Summary of the problem: Soybean yields are limited by the environment in Minnesota. One of the most
important environmental factors that consistently limits soybean yields in the region is atmospheric
drought, or evaporative demand (Zhou et al., 2020; Kimm et al., 2020). Increasing atmospheric drought
means that the desiccation power of the air increases, and soybean plants, particularly, experience this
air drying as a stress (López et al., 2021). During the soybean growing season in Minnesota, atmospheric
drying typically increases more than 600% within the day, from 0.5 to approx. 3 kPa.

This sensitivity of soybean to atmospheric drought is apparent in large yield trials conducted across the
US. For instance, variation in atmospheric drought in the first 2-3 months of the growing season was
found to be the single most important environmental driver of yields based on soybean yield trials
conducted in 27 U.S. states between 2007 and 2016. In fact, in that analysis, the impact of atmospheric
drought was even stronger than precipitation itself (Mourtzinis et al., 2019). While this negative effect
of atmospheric drought on soybean yields is real and quantifiable, a major problem for soybean
breeders is that it is not easily detectable. This is because that this ‘stealthy’ stress does not necessarily
result in visual symptoms such as wilting or rolling, but causes yield losses. This is even more
problematic for Minnesota, where atmospheric drought limits yields while the soil is well-watered
(Sinclair et al., 2010). In fact, until we have developed our methodology (see below), this effect can only
be measured based on costly, time-consuming, multiple-location, multi-year yield trials as done in the
study of Mourtzinis et al. (2019) mentioned above. Thus, breeders cannot develop a breeding program
to boost soybean yields in response to atmospheric drought, unless a screening method for tolerance to
atmospheric drying is developed. Fortunately, our lab just developed the technology to do so, which we
have recently published in an international, peer-reviewed journal (Monnens et al., 2023).

Summary of the solution: To address this problem, our lab has developed a technology that enables
quantifying the level of stress caused by atmospheric drought in soybean, without the need for such
expensive, long-term and large-scale yield trials. This system has been successfully tested in the field,
and generated yielded results that are consistent with controlled environment experiments. This
means that soybean breeders could use our new approach to screen their genetic material, and
intentionally breed for more profitable soybeans which yields are less limited by atmospheric drought.
Thus, our proposal directly addresses growers needs for better-yielding crops and the RFP
requirement for research to target soybean production profitability.

1. Goal#1. Identify superior genotypes to be used as donor parents in the U of M soybean breeding
program and identify superior commercial varieties to be recommended to farmers.

2. Goal#2. Predict state-wide yield gains and estimate profitability for Minnesota farmers
resulting from introducing such trait in farmers’ fields.

As requested by the RFP, we have structured our proposal in such a way that the three objectives
can be conducted and completed within a 12-month period. Thus, the screening experiments will have
to be conducted and replicated a number of times under controlled but naturally-fluctuating
environments to maximize chances of success and return on investment.

At the end of this year, we expect that the breeding program to have 1) identified promising
parental lines and commercial cultivars with superior nitrogen fixation tolerance to atmospheric drought 2)and
provided a quantitative estimation of profitability (yield gains) associated with this trait for
Minnesota farmers depending on where their farms are located.

D1. A mature, non-destructive and cost-saving methodology to help the U of M soybean breeding
program conduct routine screening for nitrogen fixation performance in soybean. This methodology
could be also used to screen for nitrogen fixation response to other stresses.
D2. A selection of soybean breeding lines with superior nitrogen fixation response to atmospheric
drought. These lines will be used as donor parents in the breeding program to confer higher yield
potential.
D3. A selection of commercial, elite soybean varieties to be recommended to farmers based on their
nitrogen fixation tolerance levels atmospheric drought and the resulting yield performance estimated by
our prediction.
D4. A high-resolution (0.6mi x 0.6mi) map of soybean yield potential and expected profitability resulting
from the proposed research across soybean growing regions in the state of Minnesota.

Updated August 29, 2024:
This progress report covers activities that are part of Goal#1.

As planned in the proposal, we have selected a diverse group of soybean genotypes consisting of:
1) Maturity Group I soybean genotypes that were released by the U of Minnesota breeding program over a 75-year period (between 1944 and 2023);
2) Commercial cultivars widely used by Minnesota growers and;
3) Plant productions from China, Korea, and Japan that are available from the USDA Soybean Germplasm Collection.

We have already started a first experiment where we are examining nitrogen fixation response to rising atmospheric dryness on 24 genotypes from these 3 groups. The experiment is currently ongoing and our first measurements already indicate genetic variability in nitrogen fixation. No particular challenges so far.

Updated November 28, 2024:
The experiment outlined in the previous report was completed on 10/24/2024. A total of 24 genotypes were grown for 16 weeks under controlled environment conditions that mimic the field. These genotypes representing Maturity Group I varieties released by the U of M breeding program, commercial varieties from private seed companies and landraces from highly contrasted environment collected from origin centers in Japan, Korea and China.

