To explore natural biodiversity we developed and examined introgression lines (ILs) containing chromosome segments of wild species (Solanum pennellii) in the background of the cultivated tomato (S. lycopersicum). We identified Brix9-2-5, which is a S. pennellii quantitative trait locus (QTL) that increases sugar yield of tomatoes and was mapped within a flower- and fruit-specific invertase (LIN5). QTL analysis representing five different tomato species delimited the functional polymorphism of Brix9-2-5 to an amino acid near the catalytic site of the invertase crystal, affecting enzyme kinetics and fruit sink strength. These results underline the power of diverse ILs for high-resolution perspectives on complex phenotypes.

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.floral scent ͉ phenylpropanoids ͉ phenylpropenes ͉ plant volatiles ͉ secondary compounds

Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-Å resolution. The structures confirm that SABP2 is a member of the ␣͞␤ hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well.salicylic acid ͉ salicylic-acid-binding protein ͉ systemic acquired resistance ͉ ␣͞␤ hydrolase

In nature, genetic variation usually takes the form of a continuous phenotypic range rather than discrete classes. The genetic variation underlying quantitative traits results from the segregation of numerous interacting quantitative trait loci (QTLs), whose expression is modified by the environment. To uncover the molecular basis of this variation, we characterized a QTL (Brix9-2-5) derived from the green-fruited tomato species Lycopersicon pennellii. The wild-species allele increased glucose and fructose contents in cultivated tomato fruits in various genetic backgrounds and environments. Using nearly isogenic lines for the QTL, high-resolution mapping analysis delimited Brix9-2-5 to a single nucleotide polymorphism-defined recombination hotspot of 484 bp spanning an exon and intron of a fruit-specific apoplastic invertase. We suggest that the differences between the Brix9-2-5 alleles of the two species are associated with a polymorphic intronic element that modulates sink strength of tomato fruits. Our results demonstrate a link between naturally occurring DNA variation and a Mendelian determinant of a complex phenotype for a yield-associated trait. D omesticated species represent only a small fraction of the variability available among their wild relatives. The use of saturated molecular linkage maps has enhanced our ability to study and exploit this exotic variation in plants and animals (1). The Mendelian resolution of quantitative trait loci (QTLs) was simplified with the development of nearly isogenic lines (NILs), in which a single genomic segment contains the QTL in an otherwise uniform genetic background. Such resources have enabled more accurate estimates of the number of QTLs that affect a trait, their mode of inheritance, and linkage relationships, up to a yeast artificial chromosome (YAC) resolution (2, 3).One of the major objectives in tomato breeding is to increase the content of total soluble solids (TSS or brix; mainly sugars and acids) in fruits, to improve taste and processing qualities. TSS in fruits of wild Lycopersicon species can reach up to 15% of the fruit's fresh weight, 3 times higher than in cultivated varieties. To resolve the genetic basis for this variation, a set of 50 introgression lines was developed from a cross between the green-fruited species Lycopersicon pennellii and the cultivated tomato, Lycopersicon esculentum. Each of the NILs contained a single restriction fragment length polymorphism (RFLP)-defined L. pennellii chromosome segment, and together the lines provided complete coverage of the genome. Using this resource, it was possible to map 23 QTLs that increase brix (4).One of these QTLs (Brix9-2-5) was mapped to a 9-centimorgan (cM) segment on chromosome 9 (5). Here, we further characterize this QTL with respect to its phenotypic effects and the mode of inheritance in different years of growth, environments, and genetic backgrounds. We report the map-based cloning of this QTL and show that it resides within the tomato apoplastic invertase, Lin5 (6). Agricultural pra...

