DISCUSSION
Fe is an essential element for plants and is vital for plant growth and development. Plants have evolved complicated mechanisms for regulating Fe homeostasis that involve multiple different pathways. In recent years, with the rapid development of biochemical and molecular biology technology, research on Fe absorption, transport, and utilization has yielded many new insights (Jeong & Guerinot, 2009; Kobayashi & Nishizawa, 2012; Connorton et al. 2017). Many of the upstream regulatory mechanisms have been elucidated in recent years, including the role of bHLH and MYB transcriptional factors (Palmer et al. 2013; Gao et al. 2019). Moreover, it has been shown that plant hormones, such as ABA and ethylene, as well as environmental factors, such as NO and phosphate, participate in the regulation of Fe homeostasis (Hirsch et al. 2006; Chen et al. 2010; García et al. 2010; Lei et al. 2014; Romera et al. 2017).
As a common element found in soil, nitrogen plays an important role in plant yield. Excessive use of nitrogen fertilizer may cause an imbalance of nutrients (ZhongYang. 2016). However, the exact mechanisms behind the symptoms caused by these imbalances have not been fully explored. In this study, we analyzed the effect of nitrate on the regulation of Fe homeostasis. We found that nitrate could affect Fe uptake both directly and indirectly by affecting citrate levels and ABA accumulation (Fig. 4, Fig. 7). These findings may lead to new strategies to attenuate Fe deficiency by controlling the input of other nutrients in the future.
Previous studies have indicated that plants evolved two different strategies to uptake Fe from soil, although both of these strategies share some overlap (Römheld & Marschner. 1986; Connorton et al,2017; Grillet & Schmidt. 2019). Our results further verified that apple mainly uses strategy I to uptake Fe from soil, which mainly involved differential expression of MdAHA2 and MdFRO2 (Fig. 6g, h). Moreover, the transcriptome and qRT-PCR data showed that the expression of MdCYP82C4 , which is involved in the biosynthesis of Fe-mobilizing coumarins, is repressed by HN treatment (Fig. 5b, Fig. 6e), indicating that apple also employs chelator-based Fe acquisition strategies (Schmid et al. 2014; Rajniak et al. 2018). Our results also demonstrated that low-nitrate treatment facilitates Fe activation by regulating rhizosphere pH (Fig. 2), while the expression ofMdFRO2 was higher under high-nitrate treatment (Fig. 6h). We assume that high-nitrate treatment leads to a higher pH of rhizosphere and represses Fe uptake, which pushes a higher expression level of root MdFRO2 to improve the efficiency of iron absorption. Low-nitrate treatment may also alleviate Fe deficiency by regulating citric acid and ABA accumulation in roots (Fig. 4, Fig. 7), which affects Fe reutilization and long-distance transport.
Plant Fe homeostasis regulation is a complicated process and there are likely other regulatory pathways involved which were not identified in this study. NO plays a positive role in regulating Fe deficiency, and evidence has shown that nitrate is the precursor of NO, which increases in roots upon nitrate addition (Bethke et al. 2004; García et al. 2010). Ethylene was shown to play a role in the regulation of Fe deficiency (Lucena et al. 2006; Wu et al. 2011), when Scheible et al. (2004) found that many genes involved in ethylene synthesis and perception were downregulated by nitrate addition. Nitrate addition also induces the biosynthesis of CKs by regulating expression of IPT3 . The induced CKs are then able to repress the expression of IRT1 , FRO2 , and FIT and negatively regulate iron uptake in roots (Miyawaki et al. 2004; Takei et al. 2004; Séguéla et al. 2008). Taken together, these findings indicate that the effects of nitrate on Fe homeostasis are multifaceted and highly coordinated. We utilized RNA-seq analysis and found that the nitrate signal may be connected with multiple phytohormones, including ABA, ethylene, and CKs (Fig. S5b). LN treatment significantly increased Fe content in the leaves, and significantly increased the Fe content in stem under -Fe conditions (Fig. 3a, c), indicating that this treatment can promote Fe transport from root to shoot through xylem. Many genes are likely involved in this process, including MdNRAMP1 , MdNAS1 , MdYSL1 , andMdMATE43 . NRAMP1 plays an important role in exporting Fe stored in vacuole, while NAS1 and YSL1 transport Fe (Wintz et al. 2003; Haydon & Cobbett. 2007; Kumar et al. 2017; Bastow et al. 2018; Clemens. 2019). MATE43 is a homolog of AtFRD3 which is responsible for transporting citrate into xylem (Durrett et al. 2007; Morrissey et al. 2009; Wang et al. 2018). It is well known that the chelation of Fe by metal binding compounds is the primary mechanism for long-distance transport. Upon reaching the vasculature, Fe is subsequently loaded into the xylem where it is chelated with citrate and transported to aerial plant tissues (Durrett et al. 2007; Clemens S. 2019). In this study, it was found that citrate promoted Fe transport from root to shoot, lending further support to earlier findings which showed citrate acts as an iron chelator that mobilizes iron from roots to aerial tissues. Therefore, it seems likely that LN treatment promotes the long-distance remobilization of Fe from root vacuoles, thereby attenuating the chlorosis caused by Fe deprivation.
It has been well-documented that there is mutual regulation between different plant nutrients. Gratz et al. (2019) demonstrated that Fe deficiency induced cytosolic Ca2+ concentration and Ca2+ triggered CBL1/9-mediated activation of CIPK11. This in turn led to the phosphorylation and activation of FIT proteins, enabling the Fe deficiency response. In tomato, Pi transporter geneLept2 was up regulated by nitrate treatment, suggesting a potential coordination of nitrate and Pi uptake in plant, while Pi was indicated to antagonist Fe (Wang et al. 2001; Hirsch et al. 2006; Zheng et al. 2009). In this study, we found that citrate played a dominant role in the process of nitrate-regulated Fe deficiency response in apple, while malate had no significant effect. However, malate was shown to affect Fe acquisition in Arabidopsis (Mora-Macías et al. 2017), suggesting that Fe absorption and transport are not completely conserved between different species.
Based on our Fe deficiency response data, we constructed a model to integrate the roles played by different transcription factors and transporters (Fig. 9). Under Fe deficiency conditions, LN enhanced the expression of MdAHA2 which aided rhizosphere acidification and increased the solubility of Fe, enabling more efficient uptake. Meanwhile, LN increased plant ABA content, which facilitated cell wall Fe reutilization and transport from root to shoot. Moreover, LN promoted citric acid accumulation in root and promoted Fe transport to shoot through xylem in the form of Fe3+-citrate. LN treatment also promoted the expression of MdFRD3 andMdMAE43 , which facilitated citrate-based transport of Fe into xylem. Additionally, LN enhanced the expression of MdNAS1 , which is responsible for NA synthesis and promoted Fe transport to shoot through phloem in the form of Fe2+-NA. LN also upregulated the expression of the transcription factor MdFIT , which plays an important role in the Fe homeostasis regulatory network. Further study is required to understand the exact mechanisms underlying the effect of nitrate on these different genes. Overall, this study describes a possible model by which nitrate levels affect the uptake and transportation of Fe. These finding provide new knowledge about how nitrogen, hormones, and organic acids coordinate iron balance under iron deficient conditions.