Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit.

The sugar content of Solanum lycopersicum (tomato) fruit is a primary determinant of taste and quality. Cultivated tomato fruit are characterized by near-equimolar levels of the hexoses glucose and fructose, derived from the hydrolysis of translocated sucrose. As fructose is perceived as approximately twice as sweet as glucose, increasing its concentration at the expense of glucose can improve tomato fruit taste. Introgressions of the FgrH allele from the wild species Solanum habrochaites (LA1777) into cultivated tomato increased the fructose-to-glucose ratio of the ripe fruit by reducing glucose levels and concomitantly increasing fructose levels. In order to identify the function of the Fgr gene, we combined a fine-mapping strategy with RNAseq differential expression analysis of near-isogenic tomato lines. The results indicated that a SWEET protein was strongly upregulated in the lines with a high fructose-to-glucose ratio. Overexpressing the SWEET protein in transgenic tomato plants dramatically reduced the glucose levels and increased the fructose : glucose ratio in the developing fruit, thereby proving the function of the protein. The SWEET protein was localized to the plasma membrane and expression of the SlFgr gene in a yeast line lacking native hexose transporters complemented growth with glucose, but not with fructose. These results indicate that the SlFgr gene encodes a plasma membrane-localized glucose efflux transporter of the SWEET family, the overexpression of which reduces glucose levels and may allow for increased fructose levels. This article identifies the function of the tomato Fgr gene as a SWEET transporter, the upregulation of which leads to a modified sugar accumulation pattern in the fleshy fruit. The results point to the potential of the inedible wild species to improve fruit sugar accumulation via sugar transport mechanisms.

Carotenoid biosynthesis and sequestration in red chilli pepper fruit and its impact on colour intensity traits.

The exploitation of diverse natural variation has been a key progenitor of crop breeding over the last decade. However, commercial practice is now turning to the use of accessions with less extreme phenotypes as genetic donors. In the present study, the carotenoid formation in a red-fruited discovery panel of Capsicum annuum (chilli pepper) has been characterized. The data indicated that colour intensity correlated with the amount of capsanthin and its esters, along with transcript levels of the 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and phytoene synthase-1 (PSY-1) genes. Quantification of carotenoids through development and ripening suggested the presence of separate biosynthesis and accumulation phases. Subplastid fractionation demonstrated the differential sequestration of pigments in high- and low-intensity lines and revealed the PSY protein to be most active in the membrane fractions when abundance was highest in the fibril fractions. Carotenoid accumulation was associated with the esterification of xanthophylls, expression of a putative carotenoid acyl transferase, and increased fibril content within the plastid. Interrogation of TEM images and carotenoid analysis of subplastid fractions suggest that the plastoglobuli are likely to be the progenitor of the characteristic fibrils found in pepper fruit. Collectively, these data provide an insight into the underpinning molecular, biochemical, and cellular mechanisms associated with the synthesis and sequestration of carotenoids in chromoplast-containing fruits, in addition to providing potential tools and resources for the breeding of high red colour intensity pepper varieties.

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

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.

Understanding colour retention in red chilli pepper fruit using a metabolite profiling approach.

Carotenoids are the pigments responsible for conferring the characteristic deep red colour to chilli pepper. The post-harvest retention of this colour is a key trait that governs the price of the produce. Determining colour retention and the associated underlying biochemical mechanisms are important issues that require investigation. In this present study, the ability of image analysis to determine colour change in ground chilli fruit was evaluated. This method enabled differentiation of extreme retention phenotypes whilst also reducing the duration of storage required to make accurate determinations. The analysis of volatiles indicated different levels of lipid and carotenoid derived volatiles in lines with different retention properties. Metabolite profiling of intermediary metabolism supported these findings, with increased levels of unsaturated fatty acids present in lines with low retention properties. Collectively, these data have led us to propose that in chilli fruit lipid peroxidation is one of the progenitors of carotenoid degradation.

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp.

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

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.

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 (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.

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.

Comparison of changes in fruit gene expression in tomato introgression lines provides evidence of genome-wide transcriptional changes and reveals links to mapped QTLs and described traits.

