Accumulation of glucosinolates and nutrients in pakchoi (Brassica campestris L. ssp. chinensis var. communis) two cultivar plants exposed to sulfur deficiency

Hort. Environ. Biotechnol. 52(2):121-127. 2011. DOI 10.1007/s13580-011-0097-5

Research Report

Accumulation of Glucosinolates and Nutrients in Pakchoi (Brassica campestris L. ssp. chinensis var. communis) Two Cultivar Plants Exposed to Sulfur Deficiency 1

1,2*

Keling Hu , Zhujun Zhu

2

3

, Yunxiang Zang , and Syed Azhar Hussain

1

2

Department of Horticulture, Zhejiang University, Huajiachi Campus, Kaixuan Road 268, Hangzhou 310029, China Department of Horticulture, School of Agricultural and Food Sciences, Zhejiang A & F University, Lin’an 311300, China 3 Institute of Soil and Environmental Science, University of Agriculture Faisalabad, Pakistan *Corresponding author: [email protected], [email protected]

Received May 11, 2010 / Accepted December 13, 2010 GKorean Society for Horticultural Science and Springer 2011

Abstract. To understand how sulfur nutrition affects the quality and yield of vegetable plants, we have grown two cultivars of pakchoi (Brassica campestris L. ssp. chinensis var. communis cv. Shang Hai Qing and You Dong Er) hydroponically in nutrient solution supplied with two levels of sulfur (0.0558 mM as sulfur deficiency and 1.0058 mM as sulfur sufficiency, respectively) for three weeks and their growth, nutrient uptake and glucosinolate content under these two sulfur conditions were investigated. The results showed that plant growth of both the cultivars was inhibited by sulfur deficiency. The concentrations of nitrogen and magnesium in shoots of both the cultivars were increased notably under sulfur deficiency, but there was no significant change in concentrations of sulfur, potassium and calcium. Moreover, sulfur deficiency increased phosphorus uptake in You Dong Er but not in Shang Hai Qing. In Shang Hai Qing sulfur deficiency reduced the content of all individual and total glucosinolates, while in You Dong Er this was also the case for most individual and total glucosinolates. However, in You Dong Er the total aliphatic glucosinolate concentration was not significantly influenced but the concentrations of individual aliphatic glucosinolates-glucoalyssin and gluconapin were in contrast increased under sulfur deficiency. Our data show that sulfur deficiency will decrease the yield and deteriorate the quality of pakchoi vegetable by reducing its growth and the contents of nutrients and glucosinolates. In addition, there was a significant genotypic variation in the composition and content of glucosinolates between these two pakchoi cultivars when exposed to sulfur deficiency. Additional key words: aliphatic glucosinolate, genotype, nitrogen, plant growth, sulfur sufficiency

=bhfcXiWh]cb Sulfur is an essential macronutrient for plant growth due to its presence in proteins, glutathione, phytochelatins, thioredoxins, chloroplast membrane lipids, and glucosinolates (Falk et al., 2007; Stipanuk, 2004). Sulfur deficiency is a major nutritional problem that results in decreased crop yield and quality (Hawkesford, 2000; Thomas et al., 2003). The glucosinolates are a group of sulfur rich secondary metabolites abundant in Brassica vegetables, such as cauliflower, broccoli, cabbage, etc. (Halkier and Gershenzon, 2006). Glucosinolates and their hydrolysis products (isothiocynates, thiocynates, nitriles, and epithionitriles) have many different biological activities, such as defense compounds and insect attractants. For humans some of these compounds function as chemopreventive agents, biopesticides, and flavor compounds (Fahey et al., 2001; Maruyama-Nakashita et al., 2006; Yan and Chen, 2007). All the glucosinolates

share a chemical structure comprising a sulfonated oxime moiety, a 쩁-D-thioglucose group, and a variable side chain derived from methionine, tryptophan, phenylalanine or various branched chain amino acids. Based on their precursor amino acids, glucosinolates can be categorized into three classes: aliphatic, indole and aromatic glucosinolates (Halkier and Gershenzon, 2006). It is also known that sulfur is mainly absorbed by plants in inorganic form. Each glucosinolate molecule contains at least two sulfur atoms. Changing sulfur supply is expected to have a major effect on plant glucosinolate content. Several reports have shown that sulfur supply can alter the concentrations and composition of glucosinolates in Brassica species (Asare and Scarisbtick, 1995; Schonhof et al., 2007). The glucosinolate content is significantly affected by sulfur and nitrogen fertilization (Aires et al., 2006). The nitrogen and sulfur ratio has an important role in the regulation of glucosinolate synthesis (Zhao et al., 1993). In

