HORTSCIENCE 40(5):1493–1498. 2005. in plants can be strongly infl u- enced by genotype and environment (Rosa et al., 1997). Of the environmental effects, S and Yield and N fertility have been shown to signifi cantly affect glucosinolate concentration in plant Concentrations as Affected by tissue. In an insect feeding study, nigra (L.) defi cient in S and adequate in N had Nitrogen and Sulfur Fertility lower levels of allyl and higher levels of feeding by Spodoptera eridiana than C.J. Rosen1 plants with low N and adequate S (Wolfson, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108 1982). In oilseed rape (Brassica napus L.), high glucosinolates can reduce meal quality, V.A. Fritz2 and G.M. Gardner2 and studies have shown that high N and low University of Minnesota, 1790 Folwell Avenue, St. Paul, MN 55108 S fertility can minimize glucosinolate content of the seed (Zhao et al.,1993,1994). The effect S.S. Hecht, S.G. Carmella, and P.M. Kenney of N supply on glucosinolates depended on S The Cancer Center, University of Minnesota, St. Paul, MN 55108 supply (Zhao et al., 1993). When soil S was defi cient, addition of N decreased glucosinolate Abstract. Glucosinolates are a class of nitrogen (N) and sulfur (S) containing compounds concentration. When soil S was suffi cient, ad- shown to have cancer-preventing properties in animal models and widely found in crucifer- dition of N increased glucosinolate concentra- ous plants. The overall objective of this study was to determine whether N and S fertility tion or had no effect. Under constant S supply, affects glucosinolate concentrations in cabbage (Brassica oleracea L. Capitata group). Field increasing N up to 150 kg·ha–1 increased seed studies on a sandy soil low in available N and S were conducted over a 2-year period with glucosinolates of oilseed rape, but there was both green (‘Grand Slam’) and red (‘Vorox’) cabbage cultivars. Treatments evaluated each no effect at higher N rates (Bilsborrow et al., –1 –1 year were the interactive effects of N (125 and 250 kg·ha ) and S (0, and 110 kg·ha ) fertil- 1993). Decreasing the N to S ratio in nutrient izer application. Yield of both cabbage cultivars increased with increasing N and S in the solution tended to increase the phenylethyl second year of the study, but not in the fi rst. Tissue N concentrations in heads at harvest isothiocyanate concentration (a breakdown increased with N application and tissue S concentrations increased with S application. When product of ) in watercress plants S was not applied, tissue S decreased signifi cantly as N rate increased, while N rate had no (Palaniswamy et al., 1995). Similarly, increas- effect on tissue S concentrations when S was applied. The dominant glucosinolate detected ing S fertilization increased glucoraphanin in both cabbage cultivars was glucobrassicin, with indole forms accounting for about 80% and glucobrassicin concentrations in of the total glucosinolates regardless of treatment. Tissue N was negatively correlated and fl orets (Krumbein et al., 2001). In contrast, tissue S and S to N ratio were positively correlated with total glucosinolate concentration, Vallejo et al. (2003) reported that S fertilization 2 although all correlations were generally weak (r < 0.5). Total glucosinolates and glucobras- on a clay soil, in general, had no effect on total sicin concentrations were maximized in both cultivars at the low N and high S application glucosinolates in edible portions of broccoli. rates. Except for sinigrin in one of the 2 years, all glucosinolates detected were higher in However, in that study, calcium sulfate, which Vorox than in ‘Grand Slam’. Based on these results, glucosinolates in cabbage can be ma- is only sparingly soluble, was used as the S nipulated by cultural management practices as well as genetics. source, and it was not reported whether the soil used in the study was defi cient or low in S to Glucosinolates are secondary nitrogen One major positive attribute of glucosino- begin with. Addition of S fertilizer to a soil (N) and sulfur (S) containing compounds in lates is that they have been shown to inhibit already suffi cient in S would not be expected plants that have been studied extensively in the activity of some chemical carcinogens. to have signifi cant effects on S nutrition and recent years for their effects on animal health. The plant enzyme is released upon glucosinolate synthesis. While glucosinolates are known to be present consumption of glucosinolate containing veg- Cabbage (Brassica oleracea L. Capitata in 16 families of plants, most of the edible etables and catalyzes glucosinolate hydrolysis group) is one of the most widely consumed cru- glucosinolate-containing species are found in (Fenwick et al., 1983). Products of hydrolysis ciferous plants in the U.S. (Lucier and Plummer, the family of Cruciferae (Fahey et al., 2001). depend on the structure of the glucosinolate. 