Competing interests
The named authors declare no conflict of interest, financial or
otherwise related to this work.
Figure Legend
Figure 1 . Effects of different nitrate treatment on chlorophyll
and soluble Fe content. The phenotypes (a, b), the chlorophyll content
of young leaves (c), and the soluble Fe content of young leaves (d) and
roots (e) of 6-week-old seedlings grown in vermiculite treated with 15
mM
KNO3+ 50 μM Fe, 0.5 mM KNO3 + 50 μM Fe, 15 mM
KNO3 +200 μM ferrozine (FRZ), 0.5 mM
KNO3 + 200 μM ferrozine (FRZ) for two weeks were showed.
Error bars represent standard deviation (n≥3). Different letters
represent significantly different values at P<0.05 .
Figure 2 . Effects of different nitrate treatment on Fe
deficiency responses. FCR activity ( a, b) and the pH of the treatment
solution (c, d, e) of 6-week-old seedlings treated with 15 mM
KNO3 + 50 μM Fe, 0.5 mM KNO3 + 50 μM Fe,
15 mM KNO3 +200 μM ferrozine, 0.5 mM
KNO3 + 200 μM ferrozine for the indicated time are
shown. Error bars represent standard deviation (n≥3). * represents
significantly different values at P<0.05 .
Figure 3 . Effects of different nitrate treatment on Fe
translocation from root to shoot. Total Fe content of leaves (a) and
roots (b), and the Fe concentration in stems (c) of 6-week-old seedlings
treated with 15 mM KNO3 + 50 μM Fe, 0.5 mM
KNO3 + 50 μM Fe, 15 mM KNO3 +200 μM
ferrozine, 0.5 mM KNO3 + 200 μM ferrozine for two weeks.
Error bars represent standard deviation (n≥6). Seedlings of same growth
status were treated with 0.5 mM
KNO3 + 50 µM Fe, 15
mM KNO3 + 50 µM Fe, 0.5 mM KNO3 -Fe (-Fe
solution supplemented with 200 µM ferrozine) or 15 mM
KNO3 -Fe (-Fe solution supplemented with 200 µM
ferrozine) solutions for 1, 3, 5 and 7 days, respectively. Relative
expression level of MdFRD3 (d), MdMATE43 (e),MdNAS1 (f) were detected. Different letters represent
significantly different values at P<0.05 .
Figure 4 .Nitrate alleviate iron deficiency partially through
citrate. Citrate content (a) and malate content (b) in roots. 6-week-old
seedlings were treated with 15 mM KNO3 + 50 μM Fe, 0.5
mM KNO3 + 50 μM Fe, 15 mM
KNO3 + 200 μM
ferrozine, 0.5 mM KNO3 + 200 μM ferrozine for two weeks.
Error bars represent standard deviation (n≥3). Phenotype of exogenous
0.5 mM citrate treatment seedlings(c), chlorophyll content of young
leaves (d), total Fe content of leaves (e) and roots (f) of 6-week-old
seedlings treated with 15 mM KNO3 + 200 μM ferrozine, 15
mM KNO3 + 200 μM ferrozine + 0.5 mM citrate (CIT), 0.5
mM KNO3 + 200 μM ferrozine, 0.5 mM KNO3+ 200 μM ferrozine + 0.5 mM citrate (CIT) for 3 weeks. Error bars
represent standard deviation (n≥3). Different letters represent
significantly different values at P<0.05 .
Figure 5 . Differentially expressed genes analysis of different
nitrate treatment using RNA-seq. Differently expressed gene number of
roots (a) of 6-week-old seedlings treated with 15 mM
KNO3 and 15 mM KCl for 24 hours. Fe-related differently
expressed genes of roots (b). S1, S2, S3 represent 3 biological repeats.
The data were filtered at |Foldchange|≥1.5,pval<0.05 .
Figure 6 . Relative expression level of Fe-related genes in
response to LN and HN in roots. Seedlings of same growth status were
treated with 0.5 mM
KNO3 + 50 µM Fe, 15
mM KNO3 + 50 µM Fe, 0.5 mM KNO3 -Fe (-Fe
solution supplemented with 200 µM ferrozine) or 15 mM
KNO3 -Fe (-Fe solution supplemented with 200 µM
ferrozine) solutions for 1, 3, 5 and 7 days, respectively. Relative
expression level of MdFIT (a), MdFER1 (b), MdYSL3(c), MdYSL1 (d), MdCYP82C4 (e), MdNRAMP1 (f),MdAHA2 (g) and MdFRO2 (h) were detected. MdActin was
selected as a control gene. Results were based on the average of three
replicate experiments. Different letters represent significantly
different values at P<0.05 .
Figure 7 . ABA alleviates nitrate-mediated Fe deficiency
response. ABA content in young leaves (a) and roots (b) of 6-week-old
seedlings treated with 15 mM KNO3 + 50 μM Fe, 0.5 mM
KNO3 + 50 μM Fe, 15 mM KNO3 +200 μM
ferrozine, 0.5 mM KNO3 + 200 μM ferrozine for the
indicated time are shown. Chlorophyll content of young leaves (c) and
phenotypes of exogenous 1µM ABA treatment (d). Error bars represent
standard deviation (n≥4). Different letters represent significantly
different values at P<0.05 .
Figure 8 . The regulatory mechanism on nitrate-mediated iron
deficiency response is conserved in Arabidopsis thaliana .
Phenotype (a, b), chlorophyll content (c) and total Fe content of shoot
(d) of 2-week-old seedlings treated with 20 mM KNO3 + 50
μM Fe, 0.2 mM KNO3 + 50 μM Fe, 20 mM
KNO3 +200 μM ferrozine, 0.2 mM KNO3 +
200 μM ferrozine for one week. Error bars represent standard deviation
(n≥3). Different letters represent significantly different values atP<0.05 .
Figure 9 . A model of nitrate in regulation of iron deficiency.
NO3- is a member of substrates that
affect plant iron deficiency response, both in direct and indirect ways.
On the one hand, LN treatment helped to rhizosphere acidification and
increase the solubility of Fe in rhizosphere. LN treatment increased the
expression of genes including MdFRD3 , MdMATE43 ,MdNRAMP1 , MdNRAMP6 , MdNAS1 , MdYSL1 ,MdYSL3 that are critical for Fe transport, and MdFIT which
could activate expression of downstream genes to positively regulate Fe
deficiency. On the other hand, LN treatment increased the citrate and
ABA content in roots under Fe deprivation conditions, which contribute
to Fe transportation and homeostasis. Dotted line represents the results
of previous study (Lei et al. 2014). Yellow arrows represent metabolism
pathway.
Figure S1 . Effect of applicating nitrate on the Fe deficiency
symptoms in the young leaves of apple. 2-year-old ‘Fuji’ apple trees
treated with 10g KNO3/per plant, 30g KNO3/per plant, 60g KNO3/per plant,
120g KNO3/per plant for 3 months. Phenotypes of young leaves (a, b, c,
d) and whole trees (e, f, g, h). Each line contains 11 trees. Each
treatment contains 11 plants.
Figure S2 . Chlorophyll content of young leaves of 2-year-old
‘Fuji’ apple trees treated with different concentration of nitrate.
Error bars represent standard deviation (n=5). Different letters
represent significantly different values at P<0.05 .
Figure S3 . Nitrogen content of differently treated seedlings.
NO3- content of roots (a), leaves (b)
and total nitrogen content of leaves and roots (c) of 6-week-old
seedlings treated with 15 mM KNO3 + 50 μM Fe, 0.5 mM
KNO3 + 50 μM Fe, 15 mM KNO3 + 200 μM
ferrozine, 0.5 mM KNO3 + 200 μM ferrozine for two weeks.
Error bars represent standard deviation (n=3). Different letters
represent significantly different values at P<0.05 .
Figure S4 . Effects of different nitrate treatment on citrate
content (a) and malate content (b) in leaves. 6-week-old seedlings were
treated with 15 mM KNO3 + 50 μM Fe, 0.5 mM
KNO3 + 50 μM Fe, 15 mM KNO3 +200 μM
ferrozine, 0.5 mM KNO3 + 200 μM ferrozine for two weeks.
Error bars represent standard deviation (n≥3). Different letters
represent significantly different values at P<0.05 .
Figure S5 . Nitrate regulates metal ion and phytohormone related
genes expression. Number of metal ion-related genes regulated by nitrate
(a) and number of phytohormone related genes regulated by nitrate (b) in
roots. The data were filtered at |Foldchange|≥1.5,pval<0.05 .
Fig S6 . ABA biosynthesis and signal pathway genes which are
regulated by nitrate. S1, S2, S3 represent 3 biological repeats. The
data were filtered at |Foldchange|≥1.5,pval<0.05 .
Table 1 qRT-PCR Primers used in this study