Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography-time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.Metabolites are critical in biology, and plants are especially rich in diverse biochemical compounds. It has been estimated that over 100,000 metabolites can be found in plants, and each species may contain its own chemotypic expression pattern 1 . Moreover, substantial quantitative and qualitative variation in metabolite composition is often observed within plant species 2 .Although knowledge on the regulation of metabolite formation is increasing, for thousands of metabolites, their function in the plant, their biosynthetic pathway and the regulation thereof is still unknown. QTL analysis of natural variation, which can affect metabolites 3 , in segregating populations can identify loci explaining the observed variation 4 . In recent years, a few studies have focused on identifying QTLs regulating a specific group of known metabolites using detection methods directed toward specific metabolite groups 5-9 . However, recent advances in mass spectrometry-based metabolomics and data processing techniques should now allow large-scale QTL analyses of untargeted metabolic profiles, which may uncover previously unknown regulatory functions of loci in metabolic pathways. Using dedicated alignment software, it is now possible to perform an unbiased comparison of large numbers of metabolite-derived masses detectable in large numbers of samples arising from inherently large sets of genotypes (which are required for accurate mapping of QTLs) in an RIL population 10,11 . QTL mapping will result in the localization of loci, and ultimately genes, causal for the observed variation and will allow the discovery of coregulated compounds. In this way, genomewide genetic correlative metabolic analysis now becomes feasible, as we demonstrate here. RESULTS Metabolite variation is abundant and genetically controlledTo assess the natural variation in metabolite content present in A. thaliana, we performed HPLC-QTOF MS-based untargeted metabolic fingerprinting of acidified aqueous methanol extracts fr...

SUMMARYOver the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task that is often prohibitive to the execution of large experiments. In this paper we present the GERMINATOR package: a simple, highly cost-efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The GERMINATOR package contains three modules: (i) design of experimental setup with various options to replicate and randomize samples; (ii) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (iii) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve-fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the GERMINATOR package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of recombinant inbred lines and were able to identify several quantitative trait loci for salt tolerance. GERMINATOR is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.

The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase activities than with sucrose synthase activities. The highest correlations were observed with activities in the elongating zones of roots. The genetic basis of these correlations was studied by using QTL analysis. Several loci, affecting invertase activity, colocated with loci that had an effect on root or hypocotyl length. Further fine mapping of a major locus for root length, but not for hypocotyl length (top chromosome 1), consistently showed colocation with the locus for invertase activity containing a gene coding for a vacuolar invertase. The analysis of a functional knockout line confirmed the role of this invertase in root elongation, whereas other invertase genes might play a role in hypocotyl elongation. Thus, we show the power of QTL analysis, combined for morphological and biochemical traits, followed by fine-mapping and mutant analysis, in unraveling the function of genes and their role in growth and development.Arabidopsis natural variation ͉ sucrose synthase ͉ hypocotyls T otal plant yield depends on the acquisition of raw material, i.e., photosynthesis and mineral (plus water) uptake, and on the ability of the plant to cope with stress. However, the economic yield of a crop is to a large extent also determined by the partitioning of dry matter over the harvestable and nonharvestable parts of the plant. The molecular and physiological basis of the regulation of assimilate partitioning in plants is still poorly understood. In terms of biomass, the most important components in assimilate partitioning and in total yield are carbohydrates. There is increasing evidence that a limited number of key enzymes, involved in primary (carbohydrate) metabolism, might be pivotal in this process (1).Functionally, a plant can be divided into sources (the sites of assimilate production) and sinks (the sites of use and͞or storage). Sinks can either be rapidly growing, expanding organs, such as elongating stems and roots, or storage sinks accumulating reserves, such as fruits, seeds, or tubers (2).In most plant species, carbon is transported from source to sink in the form of the disaccharide sucrose. Upon arrival in the sink, sucrose has to be hydrolyzed. In plants, two pathways are available for sucrose cleaving: via invertase (Inv), yielding glucose and fructose, and via sucrose synthase (Susy), yielding fructose and UDP-glucose. In several cases, it has been suggested that sink strength might depend on the activities of these sucrose-splitting enzymes. There is increasing evidence that in storage sinks, the predominant pathway is via Susy, whereas in growing sinks, the Inv route is most important. In potatoes, the elongating rhizomes (stolons) exhibit high Inv activity, whereas a switch toward Susycatalyzed sucrose bre...

Dehydrins accumulate in various plant tissues during dehydration. Their physiological role is not well understood, but it is commonly assumed that they assist cells in tolerating dehydration. Since in perennials the ability of the shoot apex to withstand dehydration is pivotal for survival through winter, we investigated if and how dehydrins may be involved. A first step in assessing such a role is the identification of their subcellular location. We therefore mapped the location of dehydrin homologues, abscisic acid-responsive (RAB 16-like) polypeptides, in the apex of birch (Betula pubescens Ehrh.). In non-cold-acclimated plants a single low-abundant RAB 16-member (a 33-kDa polypeptide) was produced, and localized in the cytoplasm only. During cold acclimation two additional members were produced (24 and 30 kDa) and accumulated in nuclei, storage protein bodies and starch-rich amyloplasts. Western blots of proteins isolated from purified starch granules and from protein bodies revealed the presence of the 24-kDa dehydrin. Since starch and protein reserves are gradually consumed during winter, serving cell maintenance, starch- and protein-degrading enzymes must remain locally active. We therefore investigated the hypothesis that dehydrins might create local pools of water in otherwise dehydrated cells, thereby maintaining enzyme function. In agreement with our hypothesis, enzyme assays showed that under conditions of low water activity a partially purified dehydrin fraction was able to improve the activity of alpha-amylase (EC 3.2.1.1.) relative to fractions from which dehydrin was removed by immunoprecipitation. The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest that a major function is to rescue the metabolic processes that are required for survival and re-growth.

SummaryPetunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was ef®ciently converted to non-volatile S-linalyl-b-Dglucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modi®cation, and that the compounds produced can be converted by endogenous enzymatic activity.