Chlorophyll, Carotenoid, and Visual Color Rating of Japanese-cedar Grown in the Southeastern United States

Japanese-cedar has been underused in landscapes of the United States until recent years. There are now over 100 cultivars, many of which are grown in the southeast of the United States. Performance of cultivars has been described from U.S. Department of Agriculture (USDA) Zone 6b to USDA Zone 7b; however, there are no reports on how cultivars perform in USDA Zone 8. The current study was conducted to measure chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid content and assign visual color ratings to determine if there was a relationship between pigment values and perceived greenness, which generally is regarded as a desirable and potentially heritable trait. Total chlorophyll (P = 0.0051), carotenoids (P = 0.0266), and the ratio of total chlorophyll to carotenoids (P = 0.0188) exhibited a positive relationship with greenness after accounting for season and tree effects. In contrast, the ratio of chlorophyll a to chlorophyll b did not have an effect on greenness. There was a linear relationship between total chlorophyll and carotenoid regardless of season (summer R = 0.94; winter R = 0.88) when pooled across 2 years. The observed correlation between chlorophyll and carotenoid content suggests they could be used interchangeably as predictors of greenness. There were large differences in rainfall between the 2 years that may have resulted in additional variation. Furthermore, the climate in which the evaluation was conducted differs greatly from the native distribution of japanese-cedar occurring in China and Japan. Japanese-cedar [Cryptomeria japonica (L.f.) D. Don] is a variable conifer that grows up to 60 m tall in its native range. Wild-type specimens are conical when young and become cylindrical with age (Eckenwalder, 2009). Japanese-cedar traditionally has been used as screening or specimen plantings; however, there are a large number of cultivars displaying varying forms and growth rates (Rouse et al., 2000; Tripp, 1993) that may be used in diverse landscape situations. There are estimates of over 100 different ornamental cultivars (R. Determann, personal communication; Erhardt, 2005) with 45 of these grown in the eastern United States (Rouse et al., 2000). Japanese-cedars are native to the warmtemperate zones of south China and Japan. In Japan, they generally are limited to northfacing slopes that receive 180 to 300 cm of rainfall per year (Tsukada, 1967). Japanesecedars perform well under a number of environmental and soil conditions including the hot, humid summers and heavy clay soils of the southeast United States (Tripp and Raulston, 1992). As a result of this fact, japanese-cedars have been promoted as an alternative to Leyland cypress [·Cuprocyparis leylandii (A.B.Jacks. & Dallim.) Farjon (Farjon et al., 2002)], on which numerous problems now occur including bagworms (Thyridopteryx ephemeraeformis Haworth) (Lemke et al., 2005), fungal cankers caused by Seiridium Nees ex Link spp. and Botryosphaeria dothidea (Moug.) Ces. et De Not., and cercospora needle blight (Cercosporidium sequoiae Ellis and Everh.) (Martinez et al., 2009). Japanesecedars exhibit less susceptibility to bagworm infestations, and cultivars are available that have reduced interior branch death (Tripp and Raulston, 1992); however, the species is not problem-free. Redfire (Phyllosticta aurea C.Z. Wang) is a fungal pathogen that can attack stressed japanese-cedars and cause stem death, particularly on older foliage (Cox and Ruter, 2013; Tripp, 2005). Also, as Dirr (2009) notes, there is not a fast-growing, treelike cultivar that remains green during winter. Winter browning in japanese-cedar is often unsightly and undesirable to consumers, which may have contributed to why it has remained underused in landscapes. Winter browning in japanese-cedar occurs through the conversion of chloroplasts to chromoplasts during winter (Ida, 1981). This transition takes place only in sun-exposed leaves during periods of low temperature, indicating that photoinhibition likely plays a role (Han and Mukai, 1999; Ida, 1981). Plants have several mechanisms to cope with excess light during periods of low temperature when Calvin cycle activity is