122

Keling Hu, Zhujun Zhu, Yunxiang Zang, and Syed Azhar Hussain

addition, the concentrations of glucosinolates vary depending on the varieties, cultivation conditions, and other mineral nutrients (Kushad et al., 1999; Mithen et al., 2000; Rosa et al., 1996). In general, Cruciferae and Liliaceae plants have high sulfur demand (Scherer, 2001). Pakchoi (Brassica campestris L. ssp. chinensis var. communis) is one of the common and extensively cultivated leafy vegetable in China. It is an excellent source of glucosinolates and has high iron and calcium contents which are essential for human beings (Hanson et al., 2009). The objectives of this study were to examine the effects of sulfur supply on plant growth, the nutrient and total glucosinolate content as well as on the composition of glucosinolates in shoot tissues of two pakchoi cultivar plants.

AUhYf]U`g UbX AYh\cXg D`Ubh AUhYf]U`g Two cultivars of pakchoi (Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee), cv. Shang Hai Qing and cv. You Dong Er, were used. Shang Hai Qing is a yellowish green leaf variety and You Dong Er is a dark green leaf variety. Seeds of both cultivars were sown and germinated in vermiculite. After 15 days, seedlings were transplanted into a container (40 cm × 25 cm × 15 cm) and grown hydroponically (six plants per 10 L pot). Plants were supplied with two different sulfur levels (sulfur deficiency and sulfur sufficiency). The composition of the nutrient solution used were 2.5 mM Ca(NO3)2, 1.0 mM KH2PO4, 4 mM KNO3, 0.5 mM NH4NO3, 0.05 mM MgSO4, 0.95 mM MgCl2, 3.0 쩋molL-1 MnSO4, 2.0 쩋molL-1 ZnSO4, -1 -1 -1 0.8 쩋molL CuSO4, 0.1 쩋molL H2MoO4, 10.0 쩋molL -1 H3BO3, and 40.0 쩋molL EDTA-Fe with two sulfur concentration in the form of SO42-, either 0.0558 mM for sulfur deficiency treatment or 1.0058 mM for sulfur sufficiency treatment (0.95 mM MgCl2 replaced by 0.95 mM MgSO4). Each treatment had three replicates, and a completely randomized block design. The pH of the nutrient solution was maintained close to 6.0 by adjusting with NaOH. Solution was refreshed every week. The plants were grown at a photoperiod of 14/10 h (day/night) with a photosynthetic photon flux density (PPFD) of 600 µmolm-2s-1 and temperature 25/17G(day/night). Samples were harvested after three week treatments, frozen immediately in liquid nitrogen and freeze-dried. Then the freeze-dried samples were ground and stored at -80.

