J. AMER. SOC. HORT. SCI. 117(5):852-855. 1992. Foliar Raffinose and Sucrose in Four Conifer Species: Relationship to Seasonal Temperature L.E. Hinesley1, D.M. Pharr2, L.K. Snelling3, and S.R. Funderburk4 Department of Horticultural Science and Plant Physiology Program, North Carolina State University, Raleigh, NC 27695-7609 Additional index words. Juniperus virginiana, Pinus strobus, Pinus virginiana, Cupressocyparis leylandii, Christmas trees, dormancy, cold hardiness Abstract. Foliar raffinose and sucrose concentrations in eastern white pine (Pinus strobus L.), eastern redcedar (Juniperus virginiana L.), Leyland cypress (xCupressocyparis leylandii Dallim.), and Virginia pine (Pinus virginiana L.) were measured monthly over 2 years. During cold weather, foliage of white pine and redcedar contained higher concentrations of raffinose and sucrose than did Leyland cypress and Virginia pine. Rafflnose concentrations were highest during winter and were best correlated with the frequency of occurrence of daily minima £ 1.7C during the 30 days before sampling. Sucrose concentrations, which also reached maximum levels during the winter, were best correlated with the frequency of occurrence of daily minima £ 7.2C in the prior 30 days. Sucrose concentrations were relatively high during fall and spring. Raffinose and sucrose concentrations increased in response to recurring low temperature, with correlations highest for raffinose. In the fall and winter, foliar raffinose concentration increases hybrid between Alaska cedar [Chamaecyparis nootkatensis (D. in conifers (Little, 1970; Parker, 1959). Raffinose accumulates Don) Spach] and Monterey cypress (Cupressus macrocarpa in Fraser fir [Abies fraseri (Pursh) Poir.] in response to low Hartweg.). Trees were 1.5- to 2.0-m tall, had been cultured as temperature and short days (Mitcham-Butler et al., 1986). This Christmas trees, and were growing on a uniform site in adjacent response is not confined to higher plants. A brief cold treatment rows on a research farm in Raleigh, N.C. The distance sepa- (4C) of algae (Chlorella vulgaris Beyerinck) induces an accu- rating the most widely spaced sample trees was no more than mulation of raffinose, and concentrations decrease when the 30 m. Beginning 1 Apr. 1989, and at monthly intervals there- algae are transferred to warmer temperatures (Salerno and Pon- after, foliage was collected from five trees of each species over tis, 1989). In general, high foliar raffinose concentrations are 2 years. The same trees of each species were used throughout associated with greater cold hardiness (Kandler and Hopf, 1980; the study. On each sampling date, »100 fascicles were com- Parker, 1959). posited from various positions throughout the upper crown of Fraser fir Christmas trees that are harvested after experiencing each pine tree. For redcedar and Leyland cypress, 15 to 20 small low temperatures have higher foliar raffinose concentration and branch tips (6- to 8-cm long) were used. With the pines, current- exhibit better postharvest needle retention than trees precondi- year fascicles were used beginning in June, when new needles tioned with higher temperatures (Mitcham-Butler et al., 1986). were about half the length of 1-year-old needles and could be Although raffinose increases in the foliage of several conifers removed without tearing the bark. Foliage samples were placed during the winter and might be involved in cold hardiness, there on ice, transported a short distance to North Carolina State Univ., are no studies that compare seasonal sugar levels of different and stored at –30C pending analysis. conifer species growing in the same environment. Thus, com- Samples were freeze-dried, ground through a 20-mesh screen, parisons among species from available data are difficult. Our and stored in a dessicator at 22C before analysis. Samples (50 research had two objectives: 1) determine seasonal raffinose and mg) were weighed and extracted twice for 5 min with hexane sucrose concentrations in the foliage of four conifer species that (boiling point range 37 to 55C) in a 42C water bath under a differ in hardiness and are important to the Christmas tree in- fume hood. Samples were centrifuged, supernatants discarded, dustry and 2) determine if foliar raffinose and sucrose concen- and remaining pellets were extracted three times with 80% aqueous trations are correlated with the occurrence of different baseline ethanol (v/v) at 80C to remove soluble sugars. The ethanolic (daily minimum) temperatures during the week or month pre- supernatant solutions were collected, evaporated to dryness in ceding sampling. vacuo at 45C, resolubilized in 1 ml of distilled water, and stored at –80C until analyzed. Materials and Methods Raffinose and sucrose concentrations