A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat

Abstract

Enhancing the nutritional value of food crops is a means of improving human nutrition and health. We report here the positional cloning of Gpc-B1, a wheat quantitative trait locus associated with increased grain protein, zinc, and iron content. The ancestral wild wheat allele encodes a NAC transcription factor (NAM-B1) that accelerates senescence and increases nutrient remobilization from leaves to developing grains, whereas modern wheat varieties carry a nonfunctional NAM-B1 allele. Reduction in RNA levels of the multiple NAM homologs by RNA interference delayed senescence by more than 3 weeks and reduced wheat grain protein, zinc, and iron content by more than 30%.

Fig. 1

Map-based cloning of Gpc-B1. (A) QTL for grain protein on wheat chromosome arm 6BS (11). (B) Sequenced B-genome physical contig. The position and orientation of five genes is indicated by arrows. (C) Fine mapping of Gpc-B1. The x’s indicate the positions of critical recombination events flanking Gpc-B1. Vertical lines represent polymorphism mapped in the critical lines. A single gene with three exons (green rectangles) was annotated within the 7.4 kb region flanked by the closest recombination events. The open arrowhead indicates the transcription initiation site. (D) Graphical genotypes of critical recombinant substitution lines used for fine-mapping of Gpc-B1. Blue bars represent LDN markers; red bars represent DIC markers. (E) Flag leaf chlorophyll content of recombinant substitution lines segregating for Gpc-B1 (14). Asterisks indicate significant differences (P<0.01). Phenotypes of critical recombinant substitution lines: (F) chlorophyll at 20 days after anthesis (DAA), (G) grain protein, (H) Zn, and (I) Fe concentrations. Blue and red bars indicate the presence of the LDN and DIC alleles at TtNAM-B1, respectively. (J) First 18 nucleotides of DIC and LDN TtNAM-B1 alleles and their corresponding amino acid translation. The LDN allele carries a 1-bp insertion (red T) that disrupts the reading frame (indicated by red amino acid residues). Error bars represent standard error of the means (E–I).

Fig. 2

(A) Expression profile of the different TtNAM genes relative to ACTIN in tetraploid wheat recombinant substitution line 300 carrying a functional TtNAM-B1 gene. Units are values linearized with the 2^{(−Δ ΔC_T)} method, where C_T is the threshold cycle. (B) Relative transcript level of endogenous TaNAM genes in T₂ plants (L19-54) segregating for transgenic (n = 12, white) and non-transgenic (n = 11, black) TaNAM RNAi constructs at 4 and (C) 9 days after anthesis. Asterisks indicate significant differences (P<0.05). (D) Flag leaf chlorophyll content profile of transgenic (n = 22 T₁ plants) and non-transgenic controls (n = 10 T₁ plants). (E) Representative transgenic (left) and non-transgenic (right) plants 50 DAA. (F) Main spike and peduncles of representative transgenic and non-transgenic plants 50 DAA. Error bars represent standard error of the means.