limiting, including reduction of chlorophyll, pH-dependent xanthophyll cycle, increased levels of carotenoids, and production of antioxidants or reactive oxygen species (ROS) scavenging enzymes. During winter, japanese-cedar has been shown to demonstrate two principle mechanisms to deal with excess light energy. The amount of chlorophyll decreases during winter (Han and Mukai, 1999; Ida, 1981) in both sunand shade-exposed leaves (Han et al., 2004). This occurs in both wild-type and nonbrowning mutants (Han et al., 2003), thus reducing the amount of energy absorbed. The other mechanism is the conversion of chloroplasts in sun-exposed leaves to rhodoxanthin-containing chromoplasts during winter to dissipate excess light energy as heat (Ida, 1981). In a study on 15-year-old japanese-cedar trees in Shizuoka Prefecture, Japan, Han et al. (2004) reported accumulation of rhodoxanthin in sun-exposed leaves beginning in January, reaching maximum levels in February, decreasing significantly in March, and falling to zero by April. However, timing of discoloring in winter and restoration in spring is highly variable and location-specific (personal observation). Han et al. (2003) demonstrated that wild-type leaves that accumulated rhodoxanthin maintained higher levels of photosynthesis with lower levels of zeaxanthinand antheraxanthin-dependent thermal dissipation than mutants that remained green all winter. The proposed role of rhodoxanthin is to intercept a portion of incident light to help maintain an appropriate balance among light absorption, thermal dissipation, and photosynthesis (Han et al., 2003). Japanese-cedar also accumulated substantial levels of xanthophyll cycle pigments and lutein during Received for publication 16 Sept. 2013. Accepted for publication 28 Oct. 2013. We thank Nancy Hand, Bruce Tucker, and Zhibing Xu for their technical assistance. Assistant Professor. Professor. PhD Candidate. To whom reprint requests should be addressed; e-mail [email protected]. 1452 HORTSCIENCE VOL. 48(12) DECEMBER 2013 winter (Han et al., 2003; Han and Mukai, 1999). The overarching objective of the current study was to identify an early predictor of winter foliage color (resistance to leaf browning) in japanese-cedar as a screening tool for identifying superior selections. Specifically, we assessed if quantitating pigments such as total chlorophyll (Ca+b), ratio of chlorophyll a (Ca):chlorophyll b (Cb), total carotenoids (Cx+c), and ratio of (Ca+b):(Cx+c) exhibited a strong relationship with greenness as measured by visual color rating at the University of Georgia Tifton Campus (USDA Zone 8b; USDA-ARS, 2012). Materials and Methods Plant material and growing conditions. Single plants of the following 12 taxa of japanese-cedar were randomly planted in 1997: ‘Araucariodes’, ‘Ben Franklin’, ‘Black Dragon’, ‘Cristata’, ‘Gyokruga’, ‘Rasen’, ‘Sekkan’, ‘Tansu’, ‘Tarheel Blue’, var. sinensis, ‘Yaku’, and ‘Yoshino’. These taxa were not meant to provide an evaluation of each per se as a result of single replicates; rather, they were used to provide a general sampling of the genotypic and phenotypic diversity observed in landscape forms of japanesecedar. Plants were maintained in field plots at the University of Georgia Tifton Campus (lat. 31 49’ N, long. 83 53’ W; USDA Zone 8b). Field soil was a Tifton loamy sand (fineloamy, siliceous, thermic Plinthic Paleudult), pH 5.2. Plots were fertilized in March every year after planting at a rate of 56 kg·ha nitrogen (N) using Super Rainbow 16N-1.8P6.6K plus minor elements (Agrium U.S. Inc., Denver, CO). An additional 28.5 kg·ha N was applied in late August each year after planting using the product mentioned previously. Southeast-facing branches were flagged during winter 2007–08 and material used for the duration of the study was collected from the same branches. Leaves were collected 8 Feb. 2008, 17 Aug. 2008, 9 Feb. 2009, and 5 May 2009 and frozen at –80 C until analysis. Temperature and precipitation data at the Tifton Campus for the duration of the study are included in Table 1. Supplemental irrigation was used only at the time of new plant establishment within plots. Chlorophyll and carotenoid extraction, analysis, and calculations. Three subsamples of leaf tissue were collected from the 