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.