Les glucosinolates sont des composés thio-b-D-glucopyranosiques anioniques de structure originale présentsdans de nombreux végétaux, essentiellement dans la famille des crucifères. Les glucosinolates sont hydrolysés parune enzyme appelée myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.147.) Le système myrosinase-glucosinolate est un couple enzyme-substrat que nous avons cherché à mieux comprendre et développer. Aussi l’inefficacité des méthodes classiques envers certaines molécules hétéroaromatiques et la quantité restreinte de recherches effectuées sur ce sujet nous a encouragé à vouloir développer de nouvelles voies de synthèse de glucosinolates. Les nouveaux analogues de glucosinolates avec un aglycone modifié ont été synthétisés. Un analogue hétéroaromatique soumis en test a été reconnu et hydrolysé par la myrosinase. En cette façon, une indépendance du mécanisme de reconnaissance par la myrosinase de la taille d’aglycone a été démontrée. Certains de nouveaux analogues avec un aglycone modifié, obtenus en remplaçant le thioglucose par un groupement thioéthyle, ont montré très bonne activité inhibitrice envers myrosinase. Les produits principaux de la dégradation enzymatique - des isothiocyanates et leurs thioadduits correspondants au glucosinolates obtenus auparavant ont été synthétisés. Les tests contre Plasmodium falciparum, le parasite causant le paludisme, ont montré une activité antipaludéenne de ces isothiocyanates du même rang qu’un des médicaments actuellement très largement utilisées. Une méthode de la synthèse des glucosinolates complètement nouvelle, efficace et simple a été mise au point. Cella ouvre de nouvelles possibilités pour la synthèse des glucosinolates sensibles aux conditions habituelles de la synthèse. Cette nouvelle méthode a été aussi appliquée à la synthèse des promettants et très peu étudiés. Egalement, une nouvelle voie d’approche aux glucosinolates thiofunctionalisés a été développée avec succès. / Glucosinolates are anionic thiosaccharidic compounds mainly found in plants of the family Cruciferacea, which may be hydrolysed by myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.1.). Myrosinase-glucosinolate system is a pair enzyme-substrate that we have sought to better understand and develop. Also the inefficiency of traditional methods for certain heteroaromatic molecules and the limited amount of research on this topic encouraged us to try to develop new synthetic pathways for glucosinolates. The new analogs of glucosinolates with a modified aglycone were synthesized.One heteroaromatic analogue submitted to the test was recognized and hydrolyzed by myrosinase. In this way, a mechanism of recognition by myrosinase independent of aglycon size has been demonstrated. Some new analogues with modified aglycon, obtained by replacing thioglucose by a thioethyl moiety showed very good inhibitory activity against myrosinase. The main products of enzymatic degradation of glucosinolates obtained previously - isothiocyanates and their corresponding thioadducts were synthesized. The tests against Plasmodium falciparum, the parasite causing malaria, showed antimalarial activity of these isothiocyanates of the same rank as drugs currently in wide use. A method for the synthesis of glucosinolates completely new, efficient and simple has been developed. That opens new possibilities for the synthesis of glucosinolates sensitive to usual conditions of the synthesis. This new method was also applied to the synthesis of N-oxides thioimidate - promising and little-known compounds. Also, a new way to approach glucosinolates with an external thiofunction has been successfully developed.