2003). However, relatively few studies have About 120 glucosinolate compounds have been Alkyl and arylalkyl glucosinolates yield mainly been conducted to determine environmental identifi ed in plants, although there is only lim- upon myrosinase-catalyzed conditions that affect glucosinolate concentra- ited information on the specifi c effects of each hydrolysis, whereas indolyl glucosinolates tions in edible portions of the cabbage plant. compound and their breakdown products. (glucobrassicins) yield primarily indole-3-car- Rosa and Rodrigues (1998) reported that total Activity of glucosinolates has been linked binol or related substituted indole-3-carbinols and individual glucosinolates in root and leaf to increased insect resistance (Fenwick et al., (Fahey et al., 2001; Fenwick et al., 1989; Mc- tissue of cabbage seedlings varied with time 1983; Wolfson, 1982) as well as stimulation Danell et al., 1988). Isothiocyanates as well as of day. Larger variations were found at 30 °C of insect feeding (Fenwick et al., 1983). The indole-3-carbinol are chemopreventive agents (stress temperature) compared to 20 °C (opti- compounds and breakdown products have also against carcinogenesis of the lung and other mum temperature). In a comprehensive review, been shown to be allelopathic and increase tissues in laboratory animals, and they exert Rosa et al. (1997) reported that the dominant disease resistance of plants (Angus et al., 1994; protective effects such as inhibition of car- glucosinolates in white and red cabbage are Bending and Lincoln, 1999; Rosa et al., 1997). cinogen activating enzymes, enhancement of glucobrassicin and related indole compounds, When ingested, some glucosinolates have been carcinogen detoxifying enzymes, and induction as well as the aliphatic compounds sinigrin shown to impart a bitter fl avor, cause goitro- of apoptosis (Hecht, 1999). However, indole- and glucoiberin. Other aliphatic glucosinolates genic effects, and reduce meal quality of animal 3-carbinol has also been shown to have tumor present in red cabbage include glucoraphinin, feed (Drewnowski and Gomez-Carneros, 2000; promoting activity in animal feeding studies gluconapin, and gluconapoleiferin. Hecht Fenwick et al., 1983). (Fahey and Stephenson, 1999; Hecht, 1999), et al. (2004) reported that glucobrassicans although results are often confl icting (Fahey were the major glucosinolates in cabbage as Received for publication 28 Oct. 2004. Accepted et al., 2001). Chemopreventive properties well as other grown in for publication 16 Dec. 2004. This research was of glucosinolates and their metabolites have Singapore, accounting for 70% to 93% of the conducted with support from the Minnesota Research been the motivation for recent efforts to better Fund (formerly the SOTA TEC Fund) and is grate- total glucosinolates. fully acknowledged. understand factors that affect their production Manipulating N and S fertility may be one 1Dept. of Soil, Water, and Climate. within the plant. means of altering glucosinolate concentrations 2Dept. of Horticultural Science. The concentration and chemical form of and profi les in cabbage and thereby potentially

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AAugustBook.indbugustBook.indb 11493493 66/14/05/14/05 112:25:272:25:27 PPMM increasing health benefi ts of consuming this replications. Fertilizer was the main plot and or stored at –20 °C for later analyses. Further vegetable. The overall objective of this study cabbage cultivar was the subplot. Each main washing of the columns showed no desulphated was to determine the effects of N and S fertil- plot consisted of four rows, 6 m in length, with glucosinolates, indicating complete elution. ity on yield and glucosinolate concentration the two varieties randomized in the middle of A desulphosinigrin standard curve was made profi les of green and red cabbage grown under the four rows. The fi rst and fourth rows were with 0.5 mL of sinigrin (Cat. # 85440; Sigma) fi eld conditions. border rows. Spacing within rows was 0.45 m solutions in water in fi ve concentrations ranging and between rows was 0.9 m. Urea was used from 0.6 to12.2 µg·mL–1. In total, 0.8 units of Materials and Methods as the N source for the zero S treatments, and the sulfatase was added and allowed to react ammonium sulfate was used as the N and S overnight. The next day, 100 µL of acetonitrile Field studies were conducted in 2001 and source for the N and S treatments. Urea was was added to stop the reaction, and the enzyme 2002 at the Sand Plain Research Farm in used to equalize N application as needed in the was removed by ultrafi ltration through 30,000 Becker, Minn., on a Hubbard loamy sand soil N and S treatments. Nitrogen and S fertilizers MW cut off fi lters (Amicon fi lters, cat. # 4101; (sandy, mixed, frigid Entic Hapludolls). This were applied in three equal applications at 10, Millipore, Bedford, Mass.). The fi ltered solu- site was selected because of its inherently low 17, and 24 d after planting in 2001 and 7, 15, tions were analyzed by HPLC and the standard background levels of soil N and S. For each and 24 d after planting in 2002. Because a curve was constructed. growing season, cabbage was planted in differ- small amount of N (18 kg·ha–1) was applied to Analyses were performed on a model ent areas within the farm following a previous all plots in 2001, this amount was subtracted 510 pump (Waters Corp., Milford, Mass.), crop of rye. A factorial treatment arrangement from the sidedress applications so that total a HP-1100 autosampler (Hewlett-Packard, of two cultivars (‘Vorox’ and ‘Grand Slam’), N applied within each treatment and year was Wilmington, Del.), and a SPD-10A vp variable two S rates (0 and 110 kg·ha–1), and two N the same. Irrigation was applied after each wavelength detector (Shimadzu; Wilmington, rates (125 and 250 kg·ha–1) were tested in all fertilizer application and as needed to prevent