Seeds of orchardgrass (Dactylis glomerata L. cv. Benchmark Plus) were germinated in moist paper towels under ambient conditions for 3 days. Seedlings were transplanted to 2.25 L pots (13 cm diameter × 17 cm height) containing soil mix (Metro Mix 300; SunGro, Agawam, MA, United States) with three seedlings pot^-1 (3 pots per treatment × 3 replicates). Plants were grown in the greenhouse at the Virginia Polytechnic Institute and State University (Blacksburg, VA, United States) at 28°C day/23°C night under ambient light conditions. Plants were watered daily to field capacity and supplied with 15 mL of slow-release fertilizer (Osmocote Plus; The Scotts Company, Marysville, OH, United States) twice during the establishment phase. After 10 weeks, plants were clipped to 10 cm and allowed to regrow. Plants were vernalized in the greenhouse during winter at 14–7°C under ambient light conditions for 8 weeks. Flowering was initiated with 23°C day/18°C night and supplemental lighting (14 h light/10 h dark), and plants were allowed to mature to anthesis—stage R4-R5 (Moore et al., 1991). Plants were then transferred to environmentally controlled chambers set to 20°C constant temperature for 5 days. Following this acclimatization, plants were cut to either 2.5 or 7.5 cm above the soil surface and were regrown at 20°C or 35°C constant temperature. Both chambers maintained 70% relative humidity, 14 h light/10 h dark at 400 μmol m^-2 s^-1 of photosynthetically active radiation. On days 0, 1, 3, and 11 days following cutting, stubble tissue was harvested, separated from regrowth, immediately frozen in liquid nitrogen, and stored at -80°C.

Chlorophyll florescence parameters were measured in regrowing leaf blades on days 3 and 11 using a chlorophyll fluorometer (MINI-PAM-II; WALZ, Effeltrich, Germany) and associated leaf-chip holder (2035-B). Leaf areas used for dark-adapted F_v/F_m were exposed to dark conditions for 30 min using a dark leaf clip. Light adapted F_v/F_m was determined after exposure to continuous actinic light (400 μmol m^-2 s^-1) for 5 min. Calculations for effective quantum yield of PS II photochemistry (ϕ_PSII), photochemical quenching (qP), and non-photochemical quenching (NPQ) were (F′_m-F′)/F′_m, (F′_m-F)/(F′_m-F′₀), and F_m/F′_m-1, respectively (Murchie and Lawson, 2013). Chlorophyll content was estimated in regenerating leaf blades using a portable chlorophyll meter (Opti-Sciences CCM-300, Hudson, NH, United States).

On days 3 and 11 following defoliation, gas exchange measurements were made on regrowing leaf blades. A portable photosynthesis analysis system (LI-6400XT; LI-COR Lincoln, NE, United States) was used to determine the rates of CO₂ assimilation, stomatal conductance, and transpiration. The system maintained the measurement conditions of 400 μmol CO₂ mol^-1 under 400 μmol m^-2 s^-1 of photosynthetically active radiation at 20°C and 60–70% relative humidity.

Water- and ethanol-soluble carbohydrates (WSC and ESC) and starch were analyzed using methods adapted from Fukao et al. (2012). Stubble tissue (30 mg) was homogenized in 1 mL of 80% (v/v) ethanol and incubated at 80°C for 20 min. After centrifugation, the supernatant was collected. The ethanol extraction was repeated twice more, and the three extracts were pooled. The pellet was then suspended in 1 mL water and incubated at 80°C for 20 min. Water extraction was repeated, and the supernatants from both water extractions were combined. The ethanol and water extracted solutions were dried using a vacuum concentrator and then re-dissolved in 1 mL of water. Soluble carbohydrates were measured by the anthrone method. Glucose was used as a standard. The carbohydrate extracts were incubated with 1 mL of 0.14% (w/v) anthrone solution in 100% sulfuric acid at 100°C for 20 min. Samples were cooled, and their absorbance at 620 nm was determined with a spectrophotometer. Starch content was quantified in the pellet used for soluble carbohydrate extraction. The pellet was re-suspended in 1 mL water containing 10 units of heat-resistant α-amylase. The suspension was incubated for 30 min at 95°C. The reactant was then mixed with 25 μL of 1M sodium citrate (pH 4.8) and 5 units of amyloglucosidase and incubated at 55°C for 1 h. The mixture was centrifuged for 30 min, and the glucose content of 100 μL of the supernatant was determined by the anthrone method as described above.

