Chemosystematics of Brassicales

The order Brassicales, sensu APG III, belongs to Eurosids, and comprises 17 families and 398 genera. The present work discusses the chemical features of Brassicales through the micromolecular chemical data of its taxa and selected taxonomic markers to assess pertinent affinities between its families by correlating their chemosystematic parameters. Although the chemical data of all families were obtained, the data of Brassicaceae, Capparaceae, and Cleomaceae were the most studied. The chemistry of the Brassicales species is diverse, but it reveals the chemical affinity of its families due to occurrence of flavonoids (35%) and glucosinolates (25%), which were characterized as good chemical markers. The flavonoids consist primarily of flavones and flavonols, presenting a low flavone/flavonol ratio. These micromolecules commonly contain unprotected hydroxyls, which are mainly protected by glucosilation, revealing the basal features of its taxa. In Brassicales, the predominantly allyl glucosinolates are usually found in Brassicaceae, Capparaceae, and Cleomaceae families. In the present study, the results of the chemosystematic analysis confirmed the affinity among the Brassicaceae, Capparaceae, and Cleomaceae families, and supported the concept of their monophyly in the Brassicales order. However, more chemical data of the other families is required to improve the chemosystematic conclusions.     

Piecing together the transport pathway of aliphatic glucosinolates

Glucosinolates are sulfur-rich secondary metabolites characteristic of the Brassicales order. Transport of glucosinolates was suggested more than 30 years ago through a number of studies which indicated that glucosinolates are produced in maternal tissue and subsequently transported to the seed. These observations laid the foundation for numerous studies on glucosinolate transport which have provided a wealth of information on biochemical properties of glucosinolate transport, source–sink relationships between organs and on the transport routes of glucosinolates. However, most of the conclusions and hypotheses proposed in these studies have not been discussed in context of each other to provide a complete overview of the current state of knowledge on glucosinolate transport. In this review, we are thus piecing together the glucosinolate pathway by presenting and critically analyzing all data on glucosinolate research. Furthermore, the data on glucosinolate transport is considered in the light of the newest findings on glucosinolate synthesis and distribution. The aim is to provide a comprehensive and updated set of hypotheses which may prove useful in directing future research on glucosinolate transport.     

Parallel evolution of glucosinolate biosynthesis inferred from congruent nuclear and plastid gene phylogenies

The phytochemical system of mustard-oil glucosides (glucosinolates) accompanied by the hydrolytic enzyme myrosinase (beta-thioglucosidase), the latter usually compartmented in special myrosin cells, characterizes plants in 16 families of angiosperms. Traditional classifications place these taxa in many separate orders and thus imply multiple convergences in the origin of this chemical defense system. DNA sequencing of the chloroplast rbcL gene for representatives of all 16 families and several putative relatives, with phylogenetic analyses by parsimony and maximum likelihood methods, demonstrated instead a single major clade of mustard-oil plants and one phylogenetic outlier. In a further independent test, DNA sequencing of the nuclear 18S ribosomal RNA gene for all these exemplars has yielded the same result, a major mustard-oil clade of 15 families (Akaniaceae, Bataceae, Brassicaceae, Bretschneideraceae, Capparaceae, Caricaceae, Gyrostemonaceae, Koeberliniaceae, Limnanthaceae, Moringaceae, Pentadiplandraceae, Resedaceae, Salvadoraceae, Tovariaceae, and Tropaeolaceae) and one outlier, the genus Drypetes, traditionally placed in Euphorbiaceae. Concatenating the two gene sequences (for a total of 3254 nucleotides) in a data set for 33 taxa, we obtain robust support for this finding of parallel origins of glucosinolate biosynthesis. From likely cyanogenic ancestors, the "mustard oil bomb" was invented twice.     

Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae)

Glucosinolates are biologically active secondary metabolites that display both intra- and interspecific variation in the order Brassicales. Glucosinolate profiles have not been interpreted within a phylogenic framework and little is known regarding the processes that influence the evolution of glucosinolate diversity at a macroevolutionary scale. We have analyzed leaf glucosinolate profiles from members of the Brassicaceae that have diverged from Arabidopsis thaliana within the last 15 million years and interpreted our findings relative to the phylogeny of this group. We identified several interspecific polymorphisms in glucosinolate composition. A majority of these polymorphisms are lineage-specific secondary losses of glucosinolate characters, but a gain-of-character polymorphism was also detected. The genetic basis of most observed polymorphisms appears to be regulatory. In the case of A. lyrata, geographic distribution is also shown to contribute to glucosinolate metabolic diversity. Further, we observed evidence of gene-flow between sympatric species, parallel evolution, and the existence of genetic constraints on the evolution of glucosinolates within the Brassicaceae.     

Morphological and molecular confirmation of Albugo resedae (Albuginales; Oomycota) as a distinct species from A. candida

The genus Albugo s.str. causes white blister rust on four families of the Brassicales, Brassicaceae, Capparaceae, Cleomaceae, and Resedaceae. Recent phylogenetic studies have revealed that several host specific lineages are present within Albugo on Brassicales, while it was also confirmed that Albugo candida has an exceptionally wide host range which extends from Brassicaceae to Cleomaceae and Capparaceae. The Albugo species infecting the Resedaceae was attributed in monographic studies as well as local floras to either A. resedae or, applying a broader species concept, to A. candida. In the present study, A. resedae specimens were morphologically and molecularly compared to the five Albugo species so far confirmed from Brassicales, A. candida, A. koreana, A. laibachii, A. lepidii, and A. voglmayrii. Both morphological differences of oospore ornamentation and phylogenetic analysis of cox2 mtDNA sequences provided evidence that A. resedae is distinct from A. candida and from the additional four species so far described from Brassicaceae. It thus seems possible that so far unknown factors restrict Albugo candida to Brassicaceae and its sister families, Cleomaceae and Capparaceae.     

Evolution of specifier proteins in glucosinolate-containing plants

ABSTRACT: BACKGROUND: The glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown. RESULTS: Based on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in Escherichia coli. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, but not within the AtNSP1 cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes. CONCLUSIONS: Among specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the appearance of lineage I and expanded lineage II of the Brassicaceae. TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.     

Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanism

As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.     

Glucosinolates of nine cruciferae and two capparaceae species

Autolysis products of nine species of the Cruciferae and two species of the Capparaceae were analysed by high sensitivity GC/MS. Four of the Cruciferae species were examined for glucosinolates for the first time. One new glucosinolate, 9-methylthiononylglucosinolate, was identified in Arabis purpurea and many known glucosinolates were identified for the first time in previously studied plant species. 5-Methylthiopentylglucosinolate appears to be characteristic of the genus Alyssum.     

Tipping the scales--specifier proteins in glucosinolate hydrolysis

Glucosinolates are a group of secondary plant metabolites found in the Brassicales order that are beneficial components of our diet, determine the flavor of a number of vegetables and spices and have been implicated in pest management strategies. These properties, most of the biological activities and the pungent odor and taste associated with glucosinolate-containing plants are due to the products formed from glucosinolates by their hydrolytic enzymes, myrosinases, upon tissue disruption. Specifier proteins impact the outcome of glucosinolate hydrolysis without having hydrolytic activity on glucosinolates themselves. In the presence of specifier proteins, glucosinolate hydrolysis results in nitriles, epithionitriles and organic thiocyanates whose biological functions are currently unknown. In contrast, isothiocyanates formed in the absence of specifier proteins have been demonstrated to possess a variety of biological activities and are thought to protect plants from herbivore and pathogen attack. This review discusses the current knowledge on plant and insect specifier proteins with special emphasis on their biochemical properties and possible mechanisms of action.     

