Alisdair R. Fernie

Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography–linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS–based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300–500 analytes of polar and nonpolar extracts in ∼400 experimental samples per week per machine.

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.

RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics

Recent rapid advances in next generation RNA sequencing (RNA-Seq)-based provide researchers with unprecedentedly large data sets and open new perspectives in transcriptomics. Furthermore, RNA-Seq-based transcript profiling can be applied to non-model and newly discovered organisms because it does not require a predefined measuring platform (like e.g. microarrays). However, these novel technologies pose new challenges: the raw data need to be rigorously quality checked and filtered prior to analysis, and proper statistical methods have to be applied to extract biologically relevant information. Given the sheer volume of data, this is no trivial task and requires a combination of considerable technical resources along with bioinformatics expertise. To aid the individual researcher, we have developed RobiNA as an integrated solution that consolidates all steps of RNA-Seq-based differential gene-expression analysis in one user-friendly cross-platform application featuring a rich graphical user interface. RobiNA accepts raw FastQ files, SAM/BAM alignment files and counts tables as input. It supports quality checking, flexible filtering and statistical analysis of differential gene expression based on state-of-the art biostatistical methods developed in the R/Bioconductor projects. In-line help and a step-by-step manual guide users through the analysis. Installer packages for Mac OS X, Windows and Linux are available under the LGPL licence from http://mapman.gabipd.org/web/guest/robin.

Metabolite profiling: from diagnostics to systems biology

The concept of metabolite profiling has been around for several decades, but only recent technical innovations have allowed metabolite profiling to be carried out on a large scale — with respect to both the number of metabolites measured and the number of experiments carried out. As a result, the power of metabolite profiling as a technology platform for diagnostics, and the research areas of gene-function analysis and systems biology, is now beginning to be fully realized.

GC–MS libraries for the rapid identification of metabolites in complex biological samples

Gas chromatography–mass spectrometry based metabolite profiling of biological samples is rapidly becoming one of the cornerstones of functional genomics and systems biology. Thus, the technology needs to be available to many laboratories and open exchange of information is required such as those achieved for transcript and protein data. The key‐step in metabolite profiling is the unambiguous identification of metabolites in highly complex metabolite preparations with composite structure. Collections of mass spectra, which comprise frequently observed identified and non‐identified metabolites, represent the most effective means to pool the identification efforts currently performed in many laboratories around the world. Here, we describe a platform for mass spectral and retention time index libraries that will enable this process (MSRI; www.csbdb.mpimp‐golm.mpg.de/gmd.html). This resource should ameliorate many of the problems that each laboratory will face both for the initial establishment of metabolome analysis and for its maintenance at a constant sample throughput.

Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.

That Sweet Sensation Photosynthesis in the leaf generates sucrose that must be transported via the phloem to other parts of the plant in order, for example, to be incorporated into harvestable produce. Studying Arabidopsis and rice, Chen et al. (p. 207, published online 8 December; see the Perspective by Braun) identified the SWEET family of sucrose efflux transporters that are responsible for carrying sucrose out of the leaf cells. When the transporters were disabled, sucrose accumulated in the leaves. Functioning properly, the SWEET transporters carry sucrose across the plasma membrane and other transporters move it further into the phloem. Transporters hand off sucrose from production cell to transport cell. Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose–H+ (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H+-coupled import into the sieve element–companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.

Integrated Analysis of Metabolite and Transcript Levels Reveals the Metabolic Shifts That Underlie Tomato Fruit Development and Highlight Regulatory Aspects of Metabolic Network Behavior

Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits. Finally, there was a strong relationship between ripening-associated transcripts and specific metabolite groups, such as TCA-cycle organic acids and sugar phosphates, underlining the importance of the respective metabolic pathways during fruit development.

Not just a circle: flux modes in the plant TCA cycle

The tricarboxylic acid (TCA) cycle is one of the iconic pathways in metabolism. The cycle is commonly thought of in terms of energy metabolism, being responsible for the oxidation of respiratory substrates to drive ATP synthesis. However, the reactions of carboxylic acid metabolism are embedded in a larger metabolic network and the conventional TCA cycle is only one way in which the component reactions can be organised. Recent evidence from labelling studies and metabolic network models suggest that the organisation of carboxylic acid metabolism in plants is highly dependent on the metabolic and physiological demands of the cell. Thus, alternative, non-cyclic flux modes occur in leaves in the light, in some developing oilseeds, and under specific physiological circumstances such as anoxia.

Starch as a major integrator in the regulation of plant growth

Rising demand for food and bioenergy makes it imperative to breed for increased crop yield. Vegetative plant growth could be driven by resource acquisition or developmental programs. Metabolite profiling in 94 Arabidopsis accessions revealed that biomass correlates negatively with many metabolites, especially starch. Starch accumulates in the light and is degraded at night to provide a sustained supply of carbon for growth. Multivariate analysis revealed that starch is an integrator of the overall metabolic response. We hypothesized that this reflects variation in a regulatory network that balances growth with the carbon supply. Transcript profiling in 21 accessions revealed coordinated changes of transcripts of more than 70 carbon-regulated genes and identified 2 genes (myo-inositol-1-phosphate synthase, a Kelch-domain protein) whose transcripts correlate with biomass. The impact of allelic variation at these 2 loci was shown by association mapping, identifying them as candidate lead genes with the potential to increase biomass production.

On the origins of nitric oxide

Nitric oxide (NO) is widely recognized for its role as signaling compound. However, the metabolic mechanisms that determine changes in the level of NO in plants are only poorly understood, despite this knowledge being crucial to understanding the signal function of NO. To date, at least seven possible pathways of NO biosynthesis have been described for plants, although the molecular and enzymatic components are resolved for only one of these. Currently, this represents the most significant bottleneck for NO research. In this review, we provide an overview of the multiplicity of NO production and scavenging pathways in plants. Furthermore, we discuss which areas should be focused on in future studies to investigate the origin of fluctuations in the level of NO in plants.