into another group. A similar trend has been observed in previous studies, and heveins from different plants have been divided into two groups (Saeed et al. 2016; Andreev et al. 2012; Berthelot et al. 2016a; Berthelot et al. 2016b; Porto et al. 2012; Slavokhotova et al. 2017). The gene ontology analysis showed that barley heveins play a major role in biotic and abiotic stresses. The promoter analysis showed that G-Box is the most abundant growth and development related element in the promoter of barley heveins genes. This cis-acting element plays an important role in response to light, anaerobic condition, ethylene, methyl jasmonate, and abscisic acid (Menkens et al. 1995). The elements ABRE, CGTCA-motif and TGACG-motif, which had the highest frequency of hormone-related elements in the promoter of barley heveins genes, are typically present upstream of genes involved in biotic and abiotic stresses (Pandey et al. 2015; Liu et al. 2016; Kaur et al. 2017; Wang et al. 2011). The promoter analysis showed that ARE and MBS elements are the most abundant stress-related elements in the promoter of barley heveins genes. They play vital role in response to various biotic and abiotic stresses (Kaur et al. 2017). Previous studies have reported that heveins are mainly involved in defense processes, response to environmental stresses, chitin degradation, glucose metabolism, proton transport, and signal transduction and therefore the presence of such regulatory elements in the promoter of these genes seems necessary (Wang et al. 2013; Berthelot et al. 2016b).
The tissue-specific expression analysis of barley hevein genes showed that these genes have higher expression level in root and reproductive organs of barley. In the similar study mulberry heveins, showed high expression level in roots and reproductive organs (Saeed et al. 2016). The soil is the site of most plant pathogens, and root is the first organ to encounter pathogens. Therefore, plants combat soil-borne pathogens through producing antimicrobial proteins such as heveins (Graham and Strauss 2021). In a study, the expression level and expression pattern of defensin antimicrobial peptides in hydroponic roots were significant different compared to roots grown in soil (Drikvand et al. 2019). These results indicate that heveins and other antimicrobial peptides are more expressed in susceptible tissues such as root and reproductive organs to defense against pathogens (Drikvand et al. 2019; Berthelot et al. 2016b; Saeed et al. 2016).
The barley hevein genes showed different expression level under biotic and abiotic stresses. Most of these genes were induced by both biotic and abiotic stresses. However, the expression changes under biotic stresses were higher than abiotic stresses. The salinity and temperature stresses induced the expression of hevein genes in wheat (Andreev et al. 2012). In a study, the specific regulatory elements related to response to cold, wounding, jasmonic acid and salicylic acid were found in promoter of mulberry heveins genes (Saeed et al. 2016). The over-expression of a hevein gene in transgenic rice conferred resistance against blast disease (Lee and Raikhel 1995; Van Parijs et al. 1991). The over-expression of rubber hevein gene in black mustard increased the resistance of transgenic plants to blight disease (Kanrar et al. 2002). Some plant responses to biotic or abiotic stresses are specific, while others are common between biotic or abiotic stresses. The signalling molecules and proteins, such as components of the mitogen-activated protein kinase (MAPK) cascade, may act as crossing points between biotic or abiotic stresses, induce genes such as heveins in response to both biotic and abiotic stresses (Chinnusamy et al. 2004; Andreev et al. 2012). The expression level of some barley hevein genes did not change under biotic or abiotic stresses and they may be specific for responding to biological conditions other than environmental stresses, or they may be expressed only in a specific tissue or organ. On the other hand, the expression of these genes may change under long-term stress conditions.
The results of antimicrobial activity prediction showed potential antimicrobial effects in all barley heveins. These results also showed that the antimicrobial activity of these peptides is mainly concentrated in their ChtBD1 domain and the other regions have lower antimicrobial activity than this region. Heveins have anti-fungal and antibacterial activity, and this is mainly related to their functional domain. The antifungal activity of heveins is greatly reduced by the loss of the ChtBD1 domain (Iseli et al. 1993). The ChtBD1 domain is a small chitin-binding protein and its cationic and amphipathic properties enables it to interact with negatively charged membranes of fungi and bacteria (Damme et al. 1998; Van Parijs et al. 1991; Martins et al. 1996; Xiang et al. 2004; Lipkin et al. 2005).