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.