This proposal is a continuation of research that focuses on soybean plants with increased root length and branching to boost yield and resiliency of yield in Tennessee. A strong root system is necessary for maximized plant nutrition, defense, and water use. Drought tolerance is impossible, for instance, without a robust root system, which is especially important in soybean during pod fill. Soybean plants with increased root length and branching would significantly improve water/nutrient uptake efficiency and yield potential in diverse environments. Such improvement is the primary goal of our project. Improving roots, improves crop yield.

Our previous work (TSPB 2018 progress report) identified a number of soybean genes potentially associated with root growth/development as well as promoting tolerance to drought and nutrient deficiency. We successfully overexpresses these “root genes” in soybean plants with transgenic roots and are in the process of assessing their impact on plant growth and seed production by evaluating the biomass weight, flowering time, pod setting, and seed count and weight. Here we propose to continue our research by further discovery and in-depth characterization of these identified soybean genes involved in root growth and stress tolerance to challenging environmental conditions. Discovery and in-depth study of these candidate genes would lead to a roadmap in identifying soybean genes for breeding and developing high yielding soybean varieties.

Additionally, we aim to focus on improvement of drought tolerance, which is a major abiotic stress limiting soybean growth and development. To relieve soybean from drought stress, the plant has to undergo several changes in its developmental strategies. One such strategy is an association with beneficial growth promoting microorganisms such as arbuscular mycorrhizal fungi (AMF). AMF symbiosis protects plants from drought stress by playing a key role in the water-transport process. Furthermore, it increases soil moisture retention capacity. AMF also provides its host plant with immobile micronutrients that are essential for growth and production. This supplement of nutrients significantly helps plants to better survive and increase yields in soils with limiting nutrients. AMF can also protect plants from the soil-borne pathogen.

Soybean yield greatly depends on root system architecture and nitrogen fixation. Several plants genes that are responsible for AMF symbiosis have been identified. Some of these genes are also essential for root nodule symbiosis. Root nodule symbiosis accounts for nitrogen fixation that enhances mineral nutrition of the plant. Critical symbiotic genes initiate symbiosis as well as maintain it. One such example is the aquaporin gene family, which plays roles in both AMF symbiosis and drought stress. They function as water-channel proteins stopping the water flow from the plants to soil under water stress conditions.

Objective 1: In-depth characterization of the identified soybean “root genes” and further discovery of genes involved in root growth and promoting tolerance to drought and nutrient deficiency.
We will continue to characterize the soybean plants with engineered roots for root length, lateral root branching, root surface area as well as their impact on drought tolerance following water stress treatment. We will further analyze RNA sequencing gene expression data for which the entire expression profile of the soybean root genome and stress tolerance as well as identifying the soybean genes that are essential for AMF and root nodule symbiosis to alleviate drought stress and nitrogen deficiency.

Objective 2: isolation of identified genes in Objective 1 from soybean and analyzing their efforts using the soybean hairy root method.
This is an on-going task for our candidate soybean genes. The coding sequences of novel key root genes for root architecture and stress tolerance are cloned and mobilized into transformation vectors for overexpression or knockdown of expression, based on our predicted results. For each gene, a hypothesis is developed about the phenotypic effects of producing more or less of the targeted gene product in soybean roots, with special emphasis on water stress response. We will transfer genes into soybean roots using our well-established composite plant system comprised of transgenic hairy roots grafted to wild type shoots for whole plant functional analysis.

Objective 3: Evaluation of transgenic soybean hairy roots for conferring enhanced root growth and tolerance to drought and nutrient deficiency.
We will assess soybean plants with transgenic roots for the most important root traits for promoting tolerance to drought following water stress treatment as described above. For AMF, soybean plants with transgenic roots are grown in soil/vermiculite containing Rhizophagus irregularis spores. The impact of genes on AMF colonization in plants will be calculated by counting the number of mycorrhizal hyphae compared with the control plants. To test the impact of AMF colonization on plants survival under drought condition, plants after four weeks of AMF colonization will be subjected to water stress for 15 days, and then the relative water content (RWC) of the plants and soil will be measured and compared with control. We will aslo quantify the nutrient levels of the soybean plants using inductively coupled plasma mass spectrometry (ICP-MS). Nutrient levels will be subjected to correlation analysis with frequency of AMF colonization percentage to elucidate the effect of the gene-of-interest on nutrient uptake. Ultimately, we will investigate the impact of the gene-of-interest on soybean production by measuring the number of pods per plants, number of seeds per pod, and assessment of seed quantity.

We expect that the discovery of the novel root-important genes identified in our research will lead to the production of soybean with deeper and more extensive root systems. In turn, these plants will have improved performance and yield, particularly under conditions of limited water and nutrient availability. These outcomes may be accomplished with or without being regulated as a GMOS, depending on the gene and its effect.