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn 2ϩ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. K m values of 21 and 51 m were obtained for geranyl diphosphate and Mn 2ϩ , respectively. In the presence of 18 O-labeled water, GES catalyzed the formation of 18 O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.Geraniol is an acyclic monoterpene alcohol emitted from the flowers of many species, notably roses (Rosa hybrida; Bayrak, 1994;Antonelli et al., 1997;Rao et al., 2000). It is also present in vegetative tissues of many herbs (Charles and Simon, 1992;Mallavarapu et al., 1998;Mockute and Bernotiene, 1999;Vieira et al., 2001) and is often found together with geranial and neral, which are the oxidation products of geraniol (Miyazawa and Kameoka, 1988). The mixture of geranial and neral, also called citral, imparts a "lemon" flavor, and lemongrass (Cymbopogon citratus Stapf.; Singh-Sangwan et al., 1993), ginger (Zingiber officinale Rosc.; Miyazawa and Kameoka, 1988), and some varieties of sweet basil (Ocimum basilicum; Grayer et al., 1996;Simon et al., 1999) such as basil cv Sweet Dani are particularly rich in citral (Morales and Simon, 1997). However, at present, there is no definitive proof of whether citral is synthesized from geraniol by an alcohol dehydrogenase Singh-Sangwan et al., 1993;Hallahan et al., 1995;Sekiwa-Iijima et al., 2001) or by an oxidase (Potty and Bruemmer, 1970;Banthorpe et al., 1976), nor is it known if geraniol is the only substrate whose oxidation leads to the formation of citral or whether nerol, the cis-isomer of geraniol, can also serve as a precursor (Corbier and Ehret, 1988;Ikeda et al., 1991;Hallahan e...

Medium-length methylketones (C7-C15) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C12, C14, and C16 β-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C11, C13, and C15 methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

In this study, we present the genetic analysis of a new collection of wild barley (Hordeum spontaneum) using 42 simple sequence repeat (SSR) markers that represent the seven chromosomes. The Barley1K (B1K) infrastructure consists of 1020 accessions collected in a hierarchical sampling mode (HSM) from 51 sites across Israel and represents the wide adaptive niche of the modern barley's ancestor. According to the genetic structure analysis, the sampled sites can be divided into seven groups, and sampled microsites located on opposing slopes or in different soil types did not show significant genetic differentiation. Although the genetic analysis indicates a simple isolation-by-distance model among the populations, examination of the genetic populations' structure with abiotic parameters in an ordination analysis revealed that the combination of elevation, mid-day temperature and rainfall explains a high proportion of the variance in the principal components analysis. Our findings demonstrate that the current populations have therefore been shaped and distinguished by non-selective forces such as migration; however, we suggest that aridity and temperature gradients played major roles as selective forces in the adaptation of wild barley in this part of the Fertile Crescent. This unique collection is a prelude for the investigation of the molecular basis underlying plant adaptation and responsiveness to harsh environments.

Surface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (LIS) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frameshift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes.Plants produce a large number of secondary metabolites that function in a variety of ecological contexts. Many specialized compounds are toxic and can therefore serve as defense agents against microbial pathogens and insect and animal herbivores (Wittstock and Gershenzon, 2002;Theis and Lerdau, 2003;Wink, 2003). Other compounds are volatile and serve to attract pollinators or even insects that prey on the plant's enemies or repel harmful organisms (Pare and Tumlinson, 1999;Kessler and Baldwin, 2001;Baldwin et al., 2002;Pichersky and Gershenzon, 2002).Secondary compounds with roles in defense are often sequestered in specialized cells or structures, presumably to protect the plant itself from its own toxicity (Gershenzon et al., 1989;Pare and Tumlinson, 1997;Duke et al., 2000;Dussourd and Hoyle, 2000;Hallahan, 2000;Martin et al., 2002). A common mechanism of sequestration has been the evolution of anatomical structures, termed glandular trichomes, on the surface of the aerial parts of the plants. Such structures typically contain gland cells (or a single cell) that synthesize these compounds and a cuticular sac covering the gland cells into which large amounts of the synthesized compounds are secreted. Upon damage to the tissue, or even upon mere physical pressure, the sacs rupture and release their contents. Once on the surface, secondary compounds with high vapor pressure will easily evaporate into the atmosphere.The Lamiaceae is a large plant family that includes the mints, sages, and basils and is well recognized for the diversity ...