Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062 µg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4 fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

Remusatia vivipara lectin and Sclerotium rolfsii lectin interfere with the development and gall formation activity of Meloidogyne incognita in transgenic tomato.

Root knot nematodes are serious threats to growth and yield of solaneous crops including tomato. In this study, a binary vector carrying Remusatia vivipara (rvl1) and Sclerotium rolfsii (srl1) lectin genes were introduced independently into Lycopersicon esculentum cv. Pusa Ruby via Agrobacterium tumefaciens for resistance against root knot nematode, Meloidogyne incognita. In total, one hundred and one rvl1 and srl1-transformed plants exhibiting kanamycin resistance were confirmed to carry transgenes as detected by polymerase chain reaction (PCR) with 4.59% transformation efficiency. Genetic analysis of T1 progeny confirmed Mendelian segregation of the introduced genes. Three events each of rvl1 and srl1 transgenic tomato were randomly selected for further confirmation by Southern and TAIL-PCR analyses. All three events of srl1 transgenics showed single copy transgene, whereas two rvl1 transgenic events showed single copy of transgene, while remaining event showed two copies of transgenes. Site of integration obtained for rvl1 and srl1 transgenic events by TAIL-PCR revealed that all the three events of rvl1 and srl1 transgenics differed for their site of integration and insertion sites did not contain any predicted gene. Moreover, expression of the rvl1 and srl1 transgenes was detected by haemagglutination assay in all three events of rvl1 and srl1, but not in non-transgenic tomato plant. Homozygous progenies of these events were grown and inoculated with M. incognita. Development and reproduction of M. incognita was severely affected in transgenic tomato plants expressing RVL1 and SRL1 exhibiting the high levels of resistance compared to non-transgenic plants. Therefore, these transgenic lines demonstrate a promising potential for variety development of tomato lines with enhanced resistance against M. incognita.

Overexpression of Pear (Pyrus pyrifolia) CAD2 in Tomato Affects Lignin Content.

PpCAD2 was originally isolated from the 'Wangkumbae' pear (Pyrus pyrifolia Nakai), and it encodes for cinnamyl alcohol dehydrogenase (CAD), which is a key enzyme in the lignin biosynthesis pathway. In order to verify the function of PpCAD2, transgenic tomato (Solanum lycopersicum) 'Micro-Tom' plants were generated using over-expression constructs via the agrobacterium-mediated transformation method. The results showed that the PpCAD2 over-expression transgenic tomato plant had a strong growth vigor. Furthermore, these PpCAD2 over-expression transgenic tomato plants contained a higher lignin content and CAD enzymatic activity in the stem, leaf and fruit pericarp tissues, and formed a greater number of vessel elements in the stem and leaf vein, compared to wild type tomato plants. This study clearly indicated that overexpressing PpCAD2 increased the lignin deposition of transgenic tomato plants, and thus validated the function of PpCAD2 in lignin biosynthesis.

Effective enhancement of resistance to Phytophthora infestans by overexpression of miR172a and b in Solanum lycopersicum.