L’hypothèse selon laquelle l’accumulation des métaux lourds par les plantes a évolué comme mécanisme de défense contre les herbivores a été testée avec l’hyperaccumulatrice de zinc Thlaspi caerulescens (Brassicaceae). En utilisant l’écotype métallicole (poussant sur sols métallifères) et l’écotype non métallicole (sols normaux) de T. caerulescens, nos résultats ont conduit à rejeter l’hypothèse de défense par accumulation de métaux: les plantes ont été consommées indépendamment de leur concentration en Zn dans toutes les situations expérimentales examinées (conditions contrôlées, jardin expérimental, populations naturelles). Par contre, les herbivores ont montré une préférence systématique pour les plantes de l’écotype métallicole, quelle que soit leur concentration en Zn. Lorsque l’on mesure les concentrations en métabolites secondaires défensifs (glucosinolates) des écotypes métallicole et non métallicole de T. caerulescens, on constate que les individus d’origine métallicole produisent constitutivement moins de glucosinolates que les individus non métallicoles, tant dans les populations belges que dans les populations françaises. Par ailleurs, sur les sites métallifères où ont évolué les populations métallicoles, on constate à la fois une plus faible pression d’herbivorie sur les plantes (moins de dégâts) et une plus faible densité de gastéropodes que dans les sites normaux. La diminution des défenses chez l’écotype métallicole serait la conséquence d’un relâchement de la pression d’herbivorie sur les sites métallifères. En outre, nous avons montré que la chenille spécialiste d’Issoria lathonia (Nymphalidae) est capable de se développer sur les feuilles riches en Zn de l’accumulatrice de zinc Viola calaminaria (Violaceae) en excrétant efficacement le Zn dans leurs fèces. L’ensemble de nos résultats suggère donc que l’hyperaccumulation des métaux lourds n’a pas évolué en tant que mécanisme de défense contre les herbivores.

No description available.

by Wong Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 110-114). / Abstract --- p.i / Acknowledgement --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1 --- Introduction and literature review / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Literature review --- p.5 / Chapter 1.2.1 --- General considerations --- p.5 / Chapter 1.2.2 --- Nature of glucosinolate --- p.6 / Chapter 1.2.3 --- Degradation of glucosinolates by myrosinase --- p.7 / Chapter 1.2.4 --- Toxicology of glucosinolate and hydrolysis products --- p.8 / Chapter 1.2.5 --- Plant myrosinase --- p.9 / Chapter 1.2.6 --- Fungal myrosinase --- p.11 / Chapter 1.2.7 --- Purification and properties of fungal myrosinase --- p.11 / Chapter Chapter 2 --- Screening of fungi with myrosinase activity and physiological studies of myrosinase production in Aspergillus oryzae / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Fungal strains --- p.16 / Chapter 2.2.2 --- Media --- p.16 / Chapter 2.2.3 --- Screening --- p.17 / Chapter 2.2.4 --- Enzyme assay and protein determination --- p.18 / Chapter 2.2.4.1 --- Myrosinase assay --- p.18 / Chapter 2.2.4.2 --- Definition of myrosinase unit and specific activity --- p.19 / Chapter 2.2.4.3 --- Protein determination --- p.19 / Chapter 2.2.5 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.19 / Chapter 2.2.5.1 --- Incubation time --- p.20 / Chapter 2.2.5.2 --- Inducer concentration --- p.20 / Chapter 2.3 --- Results --- p.21 / Chapter 2.3.1 --- Screening --- p.21 / Chapter 2.3.1.1 --- Degradation of sinigrin in culture medium --- p.21 / Chapter 2.3.1.2 --- Confirmation of myrosinase activity --- p.21 / Chapter 2.3.2 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.21 / Chapter 2.3.2.1 --- Incubation time --- p.21 / Chapter 2.3.2.2 --- Inducer concentration --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter 2.4.1 --- Fungi selection in screening programme --- p.23 / Chapter 2.4.2 --- Medium composition --- p.23 / Chapter 2.4.3 --- Screening --- p.24 / Chapter 2.4.4 