Team:Tacoma RAINmakers/Description

Team:ECUST/Lab/Notebook

Project Description

Inspiration:

The original pitch for our project was to take rhizobia’s nitrogen-fixing relationship with legumes and translate it to other common crops to reduce use of ecologically harmful nitrogen-based fertilizers. We found this to be a useful project due to the expansion in the field of mass agriculture that is relying on chemical fertilizers for crop efficiency and causing negative downstream effects such as dead zones in aquatic environments [1]. With further research, we learned that the use of rhizobia in agriculture comes with significant setbacks such as desiccation. Additionally, after reading and examining recent papers, we learned about the importance of legumes in developing countries and how we can improve the efficiency of the rhizobia-legume relationship. This initial research gave us a promising direction to understand how to improve the already existing applications of rhizobia and expand their application.

The Importance of Legumes and Rhizobia:

Any increase in human population translates to a greater demand for food as well as space. In many countries, legumes are a popular food choice but, like most crops, they are limited by soil conditions. However, rhizobia can increase the efficiency of legumes, which allows legumes to produce more food on the same amount of land. Additionally, nutrient-stripped land can be restored by pulse crops (such as beans, chickpeas, lentils, and peas) which can return nutrients to the soil. Our research hopes to improve rhizobial relationships and thus, increase to crops’ resistance to negative environmental conditions and simultaneously nurture the soil. Furthermore, if legume and rhizobia’s symbiotic relationship is improved, then the need for chemical fertilizers, to improve crop growth, will be greatly reduced. Unfortunately, chemical fertilizers are often overused in the hopes of improving crop growth but, only so much can be absorbed by plants and the remainder contaminates waterways. Consequently, waterways contaminated with an excess of nutrients result in mass algal growth and die-offs in bays, lakes, and coastal waters and eventually become known as dead zones. With a reduced reliance on chemical fertilizers, these harmful occurrences would be reduced and current dead zones may have a better chance of recovering..

Current Problems with Rhizobia in Agriculture:

Inoculating leguminous crops with highly efficient strains of rhizobia (elite strains) has strong positive impacts both ecologically and economically. However, under agricultural conditions, legumes inoculated with elite strains are frequently nodulated by inefficient strains of rhizobia native to the soil rather than the elite strain. The low success rates of rhizobial inoculation with elite strains is caused largely by the death of rhizobia following seed coating and present low economic incentive. Desiccation is a major contributing factor to these low survival rates. Further, rhizobia’s nitrogen-fixing benefits currently can only be applied to legume crops though research could possibly reveal additional strains presenting relationships with other crop plants.

Our Solution:

The secret to unlocking the potential of rhizobia may lie in the disaccharide trehalose. Prior research suggests a strong correlation between this sugar and anhydrobiotic organisms, organisms that can survive long dry periods and rehydrate afterward. Trehalose is an osmoprotectant that has the potential to give rhizobia a greater resistance to desiccation. Bean plants inoculated with rhizobia that overexpress otsA, a gene involved in trehalose biosynthesis, have increased levels of nitrogen fixation, higher crop yields, and raised tolerance to environmental stresses such as drought [2]. Our project will verify these findings by inoculating Phaseolus vulgaris (common bean) with R. etli overexpressing OtsA (Ox). We will test its effect on biomass, yield, and a range of environmental stressors. We will also introduce the otsA overexpression plasmid into other rhizobia strains and see if it has the same effect. In addition, we need to understand what environmental stressors the rhizobia will be tolerant against so that we can match it to challenging soils around the world. With investigation of multiple rhizobia strains and different types of legumes or crops, we can identify symbiotic relationships so that a database can be created that matches a desired crop with an optimized rhizobia.The different strains then can be used to compensate for specific soil conditions and will allow farmers to pick exactly what they need.

How it works:

otsA encodes an enzyme called trehalose-6-phosphate synthase (TPS), which catalyzes a reaction between UDP-glucose to glucose-6-phosphate that results in trehalose-6-phosphate and UDP. This compound then goes through dephosphorylation by trehalose-6-phosphate phosphatase (TPP), which leaves only trehalose.

Overexpression of the otsA gene has been shown to produce higher levels of TPS [2]. To create the overexpression strain, we will order the OtsA gene from R. etli CFN 42, subclone into the broad-host-range vector pBBR1MCS-2, and then transform into R. etli CFN 42.

It is unclear why otsA gene overexpression makes rhizobia more tolerant of drought-like conditions. When certain bacteria, like rhizobia, are subjected to osmotic pressure, trehalose tends to build up. Trehalose appears to be an osmoprotectant (small molecules that act as osmolytes), so when cells swell due to osmotic pressure, membrane channels allow osmolytes carrying water to exit the cell and thus, return the cell to a normal volume.

However, the increase in trehalose production is not significant enough to indicate that trehalose is acting as an osmoprotectant in the rhizobia. The predominant theory is that trehalose is acting as a signal molecule, triggering the plant to, for example, accumulate water in anticipation of a drought.

References

1 - US EPA. The Effects: Dead Zones and Harmful Algal Blooms. https://www.epa.gov/nutrientpollution/effects-dead-zones-and-harmful-algal-blooms. Accessed October 10, 2019.

2 - Suarez R, Wong A, Ramirez M, et al. Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia. Mol Plant Microbe Interact. 2008;21(7):958-966. doi:10.1094/MPMI-21-7-0958.