Del.) set at λ = 229 nm. The column was a possible combinations each year. water stress. Luna C18, 5 µm, 250 × 4.6 mm (Phenomenex, Selected soil properties before planting in ‘Grand Slam’ was harvested 4 Sept. 2001 Torrance, Calif.). The gradient conditions were 2001–02 were as follows (0–15 cm): organic and 17 Sept. 2002. ‘Vorox was’ harvested 24 as follows: solvent A = water and solvent B = matter, 2.4%–2.2%; pH (1 soil : 1 water) Sept. 2001 and 4 Oct. 2002. At harvest, the acetonitrile; 0 to 2 min, 5% B to 15% B; 2 to 5.7–6.8; Bray P, 45–44 mg·kg–1; ammonium middle eight heads from each harvest row 30 min, 15% B to 65% B; 30 to 35 min, 65% acetate extractable K, Ca, and Mg, 108–100, were cut, trimmed, and weighed. In 2001, one B to 90% B; 35 to 37 min, 90% B to 5% B; 527–836, and 81–174 mg·kg–1, respectively; head from each plot was stored at 2 °C for at and 37 to 60 min, 5% B. The fl ow rate was 1.0 –1 calcium phosphate extractable SO4-S, 4–5 most 2 weeks until glucosinolate extraction mL·min , and 100 µL of each sample (the 3 mg·kg–1; DTPA Zn, Fe, Cu, and Mn, 0.5–0.4, could be performed. Similar procedures were mL SAX collection) was injected. 34–15, 0.5–0.3, and 9–16 mg·kg–1 respectively; followed in 2002 except two heads from each Data were collected and integrated on Peak hot water extractable boron, 0.3–0.3 mg·kg–1. plot were stored for glucosinolate extraction. Simple software (SRI Instruments, Torrance, Water extractable nitrate-N in the top 60 cm In both years, two additional heads were Calif.). Desulphated glucosinolates were of soil in 2001 and 2002 was 15 and 9 kg·ha–1, chopped and then transferred to a forced air identifi ed by retention time, using standards respectively. All analyses were carried out dryer set at 60 °C. Dried tissue was ground kindly provided as a gift from Richard Mithen using methods described in Dahnke (1988). with a Wiley mill to pass through a 0.4-mm in Norwich, U.K. The amounts of each desul- Before planting in 2001, 18 kg·ha–1 N, 20 screen and saved for N and S determination phated glucosinolate were calculated based kg·ha–1 P, 150 kg·ha–1 K, and 2.5 kg·ha–1 B and using combustion techniques (Hern, 1984; on the desulphated sinigrin standard curve 1100 kg·ha–1 fi nely ground dolomitic limestone Horneck and Miller, 1998). and the published UV response factors of the were applied and then incorporated to a depth Glucosinolates were determined using pro- other desulphoglucosinolates relative to the of 15 cm with a fi eld cultivator. Before planting cedures described by Hecht et al. (2004) with desulphated sinigrin (Offi cial Journal of the in 2002, 20 kg·ha–1 P, 125 kg·ha–1 K, and 2.5 slight modifi cations. For glucosinolate extrac- European Communities, 1990; Lewis and kg·ha–1 B were applied and incorporated with a tion, each head in cold storage was weighed Fenwick, 1988). The following glucosinolates fi eld cultivator. Because pH was in the optimum and one quarter of the head was placed into were consistently detected in cabbage head range, lime was not needed in 2002. three times the weight per volume of boiling tissue over the 2-year study: 4-hydroxybutyl ‘Grand Slam’ green cabbage and ‘Vorox’ water and boiled for 5 min to deactivate the (4HOB), (PG), glucoraphanin (GR), red cabbage (Jordan Seed, Woodbury, Minn.) myrosinase. The hot cabbage was placed in a sinigrin (SN), gluconapin (GNP), 4-hydroxy- were seeded in 50-count transplant fl ats con- blender and ground for two minutes. Aliquots glucobrassicin (4HOGB), glucobrassicin (GB), taining moist soilless seeding media (SunGro of 100 mL were stored at –20 °C until analysis 4-methoxyglucobrassicin (4MGB), and 1- Horticulture, SunShine SB-300 Universal, could be performed. Cabbage blend (5 mL) was methoxyglucobrassicin (1MGB). Desulphated Bellevue, Wash.) and grown in the greenhouse homogenized for 2 min using a Janke-Kundel glucosinolate peaks were later confi rmed by for 5 weeks. Transplants were planted at Becker Turrax T 25 homogenizer set at 12,000 rpm, UV spectroscopy using a Waters 996 photo- on 2 July 2001 and 8 July 2002. A transplant at 4 °C. The homogenate was then centrifuged diode array detector. solution was prepared with Diazinon AG500 at 4000 rpm, 4 °C. To monitor precision of the results, aliquots at 0.16 mL·L–1 water to control root maggots Strong anion exchange (SAX) solid phase of a large sample of ‘Vorox’ from the 2001 and 11N–21P–7K at 7 g·L–1 as a liquid starter extraction cartridges (500 mg, Supelco; Sigma study was chosen as a quality control refer- fertilizer. About 250 mL of the solution was Corp., St. Louis, Mo.) were used to bind the ence standard for the 2002 study. The ‘Vorox’ applied to the base of the plants after setting glucosinolates and, after sulfatase treatment, reference standard was homogenized, aliquots in the fi eld. All planting operations were done to elute them as desulphoglucosinlates. First, made, stored at –20 °C and then included with by hand. Insects and weeds were controlled columns were conditioned with 2 mL of 0.5 each set of samples assayed in 2002. In all cases using standard commercial practices (Foster, M sodium acetate, pH 4.6, followed by 2 mL the level of total glucosinolates in the ‘Vorox’ 2002). of water. Next, 500 µL of cabbage supernatant reference standard in 2002 was within 15% Four fertilizer treatments tested were 1) was applied to the SAX columns, followed of the mean found in 2001. All