SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1.

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function.

Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates interleukin-1 receptor-associated protein splicing through the interaction with U1 small nuclear ribonucleoprotein. In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 small nuclear ribonucleoprotein but modulated by the calcium/calmodulin-dependent protein kinase signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 preferentially affected the number of inhibitory synapses. Moreover, the soluble form of PCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.

Neuron-derived neurotrophic factor protects against dexamethasone-induced skeletal muscle atrophy.

Skeletal muscle atrophy caused by various conditions including aging, nerve damage, and steroid administration, is a serious health problem worldwide. We recently reported that neuron-derived neurotrophic factor (NDNF) functions as a muscle-derived secreted factor, also known as myokine, which exerts protective actions on endothelial cell and cardiomyocyte function. Here, we investigated whether NDNF regulates skeletal muscle atrophy induced by steroid administration and sciatic denervation. NDNF-knockout (KO) mice and age-matched wild-type (WT) mice were subjected to continuous dexamethasone (DEX) treatment or sciatic denervation. NDNF-KO mice exhibited decreased gastrocnemius muscle weight and reduced cross sectional area of myocyte fiber after DEX treatment or sciatic denervation compared with WT mice. Administration of an adenoviral vector expressing NDNF (Ad-NDNF) or recombinant NDNF protein to gastrocnemius muscle of WT mice increased gastrocnemius muscle weight after DEX treatment. NDNF-KO mice showed increased expression of ubiquitin E3-ligases, including atrogin-1 and MuRF-1, in gastrocnemius muscle after DEX treatment, whereas Ad-NDNF reduced expression of atrogin-1 and MuRF-1 in gastrocnemius muscle of WT mice after DEX treatment. Pretreatment of cultured C2C12 myocytes with NDNF protein reversed reduced myotube diameter and increased expression of atrogin-1 and MuRF-1 after DEX stimulation. Treatment of C2C12 myocytes increased Akt phosphorylation. Pretreatment of C2C12 myotubes with the PI3-kinase/Akt inhibitor reversed NDNF-induced increase in myotube fiber diameter after DEX treatment. In conclusion, our findings indicated that NDNF prevents skeletal muscle atrophy in vivo and in vitro through reduction of ubiquitin E3-ligases expression, suggesting that NDNF could be a novel therapeutic target of muscle atrophy.

Distinct Expression of SLM2 Underlies Splicing-Dependent Trans-Synaptic Signaling of Neurexin Across GABAergic Neuron Subtypes.

The mammalian brain contains multiple types of neuronal cells with complex assemblies and distinct structural and functional properties encoded by divergent gene programs. There is increasing evidence that alternative splicing (AS) plays fundamental roles in transcriptomic diversity and specifying synaptic properties of each neuronal cell type. However, the mechanisms underlying AS regulation and whether it controls synapse formation across GABAergic interneurons have not been fully elucidated. Here we show the differential expression levels of Sam68-like molecule 2 (SLM2), a major splicing regulator of neurexin (NRX), in GABAergic neuronal subtypes and its contribution to GABAergic synapse specification. Cortical SLM2 is strongly expressed not only in excitatory neurons but also in a subpopulation of GABAergic interneurons, especially in VIP-positive neurons that are originated from late-born caudal ganglionic eminence (GE)- derived cells. Using artificial synapse formation assay, we found that GE containing cortices form a strong synapse with LRRTM2, a trans-synaptic receptor of the alternatively spliced segment 4 (AS4)(-) of NRX. SLM2 knock-down reduced the NRX AS4(-) isoform expression and hence weaken LRRTM2-induced synapse formation. The addition of NRX AS4(-) was sufficient to rescue the synaptic formation by LRRTM2 in SLM2 knock-down neurons. Thus, our findings suggest a novel function of SLM2 in modifying network formation of a specific population of GABAergic interneurons and contribute to a better understanding of the roles AS plays in regulating synapse specificity and neuronal molecular diversity.

Tomato E8 Encodes a C-27 Hydroxylase in Metabolic Detoxification of α-Tomatine during Fruit Ripening.

Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.

Identification of α-Tomatine 23-Hydroxylase Involved in the Detoxification of a Bitter Glycoalkaloid.

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Neuroligin-induced presynaptic differentiation through SLM2-mediated splicing modifications of neurexin in cerebellar cultures.

Neurexins (NRXs) and neuroligins (NLs) play important roles in synapse specification. The alternatively spliced segment 4 (AS4) of NRX genes (Nrxn) is a critical element in selective trans-synaptic interactions. However, the role of splicing of NRXs and NLs in synapse specification is not fully understood. To investigate the exact role of splice-dependent NRX-NL interaction in the specification of glutamatergic and gamma-aminobutyric acid (GABA)-ergic synapses in the cerebellum, we evaluated the synaptogenic receptor activity of NL1/2/3 isoforms in a neuron-fibroblast co-culture system, in which the Nrxn AS4 segments are manipulated using SLM2, a selective and dominant regulator of AS4 splicing. We show that ectopic SLM2 expression (SLM2 E/E) causes marked skipping of exon 20 of AS4 in cerebellar neuron culture. Whereas NLs can induce VAMP2+ presynaptic contacts from mainly glutamatergic neurons in both uninfected (control) and SLM2 E/E co-cultures, they induce VGAT+ GABAergic contacts in the control culture, but not properly in the SLM2 E/E culture. Furthermore, Nrxn3 is responsible for the NL-induced assembly of GABAergic synapses in co-culture. Importantly, lentivirus-based expression of Nrxn3 containing exon 20 restores the reduced NL-induced GABAergic contacts in the SLM2 E/E co-culture. Therefore, our findings may provide further insights into NRX-NL mediated synapse specification.

Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense.

Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.

Flavor characteristics of the juices from fresh market tomatoes differentiated from those from processing tomatoes by combined analysis of volatile profiles with sensory evaluation.

Various commercial tomato juices with different flavors are available at markets worldwide. To clarify the marker compounds related to the flavor characteristics of tomato juice, we analyzed 15 pure commercial tomato juices by a combination of volatile profiling and sensory evaluation. The correlations among volatiles and the relationship between volatiles and sensory descriptors were elucidated by multivariate analyses. Consequently, the tomato juices made from fresh market tomatoes (including the popular Japanese tomato variety "Momotaro") were clearly separated from other juices made from processing tomatoes, by both the volatile composition and sensory profiles. cis-3-Hexenol, hexanal, and apocarotenoids negatively contributed to the juices from fresh market tomatoes, whereas Strecker aldehydes and furfural showed positive contributions to the juices. Accordingly, the sensory characteristics of juices from fresh market tomatoes were related to cooked and fruity flavors but not to green or fresh notes.

Traumatin- and dinortraumatin-containing galactolipids in Arabidopsis: their formation in tissue-disrupted leaves as counterparts of green leaf volatiles.

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.

An application of a relational database system for high-throughput prediction of elemental compositions from accurate mass values.

SUMMARY: High-accuracy mass values detected by high-resolution mass spectrometry analysis enable prediction of elemental compositions, and thus are used for metabolite annotations in metabolomic studies. Here, we report an application of a relational database to significantly improve the rate of elemental composition predictions. By searching a database of pre-calculated elemental compositions with fixed kinds and numbers of atoms, the approach eliminates redundant evaluations of the same formula that occur in repeated calculations with other tools. When our approach is compared with HR2, which is one of the fastest tools available, our database search times were at least 109 times shorter than those of HR2. When a solid-state drive (SSD) was applied, the search time was 488 times shorter at 5 ppm mass tolerance and 1833 times at 0.1 ppm. Even if the search by HR2 was performed with 8 threads in a high-spec Windows 7 PC, the database search times were at least 26 and 115 times shorter without and with the SSD. These improvements were enhanced in a low spec Windows XP PC. We constructed a web service 'MFSearcher' to query the database in a RESTful manner. AVAILABILITY AND IMPLEMENTATION: Available for free at http://webs2.kazusa.or.jp/mfsearcher. The web service is implemented in Java, MySQL, Apache and Tomcat, with all major browsers supported. CONTACT: sakurai@kazusa.or.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato.

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

Superior high-efficiency and high-throughput volatile flavor extraction of Japanese fermented seasonings by solvent-assisted stir bar solid extraction with reverse extraction.

Fermented seasonings have pleasant flavors that stimulate our appetite. Their flavoring properties change depending on factors such as their materials and fermented conditions. Therefore, a comparative analysis of their flavor is important when evaluating their quality. However, seasonings contain high levels of various matrices such as sugars, proteins, lipids, and ethanol, making it difficult to extract aroma compounds efficiently from them. In this study, we verified a high-efficient and high-throughput volatile flavor analysis of fermented seasonings by solvent-assisted stir bar solid extraction (SA-SBSE) with reverse extraction. We applied SA-SBSE to Japanese fermented seasonings, soy sauce, miso (fermented beans), and mirin (sweet rice wine) and compared their profiles with those from other common extraction methods, headspace gas-solid-phase microextraction (HS-SPME), liquid extraction with solvent-assisted flavor evaporation (LE-SAFE), and conventional SBSE (C-SBSE). The aroma properties and profiles of extracts from SA-SBSE were close to those of the original sample, being similar to that of LE-SAFE. In addition, potent aroma compounds in each sample were extracted by SA-SBSE and LE-SAFE, which were far superior to those by C-SBSE. For quantification, SA-SBSE extracts showed a good standard curve by the standard addition method. We could quantify maltol, one of the most common potent aroma compounds in all samples, for various commercial samples by such high-throughput analysis.

Enhancement of production of eugenol and its glycosides in transgenic aspen plants via genetic engineering.

Eugenol, a volatile phenylpropene found in many plant species, exhibits antibacterial and acaricidal activities. This study attempted to modify the production of eugenol and its glycosides by introducing petunia coniferyl alcohol acetyltransferase (PhCFAT) and eugenol synthase (PhEGS) into hybrid aspen. Gas chromatography analyses revealed that wild-type hybrid aspen produced small amount of eugenol in leaves. The heterologous overexpression of PhCFAT alone resulted in up to 7-fold higher eugenol levels and up to 22-fold eugenol glycoside levels in leaves of transgenic aspen plants. The overexpression of PhEGS alone resulted in a subtle increase in either eugenol or eugenol glycosides, and the overexpression of both PhCFAT and PhEGS resulted in significant increases in the levels of both eugenol and eugenol glycosides which were nonetheless lower than the increases seen with overexpression of PhCFAT alone. On the other hand, overexpression of PhCFAT in transgenic Arabidopsis and tobacco did not cause any synthesis of eugenol. These results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene.

Long-chain free fatty acid profiling analysis by liquid chromatography-mass spectrometry in mouse treated with peroxisome proliferator-activated receptor α agonist.

A change in the free fatty acid (FFA) profile reflects an alteration in the lipid metabolism of peripheral tissue. A high-throughput quantitative analysis method for individual FFAs therefore needs to be established. We report here an optimized LC-MS assay for a high-throughput and high-sensitivity analysis of the 10 major long-chain FFAs in mouse plasma and liver. This assay enables quantification of individual FFAs by using trace amounts of samples (2 µL of plasma and 10 mg of liver tissue). We apply this method to analyze the FFA profile of plasma and liver samples from an obese mouse model treated with bezafibrate, the peroxisome proliferator-activated receptor α (PPARα) agonist, and show a change in the FFA profile, particularly in the palmitoleic and oleic acid contents. This assay is useful for quantifying individual FFAs and helpful for monitoring the condition of lipid metabolism.

Enzymatic functions of wild tomato methylketone synthases 1 and 2.

The trichomes of the wild tomato species Solanum habrochaites subsp. glabratum synthesize and store high levels of methylketones, primarily 2-tridecanone and 2-undecanone, that protect the plants against various herbivorous insects. Previously, we identified cDNAs encoding two proteins necessary for methylketone biosynthesis, designated methylketone synthase 1 (ShMKS1) and ShMKS2. Here, we report the isolation of genomic sequences encoding ShMKS1 and ShMKS2 as well as the homologous genes from the cultivated tomato, Solanum lycopersicum. We show that a full-length transcript of ShMKS2 encodes a protein that is localized in the plastids. By expressing ShMKS1 and ShMKS2 in Escherichia coli and analyzing the products formed, as well as by performing in vitro assays with both ShMKS1and ShMKS2, we conclude that ShMKS2 acts as a thioesterase hydrolyzing 3-ketoacyl-acyl carrier proteins (plastid-localized intermediates of fatty acid biosynthesis) to release 3-ketoacids and that ShMKS1 subsequently catalyzes the decarboxylation of these liberated 3-ketoacids, forming the methylketone products. Genes encoding proteins with high similarity to ShMKS2, a member of the "hot-dog fold" protein family that is known to include other thioesterases in nonplant organisms, are present in plant species outside the genus Solanum. We show that a related enzyme from Arabidopsis (Arabidopsis thaliana) also produces 3-ketoacids when recombinantly expressed in E. coli. Thus, the thioesterase activity of proteins in this family appears to be ancient. In contrast, the 3-ketoacid decarboxylase activity of ShMKS1, which belongs to the alpha/beta-hydrolase fold superfamily, appears to have emerged more recently, possibly within the genus Solanum.

Comparative and stability analyses of 9- and 13-Oxo-octadecadienoic acids in various species of tomato.

Peroxisome proliferator-activated receptor-α (PPARα) regulates lipid metabolism. We have reported that tomato fruit contains 9-Oxo-(10E,12E)-octadecadienoic acid (9-Oxo-(10E,12E)-ODA), a PPARα agonist. In this study, we found that various tomato samples contained 9-Oxo-(10E,12Z)-ODA and its 13-Oxo-ODA isomers. Furthermore, several isomers showed structural stability under hot and acidic conditions.

Processing of Airborne Green Leaf Volatiles for Their Glycosylation in the Exposed Plants.

Green leaf volatiles (GLVs), the common constituents of herbivore-infested plant volatiles (HIPVs), play an important role in plant defense and function as chemical cues to communicate with other individuals in nature. Reportedly, in addition to endogenous GLVs, the absorbance of airborne GLVs emitted by infested neighboring plants also play a major role in plant defense. For example, the exclusive accumulation of (Z)-3-hexenyl vicianoside in the HIPV-exposed tomato plants occurs by the glycosylation of airborne (Z)-3-hexenol (Z3HOL); however, it is unclear how plants process the other absorbed GLVs. This study demonstrates that tomato plants dominantly accumulated GLV-glycosides after exposure to green leaf alcohols [Z3HOL, (E)-2-hexenol, and n-hexanol] using non-targeted LC-MS analysis. Three types of green leaf alcohols were independently glycosylated without isomerization or saturation/desaturation. Airborne green leaf aldehydes and esters were also glycosylated, probably through converting aldehydes and esters into alcohols. Further, we validated these findings in Arabidopsis mutants- (Z)-3-hexenal (Z3HAL) reductase (chr) mutant that inhibits the conversion of Z3HAL to Z3HOL and the acetyl-CoA:(Z)-3-hexen-1-ol acetyltransferase (chat) mutant that impairs the conversion of Z3HOL to (Z)-3-hexenyl acetate. Exposure of the chr and chat mutants to Z3HAL accumulated lower and higher amounts of glycosides than their corresponding wild types (Col-0 and Ler), respectively. These findings suggest that plants process the exogenous GLVs by the reductase(s) and the esterase(s), and a part of the processed GLVs contribute to glycoside accumulation. Overall, the study provides insights into the understanding of the communication of the plants within their ecosystem, which could help develop strategies to protect the crops and maintain a balanced ecosystem.

Metabolic alterations in organic acids and gamma-aminobutyric acid in developing tomato (Solanum lycopersicum L.) fruits.

Salt stress improves the quality of tomato fruits. To clarify the mechanism(s) underlying this phenomenon, we investigated metabolic alterations in tomato fruits exposed to 160 mM salt, focusing on metabolism of organic acids related to the tricarboxylic acid (TCA) cycle and gamma-aminobutyric acid (GABA). Quantitative analyses revealed that most amino acids increased in response to salt stress throughout fruit development, and the effect of the stress was greater in the pericarp than in the columella, whereas organic acids did not show a remarkable tendency to salt stress. The transcript levels of 20 genes encoding enzymes of the TCA cycle and peripheral pathways were also analyzed in salt-stressed fruit. Genes responsive to salt stress could be categorized into two types, which were expressed during early development or ripening stages. During fruit development, phosphoenolpyruvate carboxylase 2 and phosphoenolpyruvate carboxykinase displayed contrasting expression patterns between early development and ripening, suggesting a switch of carbohydrate metabolism after the turning stage. Our results revealed a new metabolic pathway for GABA during the development of tomato fruits. At the start of ripening, GABA is first converted to malate via succinate semialdehyde, and it passes into a shunt through pyruvate. Then, it flows back to the TCA cycle and is stored as citrate, which contributes as a substrate for respiration during fruit maturation.

Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway.

We have modified the flavor and aroma of tomatoes by expressing the Ocimum basilicum geraniol synthase gene under the control of the tomato ripening-specific polygalacturonase promoter. A majority of untrained taste panelists preferred the transgenic fruits over controls. Monoterpene accumulation was at the expense of reduced lycopene accumulation. Similar approaches may be applicable for carotenoid-accumulating fruits and flowers of other species.