70% methanol. Then the mixtures were kept at 75Gin a water bath for 10 min. For internal standardization 100 쩋L of 5 mM sinigrin (Sigma-Aldrich Co., St. Louis, USA) were added to one of the duplicates before extraction. Then 1 mL of 0.4 molL-1 barium acetate was rapidly added and the vials were vortexed for several seconds. The homogenates were centrifuged for 10 min at 2100 g. The supernatants were decanted and stored on ice and pellets were reextracted twice with 3 mL of 70% methanol (75). The combined supernatants were applied to a 0.5 mL DEAE SephadexGA-25 column (Amersham Biosciences, Sweden) and washed with 5 mL of distilled water produced by Milli-Q system (Milli-pore Co., USA), and incubated overnight with aryl sulfatase (Sigma-Aldrich Co., St. Louis, USA) for desulfation. The resultant desulphoglucosinolates were eluted with 3 mL of ultra pure water produced by Milli-Q system and stored at -20Guntil they were analyzed by high performance liquid chromatography (HPLC). HPLC analysis was performed using an Agilent 1200 system (Santa Clara, USA) equipped with a C18 reverse-phase column (250 × 4 mm, 5 µm, Bischoff, Germany) and used the following gradient: H2O (2 min), a linear gradient of 0-20% acetonitrile (Tedia, USA) (32 min), 20% acetonitrile (6 min), followed by 100% acetonitrile and 0% acetonitrile prior to the injection of the next sample. The flow rate was 1.3 mLmin-1. 5bU`mg]g cZ A]bYfU` 9`YaYbhg The analysis of phosphorus, potassium, calcium, magnesium and sulfur was carried out following the method of Ruan et al. (2007) with minor modifications. 0.30 g of freezedried shoot powder was digested by concentrated acids mix HNO3: HClO4 = 5:2 (V: V) and analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). For total nitrogen, 0.30 g of freeze-dried powder were digested by H2SO4 (high purity grade), and then distilled in a KjelFlex K-360 (Buchi, Flawil, Switzerland) with 40% (w/v) NaOH and 2% (w/v) boric acid (methyl red and bromcresol green used as an indicator solution), then titrated with 0.02 mM H2SO4. GhUh]gh]WU` 5bU`mg]g Measurements were analyzed using SAS 8.0 (SAS Institute, Cary, NC) for the general linear models (GLM) procedure. The least significant difference (LSD) was used to compare the means.

FYgi`hg UbX 8]gWigg]cb ;`iWcg]bc`UhY 5bU`mg]g Glucosinolates were analyzed according to the method of Krumbein et al. (2005) with minor modifications. Duplicates of freeze-dried samples (0.25 g) were extracted in 4 mL of

9ZZYWh cZ Gi`Zif 8YZ]W]YbWm cb D`Ubh ;fckh\ As shown in Table 1, sulfur deficiency caused a significant reduction in the fresh and dry weight of shoots and

Hort. Environ. Biotechnol. 52(2):121-127. 2011.

123

Table 1. Effects of sulfur supply on the growth of pakchoi cultivars grown hydroponically. Fresh weight (g plant-1)

Cultivar

Shoot

Dry weight (g plant-1)

Roots

Shoot

Roots

Root/shoot ratio

Shang Hai Qing (sulfur deficiency)

8.67 ± 2.42 c

z

1.44 ± 0.37 c

0.53 ± 0.14 c

0.13 ± 0.03 b

0.24 ± 0.03 a

Shang Hai Qing (sulfur sufficiency)

12.44 ± 1.46 b

2.15 ± 0.51 b

0.79 ± 0.11 b

0.14 ± 0.02 b

0.17 ± 0.04 b

1.32 ± 0.31 c

0.58 ± 0.17 c

0.12 ± 0.02 b

0.20 ± 0.06 ab

You Dong Er (sulfur deficiency)

9.37 ± 3.25 bc

You Dong Er (sulfur sufficiency)

21.82 ± 2.64 a

2.85 ± 0.42 a

1.36 ± 0.14 a

0.21 ± 0.03 a

0.16 ± 0.03 b

F test

Sulfur level: **

Sulfur level: **

Sulfur level: **

Sulfur level: **

Sulfur level: **

Cultivar: **

Cultivar: NS

Cultivar: **

Cultivar: *

Cultivar: NS

Sulfur level × Cultivar: **

Sulfur level × Cultivar: NS

Sulfur level × Cultivar: **

Sulfur level × Cultivar: **

Sulfur level × Cultivar: NS

Data are the means ± standard deviation of three replicates; NS not significant; *p < 0.05; **p < 0.01. Means denoted by the same letter indicates no significant difference between treatments at P < 0.05 level.

z

Table 2. Effects of sulfur supply on the concentrations of phosphorus, potassium, calcium, magnesium, nitrogen and sulfur in the shoots of pakchoi cultivars grown hydroponically. Cultivar

Phosphorus -1 (mg g DW)

Potassium -1 (mg g DW)

Calcium -1 (mg g DW)

Magnesium -1 (mg g DW)

Nitrogen -1 (mg g DW)

Sulfur -1 (mg g DW)

Shang Hai Qing (sulfur deficiency)

5.78 ± 0.1 az

34.06 ± 1.28 a

5.98 ± 0.17 a

2.31 ± 0.17 a

81.29 ± 0.75 a

5.87 ± 0.08 ab

Shang Hai Qing (sulfur sufficiency)

