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.

Updated April 29, 2025:

Progress Report: First 6 Months
Project Title: Investigating the role of stigmasterol in soybean growth and development
Project Overview and Milestones:
The objective of this research is to modify stigmasterol levels in soybean to develop genotypes with improved nutritional profiles and enhanced tolerance to abiotic stresses. Successful completion of this work will result in the identification of gene networks and biological processes involved in stigmasterol biosynthesis, as well as traits such as flowering and seed development. Because stigmasterol accumulation is known to be triggered by environmental stress, it may also serve as a metabolic marker to aid in the selection of stress-resilient breeding materials.
This report outlines the progress made during the first six months of the project, with specific focus on Performance Measures 1 and 2.
Performance Measures:
1. Evaluate stigmasterol profiles in vegetative and reproductive tissues of the soybean cultivar Williams 82.
2. Characterize how stigmasterol levels change during development and under abiotic stress, such as drought.
Progress and Deliverables (First 6 Months):
1. Personnel
A postdoctoral scientist was successfully hired to lead and conduct the proposed research activities.
2. Experimental Progress
o Stigmasterol levels were measured in various soybean tissues at different developmental stages. Initial sampling was conducted at the V3 stage, and sterol profiles were analyzed.
o Gene expression data were obtained for candidate genes potentially involved in the enzymatic conversion of sitosterol to stigmasterol. These data have informed the cloning strategy.
o Several candidate genes have been successfully isolated. These will be analyzed further in Arabidopsis and will inform the development of gene silencing vectors to investigate their function in soybean growth and development.
3. Communicating science
o The postdoctoral researcher presented a poster entitled “Identification and Characterization of Genes Involved in Sitosterol to Stigmasterol Conversion in Soybean” at the 12th Annual Raymond F. Baker Plant Breeding Symposium, held at the ISU Alumni Center in Ames, IA, on March 14, 2025.

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.