J. AMER. SOC. HORT. SCI. 117(1):154-157. 1992. High Root-zone Temperatures Influence RuBisCO Activity and Pigment Accumulation in Leaves of ‘Rotundifolia’ Holly John M. Ruter 1 and Dewayne L. Ingram 2 Department of Environmental Horticulture, Institute of Food and Agricultural Sciences, Gainesville, FL 32611 Additional index words. heat stress, chlorophyll, Japanese holly, Ilex crenata Abstract. Plants of ‘Rotundifolia’ holly (Ilex crenata Thunb.) were grown for 3 weeks with root zones at 30,34,38, or 42C for 6 hours daily to evaluate the effects of supraoptimal root-zone temperatures on various photosynthetic processes. After 3 weeks, photosynthesis of plants grown with root zones at 38 or 42C was below that of plants grown at 30 or 34C. Chlorophyll and carotenoid levels decreased while leaf soluble protein levels increased as root-zone temperature increased. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity per unit protein and per unit chlorophyll responded quadratically, while RuBisCO activity per unit fresh weight increased linearly in response to increasing root-zone temperature. Results of this study suggest that ‘Rotundifolia’ holly was capable of altering metabolism or redistributing available assimilates to maintain CO 2 assimilation rates in response to increasing root- zone temperatures. Supraoptimal root-zone temperatures limit the growth of con- tainer-grown plants in the southeastern United States (Fretz, 1971; Ingram et al., 1988; Keever and Cobb, 1984). In Florida, media temperatures as high as 58C in container-grown plants have been recorded (Martin and Ingram, 1988). The critical lethal temperature (T c ) for root of ‘Rotundifolia’ holly was pre- dicted to be 48 ± 1.5C for a 30-min exposure as determined by electrolyte leakage (Ruter and Ingram, 1991). In a M-liter container under various irrigation regimes, the mean number of degree-hours exceeding 40C was in excess of 3 h·day -1 (Martin and Ingram, 1991). The T c for roots in two species of holly decreased linearly as exposure time increased exponentially (In- gram, 1986). The predicted T c for a 3-h exposure of ‘Helleri’ holly (Ilex crenata Thunb. ‘Helleri’) was 45.5 ± 0.5C. There- fore, repeated exposure of holly roots to high root-zone tem- peratures resulted in root damage that decreased the growth of container-grown holly (Ingram et al., 1988; Ruter and Ingram, 1990). Little is known about the response of photosynthetic mech- anisms to supraoptimal root-zone temperatures. Decreases in photosynthetic rates of woody plants in response to supraoptimal root-zone temperatures have been reported (Gur et al., 1972; Johnson and Ingram, 1984). Foster (1986) reported a nonsto- matal decrease in shoot carbon exchange rate when plants of ‘Rotundifolia’ holly were grown for 1 week with root zones at 36 or 40C rather than 28 or 32C. Greater recovery of carbon exchange rates than of stomatal conductance in ‘East Palatka’ holly (Ilex ×attenuata Ashe) after a 12-week exposure of the root zone to 42C indicated that regulation of photosynthesis may be nonstomatal (Martin et al., 1989). Loss of photosynthetic activity at high shoot and leaf tem- peratures has been related to decreased RuBisCO activity (Al- Received for publication 11 Feb. 1991. Accepted for publication 15 Aug. 1991. Florida Expt. Sta. J. Ser. no. R-00315. Technical assistance of Dale Haskell, Connie Lance, and Charles Guy is greatfully acknowledged. The cost of pub- lishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Former Graduate Assistant. Currently: Assistant Professor, Dept. of Horticul- ture, Coastal Plain Experiment Station, P.O. Box 748, Tifton, GA 31793. 2 Present address: Dept. of Horticulture and Landscape Architecture, Univ. of Kentucky, Lexington, KY 40546. 154 Khatib and Paulsen, 1984; Badger et al., 1982; Monson et al., 1982). Hurewitz and Janes (1987) showed that the RuBisCO activation state was decreased by increasing root-zone temper- atures and changes in root sink strength for photoassimilates. Supraoptimal root-zone temperatures influence root carbohy- drate content (Foster, 1986; Ingram et al., 1986a), photoas- similate partitioning and carbon loss (Ruter and Ingram, 1990), and root respiratory characteristics (Ruter and Ingram, 1990, 1991). Loss of chlorophyll and protein in shoots occurred in response to high root-zone temperatures (Kuroyanagi and Paulsen, 1988). Ingram et al. (1986b) found that total protein synthesis de- creased and/or protein degradation increased as treatment tem- peratures for roots of small anise tree (Illicium parviflorum L.) increased above 35C. Further research is required to document the effects of supraoptimal root-zone temperatures on physio- logical responses, particularly nonstomatal limitations related to photosynthesis. Therefore, the objectives of this research were to determine the effects of root-zone temperature on net CO 2 assimilation, RuBisCO activity, and chlorophyll and carotenoid pigment concentrations. Materials and Methods Plant material and growth conditions. Rooted, terminal stem cuttings of ‘Rotundifolia’ holly were potted in 1200-ml clear plastic bags (six drainage holes per bag) using 1000 cm 3 of Metro-Mix 300 (W.R. Grace and Co., Cambridge, Mass) as a growth medium. Plants were fertilized twice weekly with 300 mg N/liter of a soluble 20N–8.8P–16.6K fertilizer (Peters 20- 20-20, W.R. Grace and Co.). Plants were grown in a glass- house (daily air maximum, 34 ± 4C) under natural daylength conditions (Mar.–June 1989) for a minimum of 12 weeks before being transferred to a high-light growth room 3 weeks before the initiation of experiments. Plants were grown in a 3.0 × 3.6-m walk-in growth room with irradiance supplied by 12, 1000-W phosphor-coated, metal- arc HID lamps (GTE Sylvania Corp., Manchester N.H.). Pho- tosynthetic photon flux (PPF) was 850 ± 50 μmol·m -2 ·s -1 at canopy height measured with a quantum radiometer (LI-COR, Lincoln, Neb.). The photoperiod was 13 h daily (0800 to 2100 HR), with the dark period interrupted for 3 h (0100 to 0400 HR) J. Amer. Soc. Hort. Sci. 117(1):154-157. 1992.