5.90 ± 0.04 a

32.27 ± 0.45 a

5.81 ± 0.12 ab

1.04 ± 0.03 c

75.88 ± 2.28 b

6.24 ± 0.41 a

You Dong Er (sulfur deficiency)

5.20 ± 0.04 b

32.15 ± 0.58 a

5.76 ± 0.03 b

2.11 ± 0.02 b

83.3 ± 1.68 a

5.29 ± 0.19 c

You Dong Er (sulfur sufficiency)

4.56 ± 0.32 c

33.81 ± 0.88 a

5.70 ± 0.02 b

1.01 ± 0.01 c

75.00 ± 1.07 b

5.52 ± 0.08 bc

Sulfur level: NS Cultivar: NS Sulfur level × Cultivar:*

Sulfur level: NS Cultivar: * Sulfur level × Cultivar: NS

Sulfur level: ** Cultivar: * Sulfur level ×Cultivar: NS

Sulfur level:** Cultivar: NS Sulfur level × Cultivar: NS

Sulfur level: NS Cultivar: ** Sulfur level × Cultivar: NS

F test

Sulfur level: * Cultivar:** Sulfur level × Cultivar:**

Data are the means ± standard deviation of three replicates; NS not significant; *p < 0.05, **p < 0.01; z Means denoted by the same letter indicates no significant difference between treatments at P < 0.05 level.

roots in both cultivars except in Shang Hai Qing in which the root dry weight was not significantly reduced. These results show that pakchoi plants have in common plant response to sulfur depletion (Hawkesford, 2000) and in consistent with the report that Brassica oilseed crop yield was strongly affected by the sulfur fertilization (Malhi, 2007). In addition, the shoot growth responded to sulfur supply was more sensitive in You Dong Er than in Shang Hai Qing. Sulfur depletion resulted in an increased root to shoot ratio for both the cultivars Shang Hai Qing and You Dong Er. This phenomenon was seen in Arabidopsis in which sulfur deficiency led to a relative increase in the root growth as compared to the shoot growth (Kutz et al., 2002). This may be a way of plants to overcome sulfur limitation by enhancing sulfur uptake through modifying root growth or even the root architectures.

9ZZYWh cZ Gi`Zif 8YZ]W]YbWm cb D`Ubh Bihf]Ybh 7cbWYbhfUh]cb The accumulation of macronutrient (nitrogen, phosphorus, potassium, calcium, magnesium, and sulfur) in shoots of both cultivars under two sulfur supply levels was shown in Table 2. The data indicate that there was no significant difference in the sulfur concentrations between the two sulfur supply treatments in the shoots of the two cultivars. Presumably, decreasing sulfur supply decreased sulfur concentration in plants (Zhao et al., 1999). However the decrease in sulfur content of plants would be accompanied with the decrease in plant growth under sulfur deficiency, which leads to no significant decline in sulfur concentration as the concentration was expressed on the dry weight base of plants. A substantial decrease in the shoot dry weight was clearly shown in Table 1. In other words, plants can remain sulfur homeostasis under varied sulfur supply conditions by adjusting their growth rate or sulfur absorption, resulting in

Keling Hu, Zhujun Zhu, Yunxiang Zang, and Syed Azhar Hussain

124

a constant sulfur concentration in vivo. In both cultivars, sulfur deficiency led to a higher accumulation of nitrogen. It is consistent with the report that the sulfur deficiency increased nitrogen concentration in rapeseed plants (Zhao et al., 1994). However, in wheat sulfur fertilization increased the nitrogen concentration in plants and improved nitrogen use efficiency by increasing the nitrogen uptake (Salvagiotti et al., 2009). Therefore, sulfur has a synergistic effect on nitrogen uptake in wheat. It seems that the interaction between sulfur and nitrogen may exist differently in different plants. Under deficient sulfur supply, there was a difference in phosphorus uptake between Shang Hai Qing and You Dong Er. Sulfur deficiency increased phosphorus uptake in You Dong Er, but not significantly in Shang Hai Qing. Yang et al. (2009) reported that the concentration of sulfur was not significantly affected by different phosphorus supply levels in You Dong Er. Randhawa and Arora (2000) observed a synergistic relationship between phosphorus and sulfur at low level of sulfur application and antagonistic relationship at higher level of sulfur application in wheat. Sulfur deficiency had no influence on in the contents of potassium and calcium, but increased magnesium content in both the pakchoi cultivars (Table 2). Consistently, Gunes et al. (2009) also reported that applied sulfur had no significant effect on the potassium and calcium concentrations of alfalfa hay.

