Transgenic Fruit Research Articles

Abstract PR11: Medulloblastoma-associated mutations in the DEAD box RNA helicase DDX3X impair protein translation

Abstract Whole genome sequencing technologies recently identified novel recurrent mutations in childhood medulloblastomas (MB), including missense mutations in the DEAD box RNA helicase DDX3X. In the present study, we used complementary genomic approaches to illuminate normal DDX3X function and the impact of cancer-associated mutations. We used CLIPseq to identify the RNA targets of endogenous DDX3X. This analysis revealed that DDX3X primarily binds mature mRNAs and, further, that the distribution of DDX3X binding sites in targets spans the full length of these mRNAs, with enrichment in the coding regions, suggesting a role for DDX3X in translation elongation. Consistent with this observation, we detected a fraction of wild type DDX3X in polysomes by sedimentation experiments. In contrast, MB-associated DDX3X mutants appear to associate poorly with polysomes, but induce spontaneous stress granule (SG) formation in cells, suggesting a role of mutant DDX3X in triggering translation silencing. In fact, the RNA binding capabilities of DDX3X are still preserved in the context of cancer-associated mutations further supporting a role of mutant DDX3X in repressing translation of specific RNAs by retaining them within SGs. Interestingly, gene ontology analyses find mRNAs encoding translation factors themselves to be highly enriched among DDX3X RNA targets, suggesting that perturbations of DDX3X functions could have far-reaching impact on cellular translation. Indeed, knocking down DDX3X with siRNA or overexpressing a mutant led to a significant impairment in global translation as measured by S35-methionine metabolic labeling. Furthermore, assessment of whole-genome protein translation by ribosomal profiling analyses in cells overexpressing cancer-associated mutant DDX3X confirmed a decreased translation rate of direct DDX3X mRNA targets in particular, but also numerous other mRNAs. Finally, we have generated an allelic series of transgenic fruit flies expressing wild type or mutant versions of human DDX3X or its Drosophila ortholog Belle, and have captured mutation-dependent phenotypes when these transgenes are expressed in specific tissues. Together, our data show that DDX3X regulates the translation of a key subset of mRNAs and thereby indirectly influences cellular translation globally, and that this function is perturbed by cancer-associated mutations. This abstract is also presented as poster C48. Citation Format: Yasmine A. Valentin-Vega, Matthew Parker, Michael Rusch, Demelza Smeeth, Nam Chul Kim, Jinghui Zhang, Richard J. Gilbertson, Joseph P. Taylor. Medulloblastoma-associated mutations in the DEAD box RNA helicase DDX3X impair protein translation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR11.

1-Aminocyclopropane-1-Carboxylate Oxidase 2 Reduction Effects on Physical and Physiological Responses of Eksotika Papaya

The Malaysian Eksotika papaya (Carica papaya L.) has poor keeping quality due to its fast ripening attribute, which leads to post-harvest losses. This study is aimed at extending the shelf life of this perishable Eksotika fruit using antisense technology. A total of 6,000 Eksotika somatic embryogenic calli was transformed with the antisense 1-aminocyclopropane-1-carboxylic acid oxidase 2 gene (ACO2) construct, and 46 PCR-positive putative transformants were obtained. Gene expression analysis using real-time PCR on the 46 regenerated putative transgenic lines revealed that 42 showed down-regulation of the ACO2 gene with two- to five-fold differences among the lines. Out of 22 independently selected transformed lines grown under net house conditions, 16 harbored a single copy of the transgene. Physical stature of the transgenic plants was not significantly different from that of the non-transformed seed-derived papaya plants. Physiological evaluations of the transgenic fruits showed a 15-day delay in ripening compared with 4 days of the non-transformed seed-derived papaya fruits. The total soluble solid (TSS) of the transgenic fruits was comparable to that of the non-transformed seed-derived fruits with similar 11-15°Brix, implying the transgenes did not affect the TSS content. The transgenic fruits remained firm for additional 4 to 8 days at room temperature (25 ± 2°C) after achieving the full maturity index (index 6), whereas the non-transformed seed-derived fruits lost their firmness after 2 days. The outcomes of this study revealed that reduction of ethylene was successful in the Eksotika papaya by manipulating the ACO2 gene using the antisense technique.

