90 to detect N deficiencies and subsequently improve N manage- ment within the agricultural industry (Peterson et al. 1993; Smeal and Zhang 1994; Balasubramanian et al. 2000). The use of a chlorophyll SPAD meter for monitoring the N status of woody plants under field conditions has received limited attention (Loh et al. 2002). Several studies have shown that carotenoids are vitally impor- tant in protecting the leaf photosynthetic apparatus, especially photosystems I and II, against photoinhibition under prolonged environmental stress by interconversions among the xanthophyll molecules (Hall and Rao 1999; Ort 2001). In the xanthophyll cycle, violaxanthin goes through de-epoxidation to give rise to anteroxanthin and finally zeaxanthin that, together with a low pH within the photosynthetic membrane, facilitate the harmless dis- sipation of excess excitation energy directly within the light- harvesting chlorophyll antennae (Havaux 1988; Ramalho et al. 2000). Therefore, an indirect, nondestructive quantification of total leaf carotenoids will prove important for stress-related stud- ies (Torres Netto et al. 2005). Chlorophyll fluorescence, an indication of the fate of excita- tion energy in the leaf photosynthetic apparatus, has been used to provide a rapid and nondestructive diagnostic system of detect- ing and quantifying physiologic injury in tree leaves and needles (photosynthetic organs) under low temperatures, salinity, and water stress conditions (Sestak and Stiffel 1997; Percival and Fraser 2001; Percival and Sheriffs 2002; Percival and Henderson 2003). Little information associating fluorescence values as mea- sures of damage to the leaf photosynthetic system and readings with a chlorophyll SPAD meter exist (Torres Netto et al. 2005). Association between SPAD and fluorescence can be important to determine SPAD values associated with reductions in leaf pho- tosynthetic properties induced by nutrient deficiency-related dis- orders. MATERIALS AND METHODS Plant Material and Growth Conditions Mature, fully expanded leaves near the top of the canopy (gen- erally about the fourth leaf from the apex) of five sycamore (Acer pseudoplatanus), beech (Fagus sylvatica), and English oak (Quercus robur) displaying a range of visually different leaf colors indicating a range of chlorophyll concentrations were se- lected. In all cases, leaf tissue was collected and sampled in late June 3 months after leaf flush, a time when leaves show maxi- mum photosynthetic performance (Kitao et al. 1998). All trees were located in commercial plantings at the University of Read- ing campus (51°43 N, –1°08 W). The sampled leaves were transported in insulated boxes sheltered from light and all ma- terial prepared within 2 hr of collection. SPAD Readings The mean of three readings from a portable Minolta chlorophyll meter SPAD-502 (Spectrum Technologies, Inc., Plainfield, IL, U.S.) was obtained for each leaf disc from individual leaves (10 leaves per tree) and pooled to obtain one SPAD measurement per disc. The leaf disc used to obtain a SPAD value provided suffi- cient tissue for total chlorophyll and carotenoid content as well as chlorophyll fluorescence Fv/Fm quantification. In the case of total foliar N content, however, six leaf discs were required. Consequently, SPAD values for all six discs (60 leaves per tree) were pooled for statistical purposes when comparing SPAD measurements versus total leaf N content. SPAD values were ©2008 International Society of Arboriculture Percival et al.: Quantifying Nutrient Stress in Foliar Tissue measured at the midpoint of the leaf next to the main leaf vein. This position was selected because examination of the relation- ship between SPAD readings taken at different positions on a leaf concluded this position most closely correlated with total leaf N and protein content as well as plant yield (Hoel 1998). This position was also most convenient from a practical point of view (Hoel 1998). Total Leaf Chlorophyll and Carotenoid Content Quantification was obtained by measurement of absorbance at 663, 645, and 480 nm in a spectrophotometer (PU8800 Pye Unicam, Portsmouth, U.K.) after extraction with 80% v/v aque- ous acetone. Chlorophyll a, chlorophyll b, and total carotenoid concentrations were determined according to the equation of Lichtenthaler and Wellburn (1983). Nitrogen Content Assessment Six leaf discs per N analysis were thoroughly washed and then dried in a convection oven at 85°C (185°F) for 48 hr before grinding through a 0.5 mm (0.02 in) cyclone mill (Retsch, Middlesborough, U.K.). Each six discs were placed in specific groups according to the SPAD-502 reading ranges (0–10, 11–20, 21–30, 31–40, 41–50, 51–60). Samples were placed into 150 mL (4.5 fl oz) volumetric flasks and digested in 20 mL (0.6 fl oz) of 7:1 nitric/perchloric acid. After cooling, the solutions were brought to volume with deionized water and analyzed by induc- tively coupled plasma-emission spectroscopy elemental analysis. Nutrient values were expressed as percent total leaf dry weight. Chlorophyll Fluorescence Fv/Fm Measurements Chlorophyll fluorescence was determined on leaf discs using a dark-acclimated Handy Plant Efficiency Analyzer chlorophyll fluorometer (Hansatech Instruments, King’s Lynn, Norfolk, U.K.). The initial fluorescence (F0) and maximum fluorescence (Fm) were analyzed and quantum efficiency of open photosystem II centers–quantum yield (Fv/Fm) calculated. The leaf discs were previously adapted to the dark for 30 min so that all the centers of photosystem II (PSII) were at an open stage (all the primary acceptors oxidized) and energy dissipation through heat was minimal. The F0 was obtained with low-intensity light (less than 0.1 mol/m−2/s−1) not to induce any effect in the fluorescence variable. The Fm was obtained by a continuous light excitation (at 2500 mol/m2/s–1) provided by an array of six LEDs focused on the leaf surface to provide homogeneous irradiation over a 4 mm (0.16 in) diameter leaf surface. The fluorescence variable (Fv) was calculated from the difference between Fm and F0. The Fv and Fm values were used to obtain the Fv/Fm ratio. Statistical Analysis Correlation equations and coefficients of multiple determina- tions (r2) were calculated using the curve-fitting feature of Gen- stat V using quadratic polynomial, logarithmic, exponential growth or decay or simple linear models as appropriate accord- ing to which model gave the highest percentage variation, i.e., goodness of fit, accounted for. RESULTS AND DISCUSSION SPAD versus Total Chlorophyll Figure 1 shows the relationships between SPAD readings and total leaf chlorophyll concentrations in three tree species. A polynomial quadratic mathematical model best fit the relation-
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