Plants can cold-acclimate to enhance their freezing tolerance by sensing declining temperature and photoperiod cues. However, the factors influencing genotypic variation in the induction of cold acclimation are poorly understood among perennial grasses. We hypothesized that the more northern upland switchgrass (Panicum virgatum L.) ecotype develops a higher degree of freezing tolerance by initiating cold acclimation at higher temperatures as compared with the coastal and southern lowland ecotypes. First, we determined the optimal method for assessing freezing tolerance and the length of exposure to 8/4°C required to induce the maximum level of freezing tolerance in the most northern upland and most southern lowland genotypes. We characterized the maximum freezing tolerance of eight upland, three coastal and five lowland genotypes grown for 21 d at 8/4°C and a 10 or 16 h photoperiod. Next, we identified the temperature required to induce cold acclimation by exposing the 16 genotypes for 7 d at 20 to 6°C constant temperatures under a 10 or 16 h photoperiod. Cold acclimation initiated at temperatures 5 and 7°C higher in upland than coastal and lowland genotypes. Among upland genotypes the shorter photoperiod induced cold acclimation at a 1°C higher temperature. Genotypes originating from a more northern latitude initiate cold acclimation at higher temperatures and develop higher maximum freezing tolerances. An earlier response to declining temperatures may provide the upland ecotype with additional time to prepare for winter and provide an advantage when plants are subjected to the rapid changes in fall temperature associated with injurious frosts.

Injury in rhizomes frozen prior to or following excision

Northern upland DAC6 and southern lowland AP13 were held at 8/4°C for 21 d prior to assessment of injury using one of the three following tests: (1) Whole plant recovery. (2) Injury in rhizomes frozen prior to excision from whole plants. (3) Injury in rhizomes frozen following excision from whole plants.  For assessment of injury in whole plants and rhizomes frozen prior to excision from whole plants, we transferred to a programmable freezer set at 0°C for 1 h. The temperature within the freezer was cooled to -2°C over 1 h, held at -2°C for 12 h and then cooled to -4°C over 1 h. Aboveground tissues were misted with ice nucleation active bacteria to promote uniform ice nucleation among samples. The programmable freezer was then cooled 2°C h-1 to three predetermined test temperatures that were 2°C apart. Pots were transferred to a dark room set at 5°C for 24 h and then to a greenhouse room maintained at conditions for plant cultivation.  Survival was scored after 28 d as the proportion of switchgrass pots that regrew tillers. The LT50 was calculated from survival curves (Willick et al., 2021) and the experiment was repeated four times for each genotype and treatment combination. To assess freezing injury rhizomes frozen prior to excision, plants were cooled as described in the whole plant recovery test. After plants were thawed at 5°C for 24 h, a 3 cm section of rhizome associated with the newest fully developed tiller was harvested, cleaned to remove surface debris, rinsed three times in double distilled water and transferred to a test tube (1.3 x 10 cm) containing 1 mL of deionized water. To assess injury in rhizomes frozen following excision, tissue was excised from unfrozen plants and transferred to a capped test tube, cooled in a programmable freezer and thawed as described. Rhizomes frozen prior to or following excision were ice nucleated using frozen 100 μL droplets of MilliQ water. Relative electrolyte leakage was calculated as described by Lim et al. (1998)

Cold acclimation in AP13 and DAC6

To assess the physiological parameters at maximum freezing tolerance involving the most northern upland (DAC6) and southern lowland (AP13) genotypes, chamber temperature was cooled to 8/4°C with a photoperiod of 10 h. Switchgrass was sampled after 0, 7, 14, 21, 28, 35, 49 or 63 d to determine the temperature at which half of the switchgrass recovered from freezing injury (LT₅₀) and rhizome water content. The LT₅₀ was assessed and calculated as previously described. The experiment was repeated four times for each genotype and treatment combination. To assess rhizome water content, a 4 cm section of developing rhizome tissue collected from ten plants was immediately weighed to obtain fresh mass (FM). Samples were dried at 60°C for 48 h and then re-weighed to obtain the dry mass (DM). Rhizome water content was quantified as described by Willick et al., (2019) using the following formula: gH₂O gDM^-1 = (FM-DM)/DM.

Maximum freezing tolerance (LT₅₀) and threshold induction temperatures in genotypes

Genotypes were placed in a growth cabinet for 21 d set at one of four different regimes: (1) 25/20°C and a 16 h photoperiod; (2) 25/20°C and a 10 h photoperiod; (3) 8/4°C and a 16 h photoperiod; (4) 8/4°C and a 10 h photoperiod. Switchgrass genotypes were then assessed for LT₅₀ and the experiment was repeated four times for each genotype and treatment combination. To assess threshold induction temperatures, all genotypes were assessed for survival after cooling to -8°C. The threshold induction temperature was recorded as the warmest acclimation temperature at which each genotype significantly enhanced survival above 20°C.

Climate data

We retrieved data for historical maximum and minimum mean surface temperatures (1971–2000) for the months of September, October, and November from WorldClim 2.0 data sets (Fick & Hijmans, 2017). All climate data was collected at the highest available resolution (30 arc second = 1 km²) and extracted at the site of origin for each genotype using QGIS version 3.24.0 (QGIS Core Development Team, 2021).

References

• Fick, S.E. & Hijmans, R.J. (2017) WorldClim 2: new 1‐km spatial resolution climate surfaces for global land areas. International Journal of Climatology, 37 (12), 4302–4315
• Lim, C.C., Arora, R. & Townsend, E.C. (1998) Comparing Gompertz and Richards functions to estimate freezing injury in Rhododendron using electrolyte leakage. Journal of the American Society for Horticultural Science, 123 (2), 246–252
• QGIS Core Development Team. (2021). QGIS Geographic Information System. Open Source Geospatial Foundation Project. Available from: http://qgis.osgeo.org. [Accessed 12th March 2022]
• Willick, I.R., Tanino, K.K. & Gusta, L.V. (2021) The impact of global climate change on the freezing tolerance of winter cereals in Western Canada. Journal of Agronomy and Crop Science, 207, 88–99
• Willick, I.R., Gusta, L.V., Fowler, D.B. & Tanino, K.K. (2019) Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing. Plant, Cell & Environment, 42 (2), 701–716

Data can be accessed using Excel.