Fruit-specific Promoter Research Articles

The DR5 and E8 reporters are suitable systems for studying the application of the <i>Ruby</i> reporter gene in tomato

<sec><title/> Tomato (<italic>Solanum lycopersicum</italic>) fruits can be found in a range of colors, including green, yellow, red, pink and purple. The various colors are determined by the accumulation of molecules such as chlorophyll, carotenoids (including lycopene) and flavonoids. Since the DR5 promoter is induced by auxin, we can infer that the auxin responses differ at different developmental stages in tomato. Fruit-specific promoters can effectively drive gene expression in fruits, and the E8 promoter is one of the most widely used fruit-specific promoters. In this study, DR5::<italic>Ruby</italic> transgenic tomato plants were used to further clarify the auxin content at different developmental stages in tomato. The results showed that the nonuniformity of <italic>Ruby</italic> activity was consistent with the natural distribution characteristics of native auxin, and the results were also consistent with the qRT‒PCR results. Furthermore, the accumulation of endogenous auxin at the immature green stage (IMG) was higher than that at the other stages. </sec><sec><title/> In addition, E8::<italic>Ruby</italic> was transformed into different tomato varieties (red, white and yellow) to generate new tomato varieties with fruit of different colors. The results showed that the E8::<italic>Ruby-</italic>transformed plants exhibited asymmetrical pink coloration in different tomato tissues of plants from different tomato cultivars. In conclusion, all the results showed that DR5 and E8 are suitable systems for studying the application of the <italic>Ruby</italic> reporter gene in tomato. Moreover, the study will provide insight for the improvement of tomato fruit color via biotechnology. </sec>

Over-expression of GGP1 and GPP genes enhances ascorbate content and nutritional quality of tomato

L-Ascorbic acid (AsA), a strong antioxidant, serves as an enzyme cofactor and redox status marker, modulating a plethora of biological processes. As tomato commercial varieties and hybrids possess relatively low amounts of AsA, the improvement of fruit AsA represents a strategic goal for enhanced human health. Previously, we have suggested that GDP-L-Galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP) can serve as possible targets for AsA manipulation in tomato (Solanum lycopersicon L.) fruit. To this end, we produced and evaluated T3 transgenic tomato plants carrying these two genes under the control of CaMV-35S and two fruit specific promoters, PPC2 and PG-GGPI. The transgenic lines had elevated levels of AsA, with the PG-GGP1 line containing 3-fold more AsA than WT, without affecting fruit characteristics. Following RNA-Seq analysis, 164 and 13 DEGs were up- or down-regulated, respectively, between PG-GGP1 and WT pink fruits. PG-GGP1 fruit had a distinct number of up-regulated transcripts associated with cell wall modification, ethylene biosynthesis and signaling, pollen fertility and carotenoid metabolism. The elevated AsA accumulation resulted in the up regulation of AsA associated transcripts and alternative biosynthetic pathways suggesting that the entire metabolic pathway was influenced, probably via master regulation. We show here that AsA-fortification of tomato ripe fruit via GGP1 overexpression under the action of a fruit specific promoter PG affects fruit development and ripening, reduces ethylene production, and increased the levels of sugars, and carotenoids, supporting a robust database to further explore the role of AsA induced genes for agronomically important traits, breeding programs and precision gene editing approaches.

Enrichment of health-promoting lutein and zeaxanthin in tomato fruit through metabolic engineering

Carotenoids constitute a large group of natural pigments widely distributed in nature. These compounds not only provide fruits and flowers with distinctive colors, but also have significant health benefits for humans. Lutein and zeaxanthin, both oxygen-containing carotenoids, are considered to play vital roles in promoting ocular development and maintaining eye health. However, humans and mammals cannot synthesize these carotenoid derivatives, which can only be taken from certain fruits or vegetables. Here, by introducing four endogenous synthetic genes, SlLCYE, SlLCYB, SlHYDB, and SlHYDE under fruit-specific promoters, we report the metabolic engineering of lutein/zeaxanthin biosynthesis in tomato fruit. Transgenic lines overexpression of one (SlLCYE), two (SlLCYE and SlLCYB; SlLCYB and SlHYDB), and all these four synthetic genes re-established the lutein/zeaxanthin biosynthetic pathways in the ripe tomato fruit and thus resulted in various types of carotenoid riched lines. Metabolic analyses of these engineered tomato fruits showed the strategy involved expression of SlLCYE tends to produce α-carotene and lutein, as well as a higher content of β-carotene and zeaxanthin was detected in lines overexpressing SlLCYB. In addition, the different combinations of engineered tomatoes with riched carotenoids showed higher antioxidant capacity and were associated with a significantly extended shelf life during postharvest storage. This work provides a successful example of accurate metabolic engineering in tomato fruit, suggesting the potential utility for synthetic biology to improve agronomic traits in crops. These biofortified tomato fruits could be also exploited as new research subjects for studying the health benefits of carotenoid derivatives.

