1. Sulfur Fertility Influences Kale Flavor But Not Carotenoid Content David Kopsell, 1 Dean Kopsell, 1 Joanne Curran-Celentano, 2 William Randle, 3 Timothy Coolong, 3 & Carl Sams 4 1 Department of Plant Biology, The University of New Hampshire, Durham, NH; 2 Department of Animal and Nutritional Sciences, The University of New Hampshire, Durham, NH; 3 Department of Horticulture, The University of Georgia, Athens, GA; 4 The Department of Plant Sciences and Landscape Systems, The University of Tennessee, Knoxville, TN Project Summary Dietary intake of carotenoids has been associated with reduced disease risk. Kale (Brassica oleracea L. Acephala Group) has the highest reported levels of the carotenoids lutein and  -carotene. Brassica vegetables also contain glucosinolate (GS) and S-methyl-cysteine sulfoxide (MCSO) that produce bitter, acrid flavors which consumers find objectionable. Therefore, the objective of this study was to investigate the influence of sulfur (S) fertility level on GS, MCSO, and carotenoid accumulation in kale. ‘Winterbor’, ‘Redbor’, and ‘Toscano’ kale were greenhouse- grown using nutrient solution culture with S treatments of 4, 8, 16, 32, and 64 mg S·L -1. Levels of GS and MSCO decreased in response to decreasing S in nutrient solution. However, accumulation of lutein and  -carotene were unaffected by S treatment. Lowering S fertility in the production of kale should decrease the negative flavors associated with high levels of GS and MCSO without affecting carotenoid pigments levels. Introduction Sulfur (S) may be incorporated into a wide range of secondary plant compounds, such as glucosinolates (GS) and S-methyl-cysteine sulfoxide (MCSO), responsible for characteristic odors and flavors of Brassica vegetables. Carotenoid pigments protect photosynthetic plant structures by dissipating excess light energy (1) and binding singlet oxygen to inhibit oxidative damage (2). Examples of carotenoids include lutein and  -carotene. Dietary intake of lutein,  -carotene, and other carotenoids has been associated with reduced risk of lung cancer and chronic eye diseases, including cataract and age-related macular degeneration (3). Green leafy vegetables are rich in dietary carotenoids, and kale (B. oleracea L. Acephala Group) ranks high in lutein and  -carotene content. Because GS and MCSO S-containing compounds in B. oleracea can impart negative flavor attributes, understanding how they can be lowered is important for consumer acceptability. The goal of this study was to measure the effect of S fertility on: GS MCSO Lutein  -carotene accumulation in the leaf tissues of three kale cultivars. Materials and Methods Plant Culture ‘Winterbor’, ‘Redbor’, and ‘Toscano’ kale were greenhouse grown (22  C day/14  C night) under natural photoperiods (lat. 43  09’N) in 30 L of a half- strength modified Hoagland’s nutrient solution. Plants were grown under increasing S treatment concentrations at 4, 8, 16, 32, and 64 mg S·L -1. The experimental design was a split-plot, with S treatment as the main plot and kale cultivar as the subplot. Each treatment consisted of 5 plants per cultivar replicated four times. Glucosinolate and MCSO Determination Glucoiberin, glucobrassicin, neoglucobrassicin, 4- methoxyglucobrassicin, and 4-hydroxyglucobrassicin were extracted from freeze dried kale leaf tissue and quantified using high performance liquid chromatography (HPLC; 4). Methyl cysteine sulfoxide concentration was determined from dried kale leaf tissue using HPLC (5). Carotenoid & Pigment Determination Carotenoids were quantified from freeze dried tissue according to the method of Beecher and Howard (USDA Food Composition Laboratory, Beltsville, MD; 6). Literature Cited (1)Frank, H.A.; Cogdell, R.J. Carotenoids in photosynthesis. Photochem. Photobiol. 1996, 63, 257-264. (2)Tracewell, C.A.; Vrettos, J.S.; Bautista, J.A.; Frank, H.A.; Brudvig, G.W. Carotenoid photooxidation in photosystem II. Arch. Biochem. Biophysic. 2001, 385, 61-69. (3)Le Marchand, L.; Hankin, J.H.; Kolonel, L.N.; Beecher, G.R.; Wilkens, L.R.; Zhao, L.P. Intake of specific carotenoids and lung cancer risk. Cancer Epidemiol. Biomarkers Prev. 1993, 2, 183-187. (4) Hansen, M.; Møller, P.; Sørensen, H.; M. Cantwell de Trejo. Glucosinolates in broccoli stored under controlled atmosphere. J. Amer. Soc. Hort. Sci. 1995, 120, 1069-1074. (5) Edwards, S.J.; Musker, D.; Collin, H.A.; Britton, G. The analysis of the S-alk(en)yl- L-cysteine sulphoxides (flavour precursors) from species of Allium by high performance liquid chromatography. Phytochem. Anal. 1994, 5, 4-9. (6) Khachik, F.; Beecher, G.R.; Whittaker, N.F. Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J. Agr. Food Chem. 1986, 34, 603-616. (7)Drewnowski, A. Taste preferences and food intake. Annu. Rev. Nutr. 1997, 17, 237-253. Acknowledgements This project was funded in part by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under agreement No. 2001-52102-11254. This research has been accepted for publication by the Journal of Agricultural and Food Chemistry from the American Chemical Society. Conclusions Consumers often cite the bitter flavor of raw Brassica vegetables as unpleasant or objectionable. It is important to consider the sensory response to foods when developing strategies aimed at improving dietary quality (7). The lower S treatment levels reduced GS and MCSO content in kale. This should decrease the bitter and unpleasant flavors associated with eating raw Brassica. Lowering S fertility did not affect lutein and  -carotene levels, thereby preserving the health benefits of carotenoid consumption. Understanding the combined impact of production practices on flavor and nutrition may help improve consumer acceptance of phytonutritionally-enhanced vegetable crops. Methyl-Cysteine Sulfoxide 48 16 3248 mg/g dry tissue On-farm demonstration of influence of S on kale flavor at Roots & Fruits Farm, Dalton, NH. mg/100 g dry tissue 48163248 Glucosinolates 48163248 Sulfur treatment (mg/L) Leaf Tissue Sulfur % Sulfur in leaves Lutein  -carotene mg/100 g fresh tissue 48163248 48163248 Experimental Results Sulfur Compounds Percent S in kale leaf tissue increased linearly in response to increasing S treatment. In general, the concentration of most GS compounds decreased in response to decreasing S treatments. Methyl-cysteine sulfoxide decreased linearly in all the cultivars in response to decreasing S availability. Decreases in GS and MCSO compounds paralleled decreases in leaf tissue %S. Carotenoid Compounds Lutein and  -carotene pigments did not change in response to decreasing S concentrations in nutrient solutions. Values for lutein and  -carotene were within previously reported ranges for field grown kale. Lower S fertility in kale production would provide more palatable raw produce, while still providing beneficial dietary lutein and  -carotene. ‘Toscano’ kale growing at UNH Research Greenhouse. Sulfur treatment (mg/L)