Genetically encoded system to track histone modification in vivo

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

Reduced EGFR level potentially mediates the Aβ42-induced neuronal loss in transgenic fruit fly and mouse

Reduced EGFR level potentially mediates the Aβ42-induced neuronal loss in transgenic fruit fly and mouse

NON-SMOKY GLYCOSYLTRANSFERASE1 Prevents the Release of Smoky Aroma from Tomato Fruit

Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed "smoky." Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. using a combinatorial omics approach, we identified the non-smoky glycosyltransferase1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits.

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

Characterization of a bZIP gene highly expressed during ripening of the peach fruit

A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

Cytosolic monoterpene biosynthesis is supported by plastid‐generated geranyl diphosphate substrate in transgenic tomato fruits

Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O.basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids.

Construction of Expression Vector with Fruit-Specific Promoter and Genetic Transformation of Strawberry

In order to transcription and expression an exogenous gene just in transgenic strawberry fruit, the gene encoding tomato fruit-specific E8 promoter was cloned. We constructed plant expression vector with E8 promoter replace the CaMV35S of pBI121 that used for transformation of strawberry. Recombination plasmid was identified by PCR, restriction enzymes digestion and sequencing analysis, and transfered into the agrobacterium EHA105 strain. At the same time, the agrobacterium EHA105, transformation and no transformation tissue of strawberry used as the experiment materials to study the expression of reporter genegusAby the histochemical staining method. The results show that agrobacterium which containsgusAgene and strawberry organization of transformation can be dyed blue, others were not stain. So in testing the transgenic plants by agroinfiltration transient expression system can reduce or avoid false positive appearance.

Resveratrol Biosynthesis Up-Regulates the Ascorbate/Glutathione Pathway in Transgenic Tomato Fruit

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. Polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their significant antioxidant activity, important both for plant physiology and human nutrition. With the aim to verify whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues, in the present work we took advantage of the availability of two different transgenic tomato lines in which a grape stilbene synthase gene was expressed under a constitutive or a tissue specific promoter, respectively. The induction of resveratrol synthesis in tomato affected the redox status of transformed tomato fruit. In particular, ascorbate and glutathione pool increased, significantly and proportionally to the amount of the resveratrol synthesised in transformed tissues. Noteworthy, the increase in ascorbate and glutathione pool paralleled with a significant increase in the activities of the main enzymes involved in redox homeostasis, i.e. catalase, ascorbate peroxidase as well the enzymes responsive for ascorbate recycling. Furthermore, lipoxygenase activity and levels of polyunsaturated fatty acids hydroperoxides, were reduced in fruit tissues from transgenic tomato lines. The synthesis of resveratrol, together with the global redox status had an impact on the total antioxidant activity of transgenic fruits. Again the total antioxidant capability increased proportionally to the amount of synthesised resveratrol with the most remarkable increase recorded from the lipophilic-resveratrol containing-fraction of transgenic fruits. Overall, our results pointed a higher nutritional value of resveratrol synthesizing tomato fruits.

Acetyl‐CoA carboxylases are checkpoints in adipocyte differentiation

Acetyl‐CoA carboxylases (ACC) 1 and 2 depend on biotin as a coenzyme and catalyze the carboxylation of acetyl‐CoA to malonyl‐CoA. Malonyl‐CoA produced by cytoplasmic ACC1 and mitochondrial ACC2 is a precursor in fatty acid (FA) synthesis and an inhibitor of mitochondrial FA oxidation, respectively. We hypothesized that ACCs are checkpoints in adipocyte differentiation, and used human mesenchymal stem cells (hMSC) and murine 3T3‐L1 preadipocytes to test our hypothesis. The expression of ACC2 increased by 1500% in differentiating hMSC compared with non‐differentiating hMSC, judged by qRT‐PCR and western blot. This increase preceded the increase of adipocyte marker genes FABP4 and PPARy. Treatment of hMSCs with grape leaf extract (GLE) inhibited the differentiation into adipocytes judged by the abundance of PPARy/FABP4 mRNA and staining of lipid droplets with Oil Red‐O. Likewise, treatment of 3T3‐L1 cells with the microbial ACC inhibitor soraphen A inhibited differentiation, judged by decreased lipid accumulation. Treatment of transgenic fruit flies, predisposed to storing excess body lipids, with GLE decreased body lipids by ~50%. In future studies, we will use mutagenesis to determine which of the two ACCs is the critical checkpoint in differentiation. We conclude that ACCs are checkpoints in adipocyte differentiation and that manipulation of ACC activity decreases body fat.Grant Funding Source: ARD Hatch, NIFA, and NIH

