Expression profile analysis of early fruit development in iaaM-parthenocarpic tomato plants

<p>Abstract</p> <p>Background</p> <p>Fruit normally develops from the ovary after pollination and fertilization. However, the ovary can also generate seedless fruit without fertilization by parthenocarpy. Parthenocarpic fruit development has been obtained in tomato (<it>Solanum lycopersicum</it>) by genetic modification using auxin-synthesising gene(s) (<it>DefH9-iaaM</it>; <it>DefH9-RI-iaaM</it>) expressed specifically in the placenta and ovules.</p> <p>Findings</p> <p>We have performed a cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) analysis on pre-anthesis tomato flower buds (0.5 cm long) collected from <it>DefH9-iaaM </it>and <it>DefH9-RI-iaaM </it>parthenocarpic and wild-type plants, with the aim to identify genes involved in very early phases of tomato fruit development. We detected 212 transcripts differentially expressed in auxin-ipersynthesising pre-anthesis flower buds, 65 of them (31%) have unknown function. Several differentially expressed genes show homology to genes involved in protein trafficking and protein degradation via proteasome. These processes are crucial for auxin cellular transport and signaling, respectively.</p> <p>Conclusion</p> <p>The data presented might contribute to elucidate the molecular basis of the fruiting process and to develop new methods to confer parthenocarpy to species of agronomic interest. In a recently published work, we have demonstrated that one of the genes identified in this screening, corresponding to #109 cDNA clone, regulates auxin-dependent fruit initiation and its suppression causes parthenocarpic fruit development in tomato.</p

Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis.

The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence

The DefH9-iaaM auxin-sinthesizing gene increases plant fecundity and fruit production in strawberry and raspberry

Background: The DefH9-iaaM gene fusion which is expressed specifically in placenta/ovules andpromotes auxin-synthesis confers parthenocarpic fruit development to eggplant, tomato andtobacco. Transgenic DefH9-iaaM eggplants and tomatoes show increased fruit production duemainly to an improved fruit set. However, the weight of the fruits is also frequently increased.Results: DefH9-iaaM strawberry and raspberry plants grown under standard cultivation conditionsshow a significant increase in fruit number and size and fruit yield. In all three Rosaceae speciestested, Fragaria vesca, Fragaria x ananassa and Rubus idaeus, DefH9-iaaM plants have an increasednumber of flowers per inflorescence and an increased number of inflorescences per plant. Thisresults in an increased number of fruits per plant. Moreover, the weight and size of transgenic fruitswas also increased. The increase in fruit yield was approximately 180% in cultivated strawberry,140% in wild strawberry, and 100% in raspberry. The DefH9-iaaM gene is expressed in the flowerbuds of all three species. The total IAA (auxin) content of young flower buds of strawberry andraspberry expressing the DefH9-iaaM gene is increased in comparison to untransformed flowerbuds. The DefH9-iaaM gene promotes parthenocarpy in emasculated flowers of both strawberryand raspberry.Conclusions: The DefH9-iaaM gene is expressed and biologically active in Rosaceae. The DefH9-iaaM gene can be used, under cultivation conditions that allow pollination and fertilization, toincrease fruit productivity significantly in Rosaceae species. The finding that the DefH9-iaaM auxinsynthesizinggene increases the number of inflorescences per plant and the number of flowers perinflorescence indicates that auxin plays a role in plant fecundity in these three perennial Rosaceaespecies

Optimization of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes

Background: Genetic engineering of parthenocarpy confers to horticultural plants the ability toproduce fruits under environmental conditions that curtail fruit productivity and quality. TheDefH9-iaaM transgene, whose predicted action is to confer auxin synthesis specifically in theplacenta, ovules and derived tissues, has been shown to confer parthenocarpy to several plantspecies (tobacco, eggplant, tomato) and varieties.Results: UC82 tomato plants, a typical cultivar used by the processing industry, transgenic for theDefH9-iaaM gene produce parthenocarpic fruits that are malformed. UC82 plants transgenic forthe DefH9-RI-iaaM, a DefH9-iaaM derivative gene modified in its 5'ULR by replacing 53 nucleotidesimmediately upstream of the AUG initiation codon with an 87 nucleotides-long sequence derivedfrom the rolA intron sequence, produce parthenocarpic fruits of high quality. In an in vitro translationsystem, the iaaM mRNA, modified in its 5'ULR is translated 3\u20134 times less efficiently than theoriginal transcript. An optimal expressivity of parthenocarpy correlates with a reduced transgenemRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds.Consistent with the known function of the iaaM gene, flower buds transgenic for the DefH9-RI-iaaMgene contain ten times more IAA than control untransformed flower buds, but five times less thanDefH9-iaaM flower buds.Conclusions:: By using an auxin biosynthesis transgene downregulated at the post-transcriptionallevel, an optimal expressivity of parthenocarpy has been achieved in a genetic background notsuitable for the original transgene. Thus, the method allows the generation of a wider range ofexpressivity of the desired trait in transgenic plants

A review of genetic epidemiology of head and neck cancer related to polymorphisms in metabolic genes, cell cycle control and alcohol metabolism

The purpose of this report is to review the relationship between genetic polymorphisms involved in carcinogen metabolism, alcohol metabolism and cell-cycle control with the risk of head and neck cancer. The review was performed on available studies on genetic polymorphisms and head and neck cancer (HNC) published in PubMed up to September 2011. 246 primary articles and 7 meta-analyses were published. Among these, a statistically significant association was reported for glutathione S-transferases (GSTM1), glutathione S-transferases (GSTT1) and human microsomal epoxide hydrolase (EPHX1) genes. An increased risk for HNC was also associated reported for P53 codon 72 Pro/Pro, ALDH2 and three variants of the ADH gene: ADH1B (rs1229984), ADH7 (rs1573496) and ADH1C (rs698)

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs

Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation

BACKGROUND: Parthenocarpy, or fruit development in the absence of fertilization, has been genetically engineered in eggplant and in other horticultural species by using the DefH9-iaaM gene. The iaaM gene codes for tryptophan monoxygenase and confers auxin synthesis, while the DefH9 controlling regions drive expression of the gene specifically in the ovules and placenta. A previous greenhouse trial for winter production of genetically engineered (GM) parthenocarpic eggplants demonstrated a significant increase (an average of 33% increase) in fruit production concomitant with a reduction in cultivation costs. RESULTS: GM parthenocarpic eggplants have been evaluated in three field trials. Two greenhouse spring trials have shown that these plants outyielded the corresponding untransformed genotypes, while a summer trial has shown that improved fruit productivity in GM eggplants can also be achieved in open field cultivation. Since the fruits were always seedless, the quality of GM eggplant fruits was improved as well. RT-PCR analysis demonstrated that the DefH9-iaaM gene is expressed during late stages of fruit development. CONCLUSIONS: The DefH9-iaaM parthenocarpic gene is a biotechnological tool that enhances the agronomic value of all eggplant genotypes tested. The main advantages of DefH9-iaaM eggplants are: i) improved fruit productivity (at least 30-35%) under both greenhouse and open field cultivation; ii) production of good quality (marketable) fruits during different types of cultivation; iii) seedless fruit with improved quality. Such advantages have been achieved without the use of either male or female sterility genes

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.This work was funded by: FIRB RBAP10L8TY (MIUR), Fondazione Roma and PAINCAGE FP7 Collaborative Project number 603191 (RB,MD); Flagship Project InterOmics PB.05 and MIUR-PRIN-2012 (FC); Wellcome Trust Core Grant reference 092096 and Cancer Research UK Grant Reference C6946/A14492 (LP); CRUK-Cambridge Institute Core Grant reference C14303/A17197 (DB)

Assessment of antibody library diversity through next generation sequencing and technical error compensation

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.Funded by European Union Seventh Framework Program [grant No. 604102 A.C.] (Human Brain Project). https://www.humanbrainproject.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript