Art18 Journal of Applied Botany and Food Quality 82, 108 - 113 (2009) 1Section Urban Horticulture, Institute for Horticultural Sciences, Humboldt University Berlin, Berlin, Germany 2Department of Horticulture, Virginia Tech, Blacksburg, VA, USA Impact of glucosinolate structure on the performance of the crucifer pest Phaedon cochleariae (F.) M.M. Uddin1, Ch. Ulrichs1, J.G.Tokuhisa2, I. Mewis1 (Received August 28, 2008) Summary Glucosinolates (GS) are sulfur-rich secondary metabolites found in the Brassicaceae and other related families of the order Brassicales. GS consist of structurally-related compounds with different side chains. To explore the possibility that various side chain confer di- vergent biological activities to individual GS, we have investigated the performance of the specialist pest beetle, Phaedon cochleariae (F.) on Arabidopsis thaliana L. mutants and Columbia wild-type (WT) which differ in the main group of GS. Plant lines of A. thaliana altered for the expression of MAM3, because of the introduction of an over- expression construct of MAM3 (mam3+) or containing double knock- outs of CYP79B2 and CYP79B3 (cyp79B2-/cyp79B3-) were used for the study in comparison to the WT. A. thaliana genotypes differed in their GS profiles. The highest GS content was present in the WT followed by mam3+ and cyp79B2-/ cyp79B3-. A modified aliphatic GS content was detected for the mam3+ as compared to the WT lines. Furthermore, indolyl GS were completely absent in cyp79B2-/cyp79B3-. The percentage weight increase of larvae raised on each of the three plant genotypes was significant different. Larval performance was poorest on plants of cyp79B2-/cyp79B3- and best on WT, but there was no significant difference found in percentage weight increase on mam3+ and WT. There was no correlation between the weight increase of the larvae on genotypes and induced levels of aliphatic, indolyl, and total GS. However, the poor performance of beetle larvae on cyp79B2-/ cyp79B3-compared to WT and mam3+ might be explained by com- parable high aliphatic GS levels of this mutant, a different induction of secondary metabolites, and the absence of indolyl GS. Basic knowledge about the relationship of GS structures and their insect pests may help in further resistance breeding of crucifer crops. Introduction Glucosinolates (GS) are sulphur-rich β-thioglucosides present within the Brassicaceae and other related families of the order Brassicales (RODMAN et al., 1996). All GS contain a common core structure, but have variable side chains including aliphatic, aromatic, or indolic structures (WITTSTOCK and HALKIER, 2002; KLIEBENSTEIN et al., 2005). To date, more than 120 GS have been identified in plants (FAHEY et al., 2001), including 34 compounds present in the model plant Arabidopsis thaliana L. (KLIEBENSTEIN et al., 2001). In plants GS and their hydrolyzing enzymes, β-thioglucosidases, which are also called myrosinases, are stored in different cell compartments or specialized cells (LÜTHY and MATILE, 1984; KOROLEVA et al., 2000). But when the plant is damaged, hydrolysis occurs and biological active products such as isothiocyanates, thiocyanates, and nitriles are released. GS have dual roles in plant-insect interactions. Although they function as defense compounds, several insects that are specialists have life cycles that are closely linked to the presence of such secondary com- pounds in their brassicaceous host plants (RENWICK, 2002). They are known to stimulate feeding in a number of insects that feed ex- clusively on crucifers (DAVID and GARDINER, 1966; NAULT and STYER, 1972; TANTON, 1977; LARSEN et al., 1985). Brassicaceae species and individual plants vary within concentration and composition of GS (FAHEY et al., 2001; KLIEBENSTEIN et al., 2001). Additionally, many brassicaceous plants produce higher levels of indolyl GS after herbivory (KORTISAS et al., 1991; BODNARYK, 1992; GRIFFITHS et al., 1994; BAUER et al., 1998; AGRAWAL et al., 1999a). GS and/or their breakdown products have a variety of effects upon organisms that come in contact with them, but do not always control the inter- action between plants and their potential herbivore or invading microorganisms (CHEW, 1988b). Distinct GS can have different effect on the same insect herbivore. For example, p-hydroxybenzyl GS is involved in plant resistance (antixenosis) against feeding by the flea beetle, Phyllotreta cruciferae (Goeze), in seedlings of yellow mustard, Sinapis alba L. (BODNARYK, 1991).Whereas, allyl GS and indol-3- ylmethyl, which are present in seedlings of Indian mustard, Brassica juncea (L.) and oilseed rape, Brassica napus L., respectively, are not determinants of feeding behavior by P. cruciferae (BODNARYK and PALANISWAMY, 1990). The mustard leaf beetle, Phaedon cochleariae (F.) (Coleopterans: Chrysomelidae), is an insect pest of crucifer crops that is common to Europe (FINCH and JONES, 1986). When this insect emerges in large numbers, it can quickly devastate crucifer fields with especially severe effects on young seedlings. Hence, a study of the insect/host- plant interaction including the feeding behavior can lead to agri- culturally relevant management strategies. TANTON (1977) and NIELSEN (1978) found that P. cochleariae responded to a variety of GS in feeding bioassays, with distinct preferences for the aliphatic allyl GS (sinigrin) compared to the aromatic compounds: p-hydroxy- benzyl GS (sinalbin) and benzyl GS (glucotropaeolin). However, these studies have limited insights into the impact of GS structure on the larvae and beetle performance of P. cochleariae. We have taken this opportunity to investigate the effect of different GS classes on the feeding and growth of the P. cochleariae larvae by using A. thaliana genotypes with desired GS profiles. Materials and methods Plant cultivation and experimental conditions Seeds from single seed propagation were sown on petri plates containing solidified Murashige-Skoog media. After stratification for three days at 4°C, germinated seedlings were transferred to 7 x 6 cm pots filled with sterile potting mix (Gramoflor). Plants were kept in a growth chamber at 21 ± 1°C, 60 ± 5% relative humidity, at 200 µmol m-2 s-1 light intensity, and on a 10.5 : 13.5 (L : D) photo- period. Twenty-six days old plants were used for the force feeding experiments. Insect rearing The start population of P. cochleariae was obtained from Bayer Crop Science (Mohnheim, Germany). This beetle species was initially 0 50 100 150 200 250 300 350 400 Cyp79B2-/B3- MAM3+ WT Genotype a a a B 0 50 100 150 200 250 300 350 400 Cyp79B2-/B3- MAM3+ WT Genotype L a rv a lw e ig h t in c re a s e [% ] a ab b A reared on pak-choi plants (Brassica chinensis L.) following by savoy cabbage (Brassica sabauda L.) in the laboratory of the Urban Horticulture section at Humboldt University Berlin. Pak-choi plants were provided as food in insect cages. For egg laying the beetles were provided the severed stem of savoy cabbage. After the beetles laid eggs on savoy cabbage, which was transferred to a new cage for larval hatching. Larvae were fed with savoy cabbage leaves, which were replaced on alternate days until larval pupation. Pupae were transferred to another cage for beetle hatching. This procedure was repeated to obtain a sufficient number of insects all at the 1st instar stage for the bioassays. Force feeding experiments Lines (three each) of two different A. thaliana mutants with modi- fied GS profiles and the corresponding Columbia wild-type (WT) were used for the bioassays. The first lines were the double knock- out mutant of CYP79B2 and CYP79B3 (cyp79B2-/cyp79B3-, a gift from J. Chory, The Salk Institute for Biological Studies, California). Further information on the construction of the mutant can be reviewed in ZHAO et al. (2002). The second plant mutant lines are characterized by over expression of MAM3 (mam3+) and were generated as de- scribed in TEXTOR et al. (2007). First instars of P. cochleariae were weighted before release on the plants, one larva per plant. The pots were covered with small cages made from transparent plastic cylinders and fine mesh gauze. After four days pre-conditioning to their respective plant lines, the larvae were weighted and transferred to a new plant of the same genotype. After three days the final larval weight was determined. Ten replicates were made for each of the nine lines of mutants and WT. Four plants of each genotype were harvested following larval feeding at 30 days after planting (first harvest) as well as 33 days after planting (second harvest), frozen in liquid nitrogen and stored at -80°C prior to GS analysis. Data from chemical analysis and bioassays