Despite rising agricultural expenses and climate changes, millets guarantee food security and nourishment. In addition to being healthy, they have added well-being benefits and require significantly fewer efforts to grow (Bandyopadhyay et al., 2017). Amidst the mounting concern regarding climate change, these characteristics suggest millets as plants of preference for humankind. A minor pseudocereal millet crop, proso millet (Panicum miliaceum L.) has been valued as a healthy food for centuries. About ten thousand years ago, it originated in Northern China as a prime domesticated plant of the Poaceae family (Li et al., 2021). Crops are susceptible to a variety of stresses like toxic metals, fluctuating temperatures, pesticides, salinity as well as biotic stresses and millets are no exception to these stresses (Li et al., 2021; Mushtaq et al., 2021). Proso millet has the advantage of being a skillful propagator in poor environmental conditions, places where other crops have meagre chances of growth (Nazir et al., 2021). Soil salinity is a global agronomic challenge that has a detrimental influence on crop yields and thus threatens food security (Ibrahimova et al., 2021). Sodium and chloride ions accumulate in the tissues of plants causing ionic and osmotic stress (Zhao et al., 2021). In the event of ionic stress, potassium and sodium homeostasis is disrupted, inhibiting plant growth. Higher levels of sodium impede the enzymatic function and destabilize protein structure by messing with the surface charges (Singh and Roychoudhury, 2021). On the other hand osmotic stress results in more reactive oxygen species (ROS) generation in many compartments, causing oxidative damage, reduction in photosynthesis, stomatal limitations, lipid peroxidation, protein and DNA damage and reduced enzyme activity (Isayenkov and Maathuis, 2019; Ahmad et al., 2021). In this context as salinity is posing a severe danger to our food security, we must examine how the response of plants towards salt and thus identify and produce salt-tolerant plants (Ibrahimova et al., 2021). It is critical to envisage plant enhancements that allow them to live in high salt concentrations, as well as to choose plant species that can withstand salt stress. This could be achieved by using efficient crop varieties like millets and by the application of beneficial exogenous compounds. Selenium (Se) is an essential compound that has been shown to alleviate salt stress. At relatively lower concentrations of Se, plant growth and development can be improved (Hasanuzzaman et al., 2020). Se can increase the activity of antioxidant enzymes (Mushtaq et al., 2021), enhancing tolerance towards drought (Ghouri et al., 2021), salt stress (Shah et al., 2020; Taha et al., 2021), low temperature (Liu et al., 2021), toxic metals (Zhou et al., 2021), and ultraviolent light-induced stresses (Mata-Ramírez et al., 2019). Se was discovered by Swedish chemist Jacob Berzelius (1817) and the word is derived from the ancient Greek word, “Selene” meaning moon (Monesh Babu and Preetha, 2021). In the periodic table, Se is a member of group 16, and due to this reason, it has similar chemical characteristics as Sulfur (S) (Bodnar et al., 2012). In the earth’s lithosphere, Se exists in different amounts (Floor and Román-Ross, 2012) and its content and form depend on the soil type, its organic matter, the amount of rainfall and topography (Zhu et al., 2009; Gupta and Gupta, 2017). Se is abundant in soils formed by igneous rock, granite, sandstone, limestone and generally scarce in soils formed by temperate and humid climates. In mineral-enriched soils, Se levels vary by 14 mg/kg regardless of soil depth (Hossain et al., 2021). The inorganic forms of Se consist of selenate, selenite, selenide and elemental Se (Bodnar et al., 2012) while as organic forms include selenocysteine (Se-Cys) and selenomethionine (SeMet) (Wu et al., 2015; Ahmad et al., 2021). Se can cause toxicity in humans at higher concentrations, even though it is a beneficial micronutrient. However, it is still being debated whether Se is essential for plants or not, but, it has shown protective roles in several plants when applied at lower concentrations (Gupta and Gupta, 2017; Lanza and Dos Reis, 2021). Furthermore, studies report Se as a beneficial element that acts as an antioxidant, anti-senescent and provides oxidative stress tolerance to plants (Ahmad et al., 2021).