Phylogeny of Capparaceae and Brassicaceae based on chloroplast sequence data

Capparaceae and Brassicaceae have long been known to be closely related families, with the monophyly of Capparaceae more recently questioned. To elucidate the relationship between Brassicaceae and Capparaceae as well as to address infrafamilial relationships within Capparaceae, we analyzed sequence variation for a large sampling, especially of Capparaceae, of these two families using two chloroplast regions, trnL-trnF and ndhF. Results of parsimony and likelihood analyses strongly support the monophyly of Brassicaceae plus Capparaceae, excluding Forchhammeria, which is clearly placed outside the Brassicaceae and Capparaceae clade and suggest the recognition of three primary clades-Capparaceae subfamily (subf.) Capparoideae, subf. Cleomoideae, and Brassicaceae. Capparaceae monophyly is strongly contradicted with Cleomoideae appearing as sister to Brassicaceae. Two traditionally recognized subfamilies of Capparaceae, Dipterygioideae and Podandrogynoideae, are embedded within Cleomoideae. Whereas habit and some fruit characteristics demarcate the three major clades, floral symmetry, stamen number, leaf type, and fruit type all show homoplasy. Clades within Capparoideae show a biogeographical pattern based on this sampling. These results are consistent with several alternative classification schemes.     

Brassicales: an update on chromosomal evolution and ancient polyploidy

Brassicales comprise 17 families, c. 400 genera and more than 4600 species. Despite the mustard family (crucifers, Brassicaceae) continuing to be the subject of intensive research, the remaining 16 families are largely under studied. Here I summarize the available data on chromosome number and genome size variation across Brassicales in the context of a robust phylogenetic framework. This analysis has revealed extensive knowledge gaps in karyological data for non-crucifer and species-rich families in particular (i.e., Capparaceae, Cleomaceae, Resedaceae and Tropaeolaceae). A parsimonious interpretation of the combined chromosomal and phylogenetic data set suggests that the ancestral pre-Brassicales genome had 9 or 14 chromosome pairs, later multiplied by the At-β (beta) whole-genome duplication (WGD) to n?=?18 or 28. This WGD was followed by post-polyploid diploidization marked by diversification to 12 or 13 families and independent decreases in chromosome numbers. Family-specific WGDs are proposed to precede the diversification of Capparaceae, Resedaceae and Tropaeolaceae.     

Bayesian mixed models and the phylogeny of pitvipers (Viperidae: Serpentes)

The subfamily Crotalinae (pitvipers) contains over 190 species of venomous snakes distributed in both the Old and New World. We incorporated an extensive sampling of taxa (including 28 of 29 genera), and sequences of four mitochondrial gene fragments (2.3kb) per individual, to estimate the phylogeny of pitvipers based on maximum parsimony and Bayesian phylogenetic methods. Our Bayesian analyses incorporated complex mixed models of nucleotide evolution that allocated independent models to various partitions of the dataset within combined analyses. We compared results of unpartitioned versus partitioned Bayesian analyses to investigate how much unpartitioned (versus partitioned) models were forced to compromise estimates of model parameters, and whether complex models substantially alter phylogenetic conclusions to the extent that they appear to extract more phylogenetic signal than simple models. Our results indicate that complex models do extract more phylogenetic signal from the data. We also address how differences in phylogenetic results (e.g., bipartition posterior probabilities) obtained from simple versus complex models may be interpreted in terms of relative credibility. Our estimates of pitviper phylogeny suggest that nearly all recently proposed generic reallocations appear valid, although certain Old and New World genera (Ovophis, Trimeresurus, and Bothrops) remain poly- or paraphyletic and require further taxonomic revision. While a majority of nodes were resolved, we could not confidently estimate the basal relationships among New World genera and which lineage of Old World species is most closely related to this New World group.     

