DOI: 10.5061/dryad.5tb2rbp6p
title: Cold acclimation threshold induction temperatures of switchgrass ecotypes grown under a long and short photoperiod

description: 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.
description: 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 (LT<sub>50</sub>) and rhizome
 water content. The LT<sub>50</sub> 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: gH<sub>2</sub>O
 gDM<sup>-1</sup> = (FM-DM)/DM. Maximum
 freezing tolerance (LT<sub>50</sub>) 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 LT<sub>50</sub> 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
 km<sup>2</sup>) 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
description: Data can be accessed using Excel.