Identification of the Nicotianamine Synthase (NAS) Gene Family in Wheat (Triticum aestivum.L) and the Role of Its Member TaNAS4-A in Zn and Fe Transport

Identification of the Nicotianamine Synthase (NAS) Gene Family in Wheat (Triticum aestivum.L) and the Role of Its Member TaNAS4-A in Zn and Fe Transport | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of the Nicotianamine Synthase (NAS) Gene Family in Wheat (Triticum aestivum.L) and the Role of Its Member TaNAS4-A in Zn and Fe Transport Gang Liu, Yixuan Sun, Pengyuan He, Yibo Wang, Yixuan Zhang, Qingfeng Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7727623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Functional & Integrative Genomics → Version 1 posted 14 You are reading this latest preprint version Abstract Zinc (Zn) and Iron (Fe) are essential trace elements for human health, yet deficiencies in both are widespread worldwide. As a major staple crop, wheat is an important dietary source of Zn and Fe. However, the concentrations of Zn and Fe in common wheat grains are generally low, making it necessary to enhance the nutritional value of wheat. This study first elaborated that both elements are absorbed by wheat via "Strategy II" which relies on phytosiderophores (such as mugineic acids) and related transporter proteins (e.g., YSL and ZIP families). Nicotianamine (NA) plays a key chelating role in the long-distance transport of Zn and Fe. Therefore, we further analyzed the NAS gene family in wheat, which showed high genetic diversity, unique gene structures, distinct evolutionary features, and was subjected to purifying selection. Expression profiling revealed that NAS genes were tissue-specific and responsive to various stress conditions. The overexpression of TaNAS4-A in rice, as well as the silencing of TaNAS4-A in wheat using BSMV-VIGS confirmed the role of TaNAS4-A in enhancing NAS enzyme catalytic efficiency, promoting phytosiderophores secretion, and increasing the accumulation of Zn and Fe in grains. Additionally, this study suggested that NAS genes may confer other functions, such as stress resistance, which deserves further investigation. This research provides a theoretical basis for Zn and Fe biofortification in wheat. Triticum aestivum Biofortification phytosiderophores TaNAS4-A BSMV-VIGS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Zn, Fe, wheat, and human Zinc (Zn) and Iron (Fe) are essential micronutrients across all biological systems. Zn is the only metal that can serve as a cofactor for over 300 enzymes (Rink et al. 2000). Its primary role is to stabilize the structures of numerous proteins, including signaling enzymes involved in various levels of signal transduction and transcription factors. Zn is also indispensable for the structural stability of Zn finger proteins (Zfp) (Beyersmann et al. 2002). In humans, Zn is a vital micronutrient for human health, playing a fundamental role in cellular processes such as growth, differentiation, and division by regulating DNA synthesis and RNA transcription. It is also instrumental in systemic functions including immune response, prostaglandin production, taste acuity, wound healing, cognitive performance, bone mineralization, blood clotting, and fetal development (Bhowmik et al. 2010 ). Zn deficiency decreases neutrophil activation efficiency, which is reversible with Zn supplementation (Maywald et al. 2022). Additionally, during acute illnesses, plasma Zn levels often drop rapidly, leading to hypozincemia (Wessels et al. 2013 ; Besecker et al. 2011 ). Subsequently, pro-inflammatory cytokines mediate the redistribution of Zn, directing Zn ions and Zn-containing compounds into specific cellular compartments to assist in protein synthesis, neutralize free radicals, and inhibit the survival of microorganisms within human body (Maywald et al. 2022). Notably, in humans, plasma Zn levels decline with age and are closely associated with the occurrence of cardiovascular diseases. Thus, there is a link between Zn deficiency and an increased risk of cardiovascular diseases (Little et al. 2010 ). It is estimated that approximately 433,000 children under the age of five die annually due to Zn deficiency global, accounting for 4.4% of deaths in this age group (Fischer et al. 2009; Borrill et al. 2014 ). About 17% of the world’s population, roughly 1.1 billion people, are at risk of Zn deficiency (Khan et al. 2022 ). Fe is another essential nutrient for humans, functioning as a critical component of heme groups, Fe-sulfur cluster proteins, and enzymes involved in mitochondrial respiration and DNA synthesis. Thus, Fe is indispensable for cellular metabolism, survival, and proliferation (McLean et al. 2009 ). The daily production of red blood cells in the human body requires approximately 20 mg of Fe. Insufficient Fe intake can lead to Fe deficiency anemia (Ganz et al. 2012). It has been reported that around 1.2 billion people worldwide were at risk of Fe deficiency (Camaschella. 2019), and an estimated 25% of the global population suffers from Fe deficiency anemia, largely due to inadequate dietary Fe intake (Wallace. 2016). Fe deficiency increases mortality risk in patients with acute coronary syndrome and myocardial infarction (Lawler et al. 2013 ). Even mild to moderate Fe deficiency anemia may impair cognitive development (Beard. 2003) and compromise immune mechanisms (Failla. 2003). Fe deficiency anemia during pregnancy can seriously affect maternal health, leading to reduced physical performance, increased fatigue, diminished cognitive function, and a higher risk of common infections and hospitalization (Viteri. 1994). As an important staple food for humans, wheat is the source of many mineral elements. In addition to being a major source of starch and energy, it is also rich in various components essential or beneficial to health, particularly proteins, vitamins, dietary fiber, and phytochemicals (Shewry et al. 2015). It also serves as a critical source of Zn and Fe for human needs. Some studies have shown that the Zn and Fe concentrations in wheat grains range from 16 mg/kg to 142 mg/kg and from 24 mg/kg to 80 mg/kg, respectively (Velu et al. 2014 ; Borg et al. 2009 ). However, the grain Zn and Fe concentrations of bread wheat, which accounts for 95% of the wheat production at the global level (Tadesse et al. 2019 ), ranged from 25 mg/kg to 53 mg/kg and from 24 mg/kg to 51 mg/kg, respectively (Borg et al. 2009 ; Graham et al. 1999 ). The low grain Zn and Fe concentrations in bread wheat do not meet the standards for biofortification. Therefore, it is important to breed wheat with high Fe and Zn concentrations, particularly for regions relying upon wheat as a staple food, such as North Africa and Central Asia (Ling et al. 1999 ). Understanding the mechanisms of Zn and Fe transport in wheat will be critical for guiding biofortification strategies. Transport of Zn and Fe in cell There are many Zn and Fe transporter proteins in plants, most of which can bind to cell membranes or organelle membranes and play an indispensable role in the transmembrane transport of Zn and Fe (Krishna et al. 2023 ). Studies on zinc-iron transporter proteins in arabidopsis, rice, and wheat have revealed that these transporters are widely distributed on membranes. They may either transport Zn and Fe ions inward or outward, and some can even transport in both directions (Fig. 1 ).There are distinct types of zinc-iron transporter proteins. For instance, ABC transporters, MIT transporters, and ATM transporters appear to be more inclined to transport iron ions. However, it is noteworthy that proteins from the YSL, ZIP, and HMA transporter families are all involved in the transport of zinc and iron. In particular, AtYSL1, AtYSL3, and AtYSL2 from the YSL family, as well as TaHMA4 from the HMA family, have been shown to participate in Zn and Fe transport .The vacuolar transporter proteins OsVIT1/2 are also involved in the transport of both Zn and Fe (Fig. 1 ).The YSL and ZIP family transporter proteins are extensively involved in the transport of Zn and Fe, and are distributed across the plasma membrane, mitochondria, chloroplasts, and vacuolar membrane,they appear to preferentially facilitate the unidirectional efflux of either Zn or Fe ions (Fig. 1 ). Furthermore, most transporter proteins appear to only facilitate unidirectional transport of either Zn or Fe ions, as reflected in zinc and iron transport where different transporters may exhibit distinct and specialized roles.However, in this paper, we focus more on transporters that are involved in the transport of both zinc and iron, such as YSL, ZIP, and HMA, as well as small organic molecules capable of chelating zinc and iron ions to facilitate their transport within plants, such as nicotianamine (NA), mugineic acids (MAs), citrate, and phenolics to facilitate iron transport (Takahashi et al. 2003 ; Hell et al. 2003; Nishiyama et al. 2012 ; Kobayashi et al. 2019 ). The Role of NA in Zn and Fe Transport in Wheat NA, an important metal chelator in plants, plays a critical role in the long-distance transport (Ling et al. 1999 ; Mari et al. 2006 ; Douchkov et al. 2005 ) and homeostasis of metal ions such as Fe, Zn, manganese (Mn), nickel (Ni), and copper (Cu) (Takahashi et al. 2003 ; Pianelli et al. 2005 ). NA is synthesized by NA synthase (NAS) through the condensation of three S-adenosylmethionine (SAM) molecules, a process that initiates the MA biosynthetic pathway. In graminaceous plants, NA is further converted into mugineic acid (MA)-family phytosiderophores, such as deoxymugineic acid (DMA) (Fig. 3 ),it chelates iron ions in the soil through Strategy II in graminaceous crops, which is crucial for root iron absorption (Marschner et al. 1986 ). Thus, the catalytic efficiency of NAS directly influences the biosynthesis levels of both NA and MAs. NA and MAs can bind to the YSL and TOM transporter families to participate in Zn and Fe transport within plants. Molecular docking predictions suggest favorable and stable binding interactions between rice YSL/TOM transporters and the DMA. Similarly, in Arabidopsis , the YSL1 protein also exhibits stable binding with NA. These small molecules can fit into conserved transporter binding pockets to form transport-competent complexes (Nozoye et al. 2015 ; Inoue et al 2009 . Figure 2 ). Graminaceous crops typically secrete MAs into the rhizosphere to chelate metal ions, including Fe(II), Fe(III), and Zn(II), for root uptake (Koike et al. 2004 ; Inoue et al. 2009 ). Once Fe(II)-MA, Fe(III)-MA, and Zn(II)-MA complexes are absorbed into root cells, the metal ions are released under acidic conditions through reduction. These ions subsequently form chelates with NA, resulting in Fe(II)-NA, Fe(III)-NA, and Zn(II)-NA. These complexes are then transported over long distances via the phloem by transporters such as YSL (Von et al. 1999; Seregin et al. 2023). During transport, metal ions may again be released under acidic conditions and subsequently redistributed by a range of transporters, including YSL, HMA, ZIP, and FPN (Fig. 3 ). This multi-layered transport mechanism ensures highly efficient acquisition, mobilization, and utilization of metal nutrients in plants. Identification of the Wheat NAS Gene Family and Functional Validation of TaNAS4-A NA is synthesized by nicotianamine synthase (NAS). NAS genes play a key role in the regulation of Zn and Fe uptake and translocation. In rice, overexpression of HvNAS1 significantly increased the contents of NA and its downstream product DMA, thereby enhancing Zn and Fe accumulation in grains (Masuda et al. 2009 ). Similarly, overexpression of OsNAS2 and OsNAS3 also markedly elevated Zn and Fe concentrations in rice grains (Gupta et al. 2023 ; Lee et al. 2023 ). Using CRISPR-Cas9 to edit the promoter sequence of OsNAS2 led to significantly higher gene expression, and the resulting gene-edited plants showed a substantial increase in grain Zn and Fe concentrations (Ludwig et al. 2024 ). In wheat, overexpression of OsNAS2 has been shown to significantly enhance Zn and Fe concentrations in flour (Harrington et al. 2023 ). However, studies on the role of TaNAS genes in Zn and Fe accumulation in wheat grains remain limited. Bioinformatic analysis of gene families provides an effective approach for investigating target genes. In this study, we performed a comprehensive analyses of the wheat NAS gene family, examining their evolutionary relationships, sequence conservation, expression patterns, and predicted protein interactions. Particularly, function of TaNAS4-A was investigated. Results The Gene Structure and Phylogenetic Analysis of NAS In the evolutionary analysis, NAS homologs in wheat and barley exhibited significant genetic diversity, as they were classified into distinct subfamilies within the phylogenetic tree. In contrast, NAS genes in Arabidopsis were relatively conserved and clustered within the same subfamily (Fig. 4 ). Based on the phylogenetic tree, we infer the presence of well-defined orthologous and paralogous relationships among NAS genes in Poaceae species. In wheat, TaNAS1-A/B , TaNAS2-D1/D2 , and TaNAS9-A/B/D are likely paralogous. In barley, HvNAS1, HvNAS5 , and HvNAS9 may be paralogous. In maize, ZmNAS3/4/5 and ZmNAS1/2/6 are potentially paralogous. In rice, OsNAS3 and OsNAS1/2 may also share a paralogous relationship. Except for AtNAS1-4 , clear orthologous relationships were observed for the remaining NAS genes across different species (Fig. 4 ). The structure of NAS genes is highly conserved. Except for TaNAS1-A , TaNAS1-B , and TaNAS6-A , which contain two, four, and two exons, respectively, all other