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.25 / Chapter 2.4.4.1 --- Incubation time --- p.25 / Chapter 2.4.4.2 --- Inducer concentration --- p.25 / Chapter Chapter 3 --- Purification and characterization of myrosinase in Aspergillus oryzae / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Reagents --- p.35 / Chapter 3.2.2 --- Fungal propagation --- p.35 / Chapter 3.2.3 --- Purification of the fungal myrosinase --- p.36 / Chapter 3.2.3.1 --- Preparation of crude extract --- p.36 / Chapter 3.2.3.2 --- Dialysis --- p.37 / Chapter 3.2.3.3 --- DEAE-Sepharose CL-6B ion-exchange chromatography --- p.37 / Chapter 3.2.3.4 --- Sephacryl S-200 molecular sieving chromatography --- p.37 / Chapter 3.2.3.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.38 / Chapter 3.2.3.6 --- FPLC Mono P chromatofocusing --- p.38 / Chapter 3.2.4 --- Myrosinase assay and protein concentration determination --- p.39 / Chapter 3.2.4.1 --- Spot test for myrosinase activity --- p.39 / Chapter 3.2.4.2 --- Standard end-point assay --- p.40 / Chapter 3.2.4.3 --- Determination of protein concentration --- p.42 / Chapter 3.2.5 --- Physicochemical characterization of the myrosinase isozymes --- p.42 / Chapter 3.2.5.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.42 / Chapter 3.2.5.2 --- Protein staining and glycoprotein detection --- p.43 / Chapter 3.2.5.3 --- Chromatofocusing --- p.43 / Chapter 3.2.5.4 --- Gel filtration with FPLC Superose 6 --- p.44 / Chapter 3.2.6 --- Enzymatic properties --- p.44 / Chapter 3.2.6.1 --- Effect of pH on crude enzyme stability --- p.44 / Chapter 3.2.6.2 --- Effect of substrate concentration on enzyme activity --- p.45 / Chapter 3.2.6.3 --- Effect of pH on enzyme activity --- p.45 / Chapter 3.2.6.4 --- Effect of temperature on enzyme activity --- p.46 / Chapter 3.2.6.5 --- Effects of metallic ions on enzyme activity --- p.46 / Chapter 3.2.6.6 --- Effects of various compounds on enzyme activity --- p.46 / Chapter 3.2.6.7 --- Effects of various buffers on enzyme activity --- p.47 / Chapter 3.3 --- Results --- p.48 / Chapter 3.3.1 --- Fungal propagation --- p.48 / Chapter 3.3.2 --- Purification of fungal myrosinase in Aspergillus oryzae --- p.48 / Chapter 3.3.2.1 --- Extraction of the enzyme --- p.48 / Chapter 3.3.2.2 --- Dialysis --- p.49 / Chapter 3.3.2.3 --- DEAE-Sepharose ion-exchange chromatography --- p.49 / Chapter 3.3.2.4 --- Sephacryl S-200 molecular sieving chromatography --- p.50 / Chapter 3.3.2.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.50 / Chapter 3.3.2.6 --- FPLC Mono P chromatofocusing --- p.51 / Chapter 3.3.3 --- Physicochemical characterization --- p.52 / Chapter 3.3.3.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.52 / Chapter 3.3.3.2 --- Chromatofocusing --- p.53 / Chapter 3.3.3.3 --- Gel filtration --- p.53 / Chapter 3.3.4 --- Enzymatic properties --- p.53 / Chapter 3.3.4.1 --- Effect of pH on the crude enzyme stability --- p.53 / Chapter 3.3.4.2 --- Effect of substrate concentration on enzyme activity --- p.54 / Chapter 3.3.4.3 --- Effect of pH on enzyme activity --- p.54 / Chapter 3.3.4.4 --- Effect of temperature on enzyme activity --- p.55 / Chapter 3.3.4.5 --- Effects of metallic ions on enzyme activity --- p.55 / Chapter 3.3.4.6 --- Effects of various compounds on enzyme activity --- p.56 / Chapter 3.3.4.7 --- Effects of various buffers on enzyme activity --- p.57 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Purification of Aspergillus oryzae myrosinase --- p.58 / Chapter 3.4.1.1 --- Dialysis --- p.58 / Chapter 3.4.1.2 --- Enzyme purification --- p.58 / Chapter 3.4.2 --- Physicochemical properties --- p.60 / Chapter 3.4.2.1 --- Glycoprotein --- p.60 / Chapter 3.4.2.2 --- Molecular weights --- p.60 / Chapter 3.4.2.3 --- Isoelectric points --- p.61 / Chapter 3.4.3 --- Enzymatic properties --- p.61 / Chapter 3.4.3.1 --- pH and temperature optima --- p.61 / Chapter 3.4.3.2 --- Substrate affinity --- p.62 / Chapter 3.4.3.3 --- Inhibitions by various compounds and metallic ions --- p.63 / Chapter 3.4.3.4 --- Inhibitions by various buffer systems --- p.64 / Chapter Chapter 4 --- Summary --- p.106 / References --- p.110