Identification of essential element determining fruit-specific transcriptional activity in the tomato HISTIDINE DECARBOXYLASE A gene promoter.

In SlHDC-A promoter, SlHDC-A core-ES is an essential region for fruit-specific expression and interacts with GATA, HSF and AP1. Triplication of essential region was proposed as a minimal fruit-specific promoter. In plant biotechnology, fruit-specific promoter is an important tool for the improvement and utilization of tomato fruit. To expand our understanding on fruit-specific expression, it is necessary to determine the promoter region involved in fruit-specific transcriptional activity and transcriptional regulations of the promoter. In previous study, we isolated a fruit-specific SlHDC-A core promoter specifically expressed during tomato ripening stages. In this study, we identified SlHDC-A promoter region (SlHDC-A core-ES) that is essential for fruit-specific expression of the SlHDC-A. To understand the molecular mechanisms of fruit-specific expression of the SlHDC-A promoter, we first identified the putative transcription factor binding elements in the SlHDC-A core promoter region and corresponding putative transcription factors which are highly expressed during fruit maturation. Yeast one hybrid analysis confirmed that GATA, HSF, and AP1 interact with the SlHDC-A core-ES promoter region. Further transactivation analysis revealed that expression of the three transcription factors significantly activated expression of a reporter gene driven by SlHDC-A core-ES promoter. These results suggest that GATA, HSF, and AP1 are involved in the fruit-specific expression of SlHDC-A promoter. Furthermore, the synthetic promoter composed of three tandem repeats of SlHDC-A core-ES showed relatively higher activity than the constitutive 35S promoter in the transgenic tomato fruits at the orange stage. Taken together, we propose a new synthetic promoter that is specifically expressed during fruit ripening stage.

Agricultural Insights for Development of Genetically Modified Foods, Horticultural Crops and Role of Nanotechnology

Agricultural productivity under stress conditions can be accomplished by modifying cellular metabolism and molecular transformations of genetically modified plans with the help of nano-material-mediated facilitations. Horticultural crops are the main source of nutritional compounds such as proteins, carbohydrates, vitamins, minerals, and organic acids. Osmotic stress combined with temperatures over 28°C caused a 30-45 percent blossom decline in several tomato varieties. Chili too is susceptible to drought stress, which can result in production losses of as much as 50 to 60 percent. Pigment and color retention are important determinants of vegetable and fruit quality and nutritional values. Genes involved in the enhancement of anthocyanin synthesis and accumulation during postharvest storage in red-fleshed kiwi. Targeted implementation of fruit-specific promoters with distinct up and low regulated epigenetic biomarkers might be useful in the expression of genes and reduces the side effects developed by constitutive promoters. Chitosan is one kind of these nanomaterials over-coated on post-harvested mangoes to secure its edible portion from rotting, increase its vitamin C contents, maintain its freshness, and increase shelf-life. The use of nanomaterials in the food industry reduces the cost of electricity applied in refrigeration for preservation purposes alongside maintaining the original vigor and vitality of the fruits like tomatoes.

Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

SlCHYR1, a RING and CHY zinc finger domain-containing protein, promotes tomato fruit ripening by reprograming abscisic acid and ethylene signaling

During storage, transportation and sales, the deterioration of the tomato ripening fruit mainly caused by mechanical damage and microbial infection results in great economic losses. There is growing interest in fruit ripening studies and postharvest quality improvement to further enhance its already high economic value. Fruit ripening are coordinated by a complex network of endogenous and exogenous factors, including the phytohormone ethylene and abscisic acid, transcription factors, non-coding RNA, epigenetic modification, and environmental factors. However, the studies on RING-finger proteins involved in fruit development and ripening have not been reported. In this study, SlCHYR1, encoding a RING and CHY zinc finger domain-containing protein, which shows high expression levels in fruit was isolated and studied in tomato. Overexpression of SlCHYR1 with a fruit-specific promoter originating from pineapple leads to accelerated fruit ripening, reduced shelf life and repressed chlorophyll accumulation in tomato fruit. Further investigation indicate that SlCHYR1 promotes tomato fruit ripening through abscisic acid and ethylene production and signaling. This work provides novel insights into the regulation of fruit development and ripening by ZFPs (Zinc finger proteins), and would contribute to the improvement of quality and shelf life of tomato.

Different Fruit-Specific Promoters Drive AtMYB12 Expression to Improve Phenylpropanoid Accumulation in Tomato.

Tomato is an economically crucial vegetable/fruit crop globally. Tomato is rich in nutrition and plays an essential role in a healthy human diet. Phenylpropanoid, a critical compound in tomatoes, reduces common degenerative and chronic diseases risk caused by oxidative stress. As an MYB transcription factor, ATMYB12 can increase phenylpropanoid content by activating phenylpropanoid synthesis related genes, such as PAL, C4H, 4CL, CHS. However, the heterologous expression of AtMYB12 in tomatoes can be altered through transgenic technologies, such as unstable expression vectors and promoters with different efficiency. In the current study, the efficiency of other fruit-specific promoters, namely E8S, 2A12, E4, and PG, were compared and screened, and we determined that the expression efficiency of AtMYB12 was driven by the E8S promoter was the highest. As a result, the expression of phenylpropanoid synthesis related genes was regulated by AtMYB12, and the phenylpropanoid accumulation in transgenic tomato fruits increased 16 times. Additionally, the total antioxidant capacity of fruits was measured through Trolox equivalent antioxidant capacity (TEAC) assay, which was increased by 2.4 times in E8S transgenic lines. TEAC was positively correlated with phenylpropanoid content. Since phenylpropanoid plays a crucial role in the human diet, expressing AtMYB12 with stable and effective fruit-specific promoter E8S could improve tomato’s phenylpropanoid and nutrition content and quality. Our results can provide genetic resources for the subsequent improvement of tomato varieties and quality, which is significant for human health.

Engineering high levels of saffron apocarotenoids in tomato.

Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-(β-D-gentiobiosyl)-(β-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer’s disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties.

Engineering high coenzyme Q10 tomato

Coenzyme Q (CoQ) is vital for energy metabolism in living organisms. In humans, CoQ10 deficiency causes diseases and must be replenished via diet; however, CoQ content in plant foods is primarily low. Here, we report the breeding of high CoQ10 tomato lines by expressing four enzymes with a fruit-specific promoter, which modifies the chloroplast chorismate pathway, enhances cytosolic isoprenoid biosynthesis, and up-regulates the first two reactions in mitochondrion that construct the CoQ10 polyisoprenoid tail. We show that, while the level of the aromatic precursor could be markedly elevated, head group prenylation is the key to increasing the final CoQ10 yield. In the HUCD lines expressing all four transgenes, the highest CoQ10 content (0.15 mg/g dry weight) shows a seven-fold increase from the wild-type level and reaches an extraordinarily rich CoQ10 food grade. Overviewing the changes in other terpenoids by transcriptome and metabolic analyses reveals variable contents of carotenoids and α-tocopherol in the HUCD lines. In addition to the enigmatic relations among different terpenoid pathways, high CoQ10 plants maintaining substantial levels of either vitamin can be selected. Our investigation paves the way for the development of CoQ10-enriched crops as dietary supplements.

Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits.

Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in β-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in β-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.

Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits.

Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.

Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology

BackgroundPromoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development.ResultsIn this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity.ConclusionsAmong the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

The use of SlAdh2 promoter as a novel fruit-specific promoter in transgenic tomato

The use of <i>SlAdh2</i> promoter as a novel fruit-specific promoter in transgenic tomato

In silico Analysis of Polyamine Rich Transgenic Tomato Fruit Transcriptome for Salicylic Acid Biosynthesis

EFFECT of higher levels of polyamines (PAs), spermidine (SPD) and spermine (SPM), were evaluated on the salicylic acid biosynthesis genes. It has been previously reported that higher steady state levels of a pathogenesis protein pR1b1, a salicylic acid (SA) regulated protein, are present in tomato fruits with higher levels of anabolic biogenic amines, spermidine (SPD) and spermine (SPM) resulting from the expression of yeast S-adenosylmethionine decarboxylase (SAMDC), under a fruit specific E8 promoter in ripening tomato fruit. Based on this observation, we hypothesized that high SPD/SPM fruit would enhance the expression of SA- biosynthesis genes leading to enhanced SA production. In plants, SA is synthesized either through phenylalanine ammonia-lyase (PAL) or isochorismate synthase (ICS) pathway. We identified ten putative genes for the SA biosynthesis pathways in tomato and determined the relative abundance of their transcript based on RNAseq transcriptomic analyses of mature green and turning stage tomato fruits from WT and two isogenic independent SAMDC-transgenic homozygous lines with higher levels of SPD/SPM in ripening fruits. We show that at MG stage the transgenic fruits exhibited higher steady state levels of transcripts of CS, PAL1-7, ICS, IPL/PRXR1, CS, and PAL 4-10, but lower levels for CM1, CM2, EPS1, and PBS3-2. At the turning stage of tomato fruit ripening the steady state levels of only CS and PBS3-2 were upregulated whereas the transcript levels of CM1, CM2, PaL1-4AAO, PBS3-1and EPS1 were down regulated. Taken together these results suggest that SPD/SPM play role in the SA biosynthesis and higher levels of various genes especially in in PAL pathway likely increased the SA levels in the transgenic fruits.

Study on the anticancer biological mechanism of Resveratrol

Trans 3, 4 ′, 5-3 hydroxy 2 styrene (resveratrol) is a kind of naturally occurring polyphenols phytoalexin. Resveratrol has significant anti-cancer activity, mainly exists in grapes, berries, and peanuts, has anti-aging, protect the heart, antioxidant, antiproliferation, promote apoptosis and immune regulation. Resveratrol has been widely concerned in the treatment of cancer and autoimmune diseases resveratrol. Resveratrol synthase (RS) is a key enzyme in the synthesis of resveratrol synthase. In this study, RS containing genes were placed under the control of fruit-specific promoter RJ39 to transform tomatoes by transgenic method. The extraction of fruits containing RS genes showed an obvious absorption peak on the HPLC chromatographic map, and it also had an obvious inhibitory effect on the growth of Hela cells.

A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh

Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which has the dominant Aft locus combined with the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the fruit skin. Here, we report that Aft encodes a functional anthocyanin activator named SlAN2-like, while atv encodes a nonfunctional version of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light, and the functional SlAN2-like can activate the expression of both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator in the activation of anthocyanin biosynthesis. We further showed that cultivated tomatoes contain nonfunctional alleles of SlAN2-like and therefore fail to produce anthocyanins. Consistently, expression of a functional SlAN2-like gene driven by the fruit-specific promoter in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high-level accumulation of anthocyanins in both the peel and flesh. Taken together, our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

The application of the \u2018Gene-deletor\u2019 technology in banana

Banana (Musa spp.) is an important tropical crop. Banana industry is under biotic and abiotic stresses such as Fusarium wilt, typhoon, cold stress. Genetic engineering offers a powerful strategy to create germplasm of banana with enhanced resistance. The safety of genetically modified organisms has become a bottleneck restricting the popularization and application of genetically modified technology. In this study, a candidate promoter, LEAFY (LFY) for expression and flower initiation in Arabidopsis, was cloned and constructed to ‘Gene-deletor’ vector. Histochemical β-glucuronidase (GUS) staining results showed that the ‘Gene-deletor’ vector driven by LFY promoter could lead to 88.5% excision efficiency from Arabidopsis seeds based on more than 200 T3 progeny examined per event. GUS staining was found to be partially negative in transgenic bananas, however, polymerase chain reaction could still detect the presence of large fragments of the vector. These results suggest that although LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas, its efficiency of eliminating exogenous gene laid a theoretical foundation for cloning banana fruit-specific promoters, that is, ‘non-transgenic’ GM bananas.

Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters

Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used. The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.

NTRC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.