Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Plant Sciences and Plant Pathology
Non Technical Summary
Wheat and barley are important Montana crops, and are of considerable importance worldwide. Anthesis date and the following grain filling period largely determine crop yield and quality. Duration of the grain filling period is limited by plant senescence, the process by which plants turn yellow and die during late summer. Senescence leads to massive remobilization of nutrients, particularly of nitrogen, from senescing parental plants to the developing grains. Senescence timing therefore influences key agronomic traits including nitrogen use efficiency and grain protein concentration.The first part of this project will analyze two genes, with one influencing anthesis date and the other the duration of the senescence process, in improving malt barley agronomic performance. Results so far indicate that barley lines combining early anthesis with delayed senescence (long grain fill period) have lower grain protein and produce a higher percentage of plump kernels; however, no yield increase has been observed. Experiments planned for the next project period will characterize the function and interaction of the identified genes in the control of grain filling; additionally, they will identify factors which limit yield in genotypes with longer grain fill periods.A second part of this project will characterize the first steps of the process allowing nitrogen remobilization from senescing leaves to developing kernels. Knowledge and germplasm developed from this effort may allow improvement of nitrogen use efficiency, and will also allow improved control of grain protein concentration.Besides senescence, nitrogen remobilization also occurs during seed germination. The barley malting process essentially involves grain germination under standardized conditions, with nitrogen remobilization controlling important malt quality factors. A third part of this project will therefore characterize the first steps of this process, allowing for a functional comparison between senescence- and germination-associated nitrogen remobilization. Gained knowledge will be used in malt barley breeding, and may allow better control of the malting process.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Objective 1: Characterization of the effects and interactions of barley glycine-rich RNA-binding proteins and NAC transcription factors in the control of flowering, leaf senescence, and stress resistance. The influence of these genes/proteins on plant development and grain yield/quality will be analyzed using both lab methods and field experiments.Objective 2: Identification and characterization of barley papain-like cysteine proteases involved in bulk protein degradation and nitrogen recycling during leaf senescence. This objective will improve our understanding of a fundamental process which controls crop nitrogen use efficiency and grain protein concentration.Objective 3: Identification and characterization of barley papain-like cysteine proteases involved in storage protein degradation during seed germination and malting. Results from this objective will enhance our control over important malt quality parameters, particularly soluble proteins and free amino nitrogen (FAN).
Project Methods
Objective 1: Characterization of the effects and interactions of barley glycine-rich RNA-binding proteins and NAC transcription factors in the control of flowering, leaf senescence, and stress resistance1.1 Allelic distribution and variation analysis of the HvGR-RBP1 gene in the USDA barley core collectionAt least 500 additional accessions from the USDA barley core collectionwill be probed for promoter insertions. These experiments will a) produce robust data on the frequency of potentially non-functional alleles in accessions with different geographic origins, and b) provide additional information on variation in the length of inserts. We will sequence a number of inserts with different lengths to understand their type (e.g., transposons), and probe the effect of different inserts on leaf gene expression.1.2 Characterization of the barley glycine-rich RNA-binding protein familyHvGR-RBP1 belongs to a subgroup of the large family of plant glycine-rich proteins, designated as subfamily IVa. Based on preliminary BLAST searches, only two other barley proteins have the same domain structure. Using methods outlined in recent publications characterizing cereal gene families, we will first query the reference genome for any additional GR-RBPs overlooked in preliminary analyses. As HvGR-RBP1 expression has so far only been analyzed in leaves, available gene expression data for all identified genes including HvGR-RBP1 will be extracted from public gene expression databases. These experiments will identify any additional barley GR-RBP genes induced during the developmental stages and environmental conditions of interest to this project. Such genes will be probed for allelic variation (sequencing, gene expression), to identify germplasm of interest in the improvement of malt barley quality and stress tolerance.1.3 Comparison of barley lines with different HvNAM-1 and HvGR-RBP1 allele combinationsBarley lines with the 'Lewis' (functional) allele of HvGR-RB1 and the late-senescence (non-functional) 'Karl' HvNAM-1 allele perform best (longer grain fill duration, associated with increased percentage of plump kernels and higher test weight). A detailed characterization of the underlying molecular and physiological traits will shed new light on these important quality parameters, and will indicate why longer grain fill duration did not lead to increased yield.For these analyses, new crosses will be made with the aim to obtain all possible allele combinations in the var. 