data were analyzed by performing variance analysis with following mean comparison test by using SYSTAT 11.0. Glucosinolate analysis Plant samples were freeze-dried and whole plants were grounded. Samples of 20 mg were extracted in 70% methanol following the procedure described by MEWIS et al. (2005). Extraction was done in five replications for each treatment and genotype. To quantify GS content, an internal standard p-hydroxybenzyl GS (purified from Sinapis alba seeds as potassium salt) was added initially to the first methanol extract. The GS of extracts were analyzed as desulfo GS and for this purpose the extracts were desulfated on DEAE Sephadex A-25 mini columns with aryl sulfatase solution (Sigma-Aldrich Corp H 1 from Helix pomatia); column wash steps are as described in MEWIS et al. (2005). A 40 µl aliquot of each GS extracts was run on a Dionex Summit P680A HPLC system equipped with an ASI-100 auto sampler and a PDA-100 photodiode array detector. GS were separated on a narrow bore column (Acclaim TM 120, 250-2.1, RP18, Dionex) at a flow rate of 0.4 ml / min and a column temperature of 25°C. A 43 min gradient program with eluents: A) ultra pure H2O (Milli Q quality) and B) 40% acetonitrile (HPLC grade) consisted of 0 - 1 min 99.5% A, 1 - 8 min 80%, 10 - 19 min 50%, 22 - 28 min 1.0% A, 33 - 36 min 99.5% A. The eluent was monitored for ab- sorbance at 229 nm and GS were identified using purified standards, retention time, and UVVis spectra according to BROWN et al. (2003). The GS amount was calculated from HPLC peak areas using response factors of desulfo GS at 229 nm. Results Insect performance on genotypes with different glucosinolate profiles Percentage weight increase of larvae force-fed on each of the geno- types was significantly different in the first experiment (Fig. 1A). Larval performance was poorest on the cyp79B2-/cyp79B3- mutants followed by mam3+, and WT. There was no significant difference in percentage weight increase determined after feeding on mam3+ and WT. Although the same trend was observed in the second experiment (Fig. 1B), the differences observed were not significant. To explain the impact of different types of GS on P. cochleariae larval performance, simple correlation of the larval weight increase to induced total GS contents were performed. We found that there was no correlation with R = 0.136 between the weight increase of the larvae and increasing levels of induced total GS among all genotypes. The performance of larvae was comparable poor on cyp79B2-/ cyp79B3- mutants with lowest GS levels. Although a different effect of GS classes was expected, no correlation of larvae performance to induced indolyl (R = 0.231) or aliphatic GS (R = 0.032) contents of genotypes was found (Fig. 2 A and B). Fig. 1: Percentage weight increase of P. cochleariae larvae on different A. thaliana genotypes in the first (A) and second experiment (B) (Different letters indicate significant differences among treatments, Fisher’s LSD p < 0.05) Performance of Phaedon cochleariae 109 110 M.M. Uddin, Ch. Ulrichs, J.G.Tokuhisa, I. Mewis R = 0.032 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 Induced alipahtic glucosinolate content [µmol/g dry weight] Cyp79B2-/B3- MAM3+ ColWT B R = 0.213 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 30 Induced indolyl glucosinolate content [µmol/g dry weight] W e ig h t in c re a se o f P . co c h le a ri a e la rv a e [m g ] A Glucosinolate induction of Arabidopsis thaliana genotypes by Phaedon cochleariae The experimental A. thaliana mutant lines differed in their GS profile compared to the WT (Tab. 1). In the WT the major aliphatic and indolyl GS were 4-methylsulfinylbutyl GS and indol-3-ylmethyl GS, respectively. In contrast to WT, 3-methylsulfinylpropyl GS was the dominant aliphatic GS in mam3+ and levels of the long chain aliphatic GS, C7 and C8, were increased. Overall, levels of aliphatic GS were lower than in WT but with about similar levels of indolyl GS. The major GS compound detected in cyp79B2-/B3- was 4-methylsulfinyl- butyl GS followed by 3-methylsulfinylpropyl GS, as in WT, but indolyl GS were complete absent in this mutant. Similar results for constitutive and induced levels were observed for the two ex- periments. The accumulation of GS following herbivory damage differed in the three genotypes (Tab. 1). After