Heavy metals, which may be found in soil, act as co-factors, being crucial components required for optimal biological and plant cell growth. (Thomine and Vert, 2013). Bivalent cations, like copper (Cu2+), iron (Fe2+), zinc (Zn2+), manganese (Mn2+), nickel (Ni2+), and cobalt (Co2+) are such essential trace elements for plants. These ions are necessary for proper functioning of physiological processes, however when in excess they can induce toxic effects (Kolaj-Robin et al., 2015). Among others, the processes for which presence of these ions is vital are protein processing, photosynthesis, replication of DNA as well as electron transport in mitochondria and/or chloroplasts. The low level of these ions in plant tissues can also be detrimental, affecting the growth and development (Bhardwaj et al., 2020). Due to need of precisely maintaining the appropriate level of metal ions, plants have developed a complex machinery allowing them to control the uptake, trafficking, mobilization, translocation, efflux and storage of essential metal ions (Liu et al., 2019). To do so a variety of transporters can be found in plants, such as the natural resistance-associated macrophage proteins (NRAMP), cation exchanger (CAX) family or cation diffusion facilitator (CDF) proteins (Jiang et al., 2022). In our study, we focused on CDFs which are divalent cation (Zn2+, Co2+, Fe2+, Cd2+, Ni2+, and Mn2+) transporters that play crucial roles in metal homeostasis.
The CDF transporters in plants are denoted as metal-tolerance proteins (MTPs) (Lang et al., 2011, Ricachenevsky et al., 2013). They use an antiport mechanism, transporting divalent cations in exchange for H+, thus mediating metal homeostasis and tolerance controlling the outflow from the cytoplasm (Fu et al., 2017). MTPs are categorized into three different classes Mn-CDFs, Fe/Zn-CDFs, and Zn-CDFs, which is based on the substrate specificity (Montanini et al., 2007). Those three families can be further divided into seven groups based on the phylogenetic relations and annotations conducted in Arabidopsis thaliana (Gustin et al., 2011). Based on this classification, groups 1, 5 and 12 are Zn-CDFs; the groups 6 and 7 are Fe/Zn-CDFs and 8 and 9 are the Mn-CDFs (Gustin et al., 2011). In two model plant species, A. thalina and Oryza sativa, 12 and 10 MTP genes, respectively, were identified at first (Montanini et al., 2007). Since then some of them have been functionally characterized. These MTP proteins have been reported to transport a variety of metallic elements, including Fe, Mn, Cu, Zn, Ni, Co, and Cd (Das et al., 2021). For instance, AtMTP1 and AtMTP3 have been shown to localize in the vacuole in Arabidopsis, conferring Zn and/or Co tolerance by sequestering excess Zn2+ and/or Co2+ (Arrivault et al., 2006, Kobae et al., 2004). AtMTP12 interacts with AtMTP5 to form a functional complex that transports Zn2+ from the cytosol to the Golgi apparatus (Fujiwara et al., 2015). MTP8 is one of the most extensively studied Mn-CDF family members, contributing to Mn detoxification in A. thaliana and O. sativa by sequestering Mn in vacuoles (Chen et al., 2013, Takemoto et al., 2017, Eroglu et al., 2016). MTP gene family members have already been identified in several plant species, however, MTPs in Arabidopsis thaliana have received much attention (Ueno et al., 2015). In monocots, most of the characterized members of MTPs belong to rice (Chen et al., 2013, Ma et al., 2018, Menguer et al., 2013, Montanini et al., 2007, Takemoto et al., 2017, Tsunemitsu et al., 2018, Ueno et al., 2015). Therefore, a thorough understanding of MTPs in other important crop plants is of great interest.
Barley (Hordeum vulgare) is a metal-tolerant crop, tolerating Mn (Foy et al., 1988), Cu and Cd (González et al., 2017, Gvozdenac et al., 2013). As it is a member of grass family, H. vulgare is one of the most important crops, being the fourth most cultivated world-wide among grain cereals after maize, wheat and rice (Monat et al., 2019, Tanwar et al., 2022). The estimated production of barley for year 2020–2021 was 159.74 million metric tons (https://www.statista.com/statistics/271973/world-barley-production-since-2008/). Barley is a diploid with genome comprised of seven chromosomes (2n = 2x = 14) and estimated size of 5.1 Gbp, with 80% of its genome composed of repetitive elements (Wicker et al., 2017). Despite such a large genome, barley is a very convenient genetic model for Triticum aestivum – which is hexaploidy, due to simpler diploid one. Additionally, many genes in barley and wheat posses corresponding and similar functions. Therefore, the knowledge gained in barley can be swiftly utilized to predict the genes with comparable functions in wheat (Monat et al., 2019). All of this makes barley unique among crop plants as it is tremendously important for agriculture and science.
Recent advancements in genomics have enabled the generation of large-scale sequence data for many crop species (Varshney et al., 2015, Henry, 2022). Such datasets have aided in the study of genomic architecture and dynamics, as well as gene discovery. As genome sequences for more plant species become available, a number of MTP proteins have been identified genome-wide in many plant species, including Vitis vinifera (Shirazi et al., 2019), Sorghum bicolor (Ricachenevsky et al., 2013), Brassica rapa (Li et al., 2018), Nicotiana tabacum (Liu et al., 2019), Populus trichocarpa (Gao et al., 2020), Citrus sinensis (Fu et al., 2017) and Triticum aestivum (Vatansever et al., 2017). Despite the fact that numerous genomic resources have been established to examine partial or entire genomic sequences and their related functions in barley, only HvMTP8 has been studied in detail (Pedas et al., 2009, Pedas et al., 2014). In recent years, efforts have been conducted to study the physio-biochemical and molecular mechanism of heavy metal tolerance in barley; however MTP genes at the genome-scale in barley have not been well characterized until now.
In order to better understand the genome structure and fill the gaps in knowledge, we systematically explored the MTPs in barley to identify at the genome-scale level members of this family. In addition, we conducted phylogenetic relationships, gene structure, protein motifs analysis as well as homology modelling. We also studied the expression patterns of identified MTPs in response to different heavy metal stresses. Such comprehensive investigation of MTPs genes in barley would establish the base to comprehend the role of transport proteins both in terms of their structure and physiological roles. The results of this study are not only a starting point for further functional research of the molecular mechanism(s) responsible for tolerance of barley to heavy metals, but also allows for better genetic understanding of this crop.