Validation of a candidate gene for increased grain protein content in wheat
Authors: Dubcovsky Jorge , Fahima Tzion , Blechl Ann , San Miguel Phillip 
Published: March 2022

High Grain Protein Content (GPC) of wheat is important for improved nutritional value and industrial quality. However, selection for this trait is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. A gene with a large effect on GPC was detected on the short arm of chromosome 6B in a Triticum turgidum ssp. dicoccoides accession from Israel (DIC, hereafter). During the previous BARD project we constructed a half-million clones Bacterial Artificial Chromosome (BAC) library of tetraploid wheat including the high GPC allele from DIC and mapped the GPC-B1 locus within a 0.3-cM interval. Our long-term goal is to provide a better understanding of the genes controlling grain protein content in wheat. The specific objectives of the current project were to: (1) complete the positional cloning of the GPC-B1 candidate gene; (2) characterize the allelic variation and (3) expression profile of the candidate gene; and (4) validate this gene by using a transgenic RNAi approach to reduce the GPC transcript levels. To achieve these goals we constructed a 245-kb physical map of the GPC-B1 region. Tetraploid and hexaploid wheat lines carrying this 245-kb DIC segment showed delayed senescence and increased GPC and grain micronutrients. The complete sequencing of this region revealed five genes. A high-resolution genetic map, based on approximately 9,000 gametes and new molecular markers enabled us to delimit the GPC-B1 locus to a 7.4-kb region. Complete linkage of the 7.4-kb region with earlier senescence and increase in GPC, Zn, and Fe concentrations in the grain suggested that GPC-B1 is a single gene with multiple pleiotropic effects. The annotation of this 7.4-kb region identified a single gene, encoding a NAC transcription factor, designated as NAM-B1. Allelic variation studies demonstrated that the ancestral wild wheat allele encodes a functional NAC transcription factor whereas modern wheat varieties carry a non-functional NAM-B1 allele. Quantitative PCR showed that transcript levels for the multiple NAMhomologues were low in flag leaves prior to anthesis, after which their levels increased significantly towards grain maturity. Reduction in RNA levels of the multiple NAMhomologues by RNA interference delayed senescence by over three weeks and reduced wheat grain protein, Zn, and Fe content by over 30%. In the transgenic RNAi plants, residual N, Zn and Fe in the dry leaves was significantly higher than in the control plants, confirming a more efficient nutrient remobilization in the presence of higher levels of GPC. The multiple pleiotropic effects of NAM genes suggest a central role for these genes as transcriptional regulators of multiple processes during leaf senescence, including nutrient remobilization to the developing grain. The cloning of GPC-B1 provides a direct link between the regulation of senescence and nutrient remobilization and an entry point to characterize the genes regulating these two processes. This may contribute to their more efficient manipulation in crops and translate into food with enhanced nutritional value. The characterization of the GPC-B1 gene will have a significant impact on wheat production in many regions of the world and will open the door for the identification of additional genes involved in the accumulation of protein in the grain.