glucosinolate 125 kg·ha–1 N and 0 kg·ha–1 S; 2) 125 kg·ha–1 by 1 mL of 0.02 M sodium acetate, pH 4.0. data are expressed on fresh weight basis. Data N and 110 kg·ha–1 S; 3) 250 kg·ha–1 N and 0 Then 3.1 units of sulfatase (S-9626; Sigma) for 2001 are based on one head per plot, while kg·ha–1 S; 4) 250 kg·ha–1 N and 110 kg·ha–1 in 1 mL of water was added, drained to within in 2002; data are based on the average of two S. These rates were selected based on N and about 3mm of the packing, and allowed to heads per plot. S fertilizer recommendations of 200 kg·ha–1 react overnight. The next day, the column Testing for signifi cance of year, cultivar, N N and 35 kg·ha–1 S for cabbage in this re- was eluted with 3 mL water and the collected rate, S rate, and interactions among effects on all gion (Rosen and Eliason, 1996). Treatments volume was determined by weight. This col- measured variables was done using analysis of were set out as a split plot design with four lection was either analyzed directly by HPLC variance (ANOVA). Data were analyzed using

1494 HORTSCIENCE VOL. 40(5) AUGUST 2005

AAugustBook.indbugustBook.indb 11494494 66/14/05/14/05 112:25:302:25:30 PPMM the PROC GLM procedure in SAS (SAS, Cary, with increasing N rate in both cultivars, but the had signifi cantly lower tissue S than ‘Vorox’ N.C). When interactions from ANOVA were magnitude of response was greater in ‘Grand (0.47% S vs. 0.58% S), but at the high S rate, signifi cant, LSMEANS was used to determine Slam’ (2.07% vs. 2.71% N) than in ‘Vorox’ cultivar effects on tissue S were not signifi cant differences among means within each interaction (2.31% vs. 2.57% N). The generally lower tis- (average 0.73% S). The numerous interactions at the 5% probability level. Linear correlation sue N concentration in 2002 compared to 2001 make generalizations diffi cult to make, but as and regression analysis were used to examine was likely due to nitrate losses from excessive with N, lower S concentrations in head tissue relationships between tissue concentrations of N rainfall that occurred in 2002. The range of N in 2002 compared to 2001 were likely due to and S and total glucosinolate concentrations. concentrations found in head tissue is consistent losses of sulfate through leaching with exces- with a range of 1.82% to 2.28% N reported sive rainfall in 2002. Bernard et al. (1990) Results and Discussion by Peck (1981) for cabbage grown with 150 reported a range of 0.74% to 0.80% S in head and 300 kg·ha–1 N respectively. Bernard et al. tissue from plants grown with grown with 0 Yield response. Cabbage yield response to (1990) reported a range of 2.20% to 2.64% N kg·ha–1 S, 180 kg·ha–1 N and 100 kg·ha–1 S, 180 N and S rate depended on year, with the year × in head tissue from plants grown with 90 to kg·ha–1 N, respectively. The higher concentra- N rate and year × S rate interactions signifi cant 270 kg·ha–1 N, respectively. Rhoads and Olson tion of S in their 0 S treatments compared to at the 5% and 1% level, respectively (Table 1). (2001) reported ranges of 2.17% to 2.63% N the present study is likely due to higher initial Yields increased signifi cantly with increasing in head tissue from plants grown with 84 and soil S conditions, although soil test informa- N rate in 2002 (30.8 vs. 40.0 Mg·ha–1), but not 168 kg·ha–1 N, respectively. tion was not provided in their report. Rhoads in 2001 (average, 42.0 Mg·ha–1). Similarly, Sulfur concentrations in head tissue at and Olson (2001) reported an S concentration yields increased signifi cantly with increasing harvest depended on year, variety, and S rate of 0.57% to 0.69% S from plants grown with S rate in 2002 (32.3 vs. 38.5 Mg·ha–1), but not with signifi cant N rate × S rate, year × S rate, grown with 0 kg·ha–1 S, 84 kg·ha–1 N and 22 in 2001 (average, 39.8 Mg·ha–1). Differences year × cultivar, cultivar × N rate, and cultivar kg·ha–1 S, 84 kg·ha–1 N, respectively. between years in yield response to N and S × S rate interactions. When S was not applied, Glucosinolate profi les and concentrations. treatments can be explained by differences in tissue S decreased signifi cantly as N rate Indole glucosinolates accounted for about 80% rainfall during each growing season. In 2001, increased from 125 to 250 kg·ha–1 (0.57% vs. to 85% of the total glucosinolates measured in total rainfall from planting to harvest was 19.4 0.48% S), while N rate had no effect on tissue both cultivars tested (Table 2). The proportion cm. The highest event over a 24-h period was S concentrations when S was applied (average, of indole and aliphatic glucosinolates in head 3.0 cm, which occurred 26 d after transplanting. 