Total soluble solids content is a key determinant of tomato fruit quality for processing. Several tomato lines carrying defined introgressions from S. pennellii in a S. lycopersicum background produce fruit with elevated Brix, a refractive index measure of soluble solids. The genetic basis for this trait can be determined by fine-mapping each QTL to a single gene, but this is time-consuming and technically demanding. As an alternative, high-throughput analytical technologies can be used to provide useful information that helps characterize molecular changes in the introgression lines. This paper presents a study of transcriptomic changes in six introgression lines with increased fruit Brix. Each line also showed altered patterns of fruit carbohydrate accumulation. Transcriptomic changes in fruit at 20 d after anthesis (DAA) were assessed using a 12 000-element EST microarray and significant changes analysed by SAM (significance analysis of microarrays). Each non-overlapping introgression resulted in a unique set of transcriptomic changes with 78% of significant changes being unique to a single line. Principal components analysis allowed a clear separation of the six lines, but also revealed evidence of common changes; lines with quantitatively similar increases in Brix clustered together. A detailed examination of genes encoding enzymes of primary carbon metabolism demonstrated that few of the known introgressed alleles were altered in expression at the 20 DAA time point. However, the expression of other metabolic genes did change. Particularly striking was the co-ordinated up-regulation of enzymes of sucrose mobilization and respiration that occurred only in the two lines with the highest Brix increase. These common downstream changes suggest a similar mechanism is responsible for large Brix increases.

Fruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids.

A tomato line (IL9-2-5) of the cultivated species, Lycopersicon esculentum, carrying a 9 cM introgression from the wild species, Lycopersicon pennelli, produces fruit with high soluble solids content (Brix), an important determinant of fruit quality for processing. Two quantitative trait loci (QTLs) relating to fruit soluble solids content have been identified within the introgressed segment. One of these QTLs (PW-9-2-5) is silent under the growth conditions used in this study, while a second (Brix-9-2-5) has been shown to encode a fruit apoplastic invertase (Lin5) with altered kinetic properties. In this study, we have undertaken a detailed biochemical analysis of the introgression line to attempt to gain an understanding of the metabolic changes associated with increased fruit soluble solids. Increased Brix in ripe fruit was shown to be the result of increased sucrose and glucose, with a more minor contribution from aspartate and alanine. The introgression leads to a pronounced increase in apoplastic invertase activity in the columella tissue that extends throughout fruit development. Furthermore, columella tissue from IL9-2-5 fruit has a greater capacity to take up exogenously supplied sucrose, an observation that is consistent with the kinetic properties of the introgressed Lin5 allele. Apart from the increase in mature fruit sugar and increases in some amino acids, metabolite profiling revealed few other metabolic perturbations in fruit from IL9-2-5. The only other major change was a dramatic increase in starch accumulation at earlier stages of fruit metabolism. This occurred without any increase in the activity of the enzymes of sucrose metabolism or starch synthesis and may therefore be driven by increased availability of sucrose. We conclude that the major factor that leads to increased fruit sugar in IL9-2-5 is an increase in the capacity to take up sucrose unloaded from the phloem.

Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development.

Constitutive over-expression of a maize sucrose-phosphate synthase (SPS) gene in tobacco (Nicotiana tabacum) had major effects on leaf carbohydrate budgets with consequences for whole plant development. Transgenic tobacco plants flowered earlier and had greater flower numbers than wild-type plants. These changes were not linked to modified source leaf carbon assimilation or carbon export, although sucrose to starch ratios were significantly higher in leaves expressing the transgene. The youngest and oldest leaves of plants over-expressing SPS had up to 10-fold wild-type maximal extractable SPS activity, but source leaf SPS activities were only 2-3 times greater in these lines than in the wild type. In the oldest leaves, where the expression of the transgene led to the most marked enhancement in SPS activity, photosynthesis was also increased. It was concluded that these increases in the capacity for sucrose synthesis and carbon assimilation, particularly in older leaves, accelerate the whole plant development and increase the abundance of flowers without substantial changes in the overall shoot biomass.