MAIN CONCLUSION: Overexpression of miR172a and b in tomato ( Solanum lycopersicum ) Zaofen No. 2 increased resistance to Phytophthora infestans infection by suppressing of an AP2/ERF transcription factor. The miR172 family has been shown to participate in the growth phase transition, flowering time control, abiotic and biotic stresses by regulating the expression of a small group of AP2/ERF transcription factors. In this study, the precursors of miR172a and b were cloned from tomato, Solanum pimpinellifolium L3708. We used the degradome sequencing to determine the cleavage site of miR172 to a member of the AP2/ERF transcription factor family (Solyc11g072600.1.1). qRT-PCR results showed that the expression of AP2/ERF was negatively correlated with the expression of miR172 in S. pimpinellifolium L3708 infected with Phytophthora infestans. Overexpression of miR172a and b in S. lycopersicum Zaofen No. 2 conferred greater resistance to P. infestans infection, as evidenced by decreased disease index, lesion sizes, and P. infestans abundance. The SOD and POD play important roles in scavenging late massive ROS in plant-pathogen interaction. Malonaldehyde (MDA) is widely recognized as an indicator of lipid peroxidation. Membrane damage in plants can be estimated by measuring leakage of electrolytes, which is evaluated by determining relative electrolyte leakage (REL). Less H2O2 and O2-, higher activities of POD and SOD, less MDA content and REL, and higher chlorophyll content and photosynthetic rate were also shown in transgenic plants after inoculation with P. infestans. Our results constitute the first step towards further investigations into the biological function and molecular mechanism of miR172-mediated silencing of AP2/ERF transcription factors in S. lycopersicum-P. infestans interaction and provide a candidate gene for breeding to enhance biotic stress-resistance in S. lycopersicum.

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

Improved tolerance against Helicoverpa armigera in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from Capsicum annuum.

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.

Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit.

Thaumatin, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), is a promising zero-calorie sweetener for use in the food and pharmaceutical industries. Due to limited natural sources of thaumatin, its production using transgenic plants is an advantageous alternative. We report a simple protocol for purification of recombinant thaumatin II from transgenic tomato. Thaumatin was extracted from ripe tomato fruit in a low-salt buffer and purified on an SP-Sephacryl column. Recombinant thaumatin yield averaged 50 mg/kg fresh fruit. MALDI-MS analysis showed correct processing of thaumatin in tomato plants. The recombinant thaumatin was indistinguishable from the native protein in a taste test. The purified tomato-derived thaumatin had an intrinsic sweetness with a threshold value in taste tests of around 50 nM. These results demonstrate the potential of an expression system based on transgenic tomato plants for production of recombinant thaumatin for the food and pharmaceutical industries.

Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life.

Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.

Ectopic Overexpression of CsECR From Navel Orange Increases Cuticular Wax Accumulation in Tomato and Enhances Its Tolerance to Drought Stress.

Drought stress often occurred in citrus to limit its growth, distribution, and fruit quality. Cuticular waxes play an important role in regulating plant tolerance to drought stress. Plant enoyl-CoA reductase (ECR) is involved in the biosynthesis of cuticular waxes and catalyzes the last step of very long-chain fatty acids (VLCFAs) elongation. In this study, a putative ECR gene, named CsECR, was cloned from "Newhall" navel orange. CsECR protein has high identities with other plant ECR proteins and contained a conserved NADP/NAD-binding motif and three conserved functional sites. The highest expression of CsECR was observed in leaves, followed by stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals. Besides, the expression of CsECR was significantly induced by PEG6000 and ABA treatments. Ectopic overexpression of CsECR increased the contents of total waxes and aliphatic wax fractions (n-fatty acids, unsaturated fatty acids, n-alkanes, alkenes, iso-, and anteiso-alkanes) in the leaves and fruits of the transgenic tomato. Furthermore, ectopic overexpression of CsECR reduced the cuticle permeability in the leaves and fruits of the transgenic tomato and increased its tolerance to drought stress. Taken together, our results revealed that CsECR plays an important role in plant response to drought stresses by regulating cuticular wax biosynthesis.

Overexpression of Pyrus sinkiangensis HAT5 enhances drought and salt tolerance, and low-temperature sensitivity in transgenic tomato.

