The Eþect of Light and Temperature on Glucosinolate Concentration in the Leaves and Roots of Cabbage Seedlings

J Sci Food Agric 1998, 78, 208È212 The Eþect of Light and Temperature on Glucosinolate Concentration in the Leaves and Roots of Cabbage Seedlings Eduardo A S Rosa* and Paula M F Rodrigues Section of Horticulture, Department of Field Crops, Universidade de Tra s-os-montes e Alto Douro, Apartado 202, 5001 Vila Real Codex, Portugal (Received 17 April 1997; revised version received 23 January 1998; accepted 8 February 1998) Abstract: In previous studies it was shown that the concentration of total and individual glucosinolates in brassicaceous plants can vary signiðcantly over a 24-h period grown either in the Ðeld or under controlled conditions. The present study shows total and individual glucosinolate variation during a single day. Seedlings of cabbage grown under controlled conditions and at 14 and 15 days after emergence were moved to 20 C (Exp A) and 30 C (Exp B), with a constant photosynthetic photon Ñux density of 480 lmol m~2 s~1 and 75% relative humidity, over a 2-day period, during which time aerial parts and roots were sampled at regular intervals. Whilst the glucosinolate patterns of the aerial part of the plant and of the roots remained the same, the levels of major glucosinolates in the aerial part, averaged over all sampling times and 2 days, were 233 ^ 60 lmol 100 g~1 DW for 3-methylsulphinylpropyl and 72 ^ 22 for 2- propenyl; in the roots, 2-phenylethyl and 1-methoxyindol-3-ylmethyl showed the highest average concentrations, with 678 ^ 355 lmol 100 g~1 DW and 411 ^ 122, respectively. Total and individual glucosinolate levels showed very high signiðcant di erences between the two plant parts. Despite the constant temperature, light and relative humidity, glucosinolates varied within a 24-h period, showing ultradian rhythms that are common to several metabolic processes in plants. The results conðrm previous observations that at a temperature of 20 C, close to the optimum for growth and development, the diurnal variation in glucosinolate concentration, was smaller than at 30 C. ( 1998 Society of Chemical Industry J Sci Food Agric 78, 208È212 (1998) Key words: glucosinolate; temperature; light; relative humidity INTRODUCTION Glucosinolates are naturally occurring compounds in brassicaceous plants which, on hydrolysis by the enzyme myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1), liberate, in addition to sulphate ion and glucose, a range of compounds which may include isothiocyanates, nitriles and thiocyanates. These hydrolysis products are associated with several biological e ects that may be either beneðcial, such as the anticarcinogenic activity of isothiocyanates, or detrimental (eg the goitrogenic properties of thiocyanates and oxazolidine-thiones). Such e ects have been described in * To whom correspondence should be addressed. several reviews (Rosa et al 1997; Fenwick et al 1983; Duncan 1991), which also discuss the role of glucosinolates on insect behavior such as attraction, oviposition and feeding deterrence, matters of major concern in an environmental agriculture system. Glucosinolate concentrations have been shown to vary not only between plant species but also between individual parts of the same plant, throughout the growth period and between growing seasons and years (Macfarlane Smith and Grif- Ðths 1988; McGregor 1988; Clossais-Besnard and Larher 1991; Fieldsend and Milford 1994a,b; Rosa et al 1996), suggesting that climate factors are involved in such variation (Rosa et al 1994, 1996; Rosa 1997). Since brassicaceous plants are grown all year around in many parts of the world and under the most diverse climate 208 ( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain

E ect of light and temperature on glucosinolate concentration 209 conditions, it is likely that plant products might have di erent glucosinolate concentrations according to the climate in which they are produced and even the period of the day in which they are harvested. SigniÐcant changes in glucosinolate concentration between growing seasons and during a 24 h period might be reñected in the biological properties of brassicaceous plants. In previous work it was shown that glucosinolates in the leaves of young cabbage plants grown under Ðeld conditions can vary within a single day, suggesting a rapid metabolism of these compounds (Rosa et al 1994). In a more recent study