0.74%). Tissue S concentrations increased tissue varied with year and was not consistently In contrast, total rainfall in 2002 from planting signifi cantly with increasing S rate each year, affected by fertilizer treatment or cultivar. Year to harvest was 53.9 cm with four major leaching but as with N concentrations, magnitude of × S rate, year × cultivar, and year × cultivar × N events of 5.5, 3.8, 11.9, and 10.5 cm occurring response was greater in 2001 (0.66% vs. 0.95% rate interactions were signifi cant. In 2001, the 3, 17, 27, and 59 d after transplanting, respec- S) than in 2002 (0.38% vs. 0.52% S). ‘Vorox’ proportion of indole and aliphatic glucosinolates tively. The water holding capacity within the had signifi cantly higher S concentrations than was not affected by cultivar or N rate (Table 3). top 30 cm on a Hubbard loamy sand ranges ‘Grand Slam’ in 2001 (0.88% vs. 0.73% S), However, in 2002, there was a higher proportion from 3 to 4 cm. Because both nitrate and sulfate but there were no differences between the two of indole glucosinolates and lower proportion anions are susceptible to leaching losses, yield cultivars in 2002 (average, 0.45% S). Increas- of aliphatic glucosinolates in ‘Vorox’ compared response to increasing rates of these nutrients ing N rate from 125 to 250 kg·ha–1 decreased to ‘Grand Slam’. In addition, there was no would be expected in years when excessive S concentrations in ‘Vorox’ (0.70% vs. 0.63% effect of N rate on the proportion of indole or rainfall occurs, particularly on a sandy soil S), but had no effect on ‘Grand Slam’ (aver- aliphatic glucosinolates in ‘Vorox’, but in ‘Grand such as the one used in this study. The year × age, 0.59% S). At the low S rate, ‘Grand Slam’ Slam’ the proportion of indole glucosinolates cultivar interaction was also signifi cant with ‘Grand Slam’ yields signifi cantly higher than Table 1. Effect of year, cultivar, nitrogen rate, and sulfur rate on cabbage head yield, nitrogen concentrations, and sulfur concentrations. ‘Vorox’ yields each year, but the difference between the two cultivars was greater in 2002 Source (44.4 vs. 26.4 Mg·ha–1) than in 2001 (45.3 of Fresh wt N S –1 vs. 34.3 Mg·ha–1). Rhoads and Olson (2001) variation (Mg·ha ) (%) (%) reported that cabbage grown in a low S soil Year 2001 39.9 2.88 0.81 in Florida responded to N fertilization only 2002 35.4 1.94 0.45 if S was also applied. This N × S interaction Signifi cance NS ** ** was not observed in the present study and Cultivar may be due to higher organic matter contents Grand Slam 44.8 2.39 0.59 in Minnesota soils compared to Florida soils. Vorox 30.2 2.44 0.67 Some S may be released during organic matter Signifi cance ** NS ** mineralization. N rate (kg·ha–1) Tissue nitrogen and sulfur concentra- 125 34.0 2.19 0.65 tions. Nitrogen concentrations in head tissue 250 41.0 2.64 0.61 Signifi cance ** ** NS at harvest depended on year, variety, N rate, S rate (kg·ha–1) and S rate with signifi cant year × N rate, year 0 36.1 2.50 0.52 × S rate, year × cultivar, and cultivar × N rate 110 39.2 2.33 0.74 interactions (Table 1). Tissue N concentrations Signifi cance NS ** ** increased signifi cantly with increasing N rate Interactions each year, but the magnitude of response was N rate × S rate NS NS ** greater in 2001 (2.60% vs. 3.16% N) than in Year × N rate * * NS 2002 (1.77% vs. 2.12% N). Nitrogen concentra- Year × S rate ** ** ** tions decreased with increasing S rate in 2001 Year × N rate × S rate NS NS NS Year × cultivar ** ** ** (3.03% vs. 2.74% N), but were not signifi cantly Cultivar × N rate NS ** * affected by S rate in 2002 (average, 1.95% N). Year × cultivar × N rate NS NS * ‘Vorox’ had signifi cantly higher N concentra- Cultivar × S rate NS NS NS tions than ‘Grand Slam’ in 2001 (3.11% vs. Year × cultivar × S rate NS NS NS 2.66% N) but signifi cantly lower concentra- Cultivar × N rate × S rate NS NS NS tions than ‘Grand Slam’ in 2002 (1.77% vs. Year × cultivar × N rate × S rate NS NS NS 2.12% N). Tissue N concentrations increased NS,*,**Nonsignifi cant or signifi cant at P ≤ 0.05 or 0.01, respectively.

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AAugustBook.indbugustBook.indb 11495495 66/14/05/14/05 112:25:332:25:33 PPMM Table 2. Effect of year, cultivar, nitrogen rate, and sulfur rate on glucosinolate concentrations in cabbage head tissue at harvest. Proportion of Source Glucosinolatez concn (µmol/100 g fresh wt) glucosinolates of Aliphatic Indole (% of total) variation 4HOB PG GR SN GNP 4HOGB GB 4MGB 1MGB Total Aliphatic Indole Year 2001 14.2 1.4 4.3 13.4 2.1 5.2 173.6 22.4 14.4 251.0 13.5 86.5 2002 1.8 3.7 5.3 33.5 1.8 16.2 144.8 37.3 15.1 259.5 20.0 80.0 Signifi cance ** ** NS ** NS ** NS ** NS NS ** ** Cultivar Grand Slam 4.1 1.5 0.9 25.0 0.4 3.0 82.7 28.3 12.1 158.0 19.1 80.9 Vorox 11.9 3.5 8.7 21.9 3.5 18.4 235.6 31.4 17.5 352.4 14.3 85.7 Signifi cance ** ** ** * ** ** ** NS ** ** ** ** N rate (kg·ha–1) 125 9.2 2.9 5.3 25.5 2.2 9.6 180.0 31.6 17.5 283.7 17.3 82.7 250 6.9 2.2 4.3 21.4 1.7 11.8 138.4 28.1 12.1 226.8 16.2 83.8 Signifi cance ** * NS * * * * NS * * NS NS S rate (kg·ha–1) 0 7.4 2.1 3.9 20.2 1.7 10.1 137.3 29.6 13.4 225.7 16.0 84.0 110 8.6 3.0 5.7 26.7 2.2 11.3 181.0 30.1 16.2 284.8 17.4 82.6 Signifi cancey NS ** ** ** * NS * NS NS * NS NS Interactions N rate × S rate ** NS NS NS NS NS NS NS NS NS NS NS Year × N rate * NS NS NS NS ** NS NS * NS NS NS Year × S rate NS ** * ** NS NS NS NS NS NS * * Year × N rate × S rate * NS NS NS * * NS NS NS NS NS NS Year × cultivar ** NS NS ** NS ** NS ** NS * ** ** Cultivar × N rate NS NS NS NS NS NS NS NS * NS NS NS Year × cultivar × N rate NS * * * * NS NS NS NS NS * * Cultivar × S rate NS NS NS NS NS NS NS NS NS NS NS NS Year × cultivar × S rate NS NS NS NS NS NS NS NS NS NS NS NS Cultivar × N rate × S rate NS NS NS NS NS NS NS NS NS NS NS NS Year × cultivar × N rate × S rate NS NS NS NS NS NS NS NS NS NS NS NS z4HOB = 4-hydroxybutyl, PG = progoitrin, GR = glucoraphanin, SN=sinigrin , GNP = gluconapin (GNP), 4HOGB = 4-hydroxyglucobrassicin, GB = glucobras- sicin (GB), 4MGB = 4-methoxyglucobrassicin, and 1MGB = 1-methoxyglucobrassicin. NS,*,**Nonsignifi cant or signifi cant at P ≤ 0.05 or 0.01, respectively.