The homeodomain-leucine zipper protein HAT belongs to the homeodomain leucine zipper subfamily (HD-Zip) and is important for regulating plant growth and development and stress tolerance. To investigate the role of HAT5 in tolerance to drought, salt, and low temperature stress, we selected a HAT gene from Pyrus sinkiangensis Yü (Pyrus sinkiangensis T.T. Yu). The sequences were analyzed using ioinformatics, and the overexpressed tomato lines were obtained using molecular biology techniques. The phenotypes, physiological, and biochemical indexes of the wild-type and transgenic tomato lines were observed under different stress conditions. We found that the gene had the highest homology with PbrHAT5. Under drought and NaCl stress, osmotic regulatory substances (especially proline) were significantly accumulated, and antioxidant enzyme activities were enhanced. The malondialdehyde level and relative electrical conductivity of transgenic tomatoes under low temperature (freezing) stress were significantly higher than those of wild-type tomatoes. The reactive oxygen species scavenging system was unbalanced. This study found that PsHAT5 improved the tolerance of tomatoes to drought and salt stress by regulating proline metabolism and oxidative stress ability, reducing the production of reactive oxygen species, and maintaining normal cell metabolism. In conclusion, the PsHAT5 transcription factor has great potential in crop resistance breeding, which lays a theoretical foundation for future excavation of effective resistance genes of the HD-Zip family and experimental field studies.

Effect of fruit maturation on N-glycosylation of plant-derived native and recombinant miraculin.

Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.

Pterostilbene production of tomato transformed with resveratrol synthase and resveratrol O-methyltransferase genes.

Pterostilbene is a methylated derivative of resveratrol. It has been proved to be effective in preventing many human diseases. However, it is produced and accumulated in only small amounts in natural plant raw materials. Here, two genes coding for resveratrol synthase 3 (AhRS3) in Arachis hypogaea and resveratrol O-methyltransferase (VvROMT) in Vitis vinifera were artificially synthesized considering the codon preference of the tomato. They were linked by LP4/2A to form a fusion gene, controlled by cauliflower mosaic virus 35S promoter, and introduced into tomato via Agrobacterium-mediated transformation. This study aimed to obtain a tomato breeding material enriched with pterostilbene in fruits for a healthy food source. Two transgenic plants with high alien gene expression were selected from the regenerated plants using real-time polymerase chain reaction. High-performance liquid chromatography was used to detect the pterostilbene content in fruits. The highest content reached 146.701 ±â€¯47.771 µg/g dry weight, which was significantly higher than natural levels in all other species tested to date. UPLC-MS/MS was used to analyze the differences in metabolites in fruits between the transgenic and wild-type plants to understand the effect of AhRS3-LP4/2A-VvROMT gene on tomato metabolism. Results showed that the synthesis pathway of stilbenes had little influence on the flavonoid metabolic pathway in tomato fruits.

Environmental risk assessment of transgenic miraculin-accumulating tomato in a confined field trial in Japan.

The commercial use of genetically modified (GM) crops requires prior assessment of the risks to the environment when these crops are grown in the field or distributed. Assessments protocols vary across countries and GM crop events, but there is a common need to assess environmental biosafety. In this study, we conducted an environmental risk assessment in a confined field of GM tomato plants that can produce miraculin, a taste-altering protein that causes sour tastes to be perceived as sweet, for practical use in Japan. The evaluation was conducted for 1) competitiveness (the ability to compete with wild plants for nutrients, sunlight, and growing areas and prevent their growth) and 2) the production of toxic substances (the ability to produce substances that interfere with the habitat and growth of wild plants, animals, and microorganisms). Investigations of plant morphology and growth characteristics as well as tolerance to low temperature during early growth and overwintering for assessment endpoints related to competitiveness showed no biologically meaningful difference between GM tomato and non-GM tomato. In addition, harmful substances in plant residues and root secretions were assessed by the plow-in method, succeeding crop test and soil microflora tests, and it was determined that GM tomato does not exhibit an increase in harmful substances. Based on these results, it was concluded that GM miraculin-accumulating tomato is comparable to conventional tomato and is unlikely to have unintended adverse effects in the natural environment of Japan.

MADS-Box Protein Complex VvAG2, VvSEP3 and VvAGL11 Regulates the Formation of Ovules in Vitis vinifera L. cv. 'Xiangfei'.