were determined by high- Four conifers were studied: eastern redcedar, eastern white performance liquid chromatography. The system consisted of a pine, Virginia pine, and Leyland cypress. Leyland cypress is a Waters (Millipore, Waters Chromatography Division, Milford, Mass.) 6000A pump, a Waters Sugar-Pak column, and a Waters 410 Differential Refractometer. Water was used as the solvent Received for publication 20 Nov. 1991. Accepted for publication 4 May 1992. at a flow rate of 0.5 ml·min–1. The column was operated at This research was funded by the North Carolina Agricultural Research Service, 75C and was preceded by a single Waters µ-Bondapak C / Raleigh, NC 27695-7643. We gratefully acknowledge the assistance of Ka- 18 tharine B. Perry in the analysis of temperature data. Use of trade names does Corasil guard and a set of anion and cation guard cartridges not imply endorsement of products named nor criticism of similar ones not operated at 22C (Bio-Rad, Richmond, Calif., deashing guards). mentioned. The cost of publishing this paper was defrayed in part by the pay- Following dilution, samples were injected, and sugars were ment of page charges. Under postal regulations, this paper therefore must be identified and quantified using an SP4200 computing integrator hereby marked advertisement solely to indicate this fact. (Spectra-Physics, San Jose, Calif.). 1Professor. Dept. of Horticultural Science. 2Professor. Dept. of Horticultural Science and Plant Physiology Program. Daily maximum and minimum temperatures (Fig.1) were ob- 3Research Technician. Dept. of Horticultural Science. tained from a standard weather station located 200 m from the 4Research Technician. Dept. of Horticultural Science. trees and on the same contour. The frequency of days in which 852 J. Amer. Soc. Hort. Sci. 117(5):852-855. 1992. Fig. 1. Average monthly maximum ( ❍ ) and minimum ( ● ) air tem- peratures over 2 years (Apr. 1989 to Apr. 1991) on the site where Virginia pine, Leyland cypress, eastern white pine, and eastern red- cedar were sampled monthly for foliar raffinose and sucrose. Fig. 3. Monthly variation in foliar sucrose over 2 years for four Christmas tree species. (A) eastern white pine (WP, ● ) and Virginia pine (VP, ❍ ). (B) eastern redcedar (RC, ❍ ); Leyland cypress (LC, ● ). Vertical bars = SE; n = 5. Results and Discussion Foliar concentrations of raffinose increased for all species during winter (Fig. 2), with accumulation most pronounced in white pine and redcedar. Peak values for these species in De- cember and January were 0.8 to 1.1 mg/100 mg dry weight, compared to maximum values £ 0.2 mg/100 mg dry weight in Virginia pine and Leyland cypress. Concentrations were very low during the warm months of May through September (Fig. 2). The two species (white pine and redcedar) that produced the most foliar raffinose during the winter maximized foliar sucrose at the same time (Fig. 3). Similar patterns have been docu- mented for other conifers (Kozlowski and Keller, 1966; Kreuger and Trappe, 1967; Parker, 1963). In general, foliage contained more sucrose than raffinose. Compared to the other species, Fig. 2. Monthly variation in foliar raffinose over 2 years for four redcedar contained higher foliar sucrose concentrations during Christmas tree species. (A) eastern white pine (WP, ● ); Virginia late fall and early spring. pine (VP, ❍ ). (B) eastern redcedar (RC, ❍ ); Leyland cypress (LC, Foliar concentrations of raffinose in redcedar, Virginia pine, ● ). Vertical bars = SE; n = 5. and white pine were most highly correlated with the frequency of occurrence of temperatures £ 1.7C in the prior 30 days (Fig. 4A). Concentrations in Leyland cypress were only slightly more the minimum (baseline) temperature was £ 15.6, 12.8, 10.0, correlated with temperatures £ 1.1C. Except for redcedar, foliar 7.2, 5.0, 1.7, –1.1, and –5.0C was determined for the 30 sucrose levels were best correlated with the frequency of oc- days preceding each sampling date. Resulting frequencies for currence of daily minimum temperatures £ 7.2C in the previous each baseline temperature were correlated with foliar raffinose 30 days (Fig. 4B). With redcedar, the correlation was slightly and sucrose concentrations using SAS (SAS Institute, Cary, higher with a baseline temperature of 10C. N.C.). Linear regressions were developed for raffinose and su- Whether accumulation and maintenance of high foliar raffi- crose concentration as a function of frequency of occurrence of nose concentrations is a short-term response to a few cold events baseline temperature using the highest correlations. (induction) or a cumulative, longterm effect from recurring ex- J. Amer. Soc. Hort. Sci. 117(5):852-855. 1992. 853 Fig. 5. (A) Relationship of foliar raffinose concentration to the