Most plant species utilize only a little amount of Se from soil and they differ in their ability to accumulate Se in their tissues (White, 2016). Se absorption, transport, and translocation are influenced by a variety of parameters, including the quantity and type of Se in the environment, plant species, medium pH, and the presence of Sulphur and phosphorus (Rasool et al., 2022). Agricultural soils are predominantly richer in selenate than selenite and the selenate being more water soluble is readily absorbed as compared to selenite (Raina et al., 2021). As a result of the chemical analogy of selanate/selenite with sulphate and phosphate, their behavior in metabolism and transport in plants is closely related (Raina et al., 2021). A high concentration of Se seems to promote the pathway of “Starch and sucrose metabolism” as its treatment resulted in the accumulation of both forms of carbohydrates in potato (Solanum tuberosum L.) (Turakainen et al., 2004). We speculate that carbohydrate-active enzymes (CAZymes) of class glycoside hydrolases (GH) that are mostly enriched in this pathway can be the target of Selenium. The co-transport of selenate and selenite occurs with protons (Smith et al., 2000) and thus their transport occurs with the help of sulphate and phosphate transporters (Feist and Parker, 2001; White et al., 2004). In Arabidopsis thaliana Q9LIK9, a sulfur transporter controls the reduction of selenate and assists selenate and sulfur absorption and assimilation (Pilon-Smits et al., 1999). In rice, a phosphate transporter (OsPT2) is responsible for the active uptake of selenite (Zhang et al., 2014). Similar reports on the role of phosphate transporter in selenite uptake have been revealed in many studies (Li et al., 2008; Gu et al., 2016). The mechanism of selenium uptake in proso millet is not well understood and the present study aims to identify selenate and selenite transporters and their expression under salt stress and Se (Na₂SeO₃) application.

In recent years, a major focus of plant researchers has been to understand the functions of membrane transporters in plants. Recent studies in Oryza sativa L. and Arabidopsis thaliana L. have revealed that phosphate and sulphate transporters play a vital role in Se uptake (Takahashi et al., 2000; Li et al., 2008; Barberon et al., 2008; Zhang et al., 2014), however, their role in millets remains relatively unclear and to date there is little data available on their identity and function in proso millet. In this study, eight Se transporters were identified and their expression was characterized. Among these, four (RLM65282.1, RLN42222.1, RLN18407.1 and RLM74477.1) belong to sulphate transporters which have a role in selenate uptake, whereas the other four (RLN41904.1, RLN17428.1, RLN17268.1 and RLM65753.1) function in selenite uptake. The role of sulphate transporters in selenate uptake and phosphate transporters in selenite uptake has been revealed in many studies (El Mehdawi et al., 2018; Wang et al., 2019; Li et al., 2020; Peng et al., 2021). Thirty-seven selenite transporters have been identified in Pyrus malus, 23 in Camellia sinensis, 42 in populus and 26 in rice (Liu et al., 2011; Zhang et al., 2016; Sun et al., 2017; Cao et al., 2021). Whereas, 12 selenate transporters have been identified in Arabidopsis thaliana, 11 each in Populus trichocarpa and Sorghum bicolor L., 12 in Oryza sativa L., 10 in Triticum aestivum, 8 in Medicago truncatula, eight in Camellia sinensis and 6 in Astragalus racemosus (Buchner et al., 2010; Zhang et al., 2022).

In this study, the physiochemical properties of selenate transporters revealed that the amino acid lengths of these transporters ranged from 474 to 646, whereas for selenite transporters it ranged from 521 to 539. The experimentally reviewed selenate transporter amino acid sequence of Arabidopsis thaliana L. (Q9LIK9) which was used as the source of BLAST has an amino acid length of 463 and the selenate transporter amino acids sequences of proso millet have more length than Arabidopsis thaliana L. Similarly the number of amino acid in selenite transporter sequence of Oryza sativa L. (Q8GSD9) is 528, indicating a similar number of amino acids with proso millet selenite transporters. RLM65282.1 has a higher molecular weight of 72171.69 Da among selenate transporters whereas among selenite transporters the highest molecular weight was 59138.29 Da which corresponds to RLM65753.1. The theoretical isoelectric point of selenate and selenite transporters ranged from 7.4 to 8.38 and 8.1 to 8.32 respectively. From these, it is clear that all these transporters are basic in nature (Moldoveanu and David, 2017). The unstable nature of protein is determined by the presence of certain dipeptides and is related to the protein’s half-life in vivo. The protein with an instability index smaller than 40 is predicted as stable and the value greater than 40 is predicted as unstable. Owing to these values all selenate transporters were found to be unstable, whereas all selenite transporters were stable (Guruprasad et al., 1990). A measure of the relative volume occupied by aliphatic amino acids is known as the aliphatic index which is directly correlated with the thermo stability of globular proteins (Ikai, 1980). The aliphatic index of selenate transporters ranges from 80.42 to 89.85 whereas that of selenite transporters ranges from 81.17 to 92.59, indicating higher stability of selenite transporters over a wide temperature range. Similarly, the negative grand average of the hydropathicity value indicates the hydrophilicity of the protein and vice versa (Baloji et al., 2019). The calculated grand average of hydropathicity for selenate transporters revealed that these are hydrophilic in nature, whereas, all selenite transporters are hydrophobic in nature, which infer that selenate proteins are more water soluble than selenite proteins which is corroborated with a study on tea plant (Cao et al., 2021). The predicted sub-cellular localization of RLM65282.1, RLN42222.1, RLN18407.1 and RLM74477.1 were mitochondria, chloroplast, chloroplast and mitochondria respectively, whereas all four selenite transporters are localized in plasma membrane. No trans-membrane helices for selenate transporters were found, whereas selenite transporters have 11 to 12 trans-membrane helices. Comparable results for sub-cellular localization of sulphate transporters and phosphate transporter were found in Arabidopsis thaliana (Rotte and Leustek, 2000; Araie et al., 2011; Młodzińska and Zboińska, 2016; Liao et al., 2019). The chromosomal locations of these transporters indicate that all selenate transporters (RLM65282.1, RLN42222.1, RLN18407.1 and RLM74477.1) are located on chromosome number 16, 1, 2 and 15. The selenite transporters (RLN41904.1, RLN17428.1, RLN17268.1 and RLM65753.1) are located on chromosome number 1, 2, 2 and 16. To investigate the evolutionary relationships of the Se transporters, amino acid sequences from proso millet and Arabidopsis thaliana L. were used to construct the tree. Similarly, for constructing a selenite transporter tree, sequences from proso millet and Oryza sativa L. were used. Both phylogenetic trees generated two main groups with many subgroups, indicating close relation and relative likeness between them. Our study correlated with similar studies in Arabidopsis thaliana, Oryza sativa L., Camellia sinensis, Pyrus malus, and Populus trichocarpa. A phylogenetic tree of phosphate transporter genes was constructed for Arabidopsis thaliana, Oryza sativa L., Camellia sinensis, Pyrus malus, and Populus trichocarpa. Using this information, genes were grouped into five clusters showing close relationships. Similarly, a phylogenetic tree based on Arabidopsis thaliana, Oryza sativa, Populus tremula x alba, and Brassica oleracea was constructed. Based on the phylogenetic tree, eight of the sulfate transporter genes clustered within the same branch as those found in Populus tremula × alba and they had closer genetic relationships from the perspective of gene evolution (Cao et al., 2021; Zhang et al., 2022). A protein/protein interaction study revealed that 11 proteins are significantly involved in interaction with selenate transporters and 10 proteins are involved with selenite transporters, apart from this, three other selenite transporters showed interaction with each other and one transporter (A0A3L6S8F9) did not appear to interact with any protein. The selenate proteins interact with sulfite oxidase-like (catalyzes the oxidation of sulfite to sulfate), adenylyl sulfate kinase (catalyzes the synthesis of activated sulfate) and HIT domain-containing proteins. Our study correlated with various previous studies in which they revealed similar interacting proteins with these transporters (Kopriva, 2006; Lewandowska and Sirko, 2008; Takahashi et al., 2011). Among selenite transporters, A0A3L6S8X1 interacts with Actin-7-like (required for the trafficking and endocytic recycling cytoplasmic streaming, cell shape determination and cell division), H(+)/Pi cotransporter (Integral component of membrane, inorganic phosphate transmembrane transporter activity, phosphate ion transport and symporter activity), rhamnogalacturonate lyase-like isoform X1 (carbohydrate binding and lyase activity), TPT domain-containing protein (integral component of membrane, organic anion transmembrane transporter activity and organophosphate ester transmembrane transporter activity) and an uncharacterized protein which is an essential component of the cell cytoskeleton, cytoplasmic streaming, cell shape determination, cell division, organelle movement and extension growth. A0A3L6PXC9 interacts with calcium uniporter protein and putative anion transporter 3, chloroplastic. The 3D structure assessment scores were calculated and models were validated by means of the Ramachandran plot. While