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New Phytol
2020 Feb 01;2254:1667-1680. doi: 10.1111/nph.16234.
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A wheat transcription factor positively sets seed vigour by regulating the grain nitrate signal.
Li W
,
He X
,
Chen Y
,
Jing Y
,
Shen C
,
Yang J
,
Teng W
,
Zhao X
,
Hu W
,
Hu M
,
Li H
,
Miller AJ
,
Tong Y
.
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Seed vigour and early establishment are important factors determining the yield of crops. A wheat nitrate-inducible NAC transcription factor, TaNAC2, plays a critical role in promoting crop growth and nitrogen use efficiency (NUE), and now its role in seed vigour is revealed. A TaNAC2 regulated gene was identified that is a NRT2-type nitrate transporter TaNRT2.5 with a key role in seed vigour. Overexpressing TaNAC2-5A increases grain nitrate concentration and seed vigour by directly binding to the promoter of TaNRT2.5-3B and positively regulating its expression. TaNRT2.5 is expressed in developing grain, particularly the embryo and husk. In Xenopus oocyte assays TaNRT2.5 requires a partner protein TaNAR2.1 to give nitrate transport activity, and the transporter locates to the tonoplast in a tobacco leaf transient expression system. Furthermore, in the root TaNRT2.5 and TaNRT2.1 function in post-anthesis acquisition of soil nitrate. Overexpression of TaNRT2.5-3B increases seed vigour, grain nitrate concentration and yield, whereas RNA interference of TaNRT2.5 has the opposite effects. The TaNAC2-NRT2.5 module has a key role in regulating grain nitrate accumulation and seed vigour. Both genes can potentially be used to improve grain yield and NUE in wheat.
Figure 1. Overexpression of TaNAC2‐5A increases seed vigour and grain nitrate concentration. (a) Germination percentage of TaNAC2‐5A overexpression lines and wild‐type (WT) in Petri dishes. Values are means ± SE (n = 4). **, P < 0.01 (Student’s t‐test). (b) Grain nitrate concentration measured using the dry seeds. Values are means ± SE (n = 4). **, P < 0.01 (Student’s t‐test). (c) Seedling images of germination assay in pot experiment. Bars, 5 cm. (d) Shoot length (cm) distribution of (c). Values are means ± SE (n = 4).
Figure 2. TaNAC2‐5A binds to the promoter of TaNRT2.5‐3B and regulates the expression. (a) Chromatin immunoprecipitation quantitative PCR (ChIP–qPCR) assay of TaNAC2‐5A binding to TaNRT2.5‐3B promoter. P1–P4 are fragments selected in TaNRT2.5‐3B promoter for ChIP–qPCR analysis. (b) Electrophoretic mobility shift assay (EMSA) of TaNAC2‐5A binding to P4 fragment from (a). (c) The expression pattern of TaNRT2.5 in different organs of wheat at seedling stage and 14 d post‐anthesis (DPA). (d) The expression pattern of TaNRT2.5 in different parts of wheat seeds in 14 and 28 DPA. (e) The expression pattern of TaNAC2 and TaNRT2.5 in developing seeds at 7, 14, 21 and 28 DPA. (f) The expression level of TaNRT2.5 in seeds of TaNAC2‐5A overexpression lines and wild‐type (WT). Significance for the difference between the means of the transgenic lines and WT: **, P < 0.01 (Student’s t‐test). TaActin was used as an internal reference. Data represented as means ± SE (n = 4).