under a constant 14 h photoperiod (0700È2100) and two temperature regimes (20 and 30 C), the authors demonstrated variation in total and individual glucosinolate for both aerial parts and roots of developing young cabbage plants (Rosa 1997). The present work describes the e ect of two constant regimes (20 and 30 C) on total and individual glucosinolate levels throughout a 24 h period under controlled constant conditions of light at a photosynthetic photon Ñux density of 480 lmol m~2 s~1 and relative humidity at 75%. Plant material MATERIALS AND METHODS The young plants of the pointed cabbage (Brassica oleracea var capitata cv Duchy F1) were grown in polypropylene trays with alveoles of 65 cm3 Ðlled with a mixture of 1 vol sand (0É02È0É2 mm) to 3 vol peat compost (Humobact Terreau, Frans Baele, Bailleul, France). Seeds were placed at a depth of 1É5 cm and covered with vermiculite. Trays were placed in a growth cabinet (Conviron E15) under climatic controlled conditions of a 14 h photoperiod (0700È2100), corresponding to a photosynthetic photon Ñux density of 480 lmol m~2 s~1, 75% relative humidity and 25 C during the light cycle and 18 C during the dark cycle. At 12 days after emergence (DAE), light and temperature regimes were changed to a constant 480 lmol m~2 s~1 and 20 C, respectively (Exp A). After 48 h, sampling was started with samples collected at 0200, 0600, 1000, 1400, 1800 and 2200 over a period of two consecutive days (14 and 15 DAE). Another study (Exp B) was conducted in the same way as Exp A, except that temperature was kept at a constant 30 C, with light and relative humidity exactly the same as in the previous experiment. Chemical analysis In both experiments, plants were divided into aerial parts and roots and analysed separately. At harvest, the plants which were at 3È4 true leaf stage, lifted from the alveoles and the intact aerial part was cut at the soil level and immediately frozen in liquid nitrogen and freeze-dried. The roots were separated from the peat compost by soaking in water and gently washed in a continuous water Ñow to remove all peat residues with minimal damage. After removing excess water with absorbent paper, the roots were frozen in liquid nitrogen and freeze-dried. A total of six plants were used in each of three replicates. Plants were randomly selected, ensuring that at each sampling time each plant was surrounded by others to avoid border e ects. The freezedried material was reduced to a Ðne powder and samples (about 200 mg) were extracted by addition of boiling 90% methanol (3 ml plus 0É4 lmol benzyl glucosinolate as an internal standard) and maintaining boiling for 10 min, a procedure which simultaneously inactivates myrosinase. After Ðltration, the residue was re-extracted twice using boiling 70% methanol (3 ml). The extracts were combined to give a Ðnal volume of 10 ml and an aliquot (3 ml) was evaporated to dryness and taken up in water (3 ml), of which 2 ml was applied to small columns of DEAE Sephadex A 25 and the absorbed glucosinolate desulphated as described by Heaney and Fenwick (1980). Desulphoglucosinolates were eluted with water and analysed using the highperformance liquid chromatography (HPLC) procedure described by Spinks et al (1984). Glucosinolate concentration was expressed in lmol 100 g~1 DW. Statistical analysis was done using a SuperAnova 1.1 software. RESULTS AND DISCUSSION The glucosinolate patterns of the aerial part and the roots of the cabbage plants were similar in both Exp A and B, which is contrary to previous studies (Rosa 1997). The di erence between the two studies may be due to the use of di erent cultivars of the same species, which are likely to a ect glucosinolate patterns (Rosa et al 1997). The major glucosinolates in the aerial part of the cabbage seedlings (Figs 1 and 2), were 3-methylsulphinylpropyl, 2-propenyl and indol-3-ylmethyl, with minor amounts of 2-hydroxybut-3-enyl, 1-methoxyindol-3-ylmethyl, 4-hydroxyindol-3-ylmethyl and 2- phenylethyl glucosinolates. In the roots (Figs 1 and 2), 2-phenylethyl, 1-methoxyindol-3-ylmethyl, 2-propenyl and indol-3-ylmethyl were the main glucosinolates, with minor amounts of 3-methylsulphinylpropyl, 2- hydroxybut-3-enyl and 4-hydroxyindol-3-ylmethyl glucosinolates. The major glucosinolates in both parts of the plant were the same as in a previous study (Rosa 1997). As previously reported (Rosa 1997), the mean standard errors for total and individual glucosinolates in the roots were larger than for the aerial part of the plant, probably due to inevitable damage during the cleaning