Elucidating the Role of Transport Processes in Leaf Glucosinolate Distribution

In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.Feeding behavior of herbivorous insects and distribution of defense compounds in plants have been suggested to be a result of an arms race between plants and insects that has spanned millions of years (Ehrlich and Raven, 1964). Whether insects adapted first to plants or the other way around is an ongoing debate in this research field (Schoonhoven et al., 2005; Ali and Agrawal, 2012). Leaf margin accumulation of defense compounds has been demonstrated in various plant species (Gutterman and Chauser-Volfson, 2000; Chauser-Volfson et al., 2002; Kester et al., 2002; Cooney et al., 2012). In the model plant Arabidopsis (Arabidopsis thaliana), higher concentration of glucosinolates, which constitute a major part of the chemical defense system in this plant (Kliebenstein et al., 2001a; Halkier and Gershenzon, 2006), was found at the leaf midrib and margins compared with the leaf lamina (Shroff et al., 2008; Sønderby et al., 2010). This nonuniform leaf distribution of glucosinolates appeared to explain the feeding pattern of a generalist herbivore (Helicoverpa armigera), as it avoided feeding at the leaf margin and midrib (Shroff et al., 2008). A similar feeding pattern on Arabidopsis was observed for a different generalist herbivore, Spodoptera littoralis (Schweizer et al., 2013). Interestingly, S. littoralis was shown to favor feeding from Arabidopsis leaf margins in glucosinolate-deficient mutants (Schweizer et al., 2013), which could indicate an inherent preference for margin feeding and that Arabidopsis adapted to such behavior by accumulating defense compounds here. A damaged leaf margin may be more critical for leaf stability than damage to inner leaf parts (Shroff et al., 2008), further motivating protection of this tissue. The margin-feeding preference of S. littoralis might be explained by better nutritional value of the leaf margin cells (Schweizer et al., 2013), which has been shown to consist of specialized elongated cell files (Koroleva et al., 2010; Nakata and Okada, 2013).Other distribution patterns have been reported for glucosinolates in an Arabidopsis leaf. A study investigating spatiotemporal metabolic shifts during senescence in Arabidopsis reported that fully expanded mature leaves exhibited a glucosinolate gradient from base to tip, with highest level of glucosinolates at the leaf base (Watanabe et al., 2013). In contrast to the horizontal plane, less has been reported on distribution of glucosinolates in the vertical plane of a leaf. A localization study of cyanogenic glucosides, defense molecules related to glucosinolates (Halkier and Gershenzon, 2006), determined that these compounds primarily were located in the epidermis of sorghum (Sorghum bicolor; Kojima et al., 1979). Whereas epidermis-derived trichomes in Arabidopsis were recently demonstrated to contain glucosinolates and to express glucosinolate biosynthetic genes (Frerigmann et al., 2012), no studies have investigated glucosinolates in the epidermal cell layer.Based on promoter-GUS studies, biosynthesis of glucosinolates in leaves of Arabidopsis has been associated with primarily major and minor veins in leaves and silique walls (Mikkelsen et al., 2000; Reintanz et al., 2001; Tantikanjana et al., 2001; Chen et al., 2003; Grubb et al., 2004; Schuster et al., 2006; Gigolashvili et al., 2007; Li et al., 2011; Redovniković et al., 2012). The discrepancy between vasculature-associated glucosinolate biosynthesis and margin accumulation of glucosinolates suggests that transport processes must be involved in establishing the distribution pattern of glucosinolates within a leaf.Plant transport systems include the apoplastic xylem, the symplastic phloem, and plasmodesmata. Xylem transport is mainly driven by an upward pull generated by transpiration from aerial plant organs, thereby directing transport to sites of rapid evaporation (such as leaf margins; Sattelmacher, 2001). Phloem flow is facilitated by an osmosis-regulated hydrostatic pressure difference between source and sink tissue, primarily generated by Suc bulk flow (Lucas et al., 2013). Plasmodesmata are intercellular channels that establish symplasmic pathways between neighboring cells, and most cell types in a plant are symplastically connected via plasmodesmata (Roberts and Oparka, 2003). Translocation of small molecules in these channels is driven by diffusion and is regulated developmentally as well as spatially to form symplastically connected domains (Roberts and Oparka, 2003; Christensen et al., 2009). To what extent any of these transport processes are involved in establishing specific distribution patterns of glucosinolates within leaves is not known.Recently, two plasma membrane-localized, glucosinolate-specific importers, GLUCOSINOLATE TRANSPORTER1 (GTR1) and GTR2, were identified in Arabidopsis (Nour-Eldin et al., 2012). In leaf, their expression patterns were shown to be in leaf veins (GTR1 and GTR2) and surrounding mesophyll cells (GTR1; Nour-Eldin et al., 2012). Absence of aliphatic and indole glucosinolates in seeds of the gtr1gtr2 double knockout (dKO) mutant (gtr1gtr2 dKO) demonstrated that these transporters are essential for long-distance glucosinolate transport to the seeds and indicates a role in phloem loading (Nour-Eldin et al., 2012). Another study investigating long-distance transport of glucosinolates in the 3-week-old wild type and gtr1gtr2 dKO indicated that GTR1 and GTR2 were involved in bidirectional transport of aliphatic glucosinolates between root and shoot via both phloem and xylem pathways (Andersen et al., 2013). The authors suggested a role for GTR1 and GTR2 in the retention of long-chained aliphatic glucosinolates in roots by removing the compounds from the xylem (Andersen et al., 2013).Identification of the glucosinolate transporters GTR1 and GTR2 has provided a molecular tool to investigate the role of transport processes in establishing leaf glucosinolate distribution. In this study, we have performed a detailed spatial investigation of the distribution of an exogenously fed glucosinolate (sinigrin) and endogenous glucosinolates within mature wild-type and gtr1gtr2 dKO Arabidopsis leaves, achieved by collecting and analyzing leaf parts at the horizontal (y axis: petiole, base, and tip; x axis: midrib, lamina, and margin) as well as at the vertical leaf plane (z axis: abaxial epidermis). Furthermore, we analyze wild-type and gtr1gtr2 dKO root xylem sap and leaf apoplastic fluids for glucosinolates. Based on our results, we propose a model where GTR1 and GTR2 import glucosinolates from the apoplast to the symplast and where the glucosinolate distribution pattern within an Arabidopsis leaf is established via symplasmic movement of glucosinolates through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in peripheral cell layers.     