genes consist of only one exon. For genes TaNAS1-A , OsNAS1 , and HvNAS1 , accurate transcription start sites could not be predicted (Fig. 4 ). The type and arrangement of motifs in NAS genes showed high consistency with the topology of the phylogenetic tree. Most NAS protein sequences contain eight conserved motifs. In Arabidopsis, all genes except for AtNAS2 shared six common motifs (Fig. 4 ). Among the eight predicted motifs, four were most commonly shared, including three types of endocytosis-related motifs: YXXΦ, LL, and FXXF (highlighted in red boxes in Fig. 4 ). These endocytic motifs mediate internalization by interacting with receptors or other proteins on the cell membrane, thereby facilitating the uptake of extracellular substances (Pandey et al. 2009). The presence of these motifs suggests that NAS proteins, functioning as transferases, are susceptible to endocytosis, which in turn promotes catalytic reactions within the cellular matrix. The codon alignment used in this analysis comprised 391 codons, of which 390 were tested for positive selection. The results revealed that among these codons, 8 were under positive selection (indicated by red bars in Fig. 5 ), 98 were under purifying selection (green bars), and 274 were evolving neutrally (gray bars) (Fig. 5 ). The number of positively selected sites was significantly lower than those under purifying selection and neutral evolution, indicating that the evolution of this gene family is primarily influenced by purifying selection, with adaptive mutations being relatively rare. Given that NAS gene family encodes enzymatic proteins, this pattern is consistent with the dominance of purifying selection in active sites and structurally conserved protein regions, where destabilizing mutations could severely compromise function or fitness. Consequentially, natural selection tends to eliminate deleterious mutations and preserve sequence conservation. Synteny analysis of 19 TaNAS genes with identifiable chromosomal locations revealed that most genes exhibit conserved syntenic relationships across wheat subgenomes. A syntenic block was detected between the TaNAS6-B region on chromosome 4B and the TaNAS6-D region on chromosome 4D. Additionally, both regions showed synteny with the TaNAS1/3/9-A region on chromosome 2A, the TaNAS1/3/9-B region on chromosome 2B, and the TaNAS9-D region on chromosome 2D. Moreover, the TaNAS1/3/9-A , TaNAS1/3/9-B , and TaNAS9-D regions demonstrated mutual synteny, suggesting that these genes may have evolved through duplication and insertion of a common ancestral copy (Fig. 6 ). Notably, two distinct regions on chromosome 6B were found to be syntenic with the TaNAS7-A and TaNAS7-D and TaNAS2-A regions, respectively (Fig. 6 ), which may indicate the presence of previously uncharacterized NAS genes within these chromosomal segments. The remaining TaNAS genes did not show detectable synteny, potentially reflecting functional specialization or distinct evolutionary trajectories. Expression and Protein Interaction of TaNAS Genes The 18 TaNAS genes were broadly classified into three categories based on their tissue-specific expression patterns. The first category was predominantly expressed in seedlings roots. The second category showed concentrated expression in seedlings leaves. The third category was mainly expressed during the reproductive stage, such as in anthers and seeds shortly after pollination. Among them, TaNAS7-A2 exhibited the highest expression in anthers TaNAS1-A in pistils; and TaNAS9-D in seedling shoots (Fig. 7 ). Under Fe deficiency, almost all TaNAS genes were significantly upregulated by day 5 of treatment, with the exceptions of TaNAS7-D , which was downregulated, and TaNAS1-A , which was significantly downregulated. By day 7, the magnitude of upregulation began to decrease markedly, with TaNAS7-A2 and TaNAS9-D showing downregulated expressions. Among all members, TaNAS3-A and TaNAS3-B were the most responsive to Fe deficiency with upregulation fold changes greater than 5 (Fig. 7 ). Based on the phylogenetic relationships and expression profiles (FPKM) under abiotic stress conditions, the TaNAS genes were classified into four subgroups: G1, G2, G3, and G4. In subgroups G1-G3, salt stress triggered the highest responses, followed by drought and waterlogging, while little to no response was observed under dark, cold, freezing, and wounds. TaNAS7-A2 showed minimal response to abiotic stresses, with only weak reactions to drought and salt stress. Interestingly, three genes, including TaNAS9-A , TaNAS9-B , and TaNAS9-D , in subgroup G4 demonstrated the most active responses to stressful environments, reacting positively to dark, cold, freeze, wound, heat, drought, nutrient deficiency, waterlogging, and salt stress (Fig. 8 ). Proteins A0A3B6EDR2, W5BGU5_WHEAT, A0A077RTD0, A0A3B6J0A9, A0A3B6UB94, A0A3B6HUG3, A0A3B6KUD6, A0A3B6IZR0, A0A3B6JE97, and A0A3B6ILM9 were identified as co-interacting partners of proteins encoded by TaNAS genes. Among these 10 interacting proteins, most are functional proteins associated with DNA binding, DNA repair, and transcription processes. For instance, W5BGU5_WHEAT functions as a promoter recognition protein, while the remaining ones, such as A0A077RPD2, A0A3B5YTZ3, and A0A3B5ZPR6, exhibit acyltransferase activity. However, no significant interactions were detected among the TaNAS proteins. Many of the interacting proteins are linked to DNA binding and transcriptional regulation, suggesting that cells may directly couple the synthesis of secondary metabolites (enzymes) with the transcriptional regulation of their genes (DNA-binding/transcriptional proteins), forming a rapid "sensing-response" feedback loop (Fig. 9 ). Preliminary Functional Verification of TaNAS4-A Gene Involved in Zn and Fe Transport Overexpression of TaNAS4-A in rice (Nipponbare) Phenotypes of Overexpressing Rice Under Different Zn Stress Conditions When rice seeds germinated and developed their first leaf, they were maintained under hydroponic condition for 3 days. Healthy seedlings were selected, and the seeds were removed. These seedlings were then subjected to hydroponic cultivation under different Zn concentration gradients for 14 days. Under normal hydroponic conditions, we observed that the wild-type Nipponbare rice showed the most vigorous growth. The shoot height of the wild-type was greater than that of OE-3 and OE-7, and the root length and development were significantly superior to those of OE-1, OE-3, and OE-7 (Fig. 10 a). Interestingly, this trend reversed under both Zn excess and Zn deficiency. Under Zn excess, although the roots of all rice lines became thicker, shorter, and produced more root hairs, the overexpressed lines OE-1, OE-3, and OE-7 exhibited notably greater root length and density the wild-type. The shoot growth of these overexpressed lines were also superior, especially the OE-7 seedlings (Fig. 10 b). Under Zn deficiency, although the seedlings exhibited slender leaves and chlorosis, these symptoms were more severe in the wild-type compared to OE-3 and OE-7 (Fig. 10 c). Overexpression of TaNAS4-A increased NAS catalytic efficiency, MAs secretion rate, and grain Fe and Zn concentrations In both wild-type and overexpressing lines, the catalytic efficiencies of NAS enzymes in the roots wre higher than that in the leaves. The catalytic efficiencies in the roots of overexpressing lines were significantly higher than that of the wild-type, with differences were also observed among the overexpressing: OE-3 and OE-7 showed significantly higher root NAS catalytic efficiencies than OE-1. In leaves, NAS catalytic efficiencies in the overexpressing lines were also significantly higher than that in the wild-type. OE-7 exhibited significantly higher leaf NAS catalytic efficiency than OE-1 (Fig. 11 a). The day of emergence of the first true leaf was designated as Day 1. The secretion rate of MAs from root was measured on Days 7, 10, 12, and 14. On Day 7, the MAs secretion rate of the wild-type was significantly lower than that of the overexpressing lines. From Day 7 to Day 14, the wild-type consistently exhibited lower MAs secretion rates compared to the overexpressing lines. By Day 14, the secretion rate of the wild-type began to plateau, while that of the overexpressing lines still showed an increasing trend. The MA secretion rate of OE-7 remained the highest throughout the experiment. After Day 10, the MA secretion rate of OE-3 surpassed that of OE-1 (Fig. 11 b). The leaf Zn and Fe concentration of OE-3 and OE-7 were significantly higher than those of the wild-type, and the Fe concentration of OE-1 was significantly higher than that of the wild-type. No significant difference was observed in leaf Zn concentration among overexpression lines. The leaf Fe concentration of OE-3 and OE-7 reached 119.6 and 128 mg/kg, respectively, significantly higher than that of OE-1 (99.8 mg/kg) (Fig. 11c2). The grain Zn and Fe concentrations of the overexpressing lines were significantly higher than those of the wild-type. The grain Zn concentration of OE-3 and OE-7 was 45.3 and 46 mg/kg, respectively, significantly higher than that of OE-1 (40.6 mg/kg). Similarly, the grain Fe concentration of OE-3 and OE-7 was 27.9 and 26.4 mg/kg, also significantly higher than that of OE-1 (20.6 mg/kg) (Fig. 11c1). Gene silencing of TaNAS4-A in NC2 wheat mediated by BSMV-VIGS BSMV-VIGS Silencing Phenotype Phytoene desaturase (PDS, phytoene desaturase) is a key enzyme in the carotenoid biosynthesis pathway in plants, algae, and certain bacteria, playing an essential role in pigment synthesis and photoprotection. To assess the effectiveness of BSMV-VIGS, PDS was inserted into the BSMV-γ vector, and leaf whitening was used as a visual indicator. On the 11th day after rub-inoculation, wheat leaves inoculated with BSMV: TaPDS began to exhibit distinct whitening symptoms from the leaf tips to the base, while no obvious symptoms were observed in the CK (no inoculation) and Mock (buffer-only) leaves, indicating successful leaf rub-inoculation. After 5–6 days, yellow-white streaks gradually appeared on leaves inoculated with γ and BSMV: TaNAS4-A constructs (Fig. 12 a). Subsequently, during the grain-filling stage, the wheat plants were inoculated again. Pronounced whitening phenomena were observed in the whole spikes of BSMV: TaPDS inoculated lines (Fig. 12 b), demonstrating that the BSMV-VIGS system functioned effectively in wheat at both vegetative and reproductive stages. Silencing Efficiency of TaNAS4-A and Its Homologous Genes in Inoculated Leaves BSMV-VIGS achieved a silencing efficiency of 78.5% for TaNAS4-A . Homologous genes TaNAS4-D and TaNAS4-U were also significantly silenced by BSMV-VIGS, with efficiencies of 42.1% and 40%, respectively (Fig. 13 ). The expression level of TaNAS5-B was reduced by 48.7% (Fig. 14 ). The expression of TaNAS1-A and TaNAS9-A was upregulated by 1.84-fold and 2.12-fold, respectively, possibly due to functional compensation. These two genes are evolutionarily distant from TaNAS4-A (Fig. 14 ). The expression levels of other detected NAS genes were not silenced by BSMV-VIGS. It is worth noting that the expressions of TaNAS2-A , TaNAS5-B , and TaNAS9-A were significantly upregulated after inoculation with the BSMV-γ vector (Fig. 14 ). Silencing of TaNAS4-A decreased NAS catalytic efficiency and grain Zn and Fe concentrations The leaf catalytic efficiencies of NAS in wheat plants infected by BSMV: TaNAS4-A were significantly lower than that in uninfected plants (CK) and in plants infected with BSMV: TaPDS or BSMV:γ. The NAS catalytic efficiencies in plants infected with BSMV: TaPDS or BSMV:γ viral solutions was also significantly lower than that in CK (Fig. 15 a). The grain Zn and Fe concentrations of CK were 40.6 mg/kg and 31.6 mg/kg, respectively. In plants infected with the BSMV:γ viral solution, the grain Zn and Fe concentrations were 27.4 mg/kg and 28.0 mg/kg, respectively. In plants infected with BSMV: TaNAS4-A , grain Zn and Fe concentrations were 12.0 mg/kg and 15.7 mg/kg, respectively. Grain Zn and Fe concentrations in plants infected by either BSMV: TaNAS4-A or BSMV:γ were significantly lower than those in CK. Furthermore, the grain Zn and Fe concentrations in BSMV: TaNAS4-A infected plants were also significantly lower than those in BSMV:γ infected plants (Fig. 15 b). Similar trends were observed in leaf Zn and Fe concentrations (Fig. 15 c). Discussion Bioinformatic Analysis of NAS Genes Motif analysis revealed that NAS proteins contain eight conserved motifs, four of which are endocytosis-related motifs (YXXΦ, LL), implying their propensity for endocytosis, which facilitates intracellular catalytic reactions. This strongly suggests that NAS proteins may possess intrinsic roles in signal perception or membrane transport regulation. They may cycle between the plasma membrane and endosomes via endocytosis, directly participating in the cellular "sensing" of metal ion status (e.g., Fe and Zn), thereby forming a rapid "membrane perception–intracellular signaling–gene transcription feedback" loop. This extends beyond their traditional function as synthase and offers a novel perspective on their role in signal transduction. Evolutionary analysis indicates that the NAS gene family is primarily shaped by purifying selection, with very few positively selected sites. This is highly consistent with the critical enzymatic function of NAS proteins. As enzymes catalyzing the synthesis of nicotianamine, a key precursor of phytosiderophores, their active sites and three-dimensional structures must be highly conserved. Any non-neutral mutations could disrupt