INTRODUCTION
Fe is an essential element for plant growth and development, due to its role in iron sulfur (FeS) proteins, ferredoxins, and various metabolic enzymes. It impacts various cellular processes, including photosynthesis, respiration, electron transfer reactions, and others (Connorton et al. 2017). Excessive Fe causes yellow-brown spots on the tips, edge, and interveinal of old leaves and represses plant growth, while Fe deficiency triggers chlorosis and reduces fruit yields (Álvarez-Fernández et al. 2003). Although soil contains abundant Fe, it is usually not available to plants due to its insoluble nature. This problem is exacerbated by alkaline and aerobic environmental conditions. Fe deficiency is a worldwide problem, with about 30% of the world’s soil currently suffering from Fe deficiency, which severely restricts efforts to improve crop yield and quality (Marschner, 2011; Briat et al. 2015).
To cope with Fe deficiency, plants have evolved two major mechanisms to absorb Fe from the soil, known as strategy I for dicots and non-graminaceous monocots and strategy II for graminaceous monocots (Römheld & Marschner, 1986; Kobayashi & Nishizawa, 2012). Graminaceous plants, such as maize and rice, use strategy II, also known as the chelating strategy, to uptake Fe from the soil. These plants secrete phytosiderophores, which have high affinity for Fe, from their roots. Fe3+-phytosiderophore chelates are imported by YS1 (Yellow Stripe 1) transporters in maize (Curie et al. 2001) and YSL15 transporters in rice (Inoue et al. 2009). In contrast, non-graminaceous plants, such as Arabidopsis thaliana and tomato, use strategy I, also known as the reducing strategy, to absorb Fe from rhizosphere. In this strategy, plasma membrane H+-ATPases first acidify the rhizosphere and facilitate Fe solubilization by pumping protons (H+) into root rhizosphere. Next, FRO2 (Ferric Reduction Oxidase 2) at the plasma membrane reduces Fe3+ to Fe2+, which is then transported across the membrane by IRT1 (Iron-Regulated Transporter 1) (Jeong & Guerinot, 2009; Santi & Schmidt, 2009; Ivanov et al. 2012; Kobayashi and Nishizawa, 2012; Jeong et al. 2017). Finally, Fe is transported to the shoot through long-distance transportation, which requires many genes, including NAS (Nicotianamine Synthesis) (Haydon & Cobbett, 2007; Jeong and Guerinot, 2009), FRD3 (Ferric Redictase Defective3) (Durrett et al. 2007; Jeong and Guerinot, 2009),YSL (Yellow Stripe-Like) (Waters et al. 2006; Haydon & Cobbett, 2007), OPT3 (Oligopeptide Transporter3) (Stacey et al. 2008; Ivanov et al. 2012), and FPN1 (Ferroportin1) (Morrissey et al. 2009; Ivanov et al. 2012). Despite their obvious differences, evidence shows that the two strategies share several components. Strategy I plants have been found to secret chelators to rhizosphere, such as phenolics, flavonoids and flavins, as a typical feature of strategy II plants (Fourcroy et al. 2014; Connorton et al. 2017; Grillet & Schmidt, 2019). Additionally, a functional homologue of IRT1 has been found in rice, which mediates Fe2+ uptake under low oxygen conditions (Ishimaru et al. 2006).
Over the past few decades, significant progress had been made in uncovering the mitigation and regulation mechanisms of iron deficiency in plants. For strategy I plants, rhizosphere acidification and Fe3+ reduction are essential processes which enable Fe transport from the soil to roots. Upon exposure to Fe deprivation conditions, protons are released from H+-ATPase plasma membrane pumps to acidify the soil and increase the solubility of Fe. Some H+-ATPases (AHA) genes are induced under Fe deficiency and are therefore thought to function in Fe-deficiency responses (Santi et al. 2008; 2009). After acidification, free ferric Fe is reduced to ferrous ions by FRO2 (Robinson et al. 1999). InArabidopsis , the FRO family contains eight members, each of which are thought to play different functions (Mukherjee et al. 2006). Once reduced by FRO2, Fe2+ is transported into the root by IRT1, a member of the ZIP family (Guerinot. 2000). Knock-outs ofIRT1 result in Fe deficiency accompanied by cell differentiation defects, which points to the important role played by IRT1(Henriques et al. 2002). Both FRO2 and IRT1 are regulated by FIT , at the transcriptional level and the post-transcriptional level, respectively (Colangelo & Guerinot. 2004). Another process involved in strategy I is the excretion of Fe-mobilizing coumarins under high pH conditions. The biosynthesis and secretion of these coumarins involve F6’H1 (Feruloyl-CoA 6′-Hydroxylase1), S8H(Scopoletin 8-hydroxylase), CYP82C4 (Cytochrome P450, Family 82, Subfamily C, Polypeptide 4), PDR9 (Pleiotropic Drug Resistance 9), and BGLU42 (Beta Glucosidase 42) (Fourcroy et al., 2014; Zamioudis et al., 2014; Schmid et al., 2014, Rajniak et al., 2018; Siwinska et al., 2018; Tsai et al., 2018). Once Fe enters the plant, it must be safely transported to multiple parts of plant, which requires chelators, such as citrate and nicotianamine (NA) (Haydon & Cobbett. 2007; Curie et al. 2009).
Organic acids, especially citrate, are the main metal chelators in xylem and function in metal ion transportation (Brown et al, 1971; Rellan-Alvarez et al, 2010). Fe3+-citrate has been proposed to be the main form of Fe present in xylem exudates (Grotz & Guerinot. 2006). FRD3 is a MATE family member, which has been proposed to play a role in transporting citrate into xylem (Durrett et al. 2007; Morrissey et al. 2009). Arabidopsis mutants with abolished function of FRD3 show various Fe deficiency symptoms, while overexpression of FRD3 in rice enhances Fe mobility, resulting in elevated levels of Fe in the endosperm (Roschzttardtz et al. 2011; Wu et al. 2018). NA is a precursor of MA (Mugineic acid), which complexes with Fe and is required for unloading it from vascular tissues (Haydon & Cobbett. 2007; Clemens. 2019). NAS genes are responsible for NA synthesis. In Arabidopsis , AtNAS1 is involved in the synthesis of NA from SAM (S-adenosyl-L-methionine) (Haydon & Cobbett. 2007). NAS genes are also upregulated in shoots and roots under Fe deficiency (Wintz et al. 2003). YSL transporters are hypothesized to transport the Fe-NA complex from the phloem to surrounding parenchyma, and YSL1 and YSL3 are suggested to be involved in the translocation of other metals (Waters et al. 2006; Curie et al. 2009; Chu et al. 2010; Kumar et al. 2017).
Fe-deficiency induces gene expression associated with ethylene synthesis and signaling processes in roots, which causes the upregulation of Fe-related genes, such as AtFIT , AtFRO2 , AtIRT1 ,AtNAS1 , AtFRD3 , and others (Lucena et al. 2006; García et al. 2010). A similar mechanism has been found in rice, where Fe-deficiency induces abscisic acid accumulation rapidly in roots (Wu et al. 2011). Exogenous 0.5 µM ABA promotes apoplastic Fe reutilization, while treatment with ABA upregulates expression of AtFRD3 ,AtYSL2 , and AtNAS1 and increases Fe content in xylem sap, indicating that ABA promotes transport of Fe from root to shoot (Lei et al. 2014). Fe-deficiency also increases NO (Nitric Oxide) in roots, which is involved in upregulation of Fe-related genes (Chen et al. 2010; García et al. 2010). Zhu et al. (2017) found that NaCl mitigates iron deficiency by facilitating Fe reutilization and translocation from root cell wall to shoots.
Many transcription factors (TFs) participate in the regulation of Fe deficiency responses. Among them, bHLH (basic helix-loop-helix) transcription factors are the predominant family (Heim et al. 2003; Gao et al. 2019). In Arabidopsis , expression of FIT/bHLH29(Fe-Deficiency Induced Transcription Factor) is induced by Fe-deficiency in roots, which then regulates the expression of AtIRT1 andAtFRO2 , together with AtbHLH38 and AtbHLH39 (Li et al. 2016). In addition, FIT interacts with EIN3 (Ethylene Insensitive 3) to regulate Fe uptake (Lingam et al. 2011). The bHLH105/ILR3 (IAA-Leucine Resistant 3) is proposed to act as both transcriptional activator and repressor, and it may be a core TF for the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis(Tissot et al. 2019). Additionally, ILR3 also interacts with bHLH104 to modulate Fe homeostasis in Arabidopsis (Zhang et al. 2015). Zhou et al. (2019) reported that MdbHLH104 is stabilized by the SUMO E3 ligase MdSIZ1 to regulate plasma membrane H+-ATPase activity and Fe homeostasis. In apple, overexpression of theMdbHLH104 gene enhances the tolerance to Fe deficiency (Zhao et al. 2016a). Two BTB scaffold proteins, MdBT1 and MdBT2, target MdbHLH104 and negatively regulate the stability of MdbHLH104 to affect plasma membrane H+-ATPase activity and Fe homeostasis (Zhao et al. 2016b). In addition, other TFs, such as MYB and WRKY are required for plant growth under Fe-deficient conditions (Palmer et al. 2013; Yan et al. 2016; Wang et al. 2018).
Nitrogen is an indispensable nutrient for plants, with nitrate representing the main bioavailable form for land plants. Nitrate (NO3-) also acts as a vital signaling molecule that modulates various growth and development processes of plants, including seed germination, flowering, shoot branching, and root development (Bouguyon et al. 2016; Yan et al. 2016; Canales et al. 2017; Fredes et al. 2019). In potato (Sohmum tuherosum L.), interrupting the N supply increases ABA content, which slowly recovers to normal levels after N levels are restored (Krauss. 1978). In wheat seedlings, nitrogen deficiency results in the rapid accumulation of ABA, especially in roots (Teplova et al. 1998). There are also interactions between nitrate and other nutrient signaling pathways. Liu et al. (2017) found that Ca2+ signaling sensor CPKs interact with NLP7 (NIN-Like Protein 7) to modulate nitrate response. Recent research also found that nitrate acts as a signal to coordinate N-P nutrient balance in rice (Oryza sativa L.) (Hu et al. 2019).
Here, we investigated the effect of nitrate on Fe deficiency and found that the application of nitrate influenced Fe deficiency response. The regulatory mechanisms behind this relationship were also investigated, which sheds new light on the interaction between nitrate and iron in apple and other species.