The influence of hairpin RNA against lawc, on the expression of overlapping genes of the lawc/Trf2 complex in D. melanogaster

We used a genetic system of the leg-arista-wing complex/TBP related factor 2 (lawc/Trf2) gene complex as a suitable model for in vivo study of the relationship between divergent overlapping genes. We established lines of transgenic fruit flies carrying constructs capable of expressing hairpin RNA directed at post-transcriptional suppression of the lawc mRNA by way of RNA-interference. The attempt to artificially suppress lawc-transcripts caused a rise in the level of expression of the lawc gene by several times. It is shown that change in the concentration of lawc-transcripts can affect the concentration of Trf2-transcripts. Possible mechanisms of regulation of expression of the overlapping genes studied are discussed.

Reduction of Oxalate Levels in Tomato Fruit and Consequent Metabolic Remodeling Following Overexpression of a Fungal Oxalate Decarboxylase

The plant metabolite oxalic acid is increasingly recognized as a food toxin with negative effects on human nutrition. Decarboxylative degradation of oxalic acid is catalyzed, in a substrate-specific reaction, by oxalate decarboxylase (OXDC), forming formic acid and carbon dioxide. Attempts to date to reduce oxalic acid levels and to understand the biological significance of OXDC in crop plants have met with little success. To investigate the role of OXDC and the metabolic consequences of oxalate down-regulation in a heterotrophic, oxalic acid-accumulating fruit, we generated transgenic tomato (Solanum lycopersicum) plants expressing an OXDC (FvOXDC) from the fungus Flammulina velutipes specifically in the fruit. These E8.2-OXDC fruit showed up to a 90% reduction in oxalate content, which correlated with concomitant increases in calcium, iron, and citrate. Expression of OXDC affected neither carbon dioxide assimilation rates nor resulted in any detectable morphological differences in the transgenic plants. Comparative proteomic analysis suggested that metabolic remodeling was associated with the decrease in oxalate content in transgenic fruit. Examination of the E8.2-OXDC fruit proteome revealed that OXDC-responsive proteins involved in metabolism and stress responses represented the most substantially up- and down-regulated categories, respectively, in the transgenic fruit, compared with those of wild-type plants. Collectively, our study provides insights into OXDC-regulated metabolic networks and may provide a widely applicable strategy for enhancing crop nutritional value.

Fruit-specific over-expression of human S-adenosylmethionine decarboxylase gene results in polyamine accumulation and affects diverse aspects of tomato fruit development and quality

Polyamines (PAs) have been implicated in fruit ripening where they antagonize the action of ethylene: a ripening inducing phytohormone. S-adenosylmethionine decarboxylase (SAMDC) is a key enzyme involved biosynthesis of higher PAs- spermidine and spermine. Here, we report the genetic modification of tomato fruit ripening and quality by over-expressing human-SAMDC driven by fruit-specific promoter (2A11). The PA analysis of ripening fruits from these transgenics showed elevated PA levels in comparison to wild-type (WT). The increased levels of higher PAs are correlated with the accumulation of heterologous SAMDC transcripts in such fruits. Transgenic fruits exhibited reduced levels of ethylene (~50 %) production, ~10 days delay in on-vine ripening and extended post-harvest storage of ~11 days as compared to the WT fruits. As a result, these fruits showed improvement in various ripening traits like enhanced lycopene, vitamin C and total soluble solid. In Lesam fruits, an up-regulated expression of SlySAMDC, SlyEXP1, SlyTBG4, SlyDXS 1 and SlyPSY 1 was observed, while ethylene biosynthesis genes were down-regulated. Here, we have demonstrated the important role of PAs in altering the molecular and biochemical processes underlying fruit ripening by interfering with the ethylene biosynthesis.