their enzymatic activity, thereby affecting the chelation and transport of essential micronutrients such as Fe and Zn, and would likely be eliminated by natural selection. Although formed a multigene family, purifying selection suggests that each member of the NAS genes undertake indispensable or at least difficult-to-replace specific functions, such as function in different tissues, developmental stages, or stress conditions (Fig. 7 ; Fig. 8 ). Synteny and phylogenetic analyses revealed that the NAS gene family has expanded through gene duplication, including whole-genome duplication and segmental duplication, with further complexity arising from subgenome differentiation in polyploid species such as wheat. For instance, the presence of TaNAS9-A/B/D is a typical outcome of polyploidization events. Expression analysis further supported the hypothesis of functional divergence. Although homologous genes from different subgenomes (e.g., TaNAS9-A/B/D , Fig. 14 ) share sequence similarity, they exhibit tissue specific expressions (e.g., high expression of TaNAS9-D in seedling shoots) and stress specific responses (e.g., the G4 subgroup responds to nearly all stresses). This indicates that after duplication, the functions of the ancestral gene were partitioned among different copies, supporting the occurrence of subfunctionalization (Rastogi et al. 2005). Protein-protein interaction predictions revealed that TaNAS proteins do not directly interact with each other but do interact with numerous DNA-binding/transcriptional regulatory proteins and acyltransferases (Fig. 9 ). This finding reinforces a core biological concept: cells tend to directly couple the synthesis of metabolic enzymes with the transcriptional regulation of their functions (Shi et al. 2004). NAS proteins may interact with transcription factors or chromatin modification complexes, thereby feedback-regulating their own transcription or that of other downstream genes, forming an efficient homeostatic regulatory network. Functional Verification of NAS Gene Klatte et al ( 2009 ) constructed five NAS mutant lines using T-DNA insertion technology. Under moderately toxic nickel (Ni) conditions, mutant line nas4-1 exhibited more severe chlorosis than the mutant line nas3-1, and the NA level in the triple NAS mutant decreased to 30–40% of that in the wild type (Klatte et al. 2009 ). Under Fe deficiency, the chlorosis in leaves of both single and multiple mutants was more severe compared to the wild type. Among the single mutants tested, the nas4-1 mutant showed the most pronounced leaf chlorosis. These results indicate that the functions of NAS genes are not entirely redundant (Schuler et al. 2011). In rice, using endosperm-specific promoter-containing pMDC vectors, transgenic rice plants overexpressing OsNAS1 , OsNAS2 , and OsNAS3 were generated. Compared to the wild-type, all three overexpression lines showed significantly increased concentrations of NA, Zn, and Fe in grains, with OsNAS2 and OsNAS3 overexpression lines exhibiting more pronounced enhancements. Meanwhile, OsNAS2 overexpression significantly elevated Fe and Zn content in rice endosperm (Johnson et al. 2011 ). When overexpressed in wheat, OsNAS2 also markedly increased Zn concentration in wheat grains (Singh et al. 2017 ). In comparison to the wild-type, the OsNAS3 overexpression line ( OsNAS3-D1 ) accumulated higher levels of Fe and Zn in both shoots and roots, along with significantly increased contents of Fe (2.9-fold), Zn (2.2-fold), copper (1.7-fold), and NA (9.6-fold) in seeds. Concurrently, the overexpressing lines exhibited enhanced tolerance to Fe and Zn deficiency, as well as to Zn, Cu, and Ni toxicity (Lee et al. 2009 ). Under Fe excess conditions, the NA synthesized by OsNAS3 helps alleviate the toxicity of Fe overload on cells, while under normal Fe conditions, the NA produced by OsNAS1 and OsNAS2 facilitates Fe transport, suggesting distinct roles of NA under different conditions (Aung et al.2019; Nozoye et al. 2014a ). Overexpression of HvNAS1 in sweet potato resulted in leaves with NA concentrations, Fe, and Zn content that were 7.9-fold, 3-fold, and 2.9-fold higher, respectively, than those of the wild-type (Nozoye et al. 2017 ). Furthermore, transgenic soybean plants overexpressing HvNAS1 exhibited NA concentrations as high as 768 µg∙g⁻¹ dry weight, approximately 4 times that of non-transgenic soybeans, along with twice the Fe content in seeds and demonstrated tolerance to Fe deficiency in calcareous soil (Nozoye et al., 2014b ). Expression of the maize gene ZmNAS during the seedling stage is regulated by jasmonic acid, abscisic acid, and salicylic acid (Mizuno et al., 2003 ). In this study, overexpression of TaNAS4-A increased the NAS catalytic efficiencies in roots and leaves, MAs secretion rate and grain Zn and Fe concentrations, whereas TaNAS4-A silencing by BSMV-VIGS decreased the NAS catalytic efficiency and grain Zn and Fe concentration. Take together our and others’ results confirmed the critical role of NAS genes in Zn and Fe transport across plant species. Notably, the BSMV-VIGS system targeting TaNAS4-A also partially silenced its three most closely homologous genes and significantly upregulating TaNAS9-A and TaNAS1-A , which are evolutionarily more distant from TaNAS4-A . This phenomenon may stem from a functional compensation mechanism among members of the NAS gene family, whereby the expressions of certain genes is adjusted to maintain metal homeostasis when TaNAS4-A is specifically silenced. Although this compensatory effect reflects an adaptive strategy of plants, it poses challenges for elucidating the independent function of individual genes. Potential Additional Biological Functions of NAS Genes Studies have shown that overexpression of HvNAS1 enhances metal toxicity tolerance in tobacco (Takahashi et al. 2003 ). Han et al ( 2017 ) successfully cloned a TaNAS-D gene from wheat. Overexpression of this gene in Arabidopsis significantly increased NA levels and enhanced the plant's salt stress tolerance. Under alternative stress conditions, TaNAS-D transgenic Arabidopsis exhibited higher germination rates and better growth compared to the wild-type (WT). Further studies revealed that transgenic Arabidopsis plants had a higher K⁺/Na⁺ ratio, lower malondialdehyde (MDA) content, reduced ion leakage (IL), and increased activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), resulting in reduced membrane damage. Additionally, overexpression of TaNAS-D led to upregulation of AtSOS1, AtSOS2, AtSOS3, AtFAD5 , and AtSAD1 under salt stress (Han et al. 2017 ). In leguminous plants, symbiotic nitrogen fixation depends on the normal function of MtNAS2 . In the nas2-1 insertion mutant, nitrogenase activity was lost, but reintroducing a copy of the wild-type MtNAS2 gene restored nitrogenase activity in the mutant (Escudero et al. 2020 ). In our study, when wheat leaves were inoculated with the BSMV virus (γ), three genes, including TaNAS2-A , TaNAS5-B , and TaNAS9-A , were significantly upregulated, suggesting their potential roles in the antiviral responses of plant. Both our findings and previous research indicate that NAS genes may participate in biological processes beyond Zn and Fe absorption and transport. Overall This study employed bioinformatic analyses to highlight key biological characteristics of wheat NAS genes, followed by gene function validation methods to preliminarily confirm the role of TaNAS4-A in Zn and Fe transport. To the best of our knowledge, this is the first report on TaNAS4-A for its function in assisting Zn and Fe accumulation in wheat grains. Our findings provide valuable insights into the mechanisms of Zn and Fe transport in wheat. Materials and Methods Bioinformatic Analysis The NAS gene and protein sequences of wheat were obtained from IWGSC V1.1 ( https://wheat-urgi.versailles.inra.fr/Projects/IWGSC ). Gene and protein sequences of other species were sourced from NCBI ( https://www.ncbi.nlm.nih.gov ) and Ensembl Plants ( https://plants.ensembl.org/index.html ). Protein accessions were retrieved from UniProt ( https://www.uniprot.org/ ). Phylogenetic trees were constructed using the neighbor-joining method in MEGA 11. Gene structures were predicted using FGENESH ( https://www.softberry.com/berry.phtml?topic=fgenesh ), and motifs were predicted via the MEME website ( https://meme-suite.org/meme/ ). Molecular docking was performed using AutoDock Vina, and results were visualized with Pymol. Intra-species synteny analysis was conducted using the one-step MCScanX-Superfast plugin in TB-tools. Positive selection analysis was carried out using Datamonkey ( https://www.datamonkey.org/ ). Protein-protein interactions were queried via STRING ( https://cn.string-db.org/ ) and visualized using Cytoscape. Chord diagrams were generated with the circlize package in R. For some Zn and Fe transport proteins subcellular localization in silico by WoLFPSORT ( https://wolfpsort.hgc.jp/ ). Total RNA Extraction and RT-qPCR RNA was extracted from 0.1g of leaf samples using RNAiso Plus (TaKaRa, DaLian, China). High-quality RNA was used to synthesize cDNA with PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TaKaRa, DaLian, China). Fluorescent quantitative reactions were conducted using TB Green® Premix Ex Taq™ II FAST qPCR (TaKaRa, DaLian, China) in a 20 µl system. Reactions were performed on the ABI 7300 (Thermo Fisher Scientific, United States) instrument, with three biological replicates for each sample. The wheat Actin gene (Gene ID: AB181991) was used as the internal reference gene. The fluorescent quantitative data were analyzed using the 2 −ΔΔCt method. Determination of NAS Enzymatic Catalytic Efficiency Based on previous methods (Higuchi et al. 1996 ; Della et al. 1988) with certain modifications, the NAS enzymatic catalytic efficiency was determined. The detailed procedure is as follows: Approximately 0.5 g of frozen tissue (root or leaf) was ground in a mortar containing liquid nitrogen, followed by the addition of 1 mL of 0.2 M Tris/HCl buffer (pH 8.0) containing 10 mM EDTA, 5% (w/v) insoluble PVP, 5% (v/v) glycerol, 0.1 mM p-APMSF, 100 µg/mL antipain, and 10 mM DTT. The homogenate was centrifuged at 8000 × g for 20 minutes, and the supernatant was collected. The supernatant was then eluted through a 1 mL hydrophobic resin column pre-equilibrated with buffer (20 mM Tris/HCl, 1 mM EDTA, 0.1 mM p-APMSF, 3 mM DTT, 0.4 M (NH₄)₂SO₄, pH 8). The NAS enzyme was eluted using a 1% glycerol solution containing 0.1 mM p-APMSF and 3 mM DTT. The NAS enzyme reaction was carried out under the following conditions: the enzyme solution was supplemented with 20 µg/mL antipain and 50 µM SAM, incubated at 25°C for 15 minutes, and the reaction was terminated by adding ethanol to a final concentration of 50% (v/v). After termination, the pH of the solution was adjusted to 8.0. The solution was then applied to a PAX mixed-mode strong anion exchange column pre-equilibrated with 0.05 M sodium phosphate buffer (pH = 8.2). After equilibration, elution was performed three times using 0.1 M NaCl solution (pH = 7.5), and the eluate was collected. The eluate was subsequently transferred to a spectrophotometer to measure UV absorbance at 260 nm (characteristic absorption peak of adenosine from MTA/S-methyl-5'-thioadenosine). The entire process was maintained at 15°C. A standard curve was constructed using MTA as the sole solute, with concentration as the horizontal axis and absorbance as the vertical axis. Determination of Root Exudation of Mugineic Acid Family (MAS) Based on the diurnal rhythm of MAs secretion, collection began each morning after 2 hours of light exposure. The root system was first rinsed, and the exudates were collected for 4 hours into a container holding a certain volume of deionized water. The container was kept shaded and aerated. With modifications to previous determination methods (Takagi. 1993; Yu et al. 1999 ), the collection solution (containing MAs) was processed as follows: A 9.00 ml aliquot of the collection solution (MAs) was pipetted into a small plastic vial. Then, 0.50 ml of 1 mM FeCl₃ and 1.00 ml of 0.5 mol/L NaAc-HAc buffer (pH 7.0) were added. The mixture was shaken for 1 hour to fully form the Fe(Ⅲ)-MAs complex. After filtering out excess Fe(OH)₃, 8.00 ml of the filtrate (Fe(Ⅲ)-MAs) was transferred, followed by the addition of 0.20 ml of 1.5 mol/L H₂SO₄ and 0.50 ml of 8% hydroxylamine hydrochloride (HONHCl). The solution was reduced in an oven at 50°C for 20 minutes to form Fe(Ⅱ)-MAs. Then, 1.00 ml of 2 mol/L NaHAc-HAc buffer (pH 4.7) and 0.2 ml of 0.01 mol/L Ferrozin were added. The resulting Fe(Ⅱ)-Ferrozin complex (purple-red color) was measured colorimetrically at 562 nm, and the amount of solubilized Fe was calculated. The MAs secretion rate was then estimated based on the amount of solubilized Fe (i.e., Fe(Ⅱ)-Ferrozin), converted into an equivalent of Fe-chelating compounds. Determination of Zn and Fe Concentrations in Tissues The concentrations of Zn and Fe in wheat and rice grains were determined using Flame Atomic Absorption Spectroscopy (FAAS) in accordance with the GB5009.14—2017 standard. A 1-gram sample of dried grain was placed in a conical flask, to which 10 mL of nitric acid and 0.5 mL of perchloric acid were added. The mixture was digested on an adjustable electric heating plate under the following conditions: 120°C for 0.5–1 hour, increased to 180°C for 2–4 hours, and further raised to 200–220°C. If the digestate appeared brown, a small amount of additional nitric acid was added. Digestion continued until white fumes were emitted, and the solution became colorless, transparent, or slightly yellow. The digested solution was then cooled and diluted to 25 mL with water, mixed thoroughly, and set aside for analysis. Each experiment included a reagent blank control. Standard solutions and calibration curves were prepared using Zn oxide (purity > 99.8%) and ferric nitrate (purity > 99.8%). The Zn and Fe concentrations in the samples were determined by FAAS at wavelengths of 213.9 nm and 248.3 nm, respectively. A series of standard solutions with increasing mass concentrations of Zn and Fe were sequentially introduced into the flame atomizer. After atomization, their absorbance values were measured. Calibration curves were constructed with mass concentration as the x-axis and absorbance as the y-axis. Under the same experimental conditions used for the standard solutions, the blank and sample solutions were introduced into the flame atomizer. After atomization, their absorbance values were measured, and the concentrations were quantitatively determined by comparison with the standard calibration curves. Construction of Overexpression Rice Lines As described in Supplementary File 1. Construction of the BSMV-VIGS System As described in Supplementary File 2. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Clinical trial number Clinical trial number: Not applicable. Funding This research was funded by Ningxia Natural Science Foundation Project (2024AAC03096), Natural Science Project of Ningxia Institutions of Higher Education (NYG2024040), and National Natural Science Foundation of China Project (32560455). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gang Liu, Yixuan Sun, Pengyuan He and Caixia Liu. The first draft of the manuscript was written by Gang Liu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during the current study are available from the corresponding author upon reasonable request. References Aung M S, Masuda H, Nozoye T, Kobayashi T, Jeon J S, An G et al (2019) Nicotianamine synthesis by OsNAS3 is important for mitigating Fe excess stress in rice. Frontiers in plant science 10: 660.https://doi.org/10.3389/fpls.2019.00660 Ajeesh Krishna T P, Maharajan T, Victor Roch G, Ignacimuthu S, Ceasar S A (2020) Structure, function, regulation and phylogenetic relationship of ZIP family transporters of plants. 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13:05:42","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202374,"visible":true,"origin":"","legend":"","description":"","filename":"e82e5668cbe64e1586ed96f720dd331f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/1b1a23126681a5e31799012f.xml"},{"id":93937057,"identity":"312ff32d-39b2-4cdf-b810-acf7e2819294","added_by":"auto","created_at":"2025-10-20 13:05:42","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219349,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/d9948fe3279609e092f4878c.html"},{"id":93936752,"identity":"1b6435d3-a210-4149-8e2d-fbac2d744927","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":701634,"visible":true,"origin":"","legend":"\u003cp\u003eThe localizations of some transport proteins were predicted using the WoLF PSORT website (https://wolfpsort.hgc.jp/). NRAMP: Natural Resistance-Associated Macrophage Protein; MIT: Mitochondrial Fe Transporter; ATM: ABC transporters of mitochondria. Solid arrows indicate the confirmed transport direction of the transporters, double solid arrows indicate the ability to both export and import ions, and double dashed arrows indicate that the transport direction of the transporter is uncertain (Jeong. 2009; Zhang et al. 2012; Cao. 2019; Gollhofer et al. 2014; Devi et al. 2025; Chen et al. 2007; Jain et al. 2013; Nevo et al. 2006; Jalmi et al. 2022; Chen et al. 2023; Krishna et al. 2023; Seregin et al. 2021; Waters et al. 2006; Chu.2010; Ajeesh et al. 2020; Lira-Morales. 2019; Conte. 2013; Sheera. 2025; Kamaral. 2022; Singh. 2023)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/73575c975fb1e73c3ce66022.png"},{"id":93936753,"identity":"a3ee2a76-9563-4249-b546-ac212ef23941","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":790426,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular Docking Prediction of Transporter Proteins with Zn–Fe Chelate Molecules\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/c3fccf16d24df5fd09758d1d.png"},{"id":93936771,"identity":"f6b488be-50ca-4e03-b02a-d9830fd8907f","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9634260,"visible":true,"origin":"","legend":"\u003cp\u003eZn and Fe absorption process in wheat roots. Wheat roots secrete MAs (mugineic acids), which chelate with Zn and Fe ions in the rhizosphere soil. After chelation, these complexes reach the root epidermal cells and are transported into the cells by transporters such as YSL located on the cell membrane. After entering the cells, these chelates may be transported to the vacuoles by YSL transporters on the cell membrane, or they may release Zn and Fe ions in the cytoplasmic envFement. These Zn and Fe ions can then be chelated with NA (nicotianamine) for long-distance transport or directly transported into the vacuoles by transporters such as YSL, HMA, and VIT on the vacuole membrane. Fe(III) may be reduced to Fe(II) in the cytoplasm by FRO enzymes. Dashed arrows indicate possible chelation of Zn and Fe ions with chelating agents during transport; solid arrows indicate the direction of transport (Takahashi et al. 2003; Pianelli et al. 2005; Koike et al. 2004; Inoue et al. 2009; Von et al.1999; Seregin et al. 2023; Suzuki et al. 2006)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/fdf46c3a2d6a1fe25460a3bd.jpeg"},{"id":93936759,"identity":"66d94eaf-20d7-48f7-bdd1-82985630d6b3","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6705666,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree (left panel), gene structure (middle panel), and motif prediction (right panel) of NAS genes. Phylogenetic tree was categorized according to branch distances, with different background colors distinguishing these subgroups; CDSo indicates a predicted gene with only one coding segment (a single exon), CDSf indicates the first coding segment (first exon) of a predicted gene, CDSi indicates internal coding segments (internal exons) of a predicted gene, CDSl indicates the last coding segment (last exon) of a predicted gene, TSS represents the transcription start site, and PolA represents the polyadenylation site.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/83f1eaeaa2e13988d0d74e81.png"},{"id":93937036,"identity":"c6dce16a-373d-4ea1-9ab7-381332b30a36","added_by":"auto","created_at":"2025-10-20 13:05:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133183,"visible":true,"origin":"","legend":"\u003cp\u003ePositive Selection Analysis on Amino Acid Sites of 43 \u003cem\u003eNAS\u003c/em\u003e Genes\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/3474c329415ab9c283c91aa1.png"},{"id":93936762,"identity":"884520ff-bbca-400d-b6f6-65ebc2704367","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1384071,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of Syntenic Relationships Among\u003cem\u003e TaNAS\u003c/em\u003e Genes\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/03232dbcb7cf751ccc2ac93a.png"},{"id":93938023,"identity":"29ecae94-6e65-492d-b387-e256340f7225","added_by":"auto","created_at":"2025-10-20 13:13:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":358942,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eTaNAS\u003c/em\u003e Genes (Ramírez-González et al. 2018; Bonneau et al. 2016; Zhang et al. 2018).Tissue:X1, Embryo 1 day after sowing ; X2, Embryo after sowing ; X3, Radicle after sowing ; X4, Seedling roots ; X5, Seedling shoots ; X6, Seedling leaves ; X7, Immature inflorescence; X8, Lemma and palea; X9, Pistil; X10, Anther; X11, Seed 3-5 days after pollination; X12, Embryo 22 days after pollination . Days: Days of Fe deficiency treatment (the first day of soaking is counted as day 0)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/30b478a93d2896434cb43513.png"},{"id":93937039,"identity":"a87b5f35-8181-43db-b397-e0eb0d238b32","added_by":"auto","created_at":"2025-10-20 13:05:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1796004,"visible":true,"origin":"","legend":"\u003cp\u003eResponse of \u003cem\u003eTaNAS\u003c/em\u003e Genes to Abiotic Stress（Ramírez-González et al., 2018; Zhang et al., 2018）\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/0791290461bb467053046fd2.png"},{"id":93936768,"identity":"840a1656-1991-477a-abc3-0a8a0ea04a35","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":684496,"visible":true,"origin":"","legend":"\u003cp\u003eTaNAS protein interactions\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/e0424340a7967bb65280c82b.png"},{"id":93936776,"identity":"1b999f74-3331-48b5-9dd5-4fdb75de4803","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":722828,"visible":true,"origin":"","legend":"\u003cp\u003ea) at normal Zn concentration (0.6 μM); b) at excess Zn concentration (60 μM); c) at Zn deficiency concentration (0.02 μM). In a and b, from left to right are the wild-type Nipponbare, OE-1 line, OE-3 line, and OE-7 line; in c, from left to right are the wild-type Nipponbare, OE-3 line, and OE-7 line\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/f5d7b1cbcc46cfdc43ebe9af.png"},{"id":93937042,"identity":"2504c7f9-41cb-466b-b6a0-d7acdbb5aeea","added_by":"auto","created_at":"2025-10-20 13:05:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1134752,"visible":true,"origin":"","legend":"\u003cp\u003ea) Catalytic efficiency of NAS enzymes in the roots and leaves of wild-type and overexpressing lines;b) Secretion rate of MAs in the roots of wild-type and overexpressing rice;c1/2) Zn and Fe content in grains and sword leaves of overexpressing rice and wild-type Nipponbare rice\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/9c23ff4bad857650c52bbd92.png"},{"id":93937046,"identity":"aa1e2622-1baa-49bd-9a8f-81a1ab2d1614","added_by":"auto","created_at":"2025-10-20 13:05:41","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1800432,"visible":true,"origin":"","legend":"\u003cp\u003ea) Phenotype of Leaves Silenced by BSMV-VIGS, b) Phenotype of Wheat Spikes Silenced by BSMV-VIGS. The subscripts PDS, γ, and VIGS indicate that the plants were inoculated by the BSMV:\u003cem\u003eTaPDS\u003c/em\u003e, empty BSMV-γ, and BSMV:\u003cem\u003eTaNAS4-A \u003c/em\u003evectors, respectively, CK is NC2 without any treatment\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/6f5229c514ef6f281432b7c7.png"},{"id":93936769,"identity":"cfc1ef9f-7c4f-4b5a-a3c3-d0deb2de8255","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":465177,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing Efficiency of TaNAS4-A and Its Two Closely Related Homologous Genes,CK is NC2 without any treatment\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/774c6e320e95f13a70a70352.png"},{"id":93938028,"identity":"2acb6f8d-0592-4d06-b676-31217d9bbe89","added_by":"auto","created_at":"2025-10-20 13:13:41","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":666909,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing Status of Eight \u003cem\u003eTaNAS4-A\u003c/em\u003eHomologous Gene, CK is NC2 without any treatment\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/2782c900be8abe903e1ee9c6.png"},{"id":93958750,"identity":"7c613681-d9cd-41d5-8ad1-88e08ef61828","added_by":"auto","created_at":"2025-10-20 16:40:43","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":360988,"visible":true,"origin":"","legend":"\u003cp\u003ea) NAS Enzyme Efficiency in Leaves; b) Grain Zn and Fe concentrations; c) Leaf Zn and Fe concentrations . The CK, PDS, γ, and VIGS indicate NC2 uninfected plants, and plants inoculated by the BSMV:\u003cem\u003eTaPDS\u003c/em\u003e, empty BSMV-γ, and BSMV:\u003cem\u003eTaNAS4-A \u003c/em\u003evectors, respectively\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/1ce757d65379ff2fa0cf095f.png"},{"id":99172452,"identity":"8f2f49e4-817a-432a-8d95-c63f99f1ec69","added_by":"auto","created_at":"2025-12-29 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29479128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/fe934a2f-bea9-4fa6-bf45-25f02572d80b.pdf"},{"id":93938022,"identity":"95129806-3633-4e3f-aa38-6157669a7a12","added_by":"auto","created_at":"2025-10-20 13:13:41","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":526216,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile3MolecularProteinBindingfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/cd2bc77366ca76f1775ca0d2.zip"},{"id":93936758,"identity":"8e03acb6-dfa2-4709-abb8-14a50966eddc","added_by":"auto","created_at":"2025-10-20 12:57:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1395884,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/2361d9087cb88f95e9038cdb.docx"},{"id":93937038,"identity":"0b651868-2502-49f4-9407-125d859342bd","added_by":"auto","created_at":"2025-10-20 13:05:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2189454,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7727623/v1/d000d811f98761d40a4e4ae6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of the Nicotianamine Synthase (NAS) Gene Family in Wheat (Triticum aestivum.L) and the Role of Its Member TaNAS4-A in Zn and Fe Transport","fulltext":[{"header":"Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003eZn, Fe, wheat, and human\u003c/h2\u003e\u003cp\u003eZinc (Zn) and Iron (Fe) are essential micronutrients across all biological systems. Zn is the only metal that can serve as a cofactor for over 300 enzymes (Rink et al. 2000). Its primary role is to stabilize the structures of numerous proteins, including signaling enzymes involved in various levels of signal transduction and transcription factors. Zn is also indispensable for the structural stability of Zn finger proteins (Zfp) (Beyersmann et al. 2002).\u003c/p\u003e\u003cp\u003eIn humans, Zn is a vital micronutrient for human health, playing a fundamental role in cellular processes such as growth, differentiation, and division by regulating DNA synthesis and RNA transcription. It is also instrumental in systemic functions including immune response, prostaglandin production, taste acuity, wound healing, cognitive performance, bone mineralization, blood clotting, and fetal development (Bhowmik et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Zn deficiency decreases neutrophil activation efficiency, which is reversible with Zn supplementation (Maywald et al. 2022). Additionally, during acute illnesses, plasma Zn levels often drop rapidly, leading to hypozincemia (Wessels et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Besecker et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Subsequently, pro-inflammatory cytokines mediate the redistribution of Zn, directing Zn ions and Zn-containing compounds into specific cellular compartments to assist in protein synthesis, neutralize free radicals, and inhibit the survival of microorganisms within human body (Maywald et al. 2022). Notably, in humans, plasma Zn levels decline with age and are closely associated with the occurrence of cardiovascular diseases. Thus, there is a link between Zn deficiency and an increased risk of cardiovascular diseases (Little et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is estimated that approximately 433,000 children under the age of five die annually due to Zn deficiency global, accounting for 4.4% of deaths in this age group (Fischer et al. 2009; Borrill et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). About 17% of the world\u0026rsquo;s population, roughly 1.1\u0026nbsp;billion people, are at risk of Zn deficiency (Khan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFe is another essential nutrient for humans, functioning as a critical component of heme groups, Fe-sulfur cluster proteins, and enzymes involved in mitochondrial respiration and DNA synthesis. Thus, Fe is indispensable for cellular metabolism, survival, and proliferation (McLean et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The daily production of red blood cells in the human body requires approximately 20 mg of Fe. Insufficient Fe intake can lead to Fe deficiency anemia (Ganz et al. 2012). It has been reported that around 1.2\u0026nbsp;billion people worldwide were at risk of Fe deficiency (Camaschella. 2019), and an estimated 25% of the global population suffers from Fe deficiency anemia, largely due to inadequate dietary Fe intake (Wallace. 2016). Fe deficiency increases mortality risk in patients with acute coronary syndrome and myocardial infarction (Lawler et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Even mild to moderate Fe deficiency anemia may impair cognitive development (Beard. 2003) and compromise immune mechanisms (Failla. 2003). Fe deficiency anemia during pregnancy can seriously affect maternal health, leading to reduced physical performance, increased fatigue, diminished cognitive function, and a higher risk of common infections and hospitalization (Viteri. 1994).\u003c/p\u003e\u003cp\u003eAs an important staple food for humans, wheat is the source of many mineral elements. In addition to being a major source of starch and energy, it is also rich in various components essential or beneficial to health, particularly proteins, vitamins, dietary fiber, and phytochemicals (Shewry et al. 2015). It also serves as a critical source of Zn and Fe for human needs. Some studies have shown that the Zn and Fe concentrations in wheat grains range from 16 mg/kg to 142 mg/kg and from 24 mg/kg to 80 mg/kg, respectively (Velu et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Borg et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, the grain Zn and Fe concentrations of bread wheat, which accounts for 95% of the wheat production at the global level (Tadesse et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), ranged from 25 mg/kg to 53 mg/kg and from 24 mg/kg to 51 mg/kg, respectively (Borg et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Graham et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The low grain Zn and Fe concentrations in bread wheat do not meet the standards for biofortification. Therefore, it is important to breed wheat with high Fe and Zn concentrations, particularly for regions relying upon wheat as a staple food, such as North Africa and Central Asia (Ling et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Understanding the mechanisms of Zn and Fe transport in wheat will be critical for guiding biofortification strategies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTransport of Zn and Fe in cell\u003c/h2\u003e\u003cp\u003eThere are many Zn and Fe transporter proteins in plants, most of which can bind to cell membranes or organelle membranes and play an indispensable role in the transmembrane transport of Zn and Fe (Krishna et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies on zinc-iron transporter proteins in arabidopsis, rice, and wheat have revealed that these transporters are widely distributed on membranes. They may either transport Zn and Fe ions inward or outward, and some can even transport in both directions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).There are distinct types of zinc-iron transporter proteins. For instance, ABC transporters, MIT transporters, and ATM transporters appear to be more inclined to transport iron ions. However, it is noteworthy that proteins from the YSL, ZIP, and HMA transporter families are all involved in the transport of zinc and iron. In particular, AtYSL1, AtYSL3, and AtYSL2 from the YSL family, as well as TaHMA4 from the HMA family, have been shown to participate in Zn and Fe transport .The vacuolar transporter proteins OsVIT1/2 are also involved in the transport of both Zn and Fe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).The YSL and ZIP family transporter proteins are extensively involved in the transport of Zn and Fe, and are distributed across the plasma membrane, mitochondria, chloroplasts, and vacuolar membrane,they appear to preferentially facilitate the unidirectional efflux of either Zn or Fe ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, most transporter proteins appear to only facilitate unidirectional transport of either Zn or Fe ions, as reflected in zinc and iron transport where different transporters may exhibit distinct and specialized roles.However, in this paper, we focus more on transporters that are involved in the transport of both zinc and iron, such as YSL, ZIP, and HMA, as well as small organic molecules capable of chelating zinc and iron ions to facilitate their transport within plants, such as nicotianamine (NA), mugineic acids (MAs), citrate, and phenolics to facilitate iron transport (Takahashi et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hell et al. 2003; Nishiyama et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kobayashi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe Role of NA in Zn and Fe Transport in Wheat\u003c/h3\u003e\n\u003cp\u003eNA, an important metal chelator in plants, plays a critical role in the long-distance transport (Ling et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Mari et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Douchkov et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and homeostasis of metal ions such as Fe, Zn, manganese (Mn), nickel (Ni), and copper (Cu) (Takahashi et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Pianelli et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). NA is synthesized by NA synthase (NAS) through the condensation of three S-adenosylmethionine (SAM) molecules, a process that initiates the MA biosynthetic pathway. In graminaceous plants, NA is further converted into mugineic acid (MA)-family phytosiderophores, such as deoxymugineic acid (DMA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e),it chelates iron ions in the soil through Strategy II in graminaceous crops, which is crucial for root iron absorption (Marschner et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Thus, the catalytic efficiency of NAS directly influences the biosynthesis levels of both NA and MAs.\u003c/p\u003e\u003cp\u003eNA and MAs can bind to the YSL and TOM transporter families to participate in Zn and Fe transport within plants. Molecular docking predictions suggest favorable and stable binding interactions between rice YSL/TOM transporters and the DMA. Similarly, in \u003cem\u003eArabidopsis\u003c/em\u003e, the YSL1 protein also exhibits stable binding with NA. These small molecules can fit into conserved transporter binding pockets to form transport-competent complexes (Nozoye et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Inoue et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Graminaceous crops typically secrete MAs into the rhizosphere to chelate metal ions, including Fe(II), Fe(III), and Zn(II), for root uptake (Koike et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Inoue et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Once Fe(II)-MA, Fe(III)-MA, and Zn(II)-MA complexes are absorbed into root cells, the metal ions are released under acidic conditions through reduction. These ions subsequently form chelates with NA, resulting in Fe(II)-NA, Fe(III)-NA, and Zn(II)-NA. These complexes are then transported over long distances via the phloem by transporters such as YSL (Von et al. 1999; Seregin et al. 2023). During transport, metal ions may again be released under acidic conditions and subsequently redistributed by a range of transporters, including YSL, HMA, ZIP, and FPN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This multi-layered transport mechanism ensures highly efficient acquisition, mobilization, and utilization of metal nutrients in plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of the Wheat\u003c/b\u003e \u003cb\u003eNAS\u003c/b\u003e \u003cb\u003eGene Family and Functional Validation of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNA is synthesized by nicotianamine synthase (NAS). NAS genes play a key role in the regulation of Zn and Fe uptake and translocation. In rice, overexpression of \u003cem\u003eHvNAS1\u003c/em\u003e significantly increased the contents of NA and its downstream product DMA, thereby enhancing Zn and Fe accumulation in grains (Masuda et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Similarly, overexpression of \u003cem\u003eOsNAS2\u003c/em\u003e and \u003cem\u003eOsNAS3\u003c/em\u003e also markedly elevated Zn and Fe concentrations in rice grains (Gupta et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Using CRISPR-Cas9 to edit the promoter sequence of \u003cem\u003eOsNAS2\u003c/em\u003e led to significantly higher gene expression, and the resulting gene-edited plants showed a substantial increase in grain Zn and Fe concentrations (Ludwig et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In wheat, overexpression of \u003cem\u003eOsNAS2\u003c/em\u003e has been shown to significantly enhance Zn and Fe concentrations in flour (Harrington et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, studies on the role of \u003cem\u003eTaNAS\u003c/em\u003e genes in Zn and Fe accumulation in wheat grains remain limited.\u003c/p\u003e\u003cp\u003eBioinformatic analysis of gene families provides an effective approach for investigating target genes. In this study, we performed a comprehensive analyses of the wheat \u003cem\u003eNAS\u003c/em\u003e gene family, examining their evolutionary relationships, sequence conservation, expression patterns, and predicted protein interactions. Particularly, function of \u003cem\u003eTaNAS4-A\u003c/em\u003e was investigated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe Gene Structure and Phylogenetic Analysis of\u003c/b\u003e \u003cb\u003eNAS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the evolutionary analysis, \u003cem\u003eNAS\u003c/em\u003e homologs in wheat and barley exhibited significant genetic diversity, as they were classified into distinct subfamilies within the phylogenetic tree. In contrast, \u003cem\u003eNAS\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e were relatively conserved and clustered within the same subfamily (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Based on the phylogenetic tree, we infer the presence of well-defined orthologous and paralogous relationships among \u003cem\u003eNAS\u003c/em\u003e genes in Poaceae species. In wheat, \u003cem\u003eTaNAS1-A/B\u003c/em\u003e, \u003cem\u003eTaNAS2-D1/D2\u003c/em\u003e, and \u003cem\u003eTaNAS9-A/B/D\u003c/em\u003e are likely paralogous. In barley, \u003cem\u003eHvNAS1, HvNAS5\u003c/em\u003e, and \u003cem\u003eHvNAS9\u003c/em\u003e may be paralogous. In maize, \u003cem\u003eZmNAS3/4/5\u003c/em\u003e and \u003cem\u003eZmNAS1/2/6\u003c/em\u003e are potentially paralogous. In rice, \u003cem\u003eOsNAS3\u003c/em\u003e and \u003cem\u003eOsNAS1/2\u003c/em\u003e may also share a paralogous relationship. Except for \u003cem\u003eAtNAS1-4\u003c/em\u003e, clear orthologous relationships were observed for the remaining \u003cem\u003eNAS\u003c/em\u003e genes across different species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe structure of \u003cem\u003eNAS\u003c/em\u003e genes is highly conserved. Except for \u003cem\u003eTaNAS1-A\u003c/em\u003e, \u003cem\u003eTaNAS1-B\u003c/em\u003e, and \u003cem\u003eTaNAS6-A\u003c/em\u003e, which contain two, four, and two exons, respectively, all other genes consist of only one exon. For genes \u003cem\u003eTaNAS1-A\u003c/em\u003e, \u003cem\u003eOsNAS1\u003c/em\u003e, and \u003cem\u003eHvNAS1\u003c/em\u003e, accurate transcription start sites could not be predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The type and arrangement of motifs in NAS genes showed high consistency with the topology of the phylogenetic tree. Most NAS protein sequences contain eight conserved motifs. In Arabidopsis, all genes except for \u003cem\u003eAtNAS2\u003c/em\u003e shared six common motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the eight predicted motifs, four were most commonly shared, including three types of endocytosis-related motifs: YXXΦ, LL, and FXXF (highlighted in red boxes in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These endocytic motifs mediate internalization by interacting with receptors or other proteins on the cell membrane, thereby facilitating the uptake of extracellular substances (Pandey et al. 2009). The presence of these motifs suggests that NAS proteins, functioning as transferases, are susceptible to endocytosis, which in turn promotes catalytic reactions within the cellular matrix.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe codon alignment used in this analysis comprised 391 codons, of which 390 were tested for positive selection. The results revealed that among these codons, 8 were under positive selection (indicated by red bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), 98 were under purifying selection (green bars), and 274 were evolving neutrally (gray bars) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The number of positively selected sites was significantly lower than those under purifying selection and neutral evolution, indicating that the evolution of this gene family is primarily influenced by purifying selection, with adaptive mutations being relatively rare. Given that \u003cem\u003eNAS\u003c/em\u003e gene family encodes enzymatic proteins, this pattern is consistent with the dominance of purifying selection in active sites and structurally conserved protein regions, where destabilizing mutations could severely compromise function or fitness. Consequentially, natural selection tends to eliminate deleterious mutations and preserve sequence conservation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSynteny analysis of 19 \u003cem\u003eTaNAS\u003c/em\u003e genes with identifiable chromosomal locations revealed that most genes exhibit conserved syntenic relationships across wheat subgenomes. A syntenic block was detected between the \u003cem\u003eTaNAS6-B\u003c/em\u003e region on chromosome 4B and the \u003cem\u003eTaNAS6-D\u003c/em\u003e region on chromosome 4D. Additionally, both regions showed synteny with the \u003cem\u003eTaNAS1/3/9-A\u003c/em\u003e region on chromosome 2A, the \u003cem\u003eTaNAS1/3/9-B\u003c/em\u003e region on chromosome 2B, and the \u003cem\u003eTaNAS9-D\u003c/em\u003e region on chromosome 2D. Moreover, the \u003cem\u003eTaNAS1/3/9-A\u003c/em\u003e, \u003cem\u003eTaNAS1/3/9-B\u003c/em\u003e, and \u003cem\u003eTaNAS9-D\u003c/em\u003e regions demonstrated mutual synteny, suggesting that these genes may have evolved through duplication and insertion of a common ancestral copy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, two distinct regions on chromosome 6B were found to be syntenic with the \u003cem\u003eTaNAS7-A\u003c/em\u003e and \u003cem\u003eTaNAS7-D\u003c/em\u003e and \u003cem\u003eTaNAS2-A\u003c/em\u003e regions, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), which may indicate the presence of previously uncharacterized \u003cem\u003eNAS\u003c/em\u003e genes within these chromosomal segments. The remaining \u003cem\u003eTaNAS\u003c/em\u003e genes did not show detectable synteny, potentially reflecting functional specialization or distinct evolutionary trajectories.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression and Protein Interaction of\u003c/b\u003e \u003cb\u003eTaNAS\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 18 \u003cem\u003eTaNAS\u003c/em\u003e genes were broadly classified into three categories based on their tissue-specific expression patterns. The first category was predominantly expressed in seedlings roots. The second category showed concentrated expression in seedlings leaves. The third category was mainly expressed during the reproductive stage, such as in anthers and seeds shortly after pollination. Among them, \u003cem\u003eTaNAS7-A2\u003c/em\u003e exhibited the highest expression in anthers \u003cem\u003eTaNAS1-A\u003c/em\u003e in pistils; and \u003cem\u003eTaNAS9-D\u003c/em\u003e in seedling shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Under Fe deficiency, almost all \u003cem\u003eTaNAS\u003c/em\u003e genes were significantly upregulated by day 5 of treatment, with the exceptions of \u003cem\u003eTaNAS7-D\u003c/em\u003e, which was downregulated, and \u003cem\u003eTaNAS1-A\u003c/em\u003e, which was significantly downregulated. By day 7, the magnitude of upregulation began to decrease markedly, with \u003cem\u003eTaNAS7-A2\u003c/em\u003e and \u003cem\u003eTaNAS9-D\u003c/em\u003e showing downregulated expressions. Among all members, \u003cem\u003eTaNAS3-A\u003c/em\u003e and \u003cem\u003eTaNAS3-B\u003c/em\u003e were the most responsive to Fe deficiency with upregulation fold changes greater than 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on the phylogenetic relationships and expression profiles (FPKM) under abiotic stress conditions, the \u003cem\u003eTaNAS\u003c/em\u003e genes were classified into four subgroups: G1, G2, G3, and G4. In subgroups G1-G3, salt stress triggered the highest responses, followed by drought and waterlogging, while little to no response was observed under dark, cold, freezing, and wounds. \u003cem\u003eTaNAS7-A2\u003c/em\u003e showed minimal response to abiotic stresses, with only weak reactions to drought and salt stress. Interestingly, three genes, including \u003cem\u003eTaNAS9-A\u003c/em\u003e, \u003cem\u003eTaNAS9-B\u003c/em\u003e, and \u003cem\u003eTaNAS9-D\u003c/em\u003e, in subgroup G4 demonstrated the most active responses to stressful environments, reacting positively to dark, cold, freeze, wound, heat, drought, nutrient deficiency, waterlogging, and salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProteins A0A3B6EDR2, W5BGU5_WHEAT, A0A077RTD0, A0A3B6J0A9, A0A3B6UB94, A0A3B6HUG3, A0A3B6KUD6, A0A3B6IZR0, A0A3B6JE97, and A0A3B6ILM9 were identified as co-interacting partners of proteins encoded by \u003cem\u003eTaNAS\u003c/em\u003e genes. Among these 10 interacting proteins, most are functional proteins associated with DNA binding, DNA repair, and transcription processes. For instance, W5BGU5_WHEAT functions as a promoter recognition protein, while the remaining ones, such as A0A077RPD2, A0A3B5YTZ3, and A0A3B5ZPR6, exhibit acyltransferase activity. However, no significant interactions were detected among the TaNAS proteins. Many of the interacting proteins are linked to DNA binding and transcriptional regulation, suggesting that cells may directly couple the synthesis of secondary metabolites (enzymes) with the transcriptional regulation of their genes (DNA-binding/transcriptional proteins), forming a rapid \"sensing-response\" feedback loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreliminary Functional Verification of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003eGene Involved in Zn and Fe Transport\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003ein rice (Nipponbare)\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003ePhenotypes of Overexpressing Rice Under Different Zn Stress Conditions\u003c/h3\u003e\n\u003cp\u003eWhen rice seeds germinated and developed their first leaf, they were maintained under hydroponic condition for 3 days. Healthy seedlings were selected, and the seeds were removed. These seedlings were then subjected to hydroponic cultivation under different Zn concentration gradients for 14 days. Under normal hydroponic conditions, we observed that the wild-type Nipponbare rice showed the most vigorous growth. The shoot height of the wild-type was greater than that of OE-3 and OE-7, and the root length and development were significantly superior to those of OE-1, OE-3, and OE-7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). Interestingly, this trend reversed under both Zn excess and Zn deficiency. Under Zn excess, although the roots of all rice lines became thicker, shorter, and produced more root hairs, the overexpressed lines OE-1, OE-3, and OE-7 exhibited notably greater root length and density the wild-type. The shoot growth of these overexpressed lines were also superior, especially the OE-7 seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb). Under Zn deficiency, although the seedlings exhibited slender leaves and chlorosis, these symptoms were more severe in the wild-type compared to OE-3 and OE-7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003eincreased NAS catalytic efficiency, MAs secretion rate, and grain Fe and Zn concentrations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn both wild-type and overexpressing lines, the catalytic efficiencies of NAS enzymes in the roots wre higher than that in the leaves. The catalytic efficiencies in the roots of overexpressing lines were significantly higher than that of the wild-type, with differences were also observed among the overexpressing: OE-3 and OE-7 showed significantly higher root NAS catalytic efficiencies than OE-1. In leaves, NAS catalytic efficiencies in the overexpressing lines were also significantly higher than that in the wild-type. OE-7 exhibited significantly higher leaf NAS catalytic efficiency than OE-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe day of emergence of the first true leaf was designated as Day 1. The secretion rate of MAs from root was measured on Days 7, 10, 12, and 14. On Day 7, the MAs secretion rate of the wild-type was significantly lower than that of the overexpressing lines. From Day 7 to Day 14, the wild-type consistently exhibited lower MAs secretion rates compared to the overexpressing lines. By Day 14, the secretion rate of the wild-type began to plateau, while that of the overexpressing lines still showed an increasing trend. The MA secretion rate of OE-7 remained the highest throughout the experiment. After Day 10, the MA secretion rate of OE-3 surpassed that of OE-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe leaf Zn and Fe concentration of OE-3 and OE-7 were significantly higher than those of the wild-type, and the Fe concentration of OE-1 was significantly higher than that of the wild-type. No significant difference was observed in leaf Zn concentration among overexpression lines. The leaf Fe concentration of OE-3 and OE-7 reached 119.6 and 128 mg/kg, respectively, significantly higher than that of OE-1 (99.8 mg/kg) (Fig.\u0026nbsp;11c2). The grain Zn and Fe concentrations of the overexpressing lines were significantly higher than those of the wild-type. The grain Zn concentration of OE-3 and OE-7 was 45.3 and 46 mg/kg, respectively, significantly higher than that of OE-1 (40.6 mg/kg). Similarly, the grain Fe concentration of OE-3 and OE-7 was 27.9 and 26.4 mg/kg, also significantly higher than that of OE-1 (20.6 mg/kg) (Fig.\u0026nbsp;11c1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene silencing of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003ein NC2 wheat mediated by BSMV-VIGS\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eBSMV-VIGS Silencing Phenotype\u003c/h3\u003e\n\u003cp\u003ePhytoene desaturase (PDS, phytoene desaturase) is a key enzyme in the carotenoid biosynthesis pathway in plants, algae, and certain bacteria, playing an essential role in pigment synthesis and photoprotection. To assess the effectiveness of BSMV-VIGS, \u003cem\u003ePDS\u003c/em\u003e was inserted into the BSMV-γ vector, and leaf whitening was used as a visual indicator. On the 11th day after rub-inoculation, wheat leaves inoculated with BSMV:\u003cem\u003eTaPDS\u003c/em\u003e began to exhibit distinct whitening symptoms from the leaf tips to the base, while no obvious symptoms were observed in the CK (no inoculation) and Mock (buffer-only) leaves, indicating successful leaf rub-inoculation. After 5\u0026ndash;6 days, yellow-white streaks gradually appeared on leaves inoculated with γ and BSMV:\u003cem\u003eTaNAS4-A\u003c/em\u003e constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea). Subsequently, during the grain-filling stage, the wheat plants were inoculated again. Pronounced whitening phenomena were observed in the whole spikes of BSMV:\u003cem\u003eTaPDS\u003c/em\u003e inoculated lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eb), demonstrating that the BSMV-VIGS system functioned effectively in wheat at both vegetative and reproductive stages.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSilencing Efficiency of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003eand Its Homologous Genes in Inoculated Leaves\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBSMV-VIGS achieved a silencing efficiency of 78.5% for \u003cem\u003eTaNAS4-A\u003c/em\u003e. Homologous genes \u003cem\u003eTaNAS4-D\u003c/em\u003e and \u003cem\u003eTaNAS4-U\u003c/em\u003e were also significantly silenced by BSMV-VIGS, with efficiencies of 42.1% and 40%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e). The expression level of \u003cem\u003eTaNAS5-B\u003c/em\u003e was reduced by 48.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e). The expression of \u003cem\u003eTaNAS1-A\u003c/em\u003e and \u003cem\u003eTaNAS9-A\u003c/em\u003e was upregulated by 1.84-fold and 2.12-fold, respectively, possibly due to functional compensation. These two genes are evolutionarily distant from \u003cem\u003eTaNAS4-A\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e). The expression levels of other detected \u003cem\u003eNAS\u003c/em\u003e genes were not silenced by BSMV-VIGS. It is worth noting that the expressions of \u003cem\u003eTaNAS2-A\u003c/em\u003e, \u003cem\u003eTaNAS5-B\u003c/em\u003e, and \u003cem\u003eTaNAS9-A\u003c/em\u003e were significantly upregulated after inoculation with the BSMV-γ vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSilencing of\u003c/b\u003e \u003cb\u003eTaNAS4-A\u003c/b\u003e \u003cb\u003edecreased NAS catalytic efficiency and grain Zn and Fe concentrations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe leaf catalytic efficiencies of NAS in wheat plants infected by BSMV:\u003cem\u003eTaNAS4-A\u003c/em\u003e were significantly lower than that in uninfected plants (CK) and in plants infected with BSMV:\u003cem\u003eTaPDS\u003c/em\u003e or BSMV:γ. The NAS catalytic efficiencies in plants infected with BSMV:\u003cem\u003eTaPDS\u003c/em\u003e or BSMV:γ viral solutions was also significantly lower than that in CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003ea). The grain Zn and Fe concentrations of CK were 40.6 mg/kg and 31.6 mg/kg, respectively. In plants infected with the BSMV:γ viral solution, the grain Zn and Fe concentrations were 27.4 mg/kg and 28.0 mg/kg, respectively. In plants infected with BSMV:\u003cem\u003eTaNAS4-A\u003c/em\u003e, grain Zn and Fe concentrations were 12.0 mg/kg and 15.7 mg/kg, respectively. Grain Zn and Fe concentrations in plants infected by either BSMV:\u003cem\u003eTaNAS4-A\u003c/em\u003e or BSMV:γ were significantly lower than those in CK. Furthermore, the grain Zn and Fe concentrations in BSMV:\u003cem\u003eTaNAS4-A\u003c/em\u003e infected plants were also significantly lower than those in BSMV:γ infected plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eb). Similar trends were observed in leaf Zn and Fe concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eBioinformatic Analysis of\u003c/b\u003e \u003cb\u003eNAS\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMotif analysis revealed that NAS proteins contain eight conserved motifs, four of which are endocytosis-related motifs (YXXΦ, LL), implying their propensity for endocytosis, which facilitates intracellular catalytic reactions. This strongly suggests that NAS proteins may possess intrinsic roles in signal perception or membrane transport regulation. They may cycle between the plasma membrane and endosomes via endocytosis, directly participating in the cellular \"sensing\" of metal ion status (e.g., Fe and Zn), thereby forming a rapid \"membrane perception\u0026ndash;intracellular signaling\u0026ndash;gene transcription feedback\" loop. This extends beyond their traditional function as synthase and offers a novel perspective on their role in signal transduction.\u003c/p\u003e\u003cp\u003eEvolutionary analysis indicates that the NAS gene family is primarily shaped by purifying selection, with very few positively selected sites. This is highly consistent with the critical enzymatic function of NAS proteins. As enzymes catalyzing the synthesis of nicotianamine, a key precursor of phytosiderophores, their active sites and three-dimensional structures must be highly conserved. Any non-neutral mutations could disrupt their enzymatic activity, thereby affecting the chelation and transport of essential micronutrients such as Fe and Zn, and would likely be eliminated by natural selection. Although formed a multigene family, purifying selection suggests that each member of the \u003cem\u003eNAS\u003c/em\u003e genes undertake indispensable or at least difficult-to-replace specific functions, such as function in different tissues, developmental stages, or stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSynteny and phylogenetic analyses revealed that the \u003cem\u003eNAS\u003c/em\u003e gene family has expanded through gene duplication, including whole-genome duplication and segmental duplication, with further complexity arising from subgenome differentiation in polyploid species such as wheat. For instance, the presence of \u003cem\u003eTaNAS9-A/B/D\u003c/em\u003e is a typical outcome of polyploidization events. Expression analysis further supported the hypothesis of functional divergence. Although homologous genes from different subgenomes (e.g., \u003cem\u003eTaNAS9-A/B/D\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e) share sequence similarity, they exhibit tissue specific expressions (e.g., high expression of \u003cem\u003eTaNAS9-D\u003c/em\u003e in seedling shoots) and stress specific responses (e.g., the G4 subgroup responds to nearly all stresses). This indicates that after duplication, the functions of the ancestral gene were partitioned among different copies, supporting the occurrence of subfunctionalization (Rastogi et al. 2005).\u003c/p\u003e\u003cp\u003eProtein-protein interaction predictions revealed that TaNAS proteins do not directly interact with each other but do interact with numerous DNA-binding/transcriptional regulatory proteins and acyltransferases (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This finding reinforces a core biological concept: cells tend to directly couple the synthesis of metabolic enzymes with the transcriptional regulation of their functions (Shi et al. 2004). NAS proteins may interact with transcription factors or chromatin modification complexes, thereby feedback-regulating their own transcription or that of other downstream genes, forming an efficient homeostatic regulatory network.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional Verification of\u003c/b\u003e \u003cb\u003eNAS\u003c/b\u003e \u003cb\u003eGene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eKlatte et al (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) constructed five \u003cem\u003eNAS\u003c/em\u003e mutant lines using T-DNA insertion technology. Under moderately toxic nickel (Ni) conditions, mutant line nas4-1 exhibited more severe chlorosis than the mutant line nas3-1, and the NA level in the triple NAS mutant decreased to 30\u0026ndash;40% of that in the wild type (Klatte et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Under Fe deficiency, the chlorosis in leaves of both single and multiple mutants was more severe compared to the wild type. Among the single mutants tested, the nas4-1 mutant showed the most pronounced leaf chlorosis. These results indicate that the functions of \u003cem\u003eNAS\u003c/em\u003e genes are not entirely redundant (Schuler et al. 2011). In rice, using endosperm-specific promoter-containing pMDC vectors, transgenic rice plants overexpressing \u003cem\u003eOsNAS1\u003c/em\u003e, \u003cem\u003eOsNAS2\u003c/em\u003e, and \u003cem\u003eOsNAS3\u003c/em\u003e were generated. Compared to the wild-type, all three overexpression lines showed significantly increased concentrations of NA, Zn, and Fe in grains, with \u003cem\u003eOsNAS2\u003c/em\u003e and \u003cem\u003eOsNAS3\u003c/em\u003e overexpression lines exhibiting more pronounced enhancements. Meanwhile, \u003cem\u003eOsNAS2\u003c/em\u003e overexpression significantly elevated Fe and Zn content in rice endosperm (Johnson et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). When overexpressed in wheat, \u003cem\u003eOsNAS2\u003c/em\u003e also markedly increased Zn concentration in wheat grains (Singh et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In comparison to the wild-type, the \u003cem\u003eOsNAS3\u003c/em\u003e overexpression line (\u003cem\u003eOsNAS3-D1\u003c/em\u003e) accumulated higher levels of Fe and Zn in both shoots and roots, along with significantly increased contents of Fe (2.9-fold), Zn (2.2-fold), copper (1.7-fold), and NA (9.6-fold) in seeds. Concurrently, the overexpressing lines exhibited enhanced tolerance to Fe and Zn deficiency, as well as to Zn, Cu, and Ni toxicity (Lee et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Under Fe excess conditions, the NA synthesized by \u003cem\u003eOsNAS3\u003c/em\u003e helps alleviate the toxicity of Fe overload on cells, while under normal Fe conditions, the NA produced by \u003cem\u003eOsNAS1\u003c/em\u003e and \u003cem\u003eOsNAS2\u003c/em\u003e facilitates Fe transport, suggesting distinct roles of NA under different conditions (Aung et al.2019; Nozoye et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). Overexpression of \u003cem\u003eHvNAS1\u003c/em\u003e in sweet potato resulted in leaves with NA concentrations, Fe, and Zn content that were 7.9-fold, 3-fold, and 2.9-fold higher, respectively, than those of the wild-type (Nozoye et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, transgenic soybean plants overexpressing \u003cem\u003eHvNAS1\u003c/em\u003e exhibited NA concentrations as high as 768 \u0026micro;g∙g⁻\u0026sup1; dry weight, approximately 4 times that of non-transgenic soybeans, along with twice the Fe content in seeds and demonstrated tolerance to Fe deficiency in calcareous soil (Nozoye et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). Expression of the maize gene \u003cem\u003eZmNAS\u003c/em\u003e during the seedling stage is regulated by jasmonic acid, abscisic acid, and salicylic acid (Mizuno et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In this study, overexpression of \u003cem\u003eTaNAS4-A\u003c/em\u003e increased the NAS catalytic efficiencies in roots and leaves, MAs secretion rate and grain Zn and Fe concentrations, whereas \u003cem\u003eTaNAS4-A\u003c/em\u003e silencing by BSMV-VIGS decreased the NAS catalytic efficiency and grain Zn and Fe concentration. Take together our and others\u0026rsquo; results confirmed the critical role of NAS genes in Zn and Fe transport across plant species.\u003c/p\u003e\u003cp\u003eNotably, the BSMV-VIGS system targeting \u003cem\u003eTaNAS4-A\u003c/em\u003e also partially silenced its three most closely homologous genes and significantly upregulating \u003cem\u003eTaNAS9-A\u003c/em\u003e and \u003cem\u003eTaNAS1-A\u003c/em\u003e, which are evolutionarily more distant from \u003cem\u003eTaNAS4-A\u003c/em\u003e. This phenomenon may stem from a functional compensation mechanism among members of the \u003cem\u003eNAS\u003c/em\u003e gene family, whereby the expressions of certain genes is adjusted to maintain metal homeostasis when \u003cem\u003eTaNAS4-A\u003c/em\u003e is specifically silenced. Although this compensatory effect reflects an adaptive strategy of plants, it poses challenges for elucidating the independent function of individual genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePotential Additional Biological Functions of\u003c/b\u003e \u003cb\u003eNAS\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStudies have shown that overexpression of \u003cem\u003eHvNAS1\u003c/em\u003e enhances metal toxicity tolerance in tobacco (Takahashi et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Han et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) successfully cloned a \u003cem\u003eTaNAS-D\u003c/em\u003e gene from wheat. Overexpression of this gene in \u003cem\u003eArabidopsis\u003c/em\u003e significantly increased NA levels and enhanced the plant's salt stress tolerance. Under alternative stress conditions, \u003cem\u003eTaNAS-D\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e exhibited higher germination rates and better growth compared to the wild-type (WT). Further studies revealed that transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants had a higher K⁺/Na⁺ ratio, lower malondialdehyde (MDA) content, reduced ion leakage (IL), and increased activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), resulting in reduced membrane damage. Additionally, overexpression of \u003cem\u003eTaNAS-D\u003c/em\u003e led to upregulation of \u003cem\u003eAtSOS1, AtSOS2, AtSOS3, AtFAD5\u003c/em\u003e, and \u003cem\u003eAtSAD1\u003c/em\u003e under salt stress (Han et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In leguminous plants, symbiotic nitrogen fixation depends on the normal function of \u003cem\u003eMtNAS2\u003c/em\u003e. In the \u003cem\u003enas2-1\u003c/em\u003e insertion mutant, nitrogenase activity was lost, but reintroducing a copy of the wild-type \u003cem\u003eMtNAS2\u003c/em\u003e gene restored nitrogenase activity in the mutant (Escudero et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, when wheat leaves were inoculated with the BSMV virus (γ), three genes, including \u003cem\u003eTaNAS2-A\u003c/em\u003e, \u003cem\u003eTaNAS5-B\u003c/em\u003e, and \u003cem\u003eTaNAS9-A\u003c/em\u003e, were significantly upregulated, suggesting their potential roles in the antiviral responses of plant. Both our findings and previous research indicate that \u003cem\u003eNAS\u003c/em\u003e genes may participate in biological processes beyond Zn and Fe absorption and transport.\u003c/p\u003e\n\u003ch3\u003eOverall\u003c/h3\u003e\n\u003cp\u003eThis study employed bioinformatic analyses to highlight key biological characteristics of wheat \u003cem\u003eNAS\u003c/em\u003e genes, followed by gene function validation methods to preliminarily confirm the role of \u003cem\u003eTaNAS4-A\u003c/em\u003e in Zn and Fe transport. To the best of our knowledge, this is the first report on \u003cem\u003eTaNAS4-A\u003c/em\u003e for its function in assisting Zn and Fe accumulation in wheat grains. Our findings provide valuable insights into the mechanisms of Zn and Fe transport in wheat.