Transgenic Fruit Research Articles

Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato.

Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches.

Simultaneous silencing of five lipoxygenase genes increases the contents of α-linolenic and linoleic acids in tomato (Solanum lycopersicum L.) fruits.

α-Linolenic and linoleic acids are essential fatty acids (EFAs) for humans and required for maintenance of optimal health, but they cannot be synthesized by the human body and must be obtained from dietary sources. Using TomloxC fragment, TomloxD fragment, and partial TomloxA sequence that is highly identical with TomloxB and TomloxE, a RNAi expression vector was constructed. The construct was used to transform tomato cotyledon explants with the Agrobacterium-mediated co-cultivation method. The real-time reverse transcription polymerase chain reaction analysis showed that the expression of TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE in transgenic tomato plants was drastically repressed, which led to a marked decrease in the levels of lipoxygenase activity. Finally, higher accumulations of the endogenous α-linolenic and linoleic acids were detected in the transgenic tomato fruits, which were 1.65-3.99 and 2.91-4.98 times that of the non-transformed tomato fruits, respectively.

An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato.

Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.

Improving olive fruit fly Bactrocera oleae (Diptera: Tephritidae) adult and larval artificial diets, microflora associated with the fly and evaluation of a transgenic olive fruit fly strain

Research on the olive fruit fly Bactrocera oleae (Rossi) - rearing simplification, insect microflora and transgenic strain evaluation - yielded several findings: (1) incorporation of antibiotics in the adult diet is evidently not needed; (2) colonization appears to be easier when wild adults are collected from the field instead of using mature larvae emerging from field-collected infested olives; (3) a combination of standard solid starter with liquid (no cellulose powder) finisher impregnated in synthetic sponge larval diets was more promising compared with all-liquid diets; (4) molecular analysis revealed extensive differences in bacterial species associated with the fly between laboratory flies and strains from different olive varieties, as well as between strains originating from different seasons of the year; (5) when an enhanced green fluorescent protein transgenic strain was compared with the standard long mass-reared strain, it proved significantly inferior according to all quality control tests applied, i.e. egg production, egg hatch, larval-stage duration, pupal recovery, pupal weight, adult emergence and adult survival. The aforementioned findings are discussed in the context of mass rearing and quality requirements for more successful implementation of the sterile insect technique against this pest.

Increased antioxidant capacity in tomato by ectopic expression of the strawberry D-galacturonate reductase gene.

Increasing L-ascorbic acid (AsA, vitamin C) content in fruits is a common goal in current breeding programs due to its beneficial effect on human health. Attempts to increase AsA content by genetic engineering have resulted in variable success likely due to AsA's complex regulation. Here, we report the effect of ectopically expressing in tomato the D-galacturonate reductase (FaGalUR) gene from strawberry, involved in AsA biosynthesis, either under the control of the constitutive 35S or the tomato fruit-specific polygalucturonase (PG) promoters. Although transgenic lines showed a moderate increase on AsA content, complex changes in metabolites were found in transgenic fruits. Metabolomic analyses of ripe fruits identified a decrease in citrate, glutamate, asparagine, glucose, and fructose, accompanied by an increase of sucrose, galactinol, and chlorogenic acid. Significant metabolic changes also occurred in leaves of 35S-FaGalUR lines, which showed higher non-photochemical fluorescence quenching (NPQ), indicative of a higher constitutive photo-protective capacity. Overall, overexpression of FaGalUR increased total antioxidant capacity in fruits and the results suggest a tight control of AsA content, probably linked to a complex regulation of cellular redox state and metabolic adjustment.

The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

Manipulation of flavour and aroma compound sequestration and release using a glycosyltransferase with specificity for terpene alcohols.

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening.

English

Melons are good models used for explaining the physiological and biochemical changes in postharvest ripening. In this study, transgenic melons expressing apple ACC oxidase gene when treated with ethylene (AS3) were evaluated. Cell wall expression (MPG1; MPG2), ethylene synthesis ACC oxidase (ACCO1), flavour synthesis AAT (alcohol acyltransferase) and physiochemical parameters such as pulp firmness, titratable acidity (TA), soluble solid content (SSC), volatile esters, ethylene productions, antioxidant assay capacity and ascorbic acid content were evaluated. In cell wall expression analysis, MPG1 increased when fruits of transgenic melons were exposed to ethylene; showing they are ethylene- dependent. MPG2 decreased gradually when fruits were subjected to ethylene application. Fruit firmness was modified in transgenic fruits when ethylene was applied. There was a great reduction similar to that of non- transgenic fruits. However, TA in transgenic fruits remained lower than in non- transgenic fruit. The ethylene applied in transgenic fruit made the titratable acid to increase during 48 h and after it, a reduction was observed. In relation to soluble solid contents, transgenic fruits treated with or without ethylene did not reduce gradually compared to the wild type melons in all the periods. Ethylene productions in transgenic fruits were reestablished when ethylene was applied, exhibiting the same behavior as transgenic fruits. Antioxidant assay levels were more active in transgenic fruits when ethylene was applied than in control fruits, and it was only in transgenic fruits without ethylene. Ascorbic acid was kept in transgenic fruits with or without the application of ethylene. Results obtained show that the application of ethylene in transgenic ACC oxidase melons is able to change the metabolism of the cell wall, flavors and antioxidant capacity levels in fruit during the ripening process. Keywords: Esters, antioxidant, solid soluble content, ascorbic acid African Journal of Biotechnology , Vol 13(31) 3252-3261

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.

Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.

A non-climacteric fruit gene CaMADS-RIN regulates fruit ripening and ethylene biosynthesis in climacteric fruit.

MADS-box genes have been reported to play a major role in the molecular circuit of developmental regulation. Especially, SEPALLATA (SEP) group genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. However, the mechanisms underlying the regulation of SEP genes to non-climacteric fruits ripening are still unclear. Here a SEP gene of pepper, CaMADS-RIN, has been cloned and exhibited elevated expression at the onset of ripening of pepper. To further explore the function of CaMADS-RIN, an overexpressed construct was created and transformed into ripening inhibitor (rin) mutant tomato plants. Broad ripening phenotypes were observed in CaMADS-RIN overexpressed rin fruits. The accumulation of carotenoid and expression of PDS and ZDS were enhanced in overexpressed fruits compared with rin mutant. The transcripts of cell wall metabolism genes (PG, EXP1 and TBG4) and lipoxygenase genes (TomloxB and TomloxC) accumulated more abundant compared to rin mutant. Besides, both ethylene-dependent genes including ACS2, ACO1, E4 and E8 and ethylene-independent genes such as HDC and Nor were also up-regulated in transgenic fruits at different levels. Moreover, transgenic fruits showed approximately 1–3 times increase in ethylene production compared with rin mutant fruits. Yeast two-hybrid screen results indicated that CaMADS-RIN could interact with TAGL1, FUL1 and itself respectively as SlMADS-RIN did in vitro. These results suggest that CaMADS-RIN affects fruit ripening of tomato both in ethylene-dependent and ethylene-independent aspects, which will provide a set of significant data to explore the role of SEP genes in ripening of non-climacteric fruits.

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.

Tomato FRUITFULL homologs regulate fruit ripening via ethylene biosynthesis.

Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.

Advances in Transgenic Vegetable and Fruit Breeding

Vegetables and fruits are grown worldwide and play an important role in human diets because they provide vitamins, minerals, dietary fiber, and phytochemicals. Vegetables and fruits are also associated with improvement of gastrointestinal health, good vision, and reduced risk of heart disease, stroke, chronic diseases such as diabetes, and some forms of cancer. Vegetable and fruit production suffers from many biotic stresses caused by pathogens, pests, and weeds and requires high amounts of plant protection products per hectare. United States vegetables farmers are benefiting from growing transgenic squash cultivars resistant to Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus, which were deregulated and commercialized since 1996. Bt-sweet corn has also proven effective for control of some lepidopteran species and continues to be accepted in the fresh market in the USA, and Bt-fresh-market sweet corn hybrids are released almost every year. Likewise, transgenic Bt-eggplant bred to reduce pesticide use is now grown by farmers in Bangladesh. Transgenic papaya cultivars carrying the coat-protein gene provide effective protection against Papaya ring spot virus elsewhere. The transgenic “Honey Sweet” plum cultivar provides an interesting germplasm source for Plum pox virus control. Enhanced host plant resistance to Xanthomonas campestris pv. musacearum, which causes the devastating banana Xanthomonas wilt in the Great Lakes Region of Africa, was achieved by plant genetic engineering. There are other vegetable and fruit crops in the pipeline that have been genetically modified to enhance their host plant resistance to insects and plant pathogens, to show herbicide tolerance, and to improve features such as slow ripening that extends the shelf-life of the produce. Consumers could benefit further from eating more nutritious transgenic vegetables and fruits. Transgenic plant breeding therefore provides genetically enhanced seed embedded technology that contributes to integrated pest management in horticulture by reducing pesticide sprays as well as improving food safety by minimizing pesticide residues. Furthermore, herbicide-tolerant transgenic crops can help reducing plough in fields, thereby saving fuel because of less tractor use, which also protects the structure of the soil by reducing its erosion. Transgenic vegetable and fruit crops could make important contributions to sustainable vegetable production and for more nutritious and healthy food. Countries vary, however, in their market standards of acceptance of transgenic crops. Biotechnology products will be successful if clear advantages and safety are demonstrated to both growers and consumers.

A New Tomato NAC (NAM/ATAF1/2/CUC2) Transcription Factor, SlNAC4, Functions as a Positive Regulator of Fruit Ripening and Carotenoid Accumulation

Fruit ripening in tomato (Solanum lycopersicum) is a complicated development process affected by both endogenous hormonal and genetic regulators and external signals. Although the role of NOR, a member of the NAC domain family, in mediating tomato fruit ripening has been established, its underlying molecular mechanisms remain unclear. To explore further the role of NAC transcription factors in fruit ripening, we characterized a new tomato NAC domain protein, named SlNAC4, which shows high accumulation in sepal and at the onset of fruit ripening. Various stress treatments including wounding, NaCl, dehydration and low temperature significantly increased the expression of SlNAC4. Reduced expression of SlNAC4 by RNA interference (RNAi) in tomato resulted in delayed fruit ripening, suppressed Chl breakdown and decreased ethylene synthesis mediated mainly through reduced expression of ethylene biosynthesis genes of system-2, and reduced carotenoids by alteration of the carotenoid pathway flux. Transgenic tomato fruits also displayed significant down-regulation of multiple ripening-associated genes, indicating that SlNAC4 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation. Moreover, we also noted that SlNAC4 could not be induced by ethylene and may function upstream of the ripening regulator RIN and positively regulate its expression. Yeast two-hybrid assay further revealed that SlNAC4 could interact with both RIN and NOR protein. These results suggested that ethylene-dependent and -independent processes are regulated by SlNAC4 in the fruit ripening regulatory network.

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.

Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Subchromoplast Sequestration of Carotenoids Affects Regulatory Mechanisms in Tomato Lines Expressing Different Carotenoid Gene Combinations

Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.

Recent advances in genetic engineering for improvement of fruit crops

Fruits are one of the major sources of vitamins, essential nutrients, antioxidants and fibers in human diet. During the last two–three decades, genetic engineering methods based on the use of transgenes have been successfully adopted to improve fruit plants and focused mainly on enhanced tolerance to biotic and abiotic stresses, increased fruit yield, improved post harvest shelf life of fruit, reduced generation time and production of fruit with higher nutritional value. However, the development of transgenic fruit plants and their commercialization are hindered by many regulatory and social hurdles. Nowadays, new genetic engineering approaches i.e. cisgenesis or intragenesis receive increasing interest for genetic modification of plants. The absence of selectable marker gene in the final product and the introduced gene(s) derived from the same plant or plants sexually compatible with the target crop should increase consumer’s acceptance. In this article, we attempt to summarize the recent progress achieved on the genetic engineering in fruit plants and their applications in crop improvement. Challenges and opportunities for the deployment of genetic engineering in crop improvement programs of fruit plants are also discussed.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids. In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYb, EC:1.14.-.-) is a key regulatory enzyme in the catalysis of lycopene to β-carotene, an important dietary precursor of vitamin A for human nutrition. Two closely related lycopene β-cyclase cDNAs, designated CsLCYb1 and CsLCYb2, were isolated from the pulp of orange fruits (Citrus sinensis). The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopene-accumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene, and the ripe fruits displayed a bright orange colour. However, the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation. In fruits of the CsLCYb1 transgenic plants, most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g−1 DW. Unexpectedly, most transgenic tomatoes showed a reduction in total carotenoid accumulation, and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits. Collectively, these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.