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.