210 E A S Rosa, P M F Rodrigues Fig 1. Individual and total glucosinolate variation in the aerial part (K) and roots (L) throughout a 24 h period at a constant temperature of 20 C, 480 lmol m~2 s~1 and 75% relative humidity (Exp A). When SE bars are not shown, the SE is smaller than the symbols. process of the roots leading to some glucosinolate degradation. Negligible mean standard errors were determined for the aerial part which required only minimal cleaning prior to extraction. Total glucosinolates In both experiments (Figs 1 and 2), the levels of total glucosinolates in the roots, averaged over all sampling times, were signiðcantly higher (P \ 0É001) than in the leaves. Higher levels for glucosinolates in roots were Fig 2. Individual and total glucosinolate variation in the aerial part (K) and roots (L) throughout a 24 h period at a constant temperature of 30 C, 480 lmol m~2 s~1 and 75% relative humidity (Exp B). When SE bars are not shown, the SE is smaller than the symbols. also reported in previous studies (Josefsson 1967; Rosa 1997). When comparing the same plant part for total glucosinolate levels at 20 and 30 C, no signiðcant di erences were noted in leaves (386 ^ 71 lmol 100 g~1 DW in Exp A vs 409 ^ 104 in Exp B) whilst, in roots, total glucosinolate levels at 30 C (1635 ^ 684 lmol 100 g~1 DW) were signiðcantly higher (P \ 0É01) than at 20 C (1342 ^ 269 lmol 100 g~1 DW). Although these results are in agreement with previous studies conducted in the Ðeld (Rosa et al 1996) in which summer conditions tend to increase the glucosinolate levels (between 4 and 35%

E ect of light and temperature on glucosinolate concentration 211 in the leaves of Ðve Brassica species) when compared to winter seasons, they showed less magnitude of variation probably because the di erences in the temperature regimes were lower than in the Ðeld experiment and because all the other climatic factors were kept constant. In the present study, glucosinolate levels tend to be higher at higher temperature, suggesting that temperature (the only variable) could be responsible for such di erences. Consideration of total glucosinolate levels between sampling times at 20 C (Exp A) showed no signiðcant di erences in the leaves and signiðcant variation (P \ 0É05) in the roots; at 30 C (Exp B) the variation of total glucosinolate levels between sampling times in both parts of the plant was larger (P \ 0É001), probably as a result of temperature stress. These Ðndings are in agreement with previous results (Rosa 1997), further demonstrating that photoperiod is likely to have little interference in glucosinolate variation, since such variation is independent of light/dark conditions. Figure 1 shows that, in the present study, total glucosinolate concentration in the leaves in Exp A are fairly stable; in the roots, there was a sinusoidal-type trend with peaks at 0600 and 2200 following the major glucosinolates 2- phenylethyl and 1-methoxyindol-3-ylmethyl. In Exp B, at a constant 30 C (Fig 2), total glucosinolate concentration in the leaves showed an upward trend also with a sinusoidal-type curve; for concentration, in the roots, this curve type was much clearer, with peaks at 0200 and 1400. This also reñected the trend in the concentration of the major glucosinolate. This type of evolution curve corresponds to that described in the ultradian rhythms which have been reported to occur in plants for several metabolic processes such as glycolysis, sap Ñow, enzyme activity and protoplasmic streaming in less than 20 h (Salisbury and Ross 1992). This occurs even under constant illumination and steady conditions, after plants have been tuned to natural conditions. Thus, it is likely that glucosinolates and their precursors will follow a similar trend, since many photoresponses in plants become so enmeshed with the daily change in environmental factors that they become self-sustaining. Di erences between the evolution curves at both temperature regimes might reñect the sensitivity to temperature of most of the ultradian rhythms. Individual glucosinolates The concentration of the major individual glucosinolate, 3-methylsulphinylpropyl, was signiðcantly higher (P \ 0É001) in the aerial part (228 ^ 55 lmol 100 g~1 DW in Exp A and 238 ^ 67 lmol 100 g~1 DW in Exp B) than in the roots (94 ^ 39 lmol 100 g~1 DW in