Plants produce complex mixtures of secondary metabolites to defend themselves from pathogens. Among these defenses are metabolites produced de novo, phytoalexins, and constitutive metabolites, phytoanticipins. As a counter-attack, pathogenic fungi are able to transform such plant defenses utilizing detoxifying enzymes. This thesis investigates the metabolism of two important cruciferous phytoalexins (brassinin (33) and camalexin (39)) by the phytopathogenic fungus Botrytis cinerea and the metabolism of cruciferous phytoanticipins (glucosinolates and derivatives) by three economically important fungi of crucifers Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum to investigate their role in cruciferous defense. In the first part of this thesis, the transformations of brassinin (33) and camalexin (39) by B. cinerea were investigated. During these studies a number of new metabolites were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. Camalexin (39) was transformed via oxidative degradation and brassinin (33) was hydrolyzed to indoly-3-methanamine (49). The metabolic products did not show detectable antifungal activity against B. cinerea, which indicated that these transformations were detoxification processes. Camalexin (39) was found to be more antifungal than brassinin (33). In the second part of this thesis, the metabolism of glucobrassicin (86), 1-methoxyglucobrassicin (87), 4-methoxyglucobrassicin (90), phenylglucosinolate (65), and benzylglucosinolate (66), the corresponding desulfoglucosinolates and derivatives by three fungal pathogens (A. brassicicola, R. solani and S. sclerotiorum) was investigated and their antifungal activity against the same pathogens was tested. Aryl iii glucosinolates 65 and 66 were metabolized by A. brassicicola but not by R. solani or S. sclerotiorum, whereas indolylglucosinolates were not metabolized by any pathogen. Indolyl desulfoglucosinolates (159 and 233) were transformed by R. solani and S. sclerotiorum to the corresponding carboxylic acids and indolyl acetonitriles 40, 102, and 103 were also metabolized to the corresponding carboxylic acids by all pathogens. None of the glucosinolates or their desulfo derivatives showed antifungal activity, but some of their metabolites showed low to very high antifungal activities. Among these metabolites, diindolyl-3-methane (113) showed the highest antifungal activity, and benzyl isothiocyanate (170) showed higher inhibitory effect against R. solani and S. sclerotiorum, but did not inhibit the growth of A. brassicicola. The cell-free extracts of A. brassicicola, R. solani, and S. sclerotiorum were tested for myrosinase activity against several glucosinolates. The cell-free extracts of mycelia of A. brassicicola displayed higher myrosinase activity for sinigrin (131), phenyl and benzyl glucosinolates 65 and 66, but lower activities for glucobrassicin (86) and 1-methoxyglucobrassicin (87); no myrosinase activity was detected in mycelia of either R. solani or S. sclerotiorum.

Key aspects of the basic agronomy Camelina sativa were evaluated under controlled environment conditions and at multiple field locations in 2011 and 2012. Camelina is a highly adaptable crop. It germinates well even under low water availability and has a great potential for yield compensation. The line CDI007 was the most promising genotype with the highest yield potential, the lowest glucosinolate content, and the highest tolerance to downy mildew. The optimum N rate for seed yield varied by year and location: 100 kg N/ha at Truro and Canning in 2011, 120-150 kg N/ha at Canning, Truro and New Glasgow, 160-200 kg N/ha at Fredericton in 2012. N was positively correlated with protein content, but negatively correlated with oil content. Application of sulphur increased protein content at all of the sites and yield at some of the sites. In general, camelina response to S was maximized when N was sufficient.