The phenomenon of multi-carpel and multi-ovule exists in the grapevine cultivar 'Xiangfei', but the mechanism of ovule formation is seldom reported. In this study, we observed the ovule formation process by using 'Xiangfei' grapes. The role of the VvAG2 (VvAGAMOUS) gene in ovule formation was identified, and we explored the relationship between VvAG2, VvSEP3(VvMADS4) and VvAGL11(VvMADS5) proteins. The results showed that the ovule primordium appeared when the inflorescence length of 'Xiangfei' grapes were 4-5 cm long; the relative expression levels of VvAG2, VvAGL11 and VvSEP3 genes were higher during ovule formation, and the expression levels of VvAG2 gene was the highest. Transgenic tomato (Solanum lycopersicum) plants expressing VvAG2 produced higher numbers of ovules and carpels than the wild type. Moreover, yeast two-hybrid and yeast three-hybrid experiments demonstrated that VvSEP3 acts as a bridge and interacts with VvAG2 and VvAGL11 proteins, respectively. Meanwhile, a homodimer can be formed between VvSEP3 and VvSEP3, but there was no interaction between VvAG2 and VvAGL11. These findings suggest that the VvAG2 gene is involved in the formation of ovules, and VvAG2/VvSEP3 together with VvAGL11/VvSEP3 can form a tetrameric complex. In summary, our data showed that VvAG2 along with VvSEP3 and VvAGL11 jointly regulate the ovule formation of 'Xiangfei' grapes.

The accumulation of recombinant miraculin is independent of fruit size in tomato.

The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.

Functional Validation of Phytoene Synthase and Lycopene ε-Cyclase Genes for High Lycopene Content in Autumn Olive Fruit (Elaeagnus umbellata).

Lycopene is the most potent antioxidant among all carotenoids and is beneficial to human health. A ripe fruit of autumn olive (Elaeagnus umbellata Thunb.) accumulates a high level of lycopene, which is 5-20 times higher than that in an ordinary tomato fruit. During fruit ripening of autumn olive, only phytoene synthase (EutPSY) expression pattern shows a tight positive correlation with the increased lycopene content observed at four ripening stages, while the lycopene ε-cyclase (EutLCYe) transcript could not be detected throughout fruit ripening. Here, we investigated whether the two genes are important targets for engineering lycopene biosynthesis. The full-length cDNAs of EutPSY and EutLCYe were first isolated. Fruit-specific overexpression of EutPSY in tomato fruits resulted in elevated contents of lycopene and ß-carotene through feedforward regulation of carotenogenic genes, i.e., downregulation of SlLCYe and upregulation of SlLCYb and SlCYCB. These fruits were decreased in ethylene production throughout ripening. Transcript levels of genes for system-2 ethylene synthesis (SlACS2, SlACS4, SlACO1, and SlACO3), perception (SlNR/ETR3 and SlETR4), and response (SlE4 and SlE8) were also inhibited in EutPSY-overexpressing fruits. Repressing ethylene synthesis and signaling transduction delayed fruit climacteric ripening of transgenic tomato plants. Additionally, RNAi suppression of SlLCYe enhanced ß-carotene but not lycopene accumulation through altered expression of carotenogenic genes in transgenic tomato fruits by both feedforward and feedback regulatory mechanisms. Ethylene production in SlLCYe-RNAi fruits decreased, thereby delaying fruit ripening. Collectively, these results confirmed that transcriptional regulation of EutPSY and EutLCYe plays a crucial role and a part in massive lycopene accumulation in autumn olive fruits, respectively. EutPSY overexpression enhanced lycopene accumulation in tomato fruits independently of the ethylene pathway but did not influence the size and weight of tomato fruits. EutPSY can be used as an effective strategy capable of elevating the lycopene content in fruits for improving quality.

Genetic engineering of the biosynthesis of glycinebetaine enhances the fruit development and size of tomato.