9ZZYWh cZ Gi`Zif 8YZ]W]YbWm cb D`Ubh ;`iWcg]bc`UhY 7cbhYbh Seven individual glucosinolates including aliphatic glucosinolates (glucoalyssin, gluconapin and glucobrassicanapin), indole glucosinolates (glucobrassicin, neoglucobrassicin and 4-methoxyglucobrassicin) and aromatic glucosinolates (gluconasturtiin), were identified in the shoots of both the cultivars (Figs. 1 and 2). Total glucosinolate content was calculated by the sum of the seven individual glucosinolates. Total glucosinolate content was strongly influenced by sulfur supply to the shoots of both cultivars (Fig. 1A). Under sulfur sufficient condition, both cultivars had a similar level of total glucosinolate content. Whereas sulfur deficiency significantly decreased total glucosinolate content by 52% in Shang Hai Qing and 32% in You Dong Er. Thus for total glucosinolate accumulation Shang Hai Qing was more susceptible to sulfur deficiency as compared to You Dong Er. A decline in the total glucosinolate content in response to sulfur limitation has also been documented in rapeseed (Zhao et al., 1997). Glucosinolates, as a sulfur reservoir in plant tissues, may be selected for catabolic recycling of sulfur to support plant growth (Kutz et al., 2002). When sulfur supply is limited, most sulfur is incorporated into proteins, and therefore less sulfur is available for glucosinolate synthesis (Asare and Scarisbtick, 1995). The reduction in glucosinolate content under sulfur deficiency was thought to be the results of the down-regulation of gene expression of all the major

5

6

7

8

Fig. 1. Concentrations of total aliphatic, indole, aromatic/gluconasturtiin and total glucosinolates in the shoots of pakchoi (cv. Shang Hai Qing and You Dong Er) under sulfur deficiency and sufficiency. Data are the means ± standard deviation of three replicates and columns with the same letters are not significantly different from each other (P < 0.05).

Hort. Environ. Biotechnol. 52(2):121-127. 2011.

125

5

6

7

8

9

:

Fig. 2. Concentrations of individual glucosinolate in the shoots of pakchoi (cv. Shang Hai Qing and You Dong Er) under sulfur deficiency and sufficiency. Data are the means ± standard deviation of three replicates and columns with the same letters are not significantly different from each other (P < 0.05).

glucosinolate biosynthetic genes such as MAM, CYP79 and CYP83 families (Hirai et al., 2005; Maruyama-Nakashita et al., 2003). With insufficient sulfur supply, the plants not only change the total glucosinolate content, but also the composition of glucosinolates. Firstly we examined the aliphatic glucosinolates. In Shang Hai Qing the total aliphatic (Fig. 1B) and three individual aliphatic glucosinolate (Figs. 2A, B, and C) concentrations were significantly decreased. Especially gluconapin concentration was reduced by 80% under sulfur deficiency. This reduction may be due to the fact that the aliphatic glucosinolates in Brassica napus are derived from the sulfurcontaining amino acid cysteine and methionine (Halkier and Gershenzon, 2006). Sulfur deficiency conditions limited cysteine and methionine synthesis resulting in a lack of precursors for aliphatic glucosinolates synthesis (Nikiforava et al., 2005). While in You Dong Er the total aliphatic glucosinolate content and glucobrassinapin were not significantly

affected by sulfur deficiency (Figs. 1B and 2C), but in the contrast glucoalyssin and gluconapin concentrations were increased by 175% and 46% respectively (Figs. 2A and B). The increased concentrations of these two aliphatic glucosinolates in You Dong Er may be due to the substantial plant growth reduction by sulfur deficiency (Table 1), resulting in higher concentrations of these two aliphatic glucosinolates. These results showed that there was a genotypic difference in the composition of accumulating aliphatic glucosinolates between the two pakchoi cultivars. Rangkadilok et al. (2004) also found that three broccoli cultivars had different responses to sulfur application in glucoraphanin accumulation. Secondly, the content of the indole glucosinolates were analyzed. In both pakchoi cultivars the concentrations of total indole glucosinolates (Fig. 1C) and three individual indole glucosinolates (Figs. 2D, E, and F) significantly decreased by insufficient sulfur supply. This result is in agreement with the finding that in turnip roots increasing

126

Keling Hu, Zhujun Zhu, Yunxiang Zang, and Syed Azhar Hussain

increased the proportion of aliphatic glucosinolates, at the expense of the considerable decrease of indole glucosinolates. Therefore, the results suggest that genetic variability might exist to determine the composition profile of aromatic, aliphatic and indole glucosinolates in pakchoi cultivars.

7cbW`ig]cb

Fig. 3. The percentage profile of aliphatic, indole and aromatic glucosinolates to total glucosinolates in pakchoi shoots (cv. Shang Hai Qing and You Dong Er) under sulfur deficiency and sufficiency.

sulfur supply increased the indole glucosinolate concentration (Li et al., 2007). However, it is different from the observations found in that of broccoli sulfur fertilization did not affect the indole glucosinolates concentrations, but increased the aliphatic glucosinolates (Vallejo et al., 2003). There are many diverse responses of accumulating glucosinolates in plants to various sulfur and nitrogen supply. In cabbage the indole glucosinolates increased and aliphatic glucosinolates decreased with increasing nitrogen supply (Rosen et al., 2005). Li et al. (2007) also reported that total indole glucosinolate composition increased with an increasing nitrogen supply but decreased with an increasing sulfur supply. Thirdly, the composition of aromatic glucosinolates was investigated. In contrast to the fact that the most species of aliphatic and indole glucosinolates decreased in pakchoi plants grown with decreasing sulfur supply, only one of aromatic glucosinolates (Fig. 1D), the gluconasturtiin was decreased significantly in You Dong Er but not in Shang Hai Qing. It was reported that increasing sulfur supply increased gluconasturtiin concentration in turnip roots (Li et al., 2007) and the total aromatic glucosinolates in broccoli whole plants (Vallejo et al., 2003). Taken the above analysis together, the difference in the compositional change of glucosinolates between the two pakchoi cultivars under sulfur deficiency can be seen (Fig. 3). In Shang Hai Qing the percentage of aromatic glucosinolate to the total glucosinolate increased by 47% in sulfur deficiency compared with sulfur sufficiency. However, in You Dong Er there was no change in aromatic glucosinolate composition. In Shang Hai Qing, sulfur deficiency significantly decreased the percentage of aliphatic glucosinolates to total glucosinolates and increased the proportion of indole glucosinolates. On the contrary, in You Dong Er sulfur deficiency

This work shows that insufficient sulfur supply significantly limited the plant growth and altered most of the mineral nutrient content in pakchoi plants. At the same time, sulfur deficiency reduced the total glucosinolate content and the most individual glucosinolate content in the plants of both pakchoi cultivars. However, two cultivars responded to sulfur deficiency differently in the glucosinolate metabolism. Shang Hai Qing was heavily affected by sulfur deficiency with a pronounced decrease in total glucosinolates and aliphatic glucosinolates, whereas You Dong Er had not been significantly influenced. In contrast You Dong Er exhibited a larger decrease in indole glucosinolates and aromatic glucosinolate content than Shang Hai Qing. Therefore, adequate sulfur fertilization is certainly required to produce pakchoi vegetables with higher yield and quality. Moreover, a right cultivar selection for the pakchoi production under certain conditions is essential to meet different commercial purposes such as providing special glucosinolates since genotypic variation is one of main determinants in the production of functional metabolites in vegetable plants. Acknowledgements: The authors thank Mr. Lifeng Ma of Tea Research Institute of Chinese Academy of Agricultural Sciences and Key Laboratory for Tea Chemistry of The Ministry of Agriculture of China for nutrient element analyses and Mr. Xiongshun Yu of College of Environmental and Resource Sciences of Zhejiang University for nitrogen analysis. This work was supported by National Natural Science Foundation of China (30871718) and Zhejiang Provincial Natural Science Foundation of China (R3080360 & Y3090538).

