Ivana Gribaudo

A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants

INTRODUCTION RNA quality and integrity are critical for many studies in plant molecular biology. High-quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA. OBJECTIVE To develop an optimised cetyltrimethylammonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide-rich tissues of several plants. METHODOLOGY Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4 degrees C, the sample weight was decreased and the concentrations of PVP-40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines. RESULTS The rapid CTAB method gave high-quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time-consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species. CONCLUSION The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from grapevine and many woody species.

Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control

ABSTRACT A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10(-3) or 10(-4) extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

Molecular characterization of grapevine plants transformed with GFLV resistance genes: II

A collection of 127 putatively transgenic individuals of Vitis vinifera cv. Russalka was characterized by PCR and Southern hybridization. Six different constructs containing the neomycin phosphotransferase (nptII) marker gene and sequences of the Grapevine Fanleaf Virus Coat Protein (GFