Expression of an engineered acidic-subunit 11S globulin of amaranth carrying the antihypertensive peptides VY,in transgenic tomato fruits

The acidic-subunit of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr was expressed in the fruit of transgenic tomato plants. Immunoblot analyses indicate that the expressed recombinant protein was stably accumulated at levels up to 12.71 % with respect to total protein content of transgenic fruits. There was a remarkable change in total protein content (5–22 % increase) of transgenic tomato fruits compared to non-transformed samples. Specific increases of the essential amino acids valine (31–40 %), tyrosine (29–34 %), isoleucine (21–31 %), leucine (28–31 %) and phenylalanine (28–29 %) were also detected in some transgenic lines versus wild type lines. Protein hydrolysates from transgenic tomato fruits showed in vitro inhibition of the angiotensin converting enzyme, with IC₅₀ values that ranged from 0.376 to 3.241 μg ml^?1; this represents an increase of up to 13-fold in the inhibitory activity compared with the protein hydrolysates of non-transformed fruits. These results suggest the possible application of transgenic tomato fruit for massive production of this engineered version of amarantin, which could be especially useful in the prevention and control of hypertension.     

Control of the Biosynthesis of Phenylalanine and Tyrosine in Plant Tissue Cultures: Lack of Repression of Chorismate Mutase

Tobacco, rice, carrot and tomato tissue cultures were grown in liquid media containing l-phenylalanine or l-tyrosine, or both together. The addition of these amino acids increased their respective cellular levels (4–20 fold), but did not lower the level of chorismate mutase, an enzyme in the biosynthetic pathway of phenylalanine and tyrosine. These results indicate that the biosynthesis of phenylalanine and tyrosine in cultured plant cells is not regulated by repression of the synthesis of chorismate mutase by phenylalanine or tyrosine.     

Production of prenylated flavonoids in tomato fruits expressing a prenyltransferase gene from Streptomyces coelicolor A3(2)

Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.     

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.     

Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Tyamagondlu V. Venkatesh, and Balasulojini Karunanandaa have equally contributed.     

Antioxidant and anti-inflammatory properties of tomato fruits synthesizing different amounts of stilbenes

Resveratrol, a plant phenolic compound, is found in grapes and red wine, but is not widely distributed in other common food sources. The pathway for resveratrol biosynthesis is well characterized. Metabolic engineering of this compound has been achieved in tomato plants ( Lycopersicon esculentum Mill.) in order to improve their nutritional value. Tomato plants synthesizing resveratrol were obtained via the heterologous expression of a grape ( Vitis vinifera L.) cDNA encoding for the enzyme stilbene synthase (StSy), under the control of the fruit-specific promoter TomLoxB. The resulting LoxS transgenic plants accumulated trans -resveratrol and trans -piceid, in particular in the skin of the mature fruits. Quantitative analyses carried out on LoxS fruits were compared with those of a tomato line constitutively expressing the stsy gene (35SS). The LoxS fruits contained levels of trans -resveratrol that were 20-fold lower than those previously reported for the 35SS line. The total antioxidant capability and ascorbate content in transformed fruits were also evaluated, and a significant increase in both was found in the LoxS and 35SS lines. These results could explain the higher capability of transgenic fruits to counteract the pro-inflammatory effects of phorbol ester in monocyte–macrophages via the inhibition of induced cyclo-oxygenase-2 enzyme.     

Effect of prolonged root hypoxia on the antioxidant content of tomato fruit

Tomato fruit quality depends on its metabolite content, which in turn is determined by numerous metabolic changes occurring during fruit development and ripening. The aim of this work was to investigate whether flooding affects the nutritional quality of tomato fruit, focusing on compounds essential to human health: carotenoids and ascorbate. To this end, tomato plants (Solanum lycopersicum L. cv Micro-Tom) were submitted to prolonged root hypoxia (1–2% O₂) at first flower anthesis. Fruits were harvested at five stages of the ripening process and analysed for their carotenoid and ascorbate contents. Our results showed that the ripening of fruits that developed on hypoxia treated plants was not inhibited. However, root hypoxia significantly limits carotenoid and ascorbate accumulation in pericarp during fruit ripening, the strongest effects being observed at late stages of ripening. Limitation of both carotenoids and ascorbate accumulation seems to be primarily mediated by the reduced level of expression of genes of the corresponding metabolic pathway.     

Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway

The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-(14)C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits.     

Metabolic Engineering To Produce Tyrosine or Phenylalanine in a Tryptophan-Producing Corynebacterium glutamicum Strain

The aromatic amino acids are synthesized via a common biosynthetic pathway. A tryptophan-producing mutant of Corynebacterium glutamicum was genetically engineered to produce tyrosine or phenylalanine in abundance. To achieve this, three biosynthetic genes encoding the first enzyme in the common pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS), and the branch-point enzymes chorismate mutase and prephenate dehydratase were individually cloned from regulatory mutants of C. glutamicum which have either of the corresponding enzymes desensitized to end product inhibition. These cloned genes were assembled one after another onto a multicopy vector of C. glutamicum to yield two recombinant plasmids. One plasmid, designated pKY1, contains the DS and chorismate mutase genes, and the other, designated pKF1, contains all three biosynthetic genes. The enzymes specified by both plasmids were simultaneously overexpressed approximately sevenfold relative to the chromosomally encoded enzymes in a C. glutamicum strain. When transformed with pKY1 or pKF1, tryptophan-producing C. glutamicum KY10865, with the ability to produce 18 g of tryptophan per liter, was altered to produce a large amount of tyrosine (26 g/liter) or phenylalanine (28 g/liter), respectively, because the accelerated carbon flow through the common pathway was redirected to tyrosine or phenylalanine.     

Evidence for the involvement of sucrose phosphate synthase in the pathway of sugar accumulation in sucrose-accumulating tomato fruits

To better understand the mechanism of sugar unloading and sugar concentration in hexose- and sucrose-accumulating tomato fruits (Lycopersicon chmielewskii and L. esculentum, respectively) and to determine the causes of the late accumulation of sucrose present in sucrose-accumulating tomato fruits, the assimilation of [³H](fructosyl)-sucrose was studied. Key enzymes involved in carbohydrate metabolism were also assayed. The results demonstrated that the low level of sucrose present in young fruits accumulates directly without undergoing hydrolysis, suggesting a symplastic pathway for sucrose unloading. By contrast, the large quantity of the sucrose present in ripe sucrose-accumulating fruits originates from hydrolysis and resynthesis, suggesting an apoplastic pathway for sucrose unloading. The increase in sucrose level observed in sucrose-accumulating fruits is associated with a gradual decline in invertase activity and an increase in sucrose phosphate synthase activity. This latter enzyme seems to play a key biochemical role in the accumulation of sucrose and the establishment of a high sugar content in tomato fruits.     

The Solanum lycopersicum AUXIN RESPONSE FACTOR 7 (SlARF7) mediates cross-talk between auxin and gibberellin signalling during tomato fruit set and development

Transgenic tomato plants (Solanum lycopersicum L.) with reduced mRNA levels of AUXIN RESPONSE FACTOR 7 (SlARF7) form parthenocarpic fruits with morphological characteristics that seem to be the result of both increased auxin and gibberellin (GA) responses during fruit growth. This paper presents a more detailed analysis of these transgenic lines. Gene expression analysis of auxin-responsive genes show that SlARF7 may regulate only part of the auxin signalling pathway involved in tomato fruit set and development. Also, part of the GA signalling pathway was affected by the reduced levels of SlARF7 mRNA, as morphological and molecular analyses display similarities between GA-induced fruits and fruits formed by the RNAi SlARF7 lines. Nevertheless, the levels of GAs were strongly reduced compared with that in seeded fruits. These findings indicate that SlARF7 acts as a modifier of both auxin and gibberellin responses during tomato fruit set and development.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.     

Progress in the dissection and manipulation of plant vitamin E biosynthesis

Plants contain many unique biosynthetic pathways producing a diverse array of natural products that are important for plant function, agriculture, and human nutrition. The tocochromanols define one such class of compounds, comprised of four tocopherols and four tocotrienols that are collectively termed vitamin E. Tocochromanols are synthesized only by plants and other oxygenic, photosynthetic organisms, and the eight individual compounds vary widely in their vitamin E activities. Vitamin E was recognized as an essential component in mammalian diets in the 1920s and the tocochromanol biosynthetic pathway elucidated from radiotracer studies in the mid 1980s. However, it is only recently that genetic and genomics-based approaches in model photosynthetic organisms have allowed the genes and proteins for tocochromanol synthesis to be isolated, setting the stage for targeted manipulation of tocochromanol levels and types in various crops. This article reviews advancements in our molecular and genetic understanding of the tocochromanol biosynthetic pathway in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803 and highlights ongoing efforts to use this knowledge to manipulate the levels of this essential nutrient in food crops.     

Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis

Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.Mirela Raclaru and Jens Gruber contributed equally     

Phytoene desaturase is present in a large protein complex in the plastid membrane

Phytoene desaturase (PDS; EC 1.14.99.-) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants. To further characterize PDS, we isolated the PDS cDNA from cauliflower ( BoPDS ) and confirmed its function by heterologous expression in a strain of Escherichia coli containing a carotenoid-producing plasmid. The BoPDS cDNA encodes a predicted mature protein of approximately 55 kDa. In comparison with PDS from a few other plant species, BoPDS exhibited a high enzyme activity in E. coli , and its expression in plastids was independent of carotenoid levels. Plastids were purified from tissues of different plant species including cauliflower curds, tomato fruits, carrot roots and Arabidopsis leaves. By employing both Blue Native PAGE and SDS-PAGE approaches in conjunction with Western blot analysis, it was found that PDS in these plants existed in two forms. The plastid membrane form was present in a large protein complex of approximately 350 kDa, whereas the stroma version was in an approximately 660 kDa complex.     

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Characterization of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes and mutants revealed highly complex metabolite-mediated feedback regulation of the plant shikimate pathway.