@]hYfUhifY 7]hYX Aires, A., E. Rosa, and R. Carvalho. 2006. Effect of nitrogen and sulfur fertilization on glucosinolates in the leaves and roots of broccoli sprouts (Brassica oleracea var. italica). J. Sci. Food Agr. 86:1512-1516. Asare, E. and D.H. Scarisbtick. 1995. Rate of nitrogen and sulphur fertilizers on yield, yield compounds and seed quality of oilseed rape (Brassica napus L.). Field Crop Res. 44:41-46. Fahey, J.W., A.T. Zalcmann, and P. Talalay. 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5-51. Falk, K.L., J.G. Tokuhisa, and J. Gershenzon. 2007. The effect of sulfur nutrition on plant glucosinolate content: physiology and

Hort. Environ. Biotechnol. 52(2):121-127. 2011. molecular mechanisms. Plant Biol. 9:573-581. Gunes, A., A. Inal, D.J. Pilbeam, and Y.K. Kadioglu. 2009. Effect of sulfur on the yield and essential and nonessential element composition of alfalfa determined by polarized energy dispersive x-ray fluorescence. Commun. Soil Sci. Plan. 40:2264-2284. Halkier, B.A. and J. Gershenzon. 2006. Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 57:303-333. Hanson, P., R.Y. Yang, L.C. Chang, L. Ledesma, and D. Ledesma. 2009. Contents of carotenoids, ascorbic acid, minerals and total glucosinolates in leafy brassica pakchoi (Brassica rapa L. chinensis) as affected by season and variety. J. Sci. Food Agr. 89:906-914. Hawkesford, M.J. 2000. Plant responses to sulphur deficiency and the genetic manipulation of sulphate transporters to improve S-utilization efficiency. J. Exp. Bot. 51:131-138. Hirai, M.Y., M. Klein, Y. Fujikawa, M. Yano, D.B. Goodenowe, Y. Yama- zaki, S. Kanaya, Y. Nakamura, M. Kitayama, H. Suzuki, N. Sakurai, D. Shibata, J. Tokuhisa, M. Reichelt, J. Gershenzon, J. Papen-brock, and K. Saito. 2005. Elucidation of gene-to-gene and metabolite-to-gene networks in Arabidopsis by integration of metabolomics and transcriptomics. J. Biol. Chem. 280:25590-25595. Krumbein, A., I. Schonhof, and M. Schreiner. 2005. Composition and contents of phytochemicals (glucosinolates, carotenoids and chlorophylls) and ascorbic acid in selected Brassica species (B. juncea, B. rapa subsp. Nipposinica var. chinoleifera, B. rapa subsp. Chinensis and B. rapa subsp. rapa). J. App. Bot. Food Qual. 79: 168-174. Kushad, M.M., A.F. Brown, A.C. Kurilich, J.A. Juvik, B.P. Klein, M.A. Wallig, and E.H. Jeffery. 1999. Variation of glucosinolates in vegetable crops of Brassica oleracea. J. Agr. Food Chem. 47: 1541-1548. Kutz, A., A. Müller, P. Hennig, W.M. Kaiser, M. Piotrowski, and E.W. Weiler. 2002. A role for nitrilase 3 in the regulation of root morphology in sulphur-starving Arabidopsis thaliana. Plant J. 30:95-106. Li, S., I. Schonhof, A. Krumbein, L. Li, H. Stützel, and M. Schreiner. 2007. Glucosinolate concentration in turnip (Brassica rapa ssp. rapifera L.) roots as affected by nitrogen and sulfur Supply. J. Agr. Food Chem. 55:8452-8457. Malhi, S.S., Y. Gan, and J.P. Raney. 2007. Yield, seed quality, and sulfur uptake of Brassica oilseed crops in response to sulfur fertilization. Agron. J. 99:570-577. Maruyama-Nakashita, A., E. Inoue, A. Watanabe-Takahashi, T. Yamaya, and H. Takahashi. 2003. Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways. Plant Physiol. 132:597-605. Maruyama-Nakashita, A., Y. Nakamura, T. Tohge, K. Saito, and H. Takahashi. 2006. Arabidopsis slim1 is a central transcriptional regulator of plant sulfur response and metabolism. Plant Cell 18:3235-3251. Mithen, R.F., M. Dekker, R. Verkerk, S. Rabot, and I.T. Johnson. 2000. The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods. J. Sci. Food Agr. 80:967-984. Nikiforava, V.J., J. Kopha, V. Tolstikov, O. Fiehn, L. Hopkins, M.J. Hawkesford, E.H. Hess, and R. Hoefgen. 2005. Systems rebalance of mechanism in response to sulphur deprivation, as revealed by metabolome analysis of Arabidopsis plants. Plant Physiol. 138: 304-318.