Figure 3. The nitrate transport activity and subcellular localization of TaNRT2.5. (a) The nitrate transport activity of different combinations of TaNRT2.5s and TaNAR2s in pH 7.5 and 5.5 relative to water‐injected controls. Uptake from 500 µM of 15N labelled nitrate in 6 h was used for Xenopus oocytes which were injected with water or different cRNA combinations. The delta‐15N values are shown as the mean ± SE for four oocytes. Different letters above the columns indicate statistically significant differences at the P < 0.05 level according to one‐way ANOVA. (b–d) Fluorescence of green fluorescent protein (GFP) in tobacco leaf epidermal cells expressing TaNRT2.5‐3B::GFP coupled with TaNAR2.1‐6B::RFP (b), TaNRT2.5‐3B::GFP alone (c) or TaNAR2.1‐6B::RFP (d) (Bars, 20 μm). (b) Left, TaNRT2.5‐3B::GFP signal; middle, TaNAR2.1‐6B::RFP signal; right, overlap of these two signals. (c) TaNRT2.5‐3B::GFP signal. (d) TaNAR2.1‐6B::RFP signal. (e–g) Microsome from whole leaf of expressed TaNRT2.5‐3B::GFP coupled with TaNAR2.1‐6B::RFP or TaNRT2.5‐3B::GFP alone were extracted and fractionated by sucrose‐density gradient centrifugation, then the different fractions were used for Western blot. Antibodies of anti‐GFP, anti‐γ‐TIP (tonoplast marker), and anti‐H+‐ATPase (plasma membrane marker) were used (e). Relative band intensity was measured using imagej software (NIH, Bethesda, MD, USA). The ratio of different band intensity to total intensity was calculated, and three independent experiments were performed. Data represented as means ± SE (n = 3) (f, g).
Figure 4. Overexpression of TaNRT2.5‐3B increases seed germination, grain nitrate concentration and modifies the expression of some associated genes. (a) Germination percentage of TaNRT2.5 transgenic lines and wild‐type (WT). (b) Grain nitrate concentration of TaNRT2.5 transgenic lines and WT. (c) Grain nitrogen concentration of TaNRT2.5 transgenic lines and WT. (d–g) Expression levels of TaNRT2.5 (d), TaAMY (e), TaUGPase (f) and TaNR1 (g) in the germinating seeds (60 h after sowing). OE102‐6 and OE103‐1, overexpression lines; R100‐1 and R109‐2, RNAi lines. TaActin was used as an internal reference. Data represented as means ± SE (n = 4). Significance for the difference between the means of the transgenic lines and WT: *, P < 0.05; **, P < 0.01 (Student’s t‐test).
Figure 5. Overexpression of TaNRT2.5‐3B promotes wheat seedling growth. (a) Root images of TaNRT2.5‐3B overexpression lines (OE102‐6 and OE103‐1) and wild‐type (WT) grown under high and low nitrogen (N) supply conditions using a hydroponic culture system. Bars, 5 cm. (b) Shoot FW; (c) Root FW, (d) Average primary root (PR) length; (e) Total lateral root (LR) length. Data represented as means ± SE (n = 4). Different letters above the columns indicate statistically significant differences at the P < 0.05 level according to one‐way ANOVA.
Figure 6. Overexpression of TaNRT2.5‐3B promotes nitrate uptake and accumulation at the seedling stage. The seedlings of TaNRT2.5‐3B overexpression lines (OE102‐6 and OE103‐1) and wild‐type (WT) grown under high and low nitrogen (N) supply conditions using a hydroponic culture system. (a) Net nitrate uptake rate. (b) Root nitrate concentration. (c) Shoot nitrate concentration. (d) Root N concentration. (e) Shoot N concentration. (f) N uptake. Data represented as means ± SE (n = 4). Different letters above the columns indicate statistically significant differences at the P < 0.05 level by one‐way ANOVA.
Figure 7. Overexpression of TaNRT2.5‐3B increases the contribution of post‐anthesis N uptake to N accumulated in aerial parts and grains. (a) The expression levels of different TaNRT2 family genes in roots of wild‐type (WT) seedling and the plants 14 d post‐anthesis (DPA). TaActin was used as an internal reference. (b, c) Ratio of 15N‐nitrogen (N) accumulation to ANA (b) and to GNA (c) of the TaNRT2.5 overexpression (OE102‐6 and OE103‐1) and RNAi (R100‐1 and R109‐2) lines and WT. ANA, aerial N accumulation; GNA, grain N accumulation. Data represented as means ± SE (n = 4). Different letters above the columns indicate statistically significant differences at the P < 0.05 level according to one‐way ANOVA.
Figure 8. A proposed schematic model for NAC2/TaNRT2.5/TaNAR2.1 activity in nitrate grain filling and subsequent germination integrating data from Arabidopsis and wheat. Modified from Yan et al. (2016). Nitrate promotes TaNAC2 expression to activate the TaNRT2.5/TaNAR2.1 nitrate grain‐filling activity. Nitrate stored in the grain helps provide the signal promoting germination mediated by the abscisic acid (ABA) content controlled by NIN‐like protein 8 (NLP8) and CYP707A.
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