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Enhancing water stress tolerance in soybean through phytoglobin manipulations

Principal Investigator: Claudio Stasolla

Research Institution: University of Manitoba

Timeline: March 2016 – December 2019


  • Develop soybean plants with altered expression of phytoglobin (Pgb) able to tolerate soil flooding (waterlogging) and submergence (whole plant submergence). The soybean class 1 Pgb gene will be isolated and cloned into vectors to over-express or down-regulate its expression in tissues. Regenerated lines with altered levels of Pgbs will be tested for waterlogging and submergence tolerance under controlled environmental conditions. Plant survival and gas exchange parameters will be examined.
  • Determine if Pgb can be used as a molecular marker to assess waterlogging and/or submergence tolerance by assessing and correlating waterlogging and submergence tolerance in commercial soybean genotypes with expression levels of the endogenous Pgb.
  • Conduct preliminary studies to assess if Pgb can also be used to enhance drought stress tolerance under controlled environmental conditions and measure Pgb levels in cultivars characterized by different drought tolerances.


  • Tolerance to excess water can be achieved by manipulations of a single gene, Pgb. 
  • Over-expression of Pgb can be used as tool to enhance tolerance to water stress.
  • Pgb can be employed in breeding programs for selections of cultivars better able to cope with water stress.

Scientific Summary:

Phytoglobins (Pgb) are ubiquitous proteins expressed during growth, development and in response to both biotic and biotic stress in all nucleated organisms. In a previously funded ARDI project, we established that by increasing Pgb expression in alfalfa, plant survival following whole plant submergence for 3 weeks increased from approximately 20% to 75-85 %. We have recent evidence that comparable improvements in submergence tolerance can be achieved in maize. Maize plants over-expressing Pgb retain a higher photosynthetic rate and showed reduced leaf injury when waterlogged for 10 days. Similarly, survival frequency of maize plants increased from 45% to about 80% following the imposition of submergence for 6 days. Results in these two species (the former a dicot and the latter a monocot) made us very confident that Pgbs have a universal role in alleviating flooding and waterlogging stress and that similar results could be achieved in soybean. Proposed mechanisms of Pbg action under stress conditions include the ability to maintain a high energy status (i.e., ATP) and influence the developmental fate of cells through its ability to modulate key signal molecules in plants.

This project therefore sought to generate soybean plants with enhanced waterlogging and submergence tolerance via the use of Pgb which could then be utilized in future studies to introgress the “tolerance” trait into commercial varieties with tremendous benefits for farmers. Soybean yield loss due to excess moisture can be substantial, especially in Manitoba where river flooding and field inundation during torrential rainfalls can result in fields being partially or completely submerged for prolonged periods of time.

In addition to waterlogging and flooding, Pgbs are highly induced by plants under other unfavourable conditions suggesting that their protective role may be a factor in other forms of stress, including drought and environments with reduced water content. Finally, it should be noted that improved nodulation and nitrogen fixation may be a potential side benefit of this work as Pgbs are known to improve nodulation and N-fixation in soybeans.


1.  Transgenic soybean lines up-regulating or suppressing Pgb were generated.

2.  To evaluate the behavior of the transgenic lines to waterlogging the following parameters were measured: photosynthetic rate, stomatal conductivity, intercellular CO2 level, transpiration rate, and number of adventitious roots. Over-production of Pgb enhances all these parameters, while the under-production of Pgb has negative effects on all the parameters measured.

3.  The percentage of plants able to recover and grow (recovery rate) after exposure to one week of full submergence in the dark was used as an estimation of submergence response. Relative to wild type (WT) with a normal “natural” level of Pgb, plants over-producing Pgb have a much higher recovery rate while those under-producing the same protein exhibit reduced recovery rate. This behavior correlates well with the level of Pgb expression measured in these plants.

4.  The response of the 20 commercial soybean varieties to waterlogging and submergence was also further investigated by closely looking at the correlation between Pgb expression level and tolerance to each stress. For waterlogging a weak correlation was observed between Pgb in the roots or shoots and photosynthetic rate. This was in contrast to the submergence experiments where the correlation between recovery rate and Pgb expression in both roots and shoots was higher.

5.  Expression levels of aquaporins (Tonoplast intrinsic proteins, TIPs) during submergence and waterlogging was measured. TIPs facilitate movement of water between cells influencing water conductivity. In plants over-expressing Pgb the pattern of TIP expression was consistent with that observed in plants tolerant to excess moisture stress.

6. The response of plants over-producing or suppressing Pgb was examined in response to drought, simulated by applications of polyethylene glycol (PEG). Plants over-expressing Pgb had increased root length and higher number of lateral roots; this was in contrast to plants suppressing Pgbs which displayed a reduced root length and lower number of lateral roots. These results suggest a better capacity of plants over-producing Pgb to cope with water stress.

Collectively, these studies demonstrated that over-production of Pgb represents a strategy to tolerate conditions of submergence and drought, and that the natural variations of Pgb can be exploited to select lines able to cope with both types of stress.

External Funding Partners:

Manitoba Pulse and Soybean Growers

Project Related Publications:

Mira, M., Huang, S., Hill, R., Stasolla C. 2021. Tolerance to excess moisture in soybean is enhanced by over-expression of the Glycine max Phytoglobin (GmPgb1). Plant Physiol Biochem. Feb (159) : 322-334.