外源喷施萘乙酸对小白菜硫代葡萄糖苷的影响

本试验研究了不同浓度的萘乙（NAA）并结合单次喷施（NAA-1）和两次喷施（NAA．2）,对小白菜生长和硫代葡萄糖苷（简称硫苷）含量的影响。结果表明,与对照相比,不同的NAA处理浓度均显著增加了小白菜的鲜重。同时,NAA处理对总硫苷和单个硫苷含量产生了显著的影响。NAA-1处理时,总脂肪族硫苷、2．苯乙基硫苷和总硫苷在20mg·L-1时到达最大值;而总吲哚族硫苷在50mg·L-1时含量到达最高。NAA．2处理时,大部分单个硫苷和总硫苷在10mg·L-1处理即达到最大值。可见,在较低浓度两次喷施NAA试验中对大部分硫苷的诱导效果高于单次喷施NAA;但随着NAA处理浓度的提高,单次喷施NAA对硫苷的诱导效果较好。其中,吲哚-3-甲基硫苷含量在NAA-1处理,喷施50mg·L-1时显著高于其他处理：而2-苯乙基硫苷在单次和两次喷施NAA时,分别在20mg·L-1和10mg·L-达到了最大值。     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.     

Piecing together the "new" Plantaginaceae

Scrophulariaceae is one of the families that has been divided extensively due to the results of DNA sequence studies. One of its segregates is a vastly enlarged Plantaginaceae. In a phylogenetic study of 47 members of Plantaginaceae and seven outgroups based on 3561 aligned characters from four DNA regions (the nuclear ribosomal ITS region and the plastid trnL-F, rps16 intron, and matK-trnK intron regions), the relationships within this clade were analyzed. The results from parsimony and Bayesian analyses support the removal of the Lindernieae from Gratioleae to a position outside Plantaginaceae. A group of mainly New World genera is paraphyletic with respect to a clade of Old World genera. Among the New World taxa, those offering oil as a pollinator reward cluster together. Ourisia is sister to this clade. Gratioleae consist of Gratiola, Otacanthus, Bacopa, Stemodia, Scoparia, and Mecardonia. Cheloneae plus Russelia and Tetranema together constitute the sister group to a clade predominantly composed of Old World taxa. Among the Old World clade, Ellisiophyllum and Lafuentea have been analyzed for the first time in a molecular phylogenetic analysis. The former genus is sister to Sibthorpia and the latter is surprisingly the sister to Antirrhineae.