Charles Baxter

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.

The Metabolic Response of Heterotrophic Arabidopsis Cells to Oxidative Stress

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 13C-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.

Transcriptome and Metabolite Profiling Show That APETALA2a Is a Major Regulator of Tomato Fruit Ripening

This study demonstrates that the tomato APETALA2a (AP2a) transcription factor modulates fruit ripening by negatively regulating ethylene biosynthesis and signaling. Various ripening regulators are shown to act upstream of AP2a. Gene expression analysis reveals that AP2a is involved in chloroplast to chromoplast transition. Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro.

Mitochondrial uncoupling protein is required for efficient photosynthesis

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.

Decrease in Manganese Superoxide Dismutase Leads to Reduced Root Growth and Affects Tricarboxylic Acid Cycle Flux and Mitochondrial Redox Homeostasis

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves

Diurnal changes in carbohydrates and a broad range of primary metabolites were analysed through a diurnal period in potato leaves (Solanum tuberosum cv. Desiree) using an established gas chromatography-mass spectrometry based metabolic profiling protocol alongside conventional spectrophotometric technologies. In tandem, we profiled transcript levels using both a custom array containing approximately 2,500 cDNA clones predominantly representing transcripts involved in primary metabolism and commercially available arrays containing approximately 12,000 cDNA clones that gave coverage of transcript levels over a broader functional range. The levels of many metabolites and transcripts varied during the diurnal period with 56 significant differences observed in the metabolite contents and 832 significant differences recorded in transcript levels. Whilst a large number of the differences would be expected from what has been known previously, several novel changes were observed in these experiments. Notably, qualitative comparison of the combined data sets obtained from the parallel analysis of transcripts and metabolites suggests relatively few changes in gene expression strongly correlate with changes in metabolite levels during a diurnal cycle. Furthermore, these changes appear to be confined to the central metabolic pathways. However, principal component analysis of the metabolic profiles obtained here revealed that metabolite patterns change progressively through a diurnal period suggesting the operation of mechanisms for tight temporal regulation of metabolite composition.

Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits

A likely regulator of tomato ripening is identified from a gene network, its function is validated in transgenic plants, and an orthologous gene is shown to play a similar role in pepper. Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

High-Resolution Mapping of a Fruit Firmness-Related Quantitative Trait Locus in Tomato Reveals Epistatic Interactions Associated with a Complex Combinatorial Locus

Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Firs.p.QTL2.1 to Firs.p.QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Firs.p.QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Firs.p.QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

Summary Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ‐aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans‐stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast‐enriched membrane fraction. Transient expression of a SlCAT9‐YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild‐type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

Metabolic Engineering of Tomato Fruit Organic Acid Content Guided by Biochemical Analysis of an Introgression Line

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.