Frozen tissue (75 mg) was homogenized in 450 μL of 0.83 M perchloric acid on ice. After centrifugation, 300 μL of the supernatant was neutralized with 75 μL of 1 M bicin (pH 8.3) and 70 μL of 4 M potassium hydroxide. Following centrifugation, the supernatant was used for nitrate, ammonium, and amino acid assays as described in van Veen et al. (2013) and Alpuerto et al. (2016). For nitrate, the extract (10 μL) was incubated with 40 μL of 5% (w/v) salicylic acid in 100% sulfuric acid at 25°C for 20 min. The solution was mixed with 950 μL of 2 M sodium hydroxide, and the absorbance at 410 nm was determined with a spectrophotometer. Potassium nitrate was used as the standard. For ammonium, 25 μL of the extract was mixed with 375 μL of 8.8% (w/v) salicylic acid, 10 M sodium hydroxide, 21.5 mM ethylenediaminetetraacetic acid (EDTA), and 6.7 mM sodium nitroferricyanide (III) dehydrate. The mixture was added to 625 μL of 70 mM sodium phosphate, monobasic (NaH₂PO₄) and 45 mM sodium dichlorisocyanurate and incubated for 2 h at 25°C. Following incubation, the absorbance at 660 nm was determined. Ammonium sulfate was used as the standard. For total amino acid, 80 μL of the extract was added to 20 μL of 3 M magnesium oxide and incubated in an opened 1.5 mL tube for 16 h at 25°C. After incubation, 80 μL of the solution was mixed with 50 μL of 0.2 mM sodium cyanide resolved in 8 M sodium acetate and 50 μL of 168 mM ninhydrin resolved in 100% 2-methoxyethanol. The mixture was incubated at 100°C for 15 min, and 1 mL of 50% isopropanol was immediately added to the solution. After cooling, the absorbance at 570 nm was measured with a spectrophotometer. Glycine was used as the standard.

Total protein was extracted from 50 mg of stubble tissue in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 2 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) IGEPAL CA-360, and 1 mM phenylmethanesulfonyl fluoride on ice. Protein concentration was determined by Coomassie Plus protein assay reagent (Thermo Fisher Scientific, Waltham, MA, United States). Bovine serum albumin was used as the standard.

Statistical analysis was conducted using JMP Pro (ver. 11.0.0; SAS Institute, Cary, NC, United States). ANOVA was used to determine treatment differences at each time point. Tukey’s honest significant difference (HSD) was used to determine mean separation (α < 0.05).

This study broadly simulated the environmental conditions during regrowth following the first harvest of spring growth for orchardgrass hay/silage. Plants were excised at the early heading stage at 7.5 or 2.5 cm above the soil surface – a commonly recommended growth stage and cutting height range for hay harvest. Following defoliation, plants were regrown under control (20°C) or high temperature (35°C) conditions for up to 11 days. On day 11 the most vigorous regrowth was observed for the 20°C treatment cut to 7.5 cm with diminishing regrowth for the 20°C – 2.5 cm, 35°C – 7.5 cm, and 35°C – 2.5 cm treatments (Figure 1A). Consistently, the biomass of regrown tissue differed significantly with cutting height and temperature treatments (Figure 1B). At optimal temperature (20°C), 7.5 cm defoliation height supported more regrowth than 2.5 cm. Under heat stress (35°C), however, the advantage of high cutting height on regrowth vigor was not observed. When plants were cut to 7.5 cm, high temperature significantly suppressed regrowth. This temperature effect was also detected at 2.5 cm cutting height. These results indicate that heat stress affects biomass yield at both cutting heights, whereas the impact of cutting height is apparent only under non-stress temperature.

Chlorophyll abundance in regrowing laminar tissue was quantified as a determinant of photosynthetic performance (Figure 2A). Three days following defoliation, chlorophyll was significantly greater in 35°C treatments as compared to 20°C treatments, irrespective of cutting height. In addition, chlorophyll abundance was reduced by low cutting height in heat-treated plants, but not in non-stressed plants. On day 11 following defoliation, a lower chlorophyll abundance was measured in the 35°C – 2.5 cm, but the other treatments did not differ.