The influence of stages of development, soil types, dates of seeding, cultivars, and boron nutrition on glucosinolate composition and variation in rutabaga (Brassica napobrassica Mill.) and turnip (B, rapa L.) cultivars was studied by quantifying the glucosinolate hydrolysis products in these species. While in seeds of both species contents of goitrin and volatile isothiocyanates were inversely correlated, there was little variation in thiocyanate ion and total glucosinolate contents. Major synthesis or accumulation of different glucosinolates occurred at different times during the growing season in the sequence after seeding: indolyl glucosinolates yielding thiocyanate ion at the 2-week stage; glucosinolates yielding volatile isothiocyanate hydrolysis products at the 4-week stage; and progoitrin yielding goitrin at the 6- or 8-week stage. These glucosinolates were generally higher in roots of both species grown on organic soil than on loam soil. During ontogeny of both species, the content of volatile isothiocyanates generally were positively correlated with top/root ratio, while growth rate was positively correlated with thiocyanate ion content. While the contents of goitrin and volatile isothiocyanate tended to be higher in early-seeded (May 27) crops, the thiocyanate ion contents tended to be higher in late-seeded (June 21) crops. The occurrence of boron deficiency symptoms in roots of hydroponically-grown Snow Ball turnip (0.1 ppm boron treatment) was associated with an accumulation of high quantities of glucosinolates and reducing sugars.

Glucosinolates are amino acid derived secondary metabolites found in the order Capparales. It is an important class of phytochemicals involved in plant-microbe, plant-insect, plant-animal and plant-human interactions. It is, therefore, important to understand genetic mechanism of glucosinolate biosynthesis in Brassica for efficient manipulation. In this study, QTL mapping of leaf and seed glucosinolates was performed in B. rapa using two RIL populations, SR-RILs and BU-RILs. QTL mapping was performed using SR-RILs developed from a cross of Chinese cabbage and turnip rapeseed and a genetic map in B.rapa. Genetic map was developed using a total 1,579 molecular markers including 9 markers specific to glucosinolate genes, GSL-ELONG, GSL-PRO, GSL-FMOOX1, and GSL-AOP/ALK. Several QTL for progoitrin, gluconapin, glucoalyssin, glucobrassicanapin, 2-methylpropyl and 4-hydoxyglucobrassicin glucosinolates were identified with phenotype variance between 6 and 54%. Interestingly, a major QTL for 5C aliphatic glucosinolates was co-localized with a candidate Br-GSL-ELONG locus on linkage group A3, displayed co-segregation with co-dominant SCAR marker BrMAM1-1. The Br-GSL-ELONG locus was identified to regulate 20 µmole/g seed 5C glucosinolate biosynthesis. BU-RILs derived from a cross of yellow sarson and USU9 was segregated for glucoerucin, gluconapin and progoitrin 4C aliphatic glucosinolates with 4-hydoxyglucobrassicin. Phenotyping was performed in controlled and field environments for seed glucosinolates and controlled environments for leaf glucosinolates. Genetic map was developed using SRAP markers and glucosinolate gene, GSL-ELONG and GSL-PRO specific 4 loci were integrated on map. Four and three QTL were identified for seed glucoerucin and gluconapin, respectively in both environments with phenotypic variance up to 49%. Additionally, genetic manipulation of glucosinolates was performed by backcross with MAS in B. rapa. Resynthesized B. napus line was backcrossed with B. rapa genotypes, RI16, BAR6 and USU9 for replacement or introgression of glucosinolate genes, GSL-ELONG- and GSL-PRO+. In RI16 genotype, 15 to 25 µmole/g seed 5C glucosinolates reduced in 15 BC3F2 lines those were positive with GSL-ELONG- marker and negative with the A-genome and gene specific marker BrMAM1-1. This suggests that the functional allele has replaced by non-functional from B. oleracea. GSL-PRO+ positive backcross lines in RI16 genotype displayed sinigrin 3C aliphatic glucosinolate in B. rapa. This suggests introgression of GSL-PRO+ in B. rapa.