increased (proportion of aliphatic glucosinolates dominant glucosinolate in cabbage by Hecht glucobrassicin appear to be maximized under decreased) with increasing N rate (Table 3). In et al. (2004), but sinigrin was reported to be conditions of high S fertility and moderate 2002, increasing S rate increased the proportion the dominant glucosinolate by Kushad et al. levels of N fertility. The effect of increasing of aliphatic glucosinolates (18% vs. 22%) and (1999). Differences in results could be due in glucosinolate concentrations with increasing decreased the proportion of indole glucosino- part to differences in cabbage cultivars tested. S rate is consistent with previous reports on lates (82% vs. 78%), but in 2001 the effect was While glucosinolates have been associated S fertility in a number of cruciferous crops not signifi cant (13% average for aliphatic and with cancer prevention, some have also been grown on low S soils (Krumbein et al., 2001; 87% average for indole glucosinolates). The associated with bitter taste. Of interest is the Zhao et al., 1993). The decrease in glucosino- results in 2002 are similar to those of Zhao et observation that taste panelists often associated lates with increasing N rate is also consistent al. (1994). They reported that S application to bitter taste with sinigrin more than glucobras- with previous results on low S soils, but we S defi cient rapeseed plants generally resulted sicin (Drewowski and Gomez-Carneros, 2000). did not observe a signifi cant S rate × N rate in an increase in the proportion of aliphatic Newer cultivars of Brassica spp. may be lower interaction as reported by Zhao et al. (1993) glucosinolates and a decrease in the proportion in sinigrin to reduce bitterness. in oilseed rape. They found that increasing N of indole glucosinolates. They also reported Main effects of cultivar, N rate, and S rate rate increased glucosinolates when adequate that increasing N rate increased the proportion signifi cantly affected total glucosinolate and S was supplied, but decreased glucosinolates of indole glucosinolates relative to aliphatic glucobrassicin concentrations in head tissue. when S supply was defi cient. Further studies glucosinolates when S was defi cient, but N For total glucosinolates, the year × cultivar with cabbage using a wider range of N and S rate had little effect when S was suffi cient. Our effect was signifi cant, but this was due to dif- rates may be warranted to more clearly deter- study found no effect of N rate on the propor- ferences in magnitude rather than direction. mine if a signifi cant N × S interaction exists. tion of indole and aliphatic glucosinolates with In both years total glucosinolates were lower In addition, because only one or two heads varying S rate. in ‘Grand Slam’ than in ‘Vorox’, but the dif- were sampled per plot in the present study, Total and individual glucosinolate concen- ferences were greater in 2001 (133 vs. 368 composite samples of several heads may be trations in cabbage head tissue were variably µmol/100 g fresh weight) than in 2002 (183 warranted in future studies to obtain better affected by year, cultivar, N rate, and S rate and vs. 336 µmol/100 g fresh weight). For gluco- representation of each treatment. their interactions (Table 2). Of the individual brassicin, all interactions were nonsignifi cant Even though total glucosinolate concentra- glucosinolates detected, glucobrassicin was as was the effect of year. Averaged over years tions increased with increasing S fertilization, present in highest concentrations and domi- and fertilizer treatments, ‘Vorox’ had higher the overall relationship between S concentra- nated the effects on total glucosinolates. In a concentrations of glucobrassicin than ‘Grand tions (%) in head tissue and total glucosinolate study evaluating the effect of sulfur, cultivar, Slam’. Averaged over years, cultivar, and N concentrations (µmol/100 g fresh weight) was and season on glucosinolate profi les in broccoli, rate, increasing S rate signifi cantly increased weak when combined over years, cultivars, and Vallejo et al. (2003) reported numerous inter- total glucosinolate and glucobrassicin con- fertilizer treatments with an r2 of 0.04 and a actions among the variables tested on gluco- centrations. In contrast, when averaged over slope of 121 (n = 64). The reason for the lack sinolate concentrations with indole compounds years, S rate, and cultivar, increasing N rate of correlation was due to a wide disparity in found in highest concentrations regardless of decreased total glucosinolate and glucobras- tissue S over the 2 years. Tissue S concentra- treatment. Glucobrassicin was found to be the sicin concentrations. Total glucosinolates and tions in 2001 were nearly twice those found