A STAY‐GREEN protein SlSGR1 regulates lycopene and β‐carotene accumulation by interacting directly with SlPSY1 during ripening processes in tomato

As a primary source of lycopene in the human diet, fleshy fruits synthesize this compound both de novo and via chlorophyll metabolism during ripening. SlSGR1 encodes a STAY-GREEN protein that plays a critical role in the regulation of chlorophyll degradation in tomato leaves and fruits. We report that SlSGR1 can regulate tomato (Solanum lycopersicum) lycopene accumulation through direct interaction with a key carotenoid synthetic enzyme SlPSY1, and can inhibit its activity. This interaction with SlSGR1 mediates lycopene accumulation during tomato fruit maturation. We confirmed this inhibitory activity in bacteria engineered to produce lycopene, where the introduction of SlSGR1 reduced dramatically lycopene biosynthesis. The repression of SlSGR1 in transgenic tomato fruits resulted in altered accumulation patterns of phytoene and lycopene, whilst simultaneously elevating SlPSY1 mRNA accumulation and plastid conversion at the early stages of fruit ripening, resulting in increased lycopene and β-carotene (four- and nine-fold, respectively) in red ripe fruits. SlSGR1 influences ethylene signal transduction via the altered expression of ethylene receptor genes and ethylene-induced genes. Fruit shelf-life is extended significantly in SlSGR1-repressed tomatoes. Our results indicate that SlSGR1 plays a pivotal regulatory role in color formation and fruit ripening regulation in tomato, and further suggest that SlSGR1 activity is mediated through direct interaction with PSY1.

Toxicity Assessment of Transgenic Papaya Ringspot Virus of 823-2210 Line Papaya Fruits

The transgenic papaya is a valuable strategy for creating plants resistant to papaya ringspot virus (PRSV) infection and increasing production. This study was further performed to evaluate the comparative toxicity effects of the newly developed transgenic line of the fruits of two backcross transgenic papaya lines (2210 and 823) and one hybrid line (823-2210) and compare to their parent non-transgenic (TN-2) counterparts. The stability analysis of coat protein (CP) of PRSV was investigated using the digestion stability assays in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and bile salts to detect the CP fragments. Results revealed that the CP fragments were rapidly hydrolyzed in SGF and were undetectable in organs and gastrointestinal contents in rats. For the genotoxicity, three in vitro assays were conducted and exhibited that non-transgenic and backcross transgenic papaya fruits were negative. Moreover, a repeated animal feeding study was conducted by feeding 2 g/kg of body weight (bw) of non-transgenic and backcross transgenic papaya fruits for 28 days in rats. There were no biological or toxicological significances between non-transgenic and backcross transgenic papaya fruits in rats. The results demonstrated that the backcross transgenic papaya fruit can be recognized as an equivalent substitution for traditional papaya in food safety.

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

The E8 promoter-HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8-MIR-HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30-630μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8-MIR-HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113-124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.

Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells

The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function.

Biotech/GM crops in horticulture: plum cv. HoneySweet resistant to Plum pox virus

Commercialisation of Biotech/GM (Biotech) crops started in 1995. Not only field crops, but also horticultural transgenic crops are under development and are beginning to be commercialised. Genetic engineering has the potential to revolutionise fruit tree breeding. The development of transgenic fruit cultivars is in progress. Over the past 20 years an international public sector research team has collaborated in the development of HoneySweet plum which is highly resistant to Plum pox virus (PPV) the most devastating disease of plums and other stone fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven years (2002–2012) in a regulated field trial in the CzechRepublic for the resistance to PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV), all of them being serious diseases of plum. Even under the high and permanent infection pressure produced through grafting, PPV has only been detected in HoneySweet trees in several leaves and fruits situated close to the point of inoculum grafting. The lack of infection spread in HoneySweet demonstrates its high level of PPV resistance. Co-infections of PPV with PDV and/or ACLSV had practically no influence on the quantity and quality of HoneySweet fruit which are large, sweet, and of a high eating quality. In many respects, they are superior to the fruits of the well-known cultivar Stanley. Many fruit growers and fruit tree nurseries in the CzechRepublic are supportive of the deregulation of HoneySweet plum to help improve the plum production and control the spread of PPV.

FaGAST2, a Strawberry Ripening-Related Gene, Acts Together with FaGAST1 to Determine Cell Size of the Fruit Receptacle

Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H(2)O(2) or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening.