Figure LEGENDS
Figure 1. Exogenous MT treatment and overexpression ofMdASMT9 improves low-N tolerance of apple plants. (a) The
phenotype, (b) plant height, and (c) plant biomass of apple plants
pretreated with 0 mM, 0.05 mM, 0.5 mM, or 2.5 mM MT were measured after
30 d of low-N stress. (d) Relative expression patterns of MdASMT9under N-deficiency conditions. (e) Phenotypes of WT andMdASMT9 -OE apple lines under normal N supply and
N-deficiency
conditions (scale bar = 3 cm). Determination of MT contents in leaves
and roots (f), plant height, (g) and plant biomass (h) of WT andMdASMT9 -OE apple plants. Values are shown as the means ± SD.
Asterisks indicate significant differences (*P < 0.05,
**P < 0.01, and ***P < 0.001).
Figure 2. MdASMT9 transgenic plants exhibit higher
photosynthetic capacity and chlorophyll contents under N-deficiency
conditions. Changes in the net photosynthesis rate (Pn)
(a), stomatal conductance (Gs) (b), total chlorophyll content (c), and
carotenoid content (d) of WT and MdASMT9 -OE apple plants after 35
d of N-deficiency conditions. Chlorophyll fluorescence images (e) and
quantitative measurements of F0 , Fm ,FV/Fm , Y(II), Y(NO), NPQ/4, Y(NPQ), and qN (f).
Values are shown as the means ± SD. Asterisks indicate significant
differences (*P < 0.05, **P < 0.01, and
***P < 0.001).
Figure 3. Proteomic analysis of WT and OE-4 apple plants under
N-deficiency conditions. (a) PCA diagram of all quantifiable proteins.
(b) Quantitative volcano diagram of differentially expressed proteins
(DEPs). (c) Statistical diagram of the number of DEPs. (d) Subcellular
structure locations of DEPs.
Figure 4. COG/KOG and KEGG pathway analysis of DEPs. (a)
COG/KOG annotation of DEPs, and the ontology covers four domains:
Cellular process and signaling, information storage and processing,
metabolism, and poorly characterization. (b) KEGG pathway analysis of
DEPs (Q1, Q2, Q3, and Q4 respectively represent DEPs of
FC<0.667, 0.667<FC<0.769,
1.3<FC<1.5 and FC>1.5). (c) Specific
DEPs involved in C and N cycle with determination of related amino acid
contents. Heatmap shows the levels of sucrose, glucose, fructose, lysine
(Lys), methionine (Met), threonine (Thr), isoleucine (Ile), aspartate
(Asp), proline (Pro), arginine (Arg), histidine (His) and glutamate
(Glu) in WT and transgenic apple plants. Color key normalizing the amino
acid content to (-2, 2) is in the upper right corner. Redder color
indicates higher the amino acid content while bluer color indicates
lower amino acid content.
Figure 5. MdASMT9 transgenic plants maintain higher
nitrate uptake under N-deficiency conditions. (a) The content of
NO3− in roots of WT andMdASMT9 -OE plants. (b)15NO3− influx rate
in WT and MdASMT9 -OE lines. Overexpression of MdASMT9 and
exogenous MT treatment affect the expression of MdNRT2.1 (c) andMdNRT2.4 (d) in apple plants during N deficiency conditions.
Values are shown as the means ± SD. Asterisks indicate significant
differences (*P < 0.05, **P < 0.01, and
***P < 0.001).
Figure 6. MT promotes the transcription of MdHY5 under
low N stress (a). (b-c) EMSA assay of MdHY5 protein binding toMdNRT2.1 and MdNRT2.4 promoters. The recombinant protein
was incubated with biotin-labelled or mutant oligos P1, P2, P3, and P4.
(d-e) Transient expression assay of MdHY5 interacting withMdNRT2.1 and MdNRT2.4 promoters and quantitative analysis
of luminescence intensity. The value for Luc+Empty vector was set to 1.
Asterisks indicate significant differences (***P<0.001).
Figure 7. MdHY5 transgenic apple calli maintained higher
N content and higher expression of MdNRT2.1 and MdNRT2.4 .
(a) Phenotypes of WT and transgenic apple calli after N-deficiency
treatment (HY5 -OE: HY5 -overexpressing apple calli;HY5 -RNAi: HY5 -silenced apple calli). (b) Fresh weights
(FW) and (c) total N contents of WT and transgenic apple calli under
low-N treatment. (d-e) Relative expression level of MdNRT2.1 andMdNRT2.4 in WT and transgenic apple calli under low-N treatment.
Values are shown as the means ± SD. Asterisks indicate significant
differences (*P < 0.05, **P < 0.01, and
***P < 0.001).
Figure 8. Effects of exogenous MT treatment on WT andMdHY5 interfered lines (Ri-1 and Ri-2) under N-deficiency
conditions. Phenotypes (a) and total FW (b) of WT, Ri-1, and Ri-2 with
or without MT treatment under low N treatment. (c)15NO3− influx rates
of WT and MdHY5 interfered lines with or without MT treatment
under low-N conditions. (d-f) Effects of exogenous MT on the expression
of MdHY5 , MdNRT2.1 , and MdNRT2.4 in the roots of WT
and MdHY5 interfered lines under low-N stress. Values are shown
as the means ± SD. Asterisks indicate significant differences
(*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 9. Proposed model for the response ofMdASMT9 -mediated biosynthesis of MT to low-N stress in apple
plants. Solid arrows refer activation, while dashed arrows refer
indirect activation.