Biomedical subjects
Publications and source records attributed to Alisdair R Fernie.
Once viewed mainly as metabolic intermediates, small metabolites are increasingly recognized as spatially and temporally regulated signals that coordinate plant development and adaptation. Understanding these metabolite-based regulatory processes could reveal new strategies to improve crop resilience, productivity, and sustainability under changing environmental conditions.
Apricot produces climacteric fruit, which are perishable after harvest. To elucidate the regulatory role of NO treatment through DNA methylation in post-harvest senescence, apricot fruits were treated with 0.2 mmol/L sodium nitroprusside (SNP) solution for 10 min, with distilled water treatment serving as the control. Treated fruits were then stored at 25°C and 80% relative humidity. Changes in appearance quality, physiological parameters, metabolome profiles, transcriptome dynamics, and DNA methylation patterns were analyzed before and after storage. Results showed that NO treatment delayed apricot softening, increased flavonoid metabolite accumulation, and reduced lipid and abscisic acid accumulation, with these effects correlated to the expression of specific genes and transcription factors. This work reveals the epigenetic regulatory mechanism underlying NO treatment delaying ripening and senescence. Further analysis revealed that the transcription levels of ACO, PAL, UFGT-like, NCED1, PP2C, MYB21, CCoAOMT-like, CYP707A, and ZNF7-like were all correlated with DNA methylation. This indicates that SNP treatment can lead to large changes in DNA methylation levels in apricot fruits, and that the differences in gene transcription levels are associated with the occurrence of hypomethylation and hypermethylation. Collectively, these findings establish an epigenetic framework for post-harvest regulation of apricot fruit, revealing DNA methylation-mediated freshness preservation mechanisms.
Carnivorous plants such as the Venus flytrap Dionaea muscipula survive in nutrient-poor habitats by attracting and consuming animals. Upon deflection of the touch-sensitive trigger hairs, the trap closes instantly. Panicking prey repeatedly collides with trigger hairs, which activate the endocrine system: mechano- and chemosensors translate the information on the prey's nature, size, and activity into jasmonate-dependent lytic enzyme secretion. This digestive fluid gradually degrades its exoskeleton and internal tissues. The released substances are absorbed by glands covering the inner trap surface. To understand Dionaea's modification of metabolism upon prey consumption, we compared the metabolic profiles associated with secretion and insect feeding. In favor of digestive enzyme secretion, the abundance of most amino acids decreased after JA-stimulation without prey present. By contrast, insect feeding resulted in an increase in almost all amino acids within the trap. In agreement with the export of prey-derived nitrogen, the abundance of certain amino acids also increased in the petiole. In response to feeding with urea, chitin, nucleic acids, or phospholipids, the amino acid profile remained relatively unchanged. This might indicate that the alterations in the Venus flytrap's metabolism depend both on the type of substance and on its amount.
Asr genes are exclusively found in the genomes of higher plants. In many species, this gene family is expressed under abiotic stress conditions and during fruit ripening. The encoded proteins have nuclear localisation and consequently a transcription factor function has been suggested. Interestingly, yeast-one-hybrid experiments revealed that a grape ASR binds to the promoter of a hexose transporter gene (VvHT1). However, the role of these proteins in planta is still elusive. By using a reverse genetics approach in potato we found that modification of Asr1 expression has no incidence on the aerial phenotype of the plant but exerts a dramatic effect in tuber. Asr1 antisense potatoes displayed decreased tuber fresh weight whereas Asr1 overexpressors had a diminished number of tubers. Moreover, overexpression lines showed lower transcript levels of a plasma membrane hexose transporter and a concomitant decrease in glucose content in parenchyma cells of potato tubers. On the same hand glucose uptake rate was also reduced in one of the overexpressing lines. It thus seems likely that Asr1 is involved in the control of hexose uptake in heterotrophic organs. In addition, the transgenic plants were characterized by several other changes in steady state metabolite levels. Results presented here support a role for ci21A/Asr1 in glucose metabolism of potato tuber.
Gas chromatography-mass spectrometry (GC-MS) is routinely applied to the metabolite profiling of biological samples. Time-of-flight (TOF)-GC-MS metabolite profiling is based on highly reproducible electron impact ionization. Single chromatograms may comprise 200-1000 mass spectral components. The nature and composition of these mass spectral components depend on the choice of metabolite extraction, type of biological sample, and experimental condition. The components represent mass spectral tags (MSTs) of volatile metabolites or metabolite derivatives. Identification of MSTs is the major challenge in GC-MS metabolite profiling. We describe methods suitable for the automated construction of mass spectral and retention time index databases from large sets of TOF-GC-MS profiles. Application of these libraries for automated identification by pure reference compounds and classification of hitherto unidentified MSTs from biological sources is demonstrated.
Recent advances in the medical and biological sciences have been characterized by a major paradigm shift from reductionism to integrated and holistic systems approaches. Such approaches are characterized at the experimental level by the multiparallel analysis of a multitude of parameters of a given biological system at a range of different molecular levels, following the systematic perturbation of the system in question. Although a multitude of studies have been carried out to assess the transcript, protein, and metabolite complements of cells under various conditions, to date, few have been attempted that encompass the profiling of more than one of these entities. In this chapter, we describe combined analysis of data obtained from transcript and metabolic profiling, and detail advantages of using both approaches in parallel.
BACKGROUND: Microarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis. RESULTS: Here we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs.PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis.PageMan offers a complete user's guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/. CONCLUSION: PageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.
Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.
Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism.