assessing the structure of prepared models, it was found for A0A3L6PUA6, 82.23% (QMEAN -5.03) of amino acids were in Ramachandran favored region, for A0A3L6TPY9, A0A3L6SBK6 and A0A3L6QB84, 95.43% (QMEAN 0.36), 96.65% (QMEAN 0.56) and 96.48% (QMEAN 0.21) amino acids were in Ramachandran favored region with no bad angles receptively. In the same way the Ramachandran favored percentages for A0A3L6TSS2, A0A3L6S8X1, A0A3L6S8F9 and A0A3L6PXC9 were 92.14% (QMEAN -6.13), 91.95% (QMEAN -6.25), 91.02% (QMEAN -6.96) and 91.15% (QMEAN -6.31) respectively. This indicates the stability of all the models. QMEAN Z-scores around 0.0 reflect a native-like structure and, a QMEAN Z-score below 4.0 indicates a model with low quality (Benkert et al., 2011). Accordingly, A0A3L6TPY9, A0A3L6SBK6 and A0A3L6QB84 had native-like structures. The expression studies reveal that both selenate and selenite transporters are down-regulated with salt and up-regulated after applications of Se with salt. Se has a positive role in stress easing in plants (section 3.6). The up-regulation of selenate and selenite transporters under Se application can protect plants in various ways. Despite its non-essential nature, at lower doses Se has physiological benefits to plants, Se regulates the antioxidant system, enhances the chloroplast defense system, increases the ability of plants to scavenge excess ROS, promotes growth and photosynthesis, alleviates the damage to chloroplast ultrastructure, increase proline content, reduced H₂O₂ and MDA concentrations, enhanced biomass accumulation and relative water content, membrane stability index, stomatal conductance, photochemical efficiency and thus aiding them in mitigating stress (Tajima, 1993; Taha et al., 2021; Ahmad et al., 2021). In terms of the medicinal plant Iranian Borage, foliar application of sodium selenite resulted in higher levels of antioxidant activity and soluble sugars (Hosseinzadeh Rostam Kalaei et al., 2022), an effect that is very much similar to that on potato (Turakainen et al., 2004). As a consequence, the most frequent metabolic processes that follow the biosynthesis of carbohydrates exist in the pathway “Starch and sucrose metabolism” in which CAZymes acting through the double displacement catalytic mechanism are enriched towards the acquisition of energy required to cope with different biotic and abiotic stresses (Henrissat et al., 2001). Under salt stress, nano-selenium improved the antioxidant machinery and soluble sugars in groundnut (Arachis hypogaea). Studies on lettuce plants revealed the role of selenite and selenate in the alleviation of salt stress (Hawrylak-Nowak, 2015). Exogenous selenium in the form of selenate on olive reduced the negative effects of salt stress (Regni et al., 2021). In wheat, selenium and sulfur in involved in the mitigation of abiotic stress (Khan et al., 2015). Various similar studies revealed the role of selenium in abiotic stress mitigation (Hasanuzzaman et al., 2020; Shah et al., 2020; Mushtaq et al., 2021; Taha et al., 2021; Saleem et al., 2021; Rasool et al., 2022).

Both phosphate and sulphate transporters in millets have been assessed using computational and expression studies. In the present study, it can be concluded that selenite may be transported through phosphate transporters and selenate through sulfate transporters. Expression studies revealed that both transporters play a vital role in salt stress mitigation in millets. This study also revealed that selenium may have a direct impact on the plant’s response to salt stress. The study of gene expression proved the possible role of selenate and selenite under salt and in turn selenium uptake. It would be beneficial to identify and study these transporters to better understand how the millets respond and tolerate salt stress. Future research in the field of stress physiology and agriculture is required for understanding selenium accumulation, its function, and its transport mechanism in plants. This study will provide the scientific basis and theoretical framework for future studies relating to selenium, millets, and salt stress.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Conceptualization: NM, KA, SS, IT, AB, RR, and KH; data curation: NM, SS, RR, and KH; formal analysis: NM, FS, KA, SS, IT, AB, RR, and KH; funding acquisition: NM, SS, AB, RR, and KH; methodology: NM, KA, SS, FS, IT, AB, RR, and KH; software: AB, RR, and KH; validation: NM, KA, RR, and KH; visualization: RR and KH; writing – original draft: NM, KA, SS, IT, FS, AB, RR, and KH; writing – review and editing: KA, AB, RR, and KH. All authors contributed to the article and approved the submitted version.

This research work was funded by Institutional Fund Projects under grant no (IFPRC-219-130-2020). Therefore, authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia.

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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