Glycinebetaine has been widely considered as an effective protectant against abiotic stress in plants, and also found to promote plant growth under normal growing conditions, especially during the reproductive stage. Betaine aldehyde dehydrogenase (BADH) and choline oxidase (COD) are two key enzymes which have been used to confer glycinebetaine synthesis in plant which normally does not synthesis glycinebetaine. In this study, we used the tomato (Solanum lycopersicum, cv 'Moneymaker') plants of wild-type and the transgenic lines codA (L1, L2) and BADH (2, 46), which were transformed with codA and BADH, respectively, to study the impact of glycinebetaine on tomato fruit development. Our results showed that the codA and BADH transgenes induced the formation of enlarged flowers and fruits in transgenic tomato plants. In addition, the transgenic tomato plants had a higher photosynthetic rate, higher assimilates content, and higher leaf chlorophyll content than the wild-type plants. We also found that the enlargement of fruit size was related to the contents of phytohormones, such as auxin, brassinolide, gibberellin, and cytokinin. Additionally, qPCR results indicated that the expressions levels of certain genes related to fruit growth and development were also elevated in transgenic plants. Finally, transcriptome sequencing results revealed that the differences in the levels of gene expression in tomato fruit between the transgenic and wild-type plants were observed in multiple pathways, predominantly those of photosynthesis, DNA replication, plant hormone signal transduction, and biosynthesis. Taken together, our results suggest that glycinebetaine promotes tomato fruit development via multiple pathways. We propose that genetic engineering of glycinebetaine synthesis offers a novel approach to enhance the productivity of tomato and other crop plants.

Functional analysis of a tomato (Solanum lycopersicum L.) rhamnogalacturonan lyase promoter.

The enzyme rhamnogalacturonan lyase (RGL) cleaves α-1,4 glycosidic bonds located between rhamnose and galacturonic acid residues in the main chain of rhamnogalacturonan-I (RG-I), a component of the plant cell wall polymer pectin. Although the mode of action of RGL is well known, its physiological functions associated with fruit biology are less understood. Here, we generated transgenic tomato plants expressing the ß-glucuronidase (GUS) reporter gene under the control of a -504 bp or a -776 bp fragment of the promoter of a tomato RGL gene, Solyc11g011300. GUS enzymatic activity and the expression levels of GUS and Solyc11g011300 were measured in a range of organs and fruit developmental stages. GUS staining was undetectable in leaves and roots, but high GUS enzymatic activity was detected in flowers and red ripe (RR) fruits. Maximal expression levels of Solyc11g011300 were detected at the RR developmental stage. GUS activity was 5-fold higher in flowers expressing GUS driven by the -504 bp RGL promoter fragment (RGFL3::GUS) than in the isogenic line, and 1.7-fold higher when GUS gene was driven by the -776 bp RGL promoter fragment (RGLF2::GUS) or the constitutive CaMV35S promoter. Quantitative real-time polymerase chain reaction analysis showed that the highest expression of GUS was in fruits at 40 days after anthesis, for both promoter fragments. The promoter of Solyc11g011300 is predicted to contain cis-acting elements, and to be active in pollen grains, pollen tubes, flowers and during tomato fruit ripening, suggesting that the Solyc11g011300 promoter is transcriptionally active and organ-specific.

Comparison of the N-glycosylation on recombinant miraculin expressed in tomato plants with native miraculin.

Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of miraculin, recombinant miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant miraculins were much lower than those of native miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant miraculin compared to that of native miraculin should be minimized. Here, to establish the platform for functional miraculin production, we expressed miraculin in tomato plants with the same taste-modifying activity as native miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native miraculin. Our N-glycan structural analysis using purified miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the miraculins were pauci-mannosidic structures with a smaller amount of plant-specific α1,3-fucosylated and/or ß1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native miraculin from miracle fruit and recombinant miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of miraculin.

Functional analysis of tomato rhamnogalacturonan lyase gene Solyc11g011300 during fruit development and ripening.

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life.