Brassica carinata (A.) Braun and Camelina sativa (L.) Crantz are two re-emerging oilseed crops of the Brassicaceae family that are being adapted for cultivation in western Canada. Both seeds of these species reportedly accumulate considerable amounts of sulfur-containing secondary metabolites called glucosinolates. The purpose of the current work was to gain knowledge of the occurrence and distribution of glucosinolates during primary processing of these oilseeds, including during pressing and dehulling. In the first study, a reversed phase HPLC method was developed for the analysis of sinigrin, the major glucosinolate in B. carinata. Both C18 columns selected were able to separate the compound with an isocratic eluent containing 100% tetramethylammonium bromide (10 mM, pH 5) delivered at 1 mL/min at a column temperature of 25oC. These chromatographic conditions were applied and sinigrin concentration of whole B.carinata seed was estimated to be 29 μg/mg. Average matrix effect was estimated to be 104% that was caused by other components in the B. carinata seed matrix. In the second study, high concentrations of glucosinolates were detected and identified in fractions of C. sativa seeds using HPLC-ESI-MS. Methods for extraction, isolation, and purification of three individual glucosinolates from these fractions are reported. Quantitation of total glucosinolates was performed on proton NMR using DMF as an internal standard. Quantitation of individual glucosinolates was achieved by using MS extracted ion chromatogram data. Total glucosinolates were found in C. sativa whole seed at a concentration of 14 μg/mg, and glucocamelinin, the major glucosinolate, constituted 65% of the total amount. In addition, a dehulling treatment was applied to C. sativa seeds, from which both oil content and crude protein content increased after dehulling of the seeds.

Glucosinolates are amino acid derived secondary metabolites found in the order Capparales. It is an important class of phytochemicals involved in plant-microbe, plant-insect, plant-animal and plant-human interactions. It is, therefore, important to understand genetic mechanism of glucosinolate biosynthesis in Brassica for efficient manipulation. In this study, QTL mapping of leaf and seed glucosinolates was performed in B. rapa using two RIL populations, SR-RILs and BU-RILs. QTL mapping was performed using SR-RILs developed from a cross of Chinese cabbage and turnip rapeseed and a genetic map in B.rapa. Genetic map was developed using a total 1,579 molecular markers including 9 markers specific to glucosinolate genes, GSL-ELONG, GSL-PRO, GSL-FMOOX1, and GSL-AOP/ALK. Several QTL for progoitrin, gluconapin, glucoalyssin, glucobrassicanapin, 2-methylpropyl and 4-hydoxyglucobrassicin glucosinolates were identified with phenotype variance between 6 and 54%. Interestingly, a major QTL for 5C aliphatic glucosinolates was co-localized with a candidate Br-GSL-ELONG locus on linkage group A3, displayed co-segregation with co-dominant SCAR marker BrMAM1-1. The Br-GSL-ELONG locus was identified to regulate 20 µmole/g seed 5C glucosinolate biosynthesis. BU-RILs derived from a cross of yellow sarson and USU9 was segregated for glucoerucin, gluconapin and progoitrin 4C aliphatic glucosinolates with 4-hydoxyglucobrassicin. Phenotyping was performed in controlled and field environments for seed glucosinolates and controlled environments for leaf glucosinolates. Genetic map was developed using SRAP markers and glucosinolate gene, GSL-ELONG and GSL-PRO specific 4 loci were integrated on map. Four and three QTL were identified for seed glucoerucin and gluconapin, respectively in both environments with phenotypic variance up to 49%. Additionally, genetic manipulation of glucosinolates was performed by backcross with MAS in B. rapa. Resynthesized B. napus line was backcrossed with B. rapa genotypes, RI16, BAR6 and USU9 for replacement or introgression of glucosinolate genes, GSL-ELONG- and GSL-PRO+. In RI16 genotype, 15 to 25 µmole/g seed 5C glucosinolates reduced in 15 BC3F2 lines those were positive with GSL-ELONG- marker and negative with the A-genome and gene specific marker BrMAM1-1. This suggests that the functional allele has replaced by non-functional from B. oleracea. GSL-PRO+ positive backcross lines in RI16 genotype displayed sinigrin 3C aliphatic glucosinolate in B. rapa. This suggests introgression of GSL-PRO+ in B. rapa.