12 individuals and Ca+b and Cx+c were extracted by grinding 85 mg leaf tissue three times in 3.33 mL 80% aqueous acetone and the extract was transferred to a test tube and brought to a final volume of 10 mL. After the third grind in acetone, the leaf material remaining was transferred to the test tube containing the extract and maintained in the dark at 4 C for 1 h to ensure complete extraction. Two milliliters of the extract was centrifuged for 30 s at 6800 gn. The supernatant was then transferred to a cuvette and absorbance was measured at 470 nm, 646 nm, and 663 nm using a GENESYS 10 Spectrophotometer (Thermo Electron Corp., Madison, WI). Absorbance for all samples at each wavelength was between 0.2 and 0.8. Determination of Ca, Cb, and Cx+c was performed using calculations from Lichtenthaler and Wellburn (1983). Ca content was calculated using the formula: Ca (mg·L ) = (12.25 · A663) – (2.79 · A646). Cb content was calculated using the formula: Cb (mg·L ) = (22.5 · A646) – (5.1 · A663). Ca+b content was determined by summing Ca and Cb values. Total carotenoid content was determined using the formula: Cx+c (mg·L ) = [1000 · A470 – (1.82 · Ca) – (85.02 · Cb)]/198. Chlorophyll and carotenoid contents were expressed in mg·g of dry weight after being corrected for moisture content (MC) as follows: three unanalyzed leaf subsamples were collected to determine mean MC for each individual (replicate) for each harvest date using the formula {MC = [(initial weight – dry weight)/initial weight] · 100}. Color rating. Plants were observed within 1 week of the four leaf collection dates. Five evaluators assigned color ratings from 1 (very brown/yellow; off color) to 5 (very green). Ratings of 3.5 to 4 would be considered acceptable for landscape use. All plants were addressed from the southeast side, directly in front of flagged branches. Mean rating for each individual was calculated and used for statistical analysis. Design and statistical analysis. The experimental design used repeated measurements across winter and summer on each individual with subsamples taken on the individual branches. Relevant covariate information, chlorophyll and carotenoid measurements, was obtained for each subsample, whereas the target response, color rating, was characterized at the individual level. To assess the relationship between chlorophyll and carotenoid measurements with the observed color rating of the trees, data were analyzed using a mixed model framework (PROC MIXED, SAS Version 9.3; SAS Institute Inc., Cary, NC). Random intercept models (linear models with random components with each variant of tree having a different intercept, but the slopes are assumed the same) were constructed to assess the relationship between the chlorophyll and carotenoid measurements with the observed greenness of the trees. These models were of the form: Greenijk 1⁄4 mþ treei þ seasonj þ b covariateijk þ Eijk , treei ;N 0,st ð Þ, Eijk ;N 0,sE ð Þ: (1) To determine an overall best model, Akaike’s Information Criterion (AIC) was used, in which a lower number represents a better fit. Also of interest in this research was to test for differences across the seasons in color ratings and chlorophyll and carotenoid easurements. For each measured pigment, a mixed-effects analysis of variance was constructed Pigmentijk 1⁄4 mþ treei þ seasonj þ Eijk , treei ;N 0,st ð Þ, Eijk ;N 0,sE ð Þ (2) Hence, examining season term for each of these five models established whether there were seasonal differences in each pigment. The previous two statistical analyses aggregated the subsamples such that a single observation per tree per period was used. To assess whether summer greenness measures are predictive of winter greenness, a simple regression model was used, for which summer and winter greenness ratings were aggregated for each individual. Given the experimental constraints in which only a single tree from each taxon could be planted, there is no replication of taxa. Hence, statistical methods cannot be used to determine Table 1. Temperature and precipitation data at the University of Georgia Tifton Campus for the duration of a study to evaluate pigments and color of Cryptomeria japonica from Nov. 2007 through Aug. 2009. Month Avg daily maximum temp ( C) Avg daily minimum