Location: Wheat Health, Genetics, and Quality Research
Project Number: 2090-21000-039-004-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2021
End Date: Aug 31, 2026
Objective:
The objectives of this cooperative research are to: 1) Identify the enzymes and/or proteins that contribute to low falling number during wheat grain development and germination, 2) Define the underlying mechanisms that control the expression and synthesis of the enzymes and/or proteins that contribute to low falling number, 3) Develop tests that can detect enzymes to differentiate between pre-harvest sprouting and late maturity alpha-Amylase
Approach:
Hydrolytic enzymes that contribute to low falling number during wheat grain development and germination will be identified using comparative proteomics analysis. Proteins will be isolated from wheat varieties that are susceptible and resistant to pre-harvest sprouting (PHS) and late-maturity alpha-amylase (LMA). These samples will be analyzed using LC-MS/MS and proteins or peptides will be identified by comparing sequences to the UniProt and NCBI databases. Differentially expressed or accumulating proteins that are specific to PHS and LMA will be identified by comparing the proteins present and absent in these varieties. Once PHS- and LMA-specific proteins are identified, we will further filter these lists to identify the hydrolytic enzymes specific to each of these genetic conditions.
The underlying mechanisms that control the expression and synthesis of enzymes that contribute to low falling number will be investigated using multiple techniques. The identity and localization of plant hormones and/or metabolites that accumulate prior to and during PHS and LMA will be investigated using mass-spectrometry imaging. We will section wheat grains, coat the sections with a matrix, and use a laser to scan over the tissue and collect a mass spectrum using a high-resolution mass spectrometer. Metabolites or proteins in each spectrum will be identified using MS databases, and computational programs will be used to identify the location and relative intensity of each metabolite or proteins in the tissue sections. In addition, we will develop a wheat grain aleurone protoplast system for transient expression assays. First, we will isolate aleurone tissue. The aleurone tissue samples will be treated with cell-wall degrading enzymes to produce protoplasts. Then, protoplasts will be isolated and used for transformation. The protoplasts will be treated with various plant hormones, small molecules, environmental conditions and we will monitor gene expression and/or protein accumulation changes.
An immunoassay using LMA- and PHS-specific proteins will be developed. We will isolate proteins from wheat varieties that are resistant or susceptible to LMA and/or PHS. These protein samples will be analyzed using LC-MS/MS and proteins and/or peptides will be identified by comparing sequences to the UniProt and NCBI databases. We will identify LMA-/PHS-specific proteins by comparing the proteins that are present and absent in these varieties. Antibodies will be raised to selected candidate proteins, screened for effectiveness and specificity, and integrated into an immunoassay platform