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatic Analysis\u003c/h2\u003e\u003cp\u003eThe NAS gene and protein sequences of wheat were obtained from IWGSC V1.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wheat-urgi.versailles.inra.fr/Projects/IWGSC\u003c/span\u003e\u003cspan address=\"https://wheat-urgi.versailles.inra.fr/Projects/IWGSC\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene and protein sequences of other species were sourced from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Ensembl Plants (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein accessions were retrieved from UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenetic trees were constructed using the neighbor-joining method in MEGA 11. Gene structures were predicted using FGENESH (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.softberry.com/berry.phtml?topic=fgenesh\u003c/span\u003e\u003cspan address=\"https://www.softberry.com/berry.phtml?topic=fgenesh\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and motifs were predicted via the MEME website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular docking was performed using AutoDock Vina, and results were visualized with Pymol. Intra-species synteny analysis was conducted using the one-step MCScanX-Superfast plugin in TB-tools. Positive selection analysis was carried out using Datamonkey (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.datamonkey.org/\u003c/span\u003e\u003cspan address=\"https://www.datamonkey.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein-protein interactions were queried via STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized using Cytoscape. Chord diagrams were generated with the circlize package in R. For some Zn and Fe transport proteins subcellular localization in silico by WoLFPSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTotal RNA Extraction and RT-qPCR\u003c/h2\u003e\u003cp\u003eRNA was extracted from 0.1g of leaf samples using RNAiso Plus (TaKaRa, DaLian, China). High-quality RNA was used to synthesize cDNA with PrimeScript\u0026trade; FAST RT reagent Kit with gDNA Eraser (TaKaRa, DaLian, China). Fluorescent quantitative reactions were conducted using TB Green\u0026reg; Premix Ex Taq\u0026trade; II FAST qPCR (TaKaRa, DaLian, China) in a 20 \u0026micro;l system. Reactions were performed on the ABI 7300 (Thermo Fisher Scientific, United States) instrument, with three biological replicates for each sample. The wheat Actin gene (Gene ID: AB181991) was used as the internal reference gene. The fluorescent quantitative data were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of NAS Enzymatic Catalytic Efficiency\u003c/h2\u003e\u003cp\u003eBased on previous methods (Higuchi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Della et al. 1988) with certain modifications, the NAS enzymatic catalytic efficiency was determined. The detailed procedure is as follows: Approximately 0.5 g of frozen tissue (root or leaf) was ground in a mortar containing liquid nitrogen, followed by the addition of 1 mL of 0.2 M Tris/HCl buffer (pH 8.0) containing 10 mM EDTA, 5% (w/v) insoluble PVP, 5% (v/v) glycerol, 0.1 mM p-APMSF, 100 \u0026micro;g/mL antipain, and 10 mM DTT. The homogenate was centrifuged at 8000 \u0026times; g for 20 minutes, and the supernatant was collected. The supernatant was then eluted through a 1 mL hydrophobic resin column pre-equilibrated with buffer (20 mM Tris/HCl, 1 mM EDTA, 0.1 mM p-APMSF, 3 mM DTT, 0.4 M (NH₄)₂SO₄, pH 8). The NAS enzyme was eluted using a 1% glycerol solution containing 0.1 mM p-APMSF and 3 mM DTT. The NAS enzyme reaction was carried out under the following conditions: the enzyme solution was supplemented with 20 \u0026micro;g/mL antipain and 50 \u0026micro;M SAM, incubated at 25\u0026deg;C for 15 minutes, and the reaction was terminated by adding ethanol to a final concentration of 50% (v/v). After termination, the pH of the solution was adjusted to 8.0. The solution was then applied to a PAX mixed-mode strong anion exchange column pre-equilibrated with 0.05 M sodium phosphate buffer (pH\u0026thinsp;=\u0026thinsp;8.2). After equilibration, elution was performed three times using 0.1 M NaCl solution (pH\u0026thinsp;=\u0026thinsp;7.5), and the eluate was collected. The eluate was subsequently transferred to a spectrophotometer to measure UV absorbance at 260 nm (characteristic absorption peak of adenosine from MTA/S-methyl-5'-thioadenosine). The entire process was maintained at 15\u0026deg;C. A standard curve was constructed using MTA as the sole solute, with concentration as the horizontal axis and absorbance as the vertical axis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of Root Exudation of Mugineic Acid Family (MAS)\u003c/h2\u003e\u003cp\u003eBased on the diurnal rhythm of MAs secretion, collection began each morning after 2 hours of light exposure. The root system was first rinsed, and the exudates were collected for 4 hours into a container holding a certain volume of deionized water. The container was kept shaded and aerated. With modifications to previous determination methods (Takagi. 1993; Yu et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), the collection solution (containing MAs) was processed as follows: A 9.00 ml aliquot of the collection solution (MAs) was pipetted into a small plastic vial. Then, 0.50 ml of 1 mM FeCl₃ and 1.00 ml of 0.5 mol/L NaAc-HAc buffer (pH 7.0) were added. The mixture was shaken for 1 hour to fully form the Fe(Ⅲ)-MAs complex. After filtering out excess Fe(OH)₃, 8.00 ml of the filtrate (Fe(Ⅲ)-MAs) was transferred, followed by the addition of 0.20 ml of 1.5 mol/L H₂SO₄ and 0.50 ml of 8% hydroxylamine hydrochloride (HONHCl). The solution was reduced in an oven at 50\u0026deg;C for 20 minutes to form Fe(Ⅱ)-MAs. Then, 1.00 ml of 2 mol/L NaHAc-HAc buffer (pH 4.7) and 0.2 ml of 0.01 mol/L Ferrozin were added. The resulting Fe(Ⅱ)-Ferrozin complex (purple-red color) was measured colorimetrically at 562 nm, and the amount of solubilized Fe was calculated. The MAs secretion rate was then estimated based on the amount of solubilized Fe (i.e., Fe(Ⅱ)-Ferrozin), converted into an equivalent of Fe-chelating compounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of Zn and Fe Concentrations in Tissues\u003c/h2\u003e\u003cp\u003eThe concentrations of Zn and Fe in wheat and rice grains were determined using Flame Atomic Absorption Spectroscopy (FAAS) in accordance with the GB5009.14\u0026mdash;2017 standard. A 1-gram sample of dried grain was placed in a conical flask, to which 10 mL of nitric acid and 0.5 mL of perchloric acid were added. The mixture was digested on an adjustable electric heating plate under the following conditions: 120\u0026deg;C for 0.5\u0026ndash;1 hour, increased to 180\u0026deg;C for 2\u0026ndash;4 hours, and further raised to 200\u0026ndash;220\u0026deg;C. If the digestate appeared brown, a small amount of additional nitric acid was added. Digestion continued until white fumes were emitted, and the solution became colorless, transparent, or slightly yellow. The digested solution was then cooled and diluted to 25 mL with water, mixed thoroughly, and set aside for analysis. Each experiment included a reagent blank control. Standard solutions and calibration curves were prepared using Zn oxide (purity\u0026thinsp;\u0026gt;\u0026thinsp;99.8%) and ferric nitrate (purity\u0026thinsp;\u0026gt;\u0026thinsp;99.8%). The Zn and Fe concentrations in the samples were determined by FAAS at wavelengths of 213.9 nm and 248.3 nm, respectively. A series of standard solutions with increasing mass concentrations of Zn and Fe were sequentially introduced into the flame atomizer. After atomization, their absorbance values were measured. Calibration curves were constructed with mass concentration as the x-axis and absorbance as the y-axis. Under the same experimental conditions used for the standard solutions, the blank and sample solutions were introduced into the flame atomizer. After atomization, their absorbance values were measured, and the concentrations were quantitatively determined by comparison with the standard calibration curves.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of Overexpression Rice Lines\u003c/h2\u003e\u003cp\u003eAs described in Supplementary File 1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of the BSMV-VIGS System\u003c/h2\u003e\u003cp\u003eAs described in Supplementary File 2.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eClinical trial number: Not applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by Ningxia Natural Science Foundation Project (2024AAC03096), Natural Science Project of Ningxia Institutions of Higher Education (NYG2024040), and National Natural Science Foundation of China Project (32560455).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gang Liu, Yixuan Sun, Pengyuan He and Caixia Liu. The first draft of the manuscript was written by Gang Liu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAung M S, Masuda H, Nozoye T, Kobayashi T, Jeon J S, An G et al (2019) Nicotianamine synthesis by OsNAS3 is important for mitigating Fe excess stress in rice. 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The Plant Journal 72(3): 400-410. https://doi.org/10.1111/j.1365-313X.2012.05088.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triticum aestivum, Biofortification, phytosiderophores, TaNAS4-A, BSMV-VIGS","lastPublishedDoi":"10.21203/rs.3.rs-7727623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7727623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZinc (Zn) and Iron (Fe) are essential trace elements for human health, yet deficiencies in both are widespread worldwide. As a major staple crop, wheat is an important dietary source of Zn and Fe. However, the concentrations of Zn and Fe in common wheat grains are generally low, making it necessary to enhance the nutritional value of wheat. This study first elaborated that both elements are absorbed by wheat via \"Strategy II\" which relies on phytosiderophores (such as mugineic acids) and related transporter proteins (e.g., YSL and ZIP families). Nicotianamine (NA) plays a key chelating role in the long-distance transport of Zn and Fe. Therefore, we further analyzed the \u003cem\u003eNAS\u003c/em\u003e gene family in wheat, which showed high genetic diversity, unique gene structures, distinct evolutionary features, and was subjected to purifying selection. Expression profiling revealed that \u003cem\u003eNAS\u003c/em\u003e genes were tissue-specific and responsive to various stress conditions. The overexpression of \u003cem\u003eTaNAS4-A\u003c/em\u003e in rice, as well as the silencing of \u003cem\u003eTaNAS4-A\u003c/em\u003e in wheat using BSMV-VIGS confirmed the role of \u003cem\u003eTaNAS4-A\u003c/em\u003e in enhancing NAS enzyme catalytic efficiency, promoting phytosiderophores secretion, and increasing the accumulation of Zn and Fe in grains. Additionally, this study suggested that NAS genes may confer other functions, such as stress resistance, which deserves further investigation. This research provides a theoretical basis for Zn and Fe biofortification in wheat.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Identification of the Nicotianamine Synthase (NAS) Gene Family in Wheat (Triticum aestivum.L) and the Role of Its Member TaNAS4-A in Zn and Fe Transport","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 12:57:36","doi":"10.21203/rs.3.rs-7727623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T14:35:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-25T10:07:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T04:08:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T07:20:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17230162833682278167856838916707575139","date":"2025-10-13T03:46:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5221125803207928521084935844241969410","date":"2025-10-10T09:31:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97438665096724096562421815659919556965","date":"2025-10-08T04:39:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83462111538215834479083568685076591406","date":"2025-10-08T03:40:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314426677410433002552064963855441061141","date":"2025-10-08T02:33:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256327772106690228016661178296718689542","date":"2025-10-07T14:36:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T13:25:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T15:22:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T15:21:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2025-09-27T10:04:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b610654-6ef6-411c-a00a-d8bff0e33419","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:04:06+00:00","versionOfRecord":{"articleIdentity":"rs-7727623","link":"https://doi.org/10.1007/s10142-025-01798-5","journal":{"identity":"functional-and-integrative-genomics","isVorOnly":false,"title":"Functional \u0026 Integrative Genomics"},"publishedOn":"2025-12-23 15:57:34","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-10-20 12:57:36","video":"","vorDoi":"10.1007/s10142-025-01798-5","vorDoiUrl":"https://doi.org/10.1007/s10142-025-01798-5","workflowStages":[]},"version":"v1","identity":"rs-7727623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7727623","identity":"rs-7727623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}