'Hockett' malt barley background. Comparison of these lines will allow us to separate HvNAM-1 from HvGR-RBP1 effects, leading to a better functional understanding than experiments previously performed by our lab. New germplasm will be compared/analyzed using a combination of physiological and molecular approaches.1.4 Reverse genetic screens/TILLINGFor in-depth analysis of all genes investigated in this project, identification of additional mutations varying in severity will be necessary. To achieve this goal, we will utilize a recently developed resource, a highly mutagenized TILLING population in malt barley variety 'Golden Promise,' which is available from the James Hutton Institute (Dundee, UK). Methods for screening are fully described in the attached project document.Objective 2: Identification and characterization of barley papain-like cysteine proteases involved in bulk protein degradation and nitrogen recycling during leaf senescence2.1 Family C1A protease identification by activity-based protein profilingHvPap-6 has been shown to be the most prominent (active) family C1A protease in barley leaves at 8 days after girdling, using activity-based protein profiling. The same methods (activity tagging followed by mass spectrometric analysis) will be applied to senescing flag leaves at 7, 14 and 21 days past anthesis, to complement our transcriptomic analysis of developmental senescence.2.2 Biochemical characterization of identified proteasesThe most active proteases identified by activity-based protein profiling will be expressed in E. coli. If E. coli expression is unsuccessful, we will also attempt expression in the yeast Pichia pastoris. Activities of purified, active enzymes will be characterized using both artificial (fluorescent) and protein substrates isolated from barley leaves. Enzyme activities against plant protein substrates will be tested by incubation with 1) crude extracts obtained from mature green leaves; 2) lysates of isolated barley chloroplast, and 3) purified barley Rubisco. Protein degradation will be quantified using SDS-PAGE and/or immunoblotting, using commercially available antibodies against important plastidial proteins (agrisera.com). 2.3 Transient protease expression in barley protoplastsTo complement in vitro protease assays, enzyme function will also be tested by transient overexpression in isolated barley protoplasts.Methods are fully described in the attached project document.2.4 Protease knockdown/knockoutBarley lines carrying mutations in proteases identified as active during developmental leaf senescence and with demonstrated activity against plastidial proteins will be identified from a TILLING population, using the methods outlined in section 1.4.Objective 3: Identification and characterization of barley papain-like cysteine proteases involved in storage protein degradation during seed germination and malting3.1 Family C1A protease identification by transcriptomic analysis and activity-based protein profilingUsing public gene expression databases, we will identify all family C1A genes induced at the transcript level during grain germination or dedicated malting protocols. For activity-level protease detection, a widely used two-row malting variety ('AC Metcalfe') will be steeped, malted and kilned using the MSU Malt Quality Laboratory's standard protocol. Samples will also be collected after milling and mashing, with samples separately obtained after the 'protease step' (45°C), and after extraction at higher temperatures up to 70°C, to investigate up to which temperatures analyzed proteases are catalytically active. Proteases from the different steps will be visualized and identified by activity-based profiling.3.2 Biochemical characterization of identified proteasesProteases identified as active during different stages of the malting process will be subjected to biochemical characterization, using the protocols outlined in objective 2. Activities of affinity-purified enzymes will also be tested against barley hordeins extracted from 'AC Metcalfe' seeds.3.3 Protease knockdown/knockoutAs for senescence-associated proteases, barley lines carrying mutations in proteases identified as active during germination/malting and with demonstrated activity against hordeins will be identified from a TILLING population, using the methods outlined under objective 1.