Direct measurements of leaf gas exchange determined photosynthetic activity as well as stomatal conductance and transpiration rate in regrowing leaves. On day 3, the photosynthetic rate was greater at 35°C than 20°C in plants cut to 7.5 cm (Figure 2B). The same pattern was observed in plants cut to 2.5 cm. We also observed that short cutting height reduced photosynthetic activity in regrowing leaves under both non-stress and high temperature conditions. On the 11th day of recovery, photosynthesis rates were clearly augmented in plants cut to 7.5 and 2.5 cm at 20°C, and their values did not differ significantly. High temperature suppressed photosynthetic activity at both mowing heights, with a more severe reduction in plants trimmed to 2.5 cm. Stomatal conductance of regrowing leaves was significantly increased by low cutting height only at 35°C, not at 20°C on day 3 (Figure 2C). High temperature did not affect stomatal conductance at either cutting height. On day 11, heat stress repressed stomatal conductance at both mowing heights. However, the negative effect of low cutting height on stomatal conductance was detected only under high temperature. On day 3, transpiration was stimulated in response to high temperature at both defoliation levels, with higher induction in plants cut to 2.5 cm (Figure 2D). At 20°C, cutting height did not influence transpiration. On day 11, heat-induced transpiration was not observed at either mowing level; the 35°C – 2.5 cm treatment had a significantly lower transpiration rate than the other treatments.

Chlorophyll fluorescence measurements provided the insight into the performance of PSII in regrowing leaves under non-stress and heat stress conditions. Maximum quantum efficiency of dark-adapted PSII photochemistry (F_v/F_m) did not differ among treatments on day 3, but it was significantly lower in leaves of the 35°C – 2.5 cm treatment on day 11 (Figure 3A). The operating efficiency of PSII (ϕ_PSII) was enhanced by high temperature in plants cut to 7.5 and 2.5 cm on day 3 but did not differ between the cutting heights (Figure 3B). On day 11, the positive effect of high temperature on ϕ_PSII was canceled at both defoliation levels. Low cutting height suppressed ϕ_PSII under heat stress. qP did not differ among the treatments on day 3 (Figure 3C). On day 11, qP was reduced at low mowing height only at high temperature. Heat stress restrained NPQ only in plants cut to 2.5 cm on day 3 (Figure 3D). This trend was also observed on day 11.

Concentrations of three classes of carbohydrates were measured to elucidate how cutting height and heat stress influence the status of carbon reserves in the stubble during regrowth following defoliation. WSC extracts contain primarily fructan, while ESC extracts include mono- and disaccharides (Kagan et al., 2014). On days 1 and 3 following defoliation, the levels of WSC were low relative to the values on day 0 in all treatments (Figure 4A). The effect of cutting height and temperature on relative WSC concentration was not detected on day 1. However, on day 3, relative WSC content was greater under high temperature in plants cut to 2.5 cm. On day 11, no significant difference among the relative WSC concentrations was detected. The relative concentration of ESC was positively affected by high temperature only in plants cut to 7.5 cm on day 1 (Figure 4B). The effect of high temperature on ESC continued on days 3 and 11. In addition, low cutting height suppressed the relative ESC content under high temperature at these time points. For relative starch concentration, there was no significant difference among the treatments on day 1 (Figure 4C). On day 3, the relative starch concentration was greater at high temperature only when cut to 2.5 cm, which is consistent with the observation in WSC. This trend was not observed on day 11, but low defoliation height suppressed the relative starch content at high temperature, which is in accordance with the observation in ESC at the same time point.

To determine the status of nitrogen reserves in stubble tissue, the relative concentrations of total soluble protein, total free amino acids, nitrate, and ammonia were measured. At all of the three time points, high cutting height increased the relative protein content at 20°C (Figure 5A). Under high temperature, the positive effect of high cutting height was observed only on day 11. The protein content was also induced by high temperature only in plants cut to 7.5 cm on day 11. Relative total amino acids did not differ significantly among treatments on day 1 (Figure 5B). On days 3 and 11, high temperature increased the abundance of total amino acids at both mowing levels. However, low cutting height limited the accumulation of relative total amino acids under heat stress at these time points. Relative nitrate concentrations were suppressed by low cutting height under non-stress and heat stress conditions on day 1 (Figure 5C). This effect is not observed on day 3, but high temperature increased the relative nitrate content in plants cut to 7.5 cm. The positive effect of high temperature on nitrate was also detected on day 11 at both defoliation levels. Ammonium concentrations followed a similar pattern to that of nitrate (Figure 5D).