Exp A and 103 ^ 53 lmol 100 g~1 DW in Exp B) when averaged over all sampling times. This tendency had been noted in previous studies (Rosa 1997). When comparing 3-methylsulphinylpropyl glucosinolate levels within the same plant part at the two temperature regimes, no signiðcant di erences were noted. At a constant 20 C and constant light and relative humidity throughout the day (Fig 1), an inverse relationship was noted between the aerial part and roots, with no signiðcant variations between sampling times in both parts of the plant. This Ðnding suggests, as indicated for total glucosinolates, an ultradian rhythm of the 3- methylsulphinylpropyl glucosinolate associated with a turnover between roots and aerial part. When seedlings were submitted to 30 C (Exp B, constant light and relative humidity) (Fig 2), signiðcant di erences (P \ 0É001) were noted between sampling times in both parts of the plant, with levels in the leaves being characterised by an upward trend. Thus, it is likely that, at a temperatures close to stress levels, plants respond with an increase in 3-methylsulphinylpropyl glucosinolate, which is in agreement with previous Ðndings (Rosa 1997). On the other hand, when all the three climatic parameters (temperature, permanent light and relative humidity) were kept constant and under the optimum growing temperatures, 3-methylsulphinylpropyl glucosinolate levels were relatively steady. This is contrary to the results observed when plants were submitted to the same conditions except photoperiod (light cycle 0700È 2100) (Rosa 1997). Thus, light may interfere in the variation of this glucosinolate. Despite its lower concentration, 2-propenyl glucosinolate showed a similar trend to 3-methylsulphinylpropyl in both experiments. However, in this case, the levels in the roots (113 ^ 43 lmol 100 g~1 DW in Exp A and 151 ^ 83 lmol 100 g~1 DW in Exp B) were signiðcantly higher (P \ 0É001) than in the aerial part of the plant (67 ^ 18 lmol 100 g~1 DW in Exp A and 78 ^ 25 lmol 100 g~1 DW in Exp B). The levels of 2- propenyl glucosinolate in both aerial parts and roots varied signiðcantly (P \ 0É001) when temperature was changed from 20 to 30 C. At a constant 20 C and constant light and relative humidity, no signiðcant variations were noted throughout the day in either part of the plant, but, when temperatures was changed to 30 C, the variations were signiðcant (P \ 0É001), as observed in total and 3-methylsulphinylpropyl glucosinolates. These observations and previous results (Rosa 1997) indicate that, under normal growing temperatures, photoperiod has an inñuence on glucosinolate variation throughout the day; at stress temperatures, variations are larger and are almost completely dependent on temperature. This masks the e ect of photoperiod. The inverse relationship between the aerial part and roots suggested in the previous study (Rosa 1997) was only partly supported at a constant 20 C and constant light and relative humidity (Fig 1), probably as an e ect of permanent light. Indol-3-ylmethyl glucosinolate generally follows the same trend as the other two major glucosinolates in the aerial part of the plant and in the roots. When averaged

212 E A S Rosa, P M F Rodrigues over all sampling times and in both experiments, levels in the roots (127 ^ 50 lmol 100 g~1 DW) were signiðcantly higher (P \ 0É001) than in the aerial part (71 ^ 17 lmol 100 g~1 DW). SigniÐcant di erences (P \ 0É001) were noted between the two temperature regimes in the aerial parts (67 ^ 13 lmol 100 g~1 DW in Exp A and 75 ^ 21 lmol 100 g~1 DW in Exp B) and roots (89 ^ 22 lmol 100 g~1 DW in Exp A and 165 ^ 39 lmol 100 g~1 DW in Exp B). However, throughout the day, signiðcant di erences (P \ 0É05) were noted only for the aerial part of the plant at 20 C; at 30 C and despite the large variation in the roots, no signiðcant di erences were noted due to the large SE errors. The concentration of the two major glucosinolates in the roots, 2-phenylethyl and 1-methoxyindol-3-ylmethyl were signiðcantly higher (P \ 0É001) than in the aerial parts where levels were less than 7 lmol 100 g~1 DW. Levels of both glucosinolates at a constant 20 C were signiðcantly lower than at 30 C (629 ^ 181 lmol 100 g~1 DW vs 727 ^ 475 lmol 100 g~1 DW for 2- phenylethyl and 391 ^ 83 lmol 100 g~1 DW vs 431 ^ 152 lmol 100 g~1 DW for 1-methoxyindol-3- ylmethyl). When plants were submitted to 30 C, changes in levels of 2-phenylethyl glucosinolate throughout