Sterols are precursors to a vast array of signaling molecules such as steroid hormones in mammals and brassinosteroids (BR) in plants. Plants synthesize several sterols including campesterol, sitosterol, stigmasterol, and cholesterol (Schaller, 2003). Research in Arabidopsis has provided evidence that sterols are essential in plant growth and development. Campesterol is the direct precursor of BR, and several Arabidopsis sterol mutants show a dwarfed phenotype associated with BR deficiency. However, other sterol mutants show vascular patterning and embryonic defects that are not always rescued by exogenous application of BR. Further, there is striking variation in the levels of stigmasterol across different plant tissues, suggesting a specialized role in plant cell physiology (Suza and Chappell, 2016; Aboobucker and Suza, 2019). Taken together, these findings suggest that sterols have a role in plant development independent of BR. The conversion of sitosterol to stigmasterol during conditions of stress suggests that stigmasterol might modulate plasma membrane fluidity or signaling activities essential for plant growth and stress compensation.

The goal is to understand the role of stigmasterol in soybean growth and development. To identify the genes involved in stigmasterol biosynthesis in soybean we plan to
1. Measure stigmasterol content in soybean to understand how its profile changes during development and conditions of stress such as drought and salt.
2. Quantify the mRNA expression levels of candidate genes and correlate their expression with stigmasterol content in various tissues.
3. Validate candidate genes associated with the synthesis of stigmasterol by reducing their expression.
4. Evaluate VIGS plants with reduced stigmasterol for their growth and development including response to stress.

Modifying stigmasterol levels in soybeans might lead to genotypes with healthier sterols and enhanced tolerance to abiotic stress. Since stigmasterol accumulates in response to various stress factors it might serve as a metabolic marker for selecting breeding materials that are more resilient to stress.

Delivered 6 months after funding:
1. Information about stigmasterol levels in different soybean tissues throughout development.
2. Research poster to the graduate plant breeding symposium at ISU.

Delivered 12 months after funding:
1. Evaluate the expression of stigmasterol biosynthesis genes in plants treated with gene silencing.
2. Gene silencing constructs that cause changes in stigmasterol will be selected for further analysis by RT-PCR.
3. The profile of stigmasterol in soybean plants with reduced expression of stigmasterol biosynthesis genes.
4. The results will provide the first evidence of genes controlling stigmasterol in soybeans.

In the longer-term, the research will identify soybean varieties with improved stigmasterol content that are better adapted to stress conditions. Linking the proposed research with the George Washington Carver Future Hunger Fighters High School Outreach Program offers Iowa’s youth the chance to experience molecular biology research focused on a globally significant crop.