Drought and heat stresses are complex and polygenic, which makes it challenging to breed crops for increased tolerance using conventional breeding methods. Alternatively, genetic engineering with stress-responsive genes (e.g. AtGRXS17) can be used as a promising tool to enhance tolerance to a range of complex abiotic stresses (Wu et al., 2017). Hence, the proposed study using the growth chambers and unique field-based tents is a novel attempt in this direction. Following this approach can provide clues to improve resilience to harsh environmental conditions particularly during the sensitive reproductive and pod-maturing phase.
Glutaredoxins (GRXs) are small ubiquitous oxidoreductase stress responsive genes, involved in floral development signaling and known to enhance tolerance to abiotic stress during reproductive development, by detoxifying the reactive oxygen species (ROS). Proof of concept, ectopic expression of Arabidopsis glutaredoxin gene (AtGRXS17) in maize substantially increased kernel-set and yield under heat stress (37°C daytime) both in greenhouse and field conditions. Similarly, Dr. Park and his team have engineered the cultivar ‘Thorne’, [maturity group 3, developed in the 90’s from Illinois], aimed at inducing tolerance to drought and heat stress at reproductive and pod-filling stages by overexpressing the same AtGRXS17 in soybean. Three AtGRXS17-overexpressing soybean lines showed significantly higher seed-set and seed-weight under heat stress in greenhouse conditions, compared to wild-type. Hence, the major objective of the proposed project is to characterize the effectiveness of AtGRXS17-expressing soybean transformants on seed numbers and weight and seed quality under drought, heat and combined heat and drought stress using field-based heat tents and controlled-environment facilities.
Year 1: Activity 1 – Quantifying genetically-engineered soybean response to heat stress during flowering and pod-filling stages. Three stable soybean transformants carrying the AtGRXS17 gene in inbred ‘Thorne’ background and the wild-type (Fig. 1d-f) will be exposed to a range of high day-time temperatures (35/20oC, 38/20oC and 40/20oC, from start of flowering [R1] to physiological maturity [R8]) and a set of plants under control temperature (30/20oC, day/night), using large walk-in growth chambers. Chambers conditions (air temperature and relative humidity) will be monitored at 15-min intervals by installing HOBO data loggers. Flowers opening on the 2nd and 5th day after stress initiation will be sampled from all four treatments (30oC, 35oC, 38oC and 40oC) to study in vitro pollen germination and pollen tube growth. A week and 14 days after stress initiation, chlorophyll fluorescence will be measured, and leaf samples will be collected to measure membrane damage using TBARS approach and for molecular analysis including gene expression (qRT-PCR). At least 10 replicate plants will be maintained for each of the four lines (including wild-type) at each temperature, with four plants used for physiological and molecular assessment and six plants for recording agronomic parameters. Pod numbers, seed number and seed weight per plant, and quality parameters including protein, oil and oleic acid (using NIRS platform in Dr. Jagadish lab) will be recorded.
Activity 2 - Evaluate the effectiveness of transgenic soybean lines to drought stress using controlled-environment facility.
The same three transgenic soybean lines and the wild-type will be grown in the lysimeter columns to quantify the impact of drought stress covering post-flowering [R3] and pod-filling stages [R8]. For each line, twenty columns (each column [100 cm tall with 25 cm diameter] filled with 40 kg of clay loam soil) will be maintained from sowing to R3 under non-stress condition using an automated drip irrigation system. A few days prior just prior to R3 stage, ten plants will be subjected to drought treatment until physiological maturity (R8). Drought stress will be imposed following the gravimetric approach by stopping the irrigation until soil moisture content reaches the target field capacity (45 to 50% field capacity) and columns will be maintained at the target stress level until harvesting. During the treatment period, control columns will be kept at 100% field capacity (FC), while drought stress columns will be progressively exposed to 45 to 50% FC. Water loss by transpiration and moisture reaching below the target stress level will be replenished by adding back a calculated amount of water (twice a day four times per day at 0600 and 1600 h) to maintain the stress levels of 45 to 50% FC, until physiological maturity. The column surface will be covered with a circular polythene sheet to control for evaporative water loss. A slit opening in the sheet will be created to prevent heat buildup. All physiological and molecular assessments and agronomic measurements will be recorded similar to Activity 1.
Year 2: Activity 1- Validate the effectiveness of genetically engineered soybean in response to heat stress during flowering and pod-filling stages. The heat-tents (18 ft. wide x 24 ft. long x 10 ft. high at apex) are built using a galvanized steel framework and covered with a transparent polyethylene film (>90% transmittance). The tents have a movable flap (0.6 m) on the top. The thermostat will be automated to induce a maximum temperature of 38oC, and when temperatures exceed the maximum set temperature inside the heat-tents, the overhead flaps will be programmed to open to avoid excess heating. Tents are heated through latent heat generated by sunshine during the day and are documented to have the same night temperature as outside (Bergkamp et al., 2017). Temperature and relative humidity inside the tents will be monitored every 15-min using WatchDog data loggers, placed at 10cm above the crop canopy in all the tents. Similarly, three replicate sensors placed on the control plots will measure control conditions. Each transgenic line will be sown in at least six rows of eight-foot in length in each tent, with three replicate tents used for stress imposition. A similar number of replicate blocks will be maintained as true controls outside the tents. The tents will be placed on the crop at flowering [R1] and retained on the plants until physiological maturity [R8]. Functional relevance of transgene at the physiological (pollen germination and pollen tube growth, photosynthetic rate, photochemical efficiency of PSII, chlorophyll index and canopy temperature), molecular (gene expression), biochemical (membrane damage and other reactive oxygen scavengers including Catalase, SuperOxide Dismutase etc.) will be quantified after a week and 14 days after stress imposition. At maturity, all agronomic parameters including yield will be recorded on four six-foot-long rows per replicate in both stress and control plots. Seeds obtained will be used to determine the impact of heat stress on protein, oil percent and oleic content using NIRS platform, developed from support by Kansas Soybean Commission.
Activity 2- Can the engineered soybean induce combined heat and drought tolerance pod-filling stage? This hypothesis will be systematically tested by using the same set of three transgenic soybean lines and its wild-type by imposing combined heat and drought stress covering R3 and R8 stages. Replicated plants from each line will be grown (in 15 L pots) from sowing to R3 at a temperature of 30°C/20°C (day/night) and 14 h photoperiod. The plants will be divided into two groups a few days before to R3 stage: one group will be exposed to a range of high day-time temperatures (30oC, 35oC, 38oC, and 40oC) without drought stress and another group of plants will be exposed to drought stress (45 to 50% field capacity) in combination with the same set of temperatures mentioned above (combined heat and drought) using the four large walk-in growth chambers. Chambers conditions (air temperature and relative humidity) will be monitored at 15-min intervals by installing HOBO data loggers. Drought stress will be imposed following gravimetric method similar to Obj1, Activity 2. The same set of observations (Obj2, activity 1), including physiological, molecular, biochemical, agronomic and quality parameters, will be recorded in response to heat and combined heat and drought stress, imposed from post-flowering stage (R3) to physiological maturity (R8).
References
Bergkamp B., et al., 2018. Field Crops Research, 222:143-152.
Siebers, M.H., et al., 2015. Global Change Biology, 21:3114-3125.
Wu, Q., et al., 2017. Biochemical and Biophysical Research Communications, 491:1034-1039.
Zipper, S.C., et al., 2016. Environmental Research Letters, 11: 094021.