the day were larger (P \ 0É001) than at 20 C. Thus, it seems that stress temperatures induced larger 2-phenylethyl variations between sampling times, which is in agreement with previous results (Rosa 1997). For 1-methoxyindol-3-ylmethyl glucosinolate, signiðcant di erences (P \ 0É01) were noted throughout the day only at 20 C, the variation at 30 C probably being covered by the large SE. In common with other individual glucosinolates in this study, 2-phenylethyl and 1- methoxyindol-3-ylmethyl also showed the ultradian rhythms in a sinusoidal curve type with more or less variation according to temperature but with the inñuence due to light. Similar evolution trends were noted under photoperiodic conditions (0700È2100) (Rosa 1997). In comparison with the variations in the daily glucosinolate level observed either under Ðeld conditions (Rosa et al 1994) or controlled climatic conditions (Rosa 1997), the present study reveals that no large changes throughout the day could be observed when temperature, light and relative humidity are kept constant at an optimum growth and development temperature; at temperatures close to stress, variations are larger. CONCLUSIONS There were large di erences in total glucosinolate concentrations between the aerial part of the plant and the roots when growing conditions, except temperature, were kept constant. Even under constant conditions, seedlings exhibit the ultradian rhythms for glucosinolates which are more pronounced when temperatures are above the optimum for growth and development. At optimum temperatures, photoperiod inñuences the synthesis of major glucosinolates throughout the day; however, when the temperature is closer to that inducing stress, the e ect of light is almost negligible, variations being mainly due to temperature. Thus, it is likely that glucosinolates accompany the evolution of other plant constituents throughout the day as a response to external factors of environment. REFERENCES Clossais-Besnard N, Larher F 1991 Physiological role of glucosinolates in Brassica napus: concentration and distribution pattern of glucosinolates among plant organs during a complete life cycle. J Sci Food Agric 56 25È38. Duncan A J 1991 Glucosinolates. In: T oxic Substances in Crop Plants, ed Felix DÏMello J P, Du us C M & Du us J H. Royal Society of Chemistry, Cambridge, UK, pp 126È 147. Fenwick G R, Heaney R K, Mullin W J 1983 Glucosinolates and their breakdown products in food and food plants. CRC Crit Rev Food Sci Nutr 18 123È201. Fieldsend J, Milford G F J 1994a Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus). I: Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulphur within the crop. Ann Appl Biol 124 531È542. Fieldsend J, Milford G F J 1994b Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus). II: ProÐles and tissue-water concentrations in vegetative tissues and developing pods. Ann Appl Biol 124 543È555. Heaney R K, Fenwick G R 1980 Glucosinolates in Brassica vegetables: analysis of 22 varieties of Brussels sprouts (Brassica oleracea var gemmifera). J Sci Food Agric 31 785È 793. Josefsson E 1967 Distribution of thioglucosides in di erent parts of Brassica plants. Phytochemistry 6 1617È1627. Macfarlane Smith W, Griffiths D W 1988 A time-course study of glucosinolates in the ontogeny of forage rape (Brassica napus L). J Sci Food Agric 43 121È134. McGregor D I 1988 Glucosinolate content of developing rapeseed (Brassica napus L MidasÏ) seedlings. Can J Plant Sci 68 367È380. Rosa E 1997 Daily variation of glucosinolate concentration in the leaves and roots of cabbage seedlings in two constant temperature regimes. J Sci Food Agric 73 364È368. Rosa E, Heaney R K, Rego F C, Fenwick G R 1994 The variation of glucosinolate concentration during a single day in young plants of Brassica oleracea var acephala and capitata. J Sci Food Agric 66 457È463. Rosa E, Heaney, R K, Fenwick G R, Portas C A M 1996 Changes in glucosinolates concentrations in Brassica crops (B oleracea and B napus) throughout growing seasons. J Sci Food Agric 71 237È244. Rosa E, Heaney, R K, Fenwick G R, Portas C A M 1997 Glucosinolates in crop plants. Hort Rev 19 99È215. Salisbury F B, Ross C W 1992 Plant Physiology (4th edn). Wadsworth, Belmont, CA, USA. Spinks A, Sones K, Fenwick G R 1984 The quantitative analysis of glucosinolates in cruciferous vegetables, oilseeds and forage crops using high-performance liquid chromatography. Fette Seif Anstrichmitte 86 228È231.