127

Randhawa, P.S. and C.L. Arora. 2000. Phosphorus-sulfur interaction effects on dry matter yield and nutrient uptake by wheat. J. Indian Soc. Soil Sci. 48:536-544. Rangkadilok, N., M.E. Nicolas, R.N. Bennett, D.R. Eagling, R.R. Premiter, and P.W.J. Taylor. 2004. The effect of sulfur fertilizer on glucoraphanin levels in broccoli (B. oleracea L. var. italica) at different growth stages. J. Agric. Food Chem. 52:2632-2639. Rosa, E.A.S., R.K. Heaney, C.A.M., Portas, and R.G. Fenwick. 1996. Changes in glucosinolate concentrations in Brassica crops (B oleracea and B napus) throughout growing seasons. J. Sci. Food Agr. 71:237-244. Rosen, C.J., V.A. Fritz, G.M. Gardner, S.S. Hecht, S.G. Carmella, and P.M. Kenney. 2005. Cabbage yield and glucosinolate concentrations as affected by nitrogen and sulfur fertility. HortScience 40:493-1498. Ruan, J.Y., J. Gerendas, R. Haerdter, and B. Sattelmacher. 2007. Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants. Ann. Bot. 99:301-310. Salvagiotti, F., J.M. Castelları´n, D.J. Miralles, and H.M. Pedrol. 2009. Sulfur fertilization improves nitrogen use efficiency in wheat by increasing nitrogen uptake. Field Crop Res. 113:170-177. Scherer, H.W. 2001. Sulphur in crop production-invited paper. Eur. J. Agron. 14:81-111. Schonhof, I., D. Blankenburg, S. Müller, and A. Krumbein. 2007. Sulfur and nitrogen supply influence growth, product appearance, and glucosinolate concentration of broccoli. J. Plant. Nutr. Soil Sc. 170:65-72. Stipanuk, M.H. 2004. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu. Rev. Nutr. 24:539-577. Thomas, S.G., T.J. Hocking, and P.E. Bilsborrow. 2003. Effect of sulphur fertilisation on the growth and metabolism of sugar beet grown on soils of differing sulphur status. Field Crop Res. 83:223-235. Vallejo, F., F.A. TomAs-Baberh, A.G. Benavente-Garcia, and C. Garcia-Viguera. 2003. Total and individual glucosinolate contents in inflorescences of eight broccoli cultivars grown under various climatic and fertilization conditions. J. Sci. Food. Agr. 83:307-313. Yan, X.F. and S.X. Chen. 2007. Regulation of plant glucosinolate metabolism. Planta 226:1343-1352. Yang, J., Z.J. Zhu, and J. Gerendás. 2009. Interactive effects of phosphorus supply and light intensity on glucosinolates in pakchoi (Brassica campestris L. ssp. chinensis var. communis). Plant Soil 323:323-333. Zhao, F.J., E.J. Evans, P.E. Bilsborrow, and J.K. Syers. 1994. Influence of nitrogen and sulphur on the glucosinolate profile of rapeseed (Brassica napus L). J. Sci. Food Agric. 64:295-304. Zhao, F.J., E.J. Evans, P.E. Bilsborrow, and J.K. Syers. 1993. Influence of sulphur and nitrogen on seed yield and quality of low glucosinolate oilseed rape (Brassica napus L.). J. Sci. Food Agr. 63:29-37. Zhao, F.J., M.J. Hawkesford, and S.P. McGrath. 1999. Sulphur assimilation and effects on yield and quality of wheat. J. Cereal Sci. 30:1-17. Zhao, F.J., P.J.A. Withers, E.J. Evans, J. Monaghan, E.S. Salmon, R.P. Shewry, and P.S. McGrath. 1997. Sulphur nutrition: an important factor for the quality of wheat and rapeseed. Soil Sci. Plant Nutr. 43:1137-1142.