feeding of P. cochleariae, levels of total GS increased in all genotype lines, but the accumulation was only significant with the cyp79B2-/B3- mutant (Tukey’s HSD test p = 0.004). In the WT, a marked increase of indol-3-ylmethyl and 1- methoxyindol-3-ylmethyl GS levels was observed after larval feeding (Tab. 1). Also the major methylsulfinyl GS increased after herbivory of P. cochleariae. In the mam3+ mutant levels of nearly all aliphatic (except 4-methylthiobutyl GS and 8-methylsulfinyloctyl GS) and indolyl GS increased after feeding by larvae. The major aliphatic GS, 4-methylsulfinylbutyl GS increased about two/three-fold. As with the WT response, a strong increase of indol-3-ylmethyl and 1- methoxyindol-3-ylmethyl GS was observed after beetle herbivory. Only methylsulfinyl GS were induced by larvae feeding in cyp79B2- /B3-, wherein this GS increased about two-fold in this mutant. Discussion It has been shown that GS and their corresponding hydrolysis products determine plant resistance in crucifers such as A. thaliana and Brassica sp., especially against generalist insects (LAMBRIX et al., 2001, reviewed in KLIEBENSTEIN et al., 2004). However, GS are known to influence the behavior of insects that specialize on crucifer plants (RENWICK, 2002; MEWIS et al., 2002). The effects of GS are assumed to vary depending on the attacking insect herbi- vore as well as the concentration and composition of GS present in the plants. Many studies have demonstrated that GS and their hydrolysis products stimulate feeding in crucifer specialists (HICKS, 1974; CHEW, 1988a; BARLET and WILLIAMS, 1991; LI et al., 2000). That GS and their corresponding hydrolysis products can adversely affect the performance of specialists as well is reported by AGRAWAL and KURASHIGE (2003). They reported a negative effect of higher GS concentrations on the lepidopteron Pieris rapae L. To study the effect of GS classes on the crucifer specialist P. coch- leariae, we used different mutant lines of A. thaliana with modified GS profiles and their corresponding WT. Larval performance in the experiments, measured as percentage weight increase, was best on Columbia WT, followed by mam3+, and the double knock out mutant cyp79B2-/cyp79B3-. This was opposite to detected constitutive and induced GS contents in genotypes, whereby levels were highest in the WT and lowest in cyp79B2-/cyp79B3-. Furthermore, we did not find a negative correlation of larval performance to increasing total GS contents of genotypes. This differs to other studies, where weight increase of crucifer specialist larvae was slower on plants with higher GS contents (NAYAR and THORSTEINSON, 1963; AHMAN, 1986; LOUDA and COLLINGE, 1992; WOLFSON, 1982; AGRAWAL and KURASHIGE, 2003; ROHR et al., 2006). For example, LI et al. (2000) reported that there was a negative correlation of larvae weight increase in Plutella xyllostella (L.) and Spodoptera eridania (Stoll) to an increasing allyl GS concentration. Also ROHR et al. (2006) found that larval per- formance of Pieris brassicae L. was negatively related to increasing GS contents in A. thaliana ecotypes. In contrast, KLIEBENSTEIN et al. (2005) demonstrated that plant feeding damage was positively correlated to increasing levels of aliphatic GS in the crucifer specialist P. xylostella, whereas negative relationships of larval performance and increasing GS levels were detected for the generalist caterpillars Trichoplusia ni (Hübner) and Spodoptera exigua (Hübner). In our present study the insect-plant interaction seems to be complex, but it can be noted that the P. cochleariae performance was worse on the mutant which contain only aliphatic GS. Furthermore, beetles feeding induced aliphatic GS to a greater extent in cyp79B2-/cyp79B3- and mam3+ compared to the WT at the end of the experiment (second harvest, Tab. 1). The study of SIEMENS and MITCHELLOLDS (1996) with Phyllotreat cruciferae (Goeze) and P. xylostella showed that herbivory of these insects varied curvilinearly with GS levels in Brassica campestris (L.), with maximum herbivory at intermediate GS levels. Such relationship could be also true for P. cochleariae. A. thaliana ecotypes differ especially in their aliphatic but not their indolyl GS structures (KLIEBENSTEIN et al., 2001; REICHELT et al., 2002). From the high variability of aliphatic GS it is assumed that