Each week, nitrogen fixation, canopy water use and photosynthesis were measured 3 times under two levels of atmospheric drought (or vapor pressure deficit-- VPD): low VPD and high VPD conditions. At the end of the experiment, plant shoots, leaves roots and nodules were harvested to estimate their dry mass and in the case of nodules, their number. The experiment yielded a large data set consisting of >1400 time courses of nitrogen fixation data and >2400 measurements of water use traits. This data set is currently being analyzed to characterize the extent of the genetic variability in nitrogen fixation response to rising atmospheric drying. We are on track to complete the data analysis by the end of the year as planned in the proposal. No challenges so far.

Updated February 28, 2025:
Goal#1: During the last quarter, we made strong progress in the data analysis pipeline. The latest round of analysis has revealed that out of the 24 genotypes, 9 expressed an increase in nitrogen fixation in response to increasing VPD. This indicates that there is a genetic variability in this trait, and therefore that it is amenable to breeding. Furthermore, our experiments have shown that, surprisingly, commercial varieties do not exhibit a positive nitrogen fixation response to atmospheric drought. However, landraces, and some of the varieties released by the U of M breeding program exhibit this trait. This indicates that farmers will benefit from research aiming at introgression of tolerance to atmospheric drought into soybean commercial varieties. The next steps will be to evaluate the dependency of this trait on the phenology of the crop and its potential association with crop water use.

Goal#2. We have started our crop modeling effort. The analysis conducted so far indicate potential soybean yield benefits to Minnesota farmers that would arise from the introduction of this trait in commercially available varieties. Over the next few weeks we will fine-tune the analysis to better characterize the spatial patterns of yield gradients.

Updated May 30, 2025:
Goal#1. The data analysis of nitrogen fixation response to atmospheric drought across multiple phenological windows was successfully completed. It revealed that the sensitivity of nitrogen fixation to atmospheric drought is dependent on the growth stage, but that this variation is also dependent on the genotype. We specifically detected four different seasonal phenotypes. These consist of: (1) lines that would increase their nitrogen fixation in response to atmospheric drought early in the season but not later, (2) lines that increase their nitrogen fixation in response to atmospheric drying later during the season, (3) lines that increase their nitrogen fixation essentially across all growing stages and finally (4) lines that do not exhibit an increase in nitrogen fixation under increasing atmospheric drought regardless of the growth stage. These results are important in that they show that the expression of this favorable trait is a function of the growth stage, which means that to be successful, high throughput phenotyping efforts will need to be conducted across multiple windows during the growing season in order to successfully identify the genetic basis of this promising trait. We suggest that such efforts be conducted at the V4, R2 and R6.

Overall, our research has successfully identified, and for the fist time, an extensive genetic variability in nitrogen fixation response to increasing atmospheric drought, and uncovered different phenotypes for this response sensitivity to growth stages. The experiment demonstrated that in some soybean genotypes, atmospheric drought has a positive effect on nitrogen fixation. However, this favorable response was not detected in the tested commercial germplasm. It was, in contrast, detected in breeding material and in landraces. This is a very encouraging result pointing to the possibility for using this genetic material to breed for boosted soybean yields in response to this stressor that is prevalent under Minnesota conditions.

Goal#2. The results obtained indicate that if nitrogen fixation increase in response to atmospheric drought is introgressed successfully in varieties released to farmers, then yield increases are expected to occur with high probability. Importantly, these increases are expected to maximize in state regions with the highest soybean acreage, in the western part of the state (north west, west central and south west). These results confirm the idea that nitrogen fixation response to atmospheric drought is a valuable trait with a strong potential to increase soybean yields for Minnesota farmers.

Atmospheric drought is a type of drought that is predominant in continental climates such as Minnesota. This atmospheric drought determines how much water evaporation takes place from the crop. Nation-wide studies have shown that variation in this atmospheric drought has the strongest effect on non-irrigated soybean yields across the nation.

We discovered that a key physiological process that determines yield, namely nitrogen fixation, is responsive to this stressor. We discovered that some soybean genotypes, actually increase their nitrogen fixation in response to this stressor. We found that this sensitivity is dependent on the growth stage, that is, some genotypes express it across the entire growing season while others do not. We also discovered that commercial varieties do not express this favotable trait, but that it exists in breeding lines and landraces. Modeling efforts, while still in progress, indicate significant yield gains for the Minnesota farmer if this trait is introgressed in varieties released to farmers.

We conclude that there is an untapped opportunity to further increase soybean tolerance to drought in ways that are profitable for the farmer across Minnesota. Soybean breeding programs would greatly benefit from actively breeding for this trait and release better yielding, more drought tolerant varieties to Minnesota farmers.

B1. Better-yielding varieties for Minnesota soybean growers. Atmospheric drought causes stress for the
soybean crop and limits its yield potential across the nation, including Minnesota, which is already
challenged by a relatively short growing season. By breeding for improved yield under atmospheric
drought, this proposal ultimately seeks to help the U of M soybean breeding program deliver more
productive and therefore more profitable soybean varieties to Minnesota growers.
B2. Making Minnesota soybeans more competitive. The U of M breeding
program will be in a position to be the first to deliver better-yielding varieties under atmospheric drought, a problem that
faces soybean production across the entire nation.
B3. A new technology to help Minnesota growers monitor the ability of their soybean in fixing nitrogen.
The proposed research is based on a methodology that can be made available to farmers so that they
can evaluate the nitrogen fixing performance of their crop in real time.