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AAugustBook.indbugustBook.indb 11496496 66/14/05/14/05 112:25:362:25:36 PPMM Table 3. Proportion of aliphatic and indole glucosinolates in cabbage as affected by the year × cultivar × J.M. Desmarchelier. 1994. Biofumigation: nitrogen rate interaction. Isothiocynates released from Brassica roots Variable Glucosinolate (%) inhibit growth of take-all fungus. Plant and Soil. 162:107–112. –1 Year Cultivar N rate (kg·ha ) Aliphatic Indole Bending, G.D. and S.D. Lincoln. 1999. Characterisa- 2001 Grand Slam 125 11.5 cz 88.5 a tion of volatile sulphur containing compounds 2001 Grand Slam 250 11.6 c 88.4 a produced during decomposition of Brassica 2001 Vorox 125 15.8 c 84.2 a juncea tissues in soil. Soil Biol. Biochem. 2001 Vorox 250 15.0 c 85.0 a 31:695–703. 2002 Grand Slam 125 29.6 a 70.4 c Berard, L.S., M. Senecal, and B. Vigier. 1990. Effects 2002 Grand Slam 250 23.9 b 76.1 b of nitrogen fertilization on stored cabbage. II. 2002 Vorox 125 12.1c 87.9 a Mineral composition in midrib and head tissues 2002 Vorox 250 14.3c 85.7 a of two cultivars. J. Hort. Sci. 65:409–416. zMeans within a column followed by the same letter are not signifi cantly different at P = 0.05. Bilsborrow, P.E., E.J. Evans, and F.J. Zhao. 1993. The infl uence of spring nitrogen on yield, yield in 2002, yet total glucosinolate concentrations interactions. In 2002, ‘Grand Slam’ had higher components and glucosinolate content of autumn were similar. Similarly, the overall relationship concentrations than ‘Vorox’. This was the only sown oilseed rape (Brassica napus). J Agr. Sci. between total glucosinolate concentrations and case where a glucosinolate was found in higher Cambr. 120:219–224. N concentrations was also weak with an r2 of concentrations in ‘Grand Slam’ than in ‘Vorox’. Dahnke, W.C. 1988. Recommended chemical soil test procedures for the north central region. N.Dak. 0.01 and a slope of –22 (n = 64). The relation- Also in 2002, the higher N rate resulted in lower Agr. Expt. Sta. Bul. 499. ship between total glucosinolate concentrations SN concentrations in ‘Grand Slam’, but N rate Drewnowski, A. and C. Gomez-Carneros. 2000. and S to N ratio was higher than the individual had no effect in ‘Vorox’ (data not presented). Bitter taste, phytonutrients, and the consumer: A elements with an r2 of 0.12 and a slope of 608 In 2001, ‘Vorox’ had higher SN concentrations review. Amer. J. Clinical Nutr. 72:1424–1435. (n = 64), but was still relatively weak. Stronger than ‘Grand Slam’ with no effect due to N rate in Fahey, J.W. and K.K Stephenson. 1999. Cancer che- relationships between total glucosinolates and either cultivar. Sinigrin increased with increas- moprotective effects of cruciferous vegetables. tissue S, N and S to N were obtained when ing S rate in 2002, but was not signifi cantly HortScience 34:1159–1163. they were calculated using individual cultivars affected by S rate in 2001 (data not presented). Fahey, J.W., A.T. Zalemann, and P. Talalay. 2001. within years (data not presented). All slopes 4-hydroxybutyl glucosinolate was strongly The chemical diversity and distribution of glu- cosinolates and isothiocynates among plants. were positive for relationships between total affected by year with higher concentrations Phytochemistry 56:5–51. glucosinolates and tissue S and S to N ratios found in 2001 compared to 2002. Cultivar Fenwick, G. R., R. K. Heaney, and W. J. Mullin. 1983. and negative for relationships with tissue N. effects were only signifi cant in 2001 with Glucosinolates and their break down products The highest r2 obtained was 0.50 for ‘Grand higher 4-HOB concentrations found in ‘Vorox’ in food and food plants. Crit. Rev. Food Sci. Slam’ in 2001 for the relationship between total compared to ‘Grand Slam’. The N rate × S rate Nutr. 18:123–201. glucosinolates and S to N ratio (n = 16, slope interaction was signifi cant in 2001 but not in Fenwick, G.R., R.K. Heaney, and R. Mawson. 1989. of 338 and intercept of 36). While some trends 2002. In 2001, increasing N decreased 4HOB Glucosinolates, p. 2–41. In: P.R. Cheeke (ed.). were evident, these results suggest that use of glucosinolate concentrations at low S (18 vs. 9 Toxicants of plant origin. vol 2. . CRC tissue N and S alone cannot be used to predict µmol/100 g fresh weight) but N rate had no ef- Press, Boca Raton, Fla. Foster, R. 2002. Midwest vegetable production guide total glucosinolate levels in cabbage. fect when S was applied (average 15 µmol/100 for commercial growers. Univ. Minn. Ext. Serv. The other indole glucosinolates were g fresh weight). Glucoraphinin, gluconapin, BU-07094-S. p. 49–56. present at much lower concentrations than and progoitrin were present at relatively low Hecht, S.S. 1999. Anticarcinogenesis by isothiocy- glucobrassicin. 1-Methoxyglucobrassicin con- concentrations both years and generally fol- nates, indole-3-carbinol, and Allium thiols, p. centrations were higher in ‘Vorox’ than ‘Grand lowed trends of the other glucosinolates with 306–333. Proc. DFG Sympo. Carcinogenic/Anti- Slam’ and increasing N rate from 125 to 250 higher concentrations in ‘Vorox’ compared to carcinogenic Factors in Foods: Novel Concepts. kg·ha–1 decreased concentrations in ‘Vorox’ ‘Grand Slam’ and lowest concentrations with Wiley VCH. (22 vs. 13 µmol/100 g fresh weight), but not low S and high N fertility. Hecht S.S., S.G. Carmella, P.M. Kenney, S. Low, in ‘Grand Slam’ (average, 12 µmol/100 g fresh K. Arakawa K, and M.C. Yu . 2004. Effects of cruciferous vegetable consumption on urinary weight). Nitrogen rate also tended to decrease Conclusions metabolites of the tobacco-specifi c lung carcino- 1MGB concentrations more in 2001 than in gen 4-(methylnitrosamino)-1-(3-pyridyl)-1-bu- 2002 (data not presented). 4-Methoxygluco- Cabbage yield increased with N and S tanone in Singapore Chinese. Cancer Epidemiol. brassicin concentrations were higher in ‘Vorox’ fertilizer applications in one of the 2 years of Biomarkers Prev. 13:997–1003. than ‘Grand Slam’ in 2002 (27 vs. 18 µmol/100 the study. The responsive year occurred with Hern, J.L. 1984. Determination of total sulfur in plant g fresh weight), but both cultivars had similar excessive rainfall through the growing period. materials using an automated sulfur analyzer. concentrations in 2001 (average 37 µmol/100 The results of this research clearly demonstrate Commun. Soil Sci. Plant Anal.15:99–107. g fresh weight). 4-Hydroxyglucobrassicin that glucosinolate concentrations in cabbage Horneck, D.A. and R.O. Miller. 1998. Determination concentrations depended on year, N rate, and of total nitrogen in plant tissue. In: Y.P. Kalra grown on a low S and N soil can be manipulated (ed.). Handbook of reference methods for plant cultivar with signifi cant year × cultivar, year × by soil fertility and genetics. ‘Vorox’, a red analysis. CRC Press, Boca Raton, Fla. N rate and year × N rate × S rate interactions. cabbage, consistently had higher glucosinolate Krumbein, A., I. Schonhof, J. Ruhlmann, and S. ‘Vorox’ had higher 4HOGB concentrations concentrations compared to ‘Grand Slam’, a Widell. 2001. Infl uence of sulphur and nitrogen than ‘Grand Slam’ in both years but the mag- green cabbage. Under conditions of this study, supply on fl avour and health-affecting com- nitude of the differences was greater in 2002 indole forms were the dominant glucosino- pounds in , p. 294–295. In: W.J. than in 2001 (data not presented). In 2001, N lates detected and maximized at the low N Horst et al. (eds). Plant nutrition—Food security rate had no effect on 4HOGB concentrations fertilizer application rate and high S fertilizer and sustainability of agro-ecosystems through at either S rate. In 2002, 4HOGB increased application rate. Interactions between N and S basic and applied research. Kluwer, Dordrecht, with increasing S rate at the high N rate, but The Netherlands. fertility on glucosinolate concentrations were Kushad, M.M., A.F. Brown, A.C. Kurilich, J.A. it was not affected by S rate at the low N rate not signifi cant; however further studies with Juvik, B.P. Klein, M.A. Wallig, and E.H. Jeffery. (data not presented). cabbage using a wider range of N and S rates 1999. Variation in glucosinolates in vegetable Of the aliphatic glucosinolates, sinigrin was may be warranted to more clearly determine if crops of Brassica oleracea. J. Agr. Food Chem. consistently found in highest concentrations a signifi cant N × S interaction exists. 47:1541–1548. over the 2-year study. Sinigrin concentrations Lewis, J. and G.R. Fenwick. 1988. Glucosinolate con- were signifi cantly affected by year, cultivar, N Literature Cited tent of Brassica vegetables—Chinese Pe-tsi (Brassica pekinensis) and Pak-choi (Bras- rate, and S rate with signifi cant year × S rate, Angus, J.F., P.A. Gardner, J.A. Kirkegaard, and year × cultivar, and year × cultivar × N rate sica chinensis). J. Sci. Food Agr. 45:379–386.

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AAugustBook.indbugustBook.indb 11497497 66/14/05/14/05 112:25:422:25:42 PPMM Lucier, G. and C. Plummer. 2003. Vegetables and and Health. Amer. Soc. Plant Physiol. Benavente-Garcia and C. Garcia-Viguera. 2003. melons outlook. Electronic outlook report from Peck, N.H. 1981. Cabbage plant responses to nitrogen Total and individual glucosinolate contents in the economic research service. USDA VGS-297. fertilization. Agon. J. 73:679–684. infl orescences of eight broccoli cultivars grown http://www.ers.usda.gov/publications/vgs/ Rhoads, F.M. and S.M. Olson. 2001. Cabbage re- under various climatic and fertilization condi- jun03/vgs297.pdf. sponse to sulfur source and nitrogen rate. Soil tions. J. Sci. Food Agr. 83:307–313. McDanell, R., A.E. McLean, A.B. Hanley, R.K. Crop Sci. Soc. Flla. Proc. 60:37–40. Wolfson, J.L. 1982. Developmental responses of Heaney, and G.R. Fenwick. 1988. Chemical Rosa, E.A., R.K. Heaney, G.R. Fenwick, and C.A. and Spodoptera eridania to envi- and biological properties of indole glucosino- M. Portas. 1997. Glucosinolates in crop plants. ronmentally induced variation in Brassica nigra. lates (glucobrassicins): A review. Food Chem. Hort. Rev. 19:99–215. Environ. Entomol. 11:207–213. Toxicol. 26:57–90. Rosa, E. A. and P. M. Rodrigues. 1998. The effect of Zhao, F., E.J. Evans, P.E. Bilsborrow, and J.K. Offi cial Journal of the European Communities. 1990. light and temperature on glucosinolate concentra- Syers. 1993. Infl uence of sulphur and nitrogen L170, 03.07.27-34. Offi c. J. Euro. Commun. tion in the leaves and roots of cabbage seedlings. on seed yield and quality of low glucosinolate Palaniswamy, U., R. McAvoy, B. Bible, S. Singha, J. Sci. Food Agr. 78:208–212. oilseed rape (Brassica napus L). J. Sci. Food and D. Hill. 1995. Phenylethyl isothiocyanate Rosen, C.J. and R.D. Eliason. 1996. Nutrient man- Agr. 63:29–37. concentration in watercress (Nastutium offi cinale agement for commercial fruit and vegetable Zhao, F., E.J. Evans, P.E. Bilsborrow, and J.K. Syers. R.Br.) is altered by the nitrogen and sulfur ratio crops in Minnesota. Univ. Minn. Ext. Ser. Bul. 1994. Infl uence of nitrogen and sulphur on the in hydroponic solution, p. 280–284. In: DL BU-05886. glucosinolate profi le of rapeseed (Brassica napus Gustine and HE Flores (eds.). Phytochemicals Vallejo, F., F.A. Tomas-Barberan, A. Gonzalez L). J. Sci. Food Agr. 64:295–304.

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