structurally different GS and their hydrolysis products have different biological effects. For example, higher oviposition preference by Fig. 2: Correlation between weight increase of P. cochleariae larvae and induced aliphatic (A) and indolyl glucosinolate (B) content of A. thaliana genotypes (1st experiment) Performance of Phaedon cochleariae 111 Tab. 1: Glucosinolate content of Arabidopsis thaliana genotypes in the first experiments, control plants and plants after P. cochleariae feeding (induction of single compounds is in grey) Genotype Glucosinolate Glucoinolate content (µmol/g dry weight) First harvest Second harvest Control Phaedon Control Phaedon 3-Methylsulfinylpropyl 4.34 5.04 4.77 5.76 4-Methylsulfinylbutyl 26.07 27.47 24.72 36.96 5-Methylsulfinylpentyl 0.71 0.86 0.72 1.08 6-Methylsulfinylhexyl 0.13 0.21 0.17 0.26 4-Hydroxyindol-3-ylmethyl 0.10 0.19 0.27 0.32 7-Methylsulfinylheptyl 0.97 0.96 0.93 1.12 4-Methylthiobutyl 1.30 1.29 1.18 1.10 Indol-3-ylmethyl 0.86 6.51 5.24 10.14 8-Methylsulfinyloctyl 0.00 0.00 0.00 0.64 4-Methoxyindol-3-ylmethyl 1.28 1.24 0.32 0.90 1-Methoxyindol-3-ylmethyl 0.57 1.13 0.00 1.20 7-Methylthioheptyl 0.10 0.14 0.05 0.09 8-Methylthiooctyl 0.19 0.22 0.13 0.20 Total 36.63a 45.27a 38.48a 59.76a 3-Methylsulfinylpropyl 11.53 12.78 11.46 15.31 4-Methylsulfinylbutyl 4.22 7.07 3.61 10.13 5-Methylsulfinylpentyl 0.24 0.42 0.25 0.49 6-Methylsulfinylhexyl 0.38 0.66 0.58 1.14 4-Hydroxyindol-3-ylmethyl 0.07 0.14 0.12 0.33 7-Methylsulfinylheptyl 1.77 2.90 2.14 3.23 4-Methylthiobutyl 0.35 0.39 0.16 0.34 Indol-3-ylmethyl 5.59 7.59 6.60 12.79 8-Methylsulfinyloctyl 1.22 0.29 0.00 0.04 4-Methoxyindol-3-ylmethyl 1.34 1.42 1.10 1.83 1-Methoxyindol-3-ylmethyl 0.47 1.94 0.08 0.49 7-Methylthioheptyl 0.47 0.93 0.63 0.73 8-Methylthiooctyl 0.75 1.29 0.81 1.54 Total 28.38a 37.81a 27.53a 48.38b 3-Methylsulfinylpropyl 1.68 2.34 2.19 3.68 4-Methylsulfinylbutyl 9.36 14.91 10.79 21.73 5-Methylsulfinylpentyl 0.28 0.49 0.32 0.75 6-Methylsulfinylhexyl 0.07 0.13 0.06 0.19 4-Hydroxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 7-Methylsulfinylheptyl 0.32 0.67 0.32 0.86 4-Methylthiobutyl 1.10 1.03 0.96 0.85 Indol-3-ylmethyl 0.00 0.00 0.00 0.00 8-Methylsulfinyloctyl 1.22 2.73 1.68 3.65 4-Methoxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 1-Methoxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 7-Methylthioheptyl 0.06 0.10 0.06 0.06 8-Methylthiooctyl 0.14 0.22 0.17 0.15 Total 14.21a* 22.60b 16.53a 31.93b *different letters indicate significant differences among treatments, Tukey’s HSD test p < 0.05 W T m am 3+ cy p7 9B 2- /B 3- 112 M.M. Uddin, Ch. Ulrichs, J.G.Tokuhisa, I. Mewis Hellula undalis (F.) was noted for aliphatic alkenyl GS than indolyl GS (MEWIS et al., 2002). Also KLIEBENSTEIN et al. (2002b) found evidence for diverse biological activities of GS by studying their variation and impact on plant resistance against the generalist T. ni. The authors suggested that GS with alkenyl side chains were more of a deterrent to the test insect than GS with non-alkenyl side chains. ROHR et al. (2006) observed a correlation of side chain structure of GS in A. thaliana ecotypes and the performances of a generalist and specialist lepidopteron, S. exigua and P. brassicae, respectively. Here larval weight gain was greater on the 3-hydroxypropyl GS-containing ecotypes than on methylsulphinyl GS-producing ecotypes. Further- more, GIAMOUSTARIS and MITHEN (1995) reported an increasing damage of Brassica napus L. plants by the cabbage stem flea beetle, Psylliodes chrysocephala (L.), with plants containing GS with shorter side chains and increased levels of hydroxylation. Our initial results with P. cochleariae do not support the notion of different responses to structurally distinct classes of GS by a specialist. However, in this study P. cochleariae fed on only a limited number of GS, which probably represents a subset of all the GS that the species may encounter. However, further studies with different specialist insects are needed to identify general effects of GS structures on feeding behavior that could be employed by plant breeders to develop insect resistance crop plants. Acknowledgement We thank Dr. Joanne Chory (The Salk Institute for Biological Studies, California, US) for providing the cyp79B2-/cyp79B3- mutant lines. 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