Rhizobium Tropici-Produced Biopolymer—Analyzing Its Impact on the Phenotype and Genotype of Arabidopsis Thaliana

Rhizobium Tropici is known to form symbiotic relationships with leguminous plants to promote overall plant growth. It is hypothesized that the active component is the EPS (extracellular polymeric substance) secreted by the bacteria. We extracted the EPS directly by precipitation (EPM) and studied its effects on plant growth. Arabidopsis Thaliana (At) was selected because of its well-characterized genomic background. As its entire genome has been sequenced, it has commonly been used as a model organism to analyze molecular mechanisms. This study aims to investigate how EPM promotes growth in At plants by analyzing both the phenotypic and genotypic expression.

At plants were grown for 4 weeks in soil under varying concentrations of EPM (control, 12.5 mg/kg, and 125 mg/kg). The samples were then weighed to determine the root mass and total mass. Compared to the control, the samples treated with 12.5 and 125 mg/kg EPM had a greater root mass and total mass, indicating that EPM promotes both root and overall plant development. To determine its exact mechanism, the plants were then grown in agar to eliminate the influence of external factors. The percentage of sprouts per dish of agar was recorded each day, and samples treated with EPM yielded higher germination rates.

Genetic analysis was performed to determine how EPM influences the genetic expression of At samples. After At plants were germinated in soil, they were transferred into separate pots with EPM-treated sand. Every 3-4 days, under sterile conditions, some of the plants would be removed from the soil, have their roots separated from upper vegetative growth, washed with water, quickly immersed into liquid nitrogen to prevent degradation of RNA by ribonucleases, and then stored in -80°C. Then, RNA extraction and qRT-PCR (quantitative reverse transcription polymerase chain reaction) were performed. Compared to the control, samples treated with 125 mg/kg EPM had a higher expression of the genes WOX2 and WOX8. These genes are initially coexpressed in the zygote and act as complementary cell fate regulators. WOX2 influences the apical lineage and promotes the formation of the shoot system, while WOX8 influences the basal lineage, which promotes root development and root apical meristem (RAM) activity.

Another series of studies was conducted with At in agar mediums, some plates infused with EPM (5%, 10%, and 20%), others with H2O for control. The samples were grown in agar, which provides necessary nutrients for growth, eliminates the complex factors involved with soil. and is easier for microscopic imaging. Comparing the optical images, we observed a dramatic increase in lateral root growth for the 10% samples immediately at the air-agar interface. At this interface, conditions are less ideal for growth, as there is simply an accumulation of vapor. Further RNA extraction and qRT-PCR analysis are currently being conducted for these samples and the results will be reported as a function of time in culture and EPM concentration.

In conclusion, this study suggests that EPM promotes phenotypic growth in At plants as a result of not just growth medium rheology but also influences gene expression at the molecular level. The increased levels of WOX2 and WOX8 demonstrates EPM’s genetic mechanism to enhance both shoot and root development. In particular, the upregulation of WOX8 directly aligns with the observed increase in root mass for EPM treated samples, highlighting its role in the development and maintenance of the root system. The increase in lateral root density for EPM-treated samples in agar may be part of the mechanism that allows the plant to grow in non-ideal conditions. This will be studied further to determine whether this is a common phenomenon in other plants such as Bermuda Grass.

The authors would like to thank the US Army Corps of Engineers (ERDC) and the Louis Morin Charitable Trust for their support (W912HZ-20-2-0054) in this research.