To cope with oxidative stress, the metabolic network of plant cells must be reconfigured either to bypass damaged enzymes or to support adaptive responses. To characterize the dynamics of metabolic change during oxidative stress, heterotrophic Arabidopsis (Arabidopsis thaliana) cells were treated with menadione and changes in metabolite abundance and (13)C-labeling kinetics were quantified in a time series of samples taken over a 6 h period. Oxidative stress had a profound effect on the central metabolic pathways with extensive metabolic inhibition radiating from the tricarboxylic acid cycle and including large sectors of amino acid metabolism. Sequential accumulation of metabolites in specific pathways indicated a subsequent backing up of glycolysis and a diversion of carbon into the oxidative pentose phosphate pathway. Microarray analysis revealed a coordinated transcriptomic response that represents an emergency coping strategy allowing the cell to survive the metabolic hiatus. Rather than attempt to replace inhibited enzymes, transcripts encoding these enzymes are in fact down-regulated while an antioxidant defense response is mounted. In addition, a major switch from anabolic to catabolic metabolism is signaled. Metabolism is also reconfigured to bypass damaged steps (e.g. induction of an external NADH dehydrogenase of the mitochondrial respiratory chain). The overall metabolic response of Arabidopsis cells to oxidative stress is remarkably similar to the superoxide and hydrogen peroxide stimulons of bacteria and yeast (Saccharomyces cerevisiae), suggesting that the stress regulatory and signaling pathways of plants and microbes may share common elements.
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.
While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
Metabolite profiling is a fast growing technology and is useful for phenotyping and diagnostic analyses of plants. It is also rapidly becoming a key tool in functional annotation of genes and in the comprehensive understanding of the cellular response to biological conditions. Metabolomics approaches have recently been used to assess the natural variance in metabolite content between individual plants, an approach with great potential for the improvement of the compositional quality of crops. Here, we assess the contribution of metabolite profiling to these areas.
Despite much study of the role of S-adenosylmethionine (SAM) in the methylation of DNA, RNA, and proteins, and as a cofactor for a wide range of biosynthetic processes, little is known concerning the intracellular transport of this essential metabolite. Screening of the Arabidopsis (Arabidopsis thaliana) genome yielded two potential homologs of yeast (Saccharomyces cerevisiae) and human SAM transporters, designated as SAMC1 and SAMC2, both of which belong to the mitochondrial carrier protein family. The SAMC1 gene is broadly expressed at the organ level, although only in specialized tissues of roots with high rates of cell division, and appears to be up-regulated in response to wounding stress, whereas the SAMC2 gene is very poorly expressed in all organs/tissues analyzed. Direct transport assays with the recombinant and reconstituted SAMC1 were utilized to demonstrate that this protein displays a very narrow substrate specificity confined to SAM and its closest analogs. Further experiments revealed that SAMC1 was able to function in uniport and exchange reactions and characterized the transporter as highly active, but sensitive to physiologically relevant concentrations of S-adenosylhomocysteine, S-adenosylcysteine, and adenosylornithine. Green fluorescent protein-based cell biological analysis demonstrated targeting of SAMC1 to mitochondria. Previous proteomic analyses identified this protein also in the chloroplast inner envelope. In keeping with these results, bioinformatics predicted dual localization for SAMC1. These findings suggest that the provision of cytosolically synthesized SAM to mitochondria and possibly also to plastids is mediated by SAMC1 according to the relative demands for this metabolite in the organelles.
PPi has previously been implicated specifically in the co-ordination of the sucrose-starch transition and in the broader context of its role as co-factor in heterotrophic plant metabolism. In order to assess the compartmentation of pyrophosphate (PPi) metabolism in the potato tuber we analysed the effect of expressing a bacterial pyrophosphatase in the amyloplast of wild type tubers or in the cytosol or amyloplast of invertase-expressing tubers. The second and third approaches were adopted since we have previously characterized the invertase expressing lines to both exhibit highly altered sucrose metabolism and to contain elevated levels of PPi (Farré et al. (2000a) Plant Physiol 123:681) and therefore this background rendered questions concerning the level of communication between the plastidic and cytosolic pyrophosphate pools relatively facile. In this study we observed that the increase in PPi in the invertase expressing lines was mainly confined to the cytosol. Accordingly, the expression of a bacterial pyrophosphatase in the plastid of either wild type or invertase-expressing tubers did not lead to a decrease in total PPi content. However, the expression of the heterologous pyrophosphatase in the cytosol of cytosolic invertase-expressing tubers led to strong metabolic changes. These results are discussed both with respect to our previous hypotheses and to current models of the compartmentation of potato tuber metabolism.
The aim of this work was to investigate the importance of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) in potato carbohydrate metabolism. For this purpose, the cytosolic isoform of phosphorylating GAPC was cloned and used for an antisense approach to generate transgenic potato plants that exhibited constitutively decreased GAPDH activity. Potato lines with decreased activities of phosphorylating GAPC exhibited no major changes in either whole-plant or tuber morphology. However, the levels of 3-phosphoglycerate were decreased in leaves of the transformants. A broad metabolic phenotyping of tubers from the transformants revealed an increase in sucrose and UDPglucose content, a decrease in the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate but little change in the levels of other metabolites. Moreover, the transformants displayed no differences in cold sweetening with respect to the wild type. Taken together these data suggest that phosphorylating GAPC plays only a minor role in the regulation of potato metabolism. The results presented here are discussed in relation to current models regarding primary metabolism in the potato tuber parenchyma.
Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants.
An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADC1A or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2-phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene.