Fig 1.
The expression patterns of TaAAP2, TaAAP13, and TaAAP21.
(a-c), Expression of TaAAP2 (a), TaAAP13 (b), and TaAAP21 (c) in grain cells: endosperm transfer cells (ETC), starchy endosperm (SE), aleurone cells (AL) of Chinese Spring at 20DPA (days post anthesis) by RNA-seq data [10, 32]. (d-f), Expression of TaAAP2B (d), TaAAP13D (e), and TaAAP21A (f) in different organs of wheat cultivar Hereward from whole caryopses at 5, 10, 14, 17, 21, and 28 DPA (days post anthesis), roots, leaves and stems at Zadoks 23 (2–3 tillers stage) and Zadoks 45 (booting stage) at two nitrogen levels, 0kg N/ha and 200kg N/ha, by RT-qPCR. Error bars represent standard errors (SE).
Fig 2.
Localization of TaAAP2B, TaAAP13D, and TaAAP21A by promoter::GUS expression.
The GUS expression patterns driven by promoters of TaAAP2B, TaAAP13D, and TaAAP21A in T2 developing grains at 7, 14, 21, 28 and 33DPA (days post anthesis). The GUS staining was visualised directly for TaAAP2B and TaAAP13D or after serial ethanol de-staining for TaAAP21A under stereomicroscope Leica MA250. Scale bars represent 500 μm except TaAAP2B (21D enlarged) and TaAAP13D (7D) for 250 μm. ETC: endosperm transfer cells, EM: embryo, AL: aleurone cells, ES: epithelium of scutellum, SE: starchy endosperm, RP: root primordia.
Fig 3.
TaAAP13 expression and nitrogen concentrations in RNAi lines (SE-1R, SE-9R, SE-10R), null lines (SE-3N, SE-11N), and non-transgenic line SE-24WT (Cadenza, wild type).
(a), TaAAP13 gene expression at 14DPA of wheat developing grain. Asterisks (***) indicated the significant differences between RNAi lines and nulls or wild type detected by ANOVA (P<0.001, F-test). Error bars represent standard errors. (b), Nitrogen concentrations of wholemeal flour and white flour. The differences between RNAi lines and nulls were not statistically significant (P<0.05, F-test).
Fig 4.
Metabolite data analysis of RNAi and null lines by 1H-NMR.
(a), OPLS-DA of polar metabolite profiles of wholemeal flour in RNAi line SE-1R and null SE-3N by 1H-NMR. (b), Contribution plot comparing significant component changes from two lines SE-1R and SE-3N. The red colour represents more elevated in SE-3N on the top, and in SE-1R at the bottom. The amino acids (glutamine and glycine) were most elevated in SE-1R, and are indicated by blue arrows. (c), PCA scores plot of pearling fractions: F1, F2, F3, F4, F5, core and wholemeal flour of SE-1R and SE-3N. (d), The loading plot of PCA for pearling fractions. The coloured circles were drawn by separation of different lines in (a) and different fractions in (c), rather than statistically significant. F1, F2, F3, F4, F5 and core are mainly enriched in bran (F1), aleurone cells (F2), sub-aleurone cells (F3), two progressive inner endosperm fractions (F4, F5) and core.
Fig 5.
The gene expression and phenotype of overexpression TaAAP13D under the control of endosperm–specific promoter HMW-GS 1Dx5.
(a), Gene expression of TaAAP13 at 21DPA in developing grains of overexpression (OE) lines: P22-OE and P16-OE (6 copies), P25-OE (16 copies), P26-OE (14copies), P23-OE (26 copies), P24 (12 copies), null (P15-null), and non-transgenic (P29-WT, Cadenza). Significant differences between OE lines and null or wild type were detected (F2,14 = 20.85, p<0.001) (P<0.001, F-test) and indicated with asterisk (***). (b), N concentration % of wholemeal flour. (c), Thousand grain weight (TGW) at 15% moisture content (g). (d), Nitrogen content (mg) per grain. (e), Grain morphology of T3 grains. Scale bar represents 1 cm. Significant differences between OE lines and null were detected using ANOVA (P<0.05, F-test) and indicated with asterisk (*).
Fig 6.
The protein distribution of mature grain resin sections of TaAAP13 overexpression line.
(a), Dorsal of P23-OE. (b), Dorsal of P15-null. (c), Lobe of P23-OE. (d), Lobe of P15-null. (e), Whole grain section of P23-OE. (f), Whole grain section of P15-null. Resin sections with 1 μm thickness in (a)-(d) and 0.5μm thickness in (e) and (f) were stained with 1% (w/v) Naphthol Blue Black in 7% (w/v) acetic acid. Scale bars represent 50 μm in (a)-(d) and 1mm in (e) and (f). Red arrows indicated large protein body matrix (PB). AL: aleurone cells, SA: sub-aleurone cells, SG: starch granules.
Fig 7.
Schematic view of three amino acid transporters in nitrogen transport.
(a), Wheat grain transverse section. (b), Wheat grain longitudinal section. VB: vascular bundle; EC: endosperm cavity for amino acids delivered to endosperm from vascular bundle; NPTC: nucellar projection transfer cells; ETC: endosperm transfer cells, TaAAP2 expression localization for uptake of amino acids from endosperm cavity; SE: starch endosperm; SA: sub-aleurone cells for proteins (mainly ω and α-gliadins, LMW-glutenin subunits), and TaAAP13 expression localization; AL: aleurone cells for TaAAP21 expression localization. ES: epithelium of scutellum for TaAAP21 expression localization. SC: scutellum. EM: embryo.