2011 Annual Report
1a.Objectives (from AD-416)
Objective 1: Develop transformation expression vectors to target transgene expression specifically to tissues that are initially infected by Fusarium graminearum.
Objective 2: Develop antifungal candidate genes and gene constructs that can be used in targeted expression system to develop Fusarium-resistant barley.
Objective 3: Identify components of the GA response that can be used as predictors of malting.
1b.Approach (from AD-416)
Produce gene macroarrays from our lemma-specific gene library and a new epicarpspecific library. Probe libraries with cDNA from Fusarium-infected lemma and epicarp. Clone and identify the upregulated genes, and confirm tissue-specificity with RNA blots. A modified inverse PCR will be used to clone their promoters from barley. Promoter (upstream) regions will be ligated upstream of the green fluorescent protein gene in an expression vector and functionally confirmed in transient bombardment assays where tissues will be examined for fluorescence before and after infection with Fusarium. If successful, barley will be stably transformed with antifungal protein genes driven by these promoters using the Agrobacterium vector pRSHyg. These genes (cloned in this lab) include lemma thionin, Ltp, and germin. Transformants will be tested for Fusarium resistance. The gene for the barley gibberellin (GA) hormone receptor will be cloned using homologies to the rice receptor. The gene will be compared in GA response mutants. Receptor sequence and mRNA levels will be analyzed in barleys of varying malting qualities. The Barley1 GeneChip will be used to examine transcripts in 7 malting barleys and GA response mutants. The differences in transcript profiles will provide insights into the relationship of the GA signal transduction pathway and malting quality.
Briggs' studies in the 1960s determined that an “embryo factor” released from the germinating barley embryo at a specific time interval was correlated with subsequent alpha-amylase enzyme production in the aleurone. The “embryo” in those studies consisted of the scutellum + root/shoot axis. We recently determined.
1)that the axis alone releases the embryo factor and that.
2)this factor is released between 24 and 48 hours from the start of seed imbibition. This factor is likely to be a gibberellin (GA) hormone produced in the first 3 days of germination (companion project – Specific Cooperative Agreement with a GA expert at the University of Calgary, Canada). Initial analyses of seedling organs show that the aleurone already has enough GA1 to turn on alpha-amylase genes at 8 hours. All dissected seedling organs (aleurone, sub-aleurone, scutellum and root/shoot axis) have been collected and sent to cooperator. GA turns on genes for the production of enzymes that are essential to malting. “Della” proteins inhibit the action of GA. We found a single base sequence change in the Della protein gene of Steptoe, a feed barley with no malting quality. The change does not occur in Morex, a high malting quality barley. The same change occurs in rice and causes dwarfing because it occurs at a critical position involved in the binding of Della to the GA receptor. Steptoe appears to have two Della genes, the normal and the variant, even though barley is thought to have only one Della gene. We are exploring the possibility that the normal gene controls vegetative growth, while the second (variant) gene controls GA response in the aleurone and, thus, may influence malting quality. Studies related to transformation of barley for Fusarium resistance are being completed. Transformants segregating for the presence or absence of the introduced thionin gene will be used in a final resistance test. All samples were sent to a research lab at the University of California, Davis for a collaborative study on profiling metabolite changes during infection by Fusarium.
How does a seed turn on its genes? Germinating barley seeds produce a great variety of enzymes that break down starches, proteins and other stored molecules in order to nourish the growing seedling. After decades of research, we still don’t know how this complex process is regulated. ARS researchers at Madison, Wisconsin, have revisited old studies from other labs that pointed to the “embryo” as the source of a signal that dictates the types and amounts of enzymes that will be produced. The embryos in the previous studies actually consisted of the minute root and shoot – both embedded in the scutellum. We recently determined that the signal comes from the root/shoot axis alone. This signal (probably a gibberellin hormone) leaves the root/shoot by 1 ½ days from the time the seed is immersed in water. When this factor is identified, we can compare its levels in good versus poor malting barleys. It is important to know what distinguishes seeds with strong germination from those that are weaker. This will lead to stronger stands of cereal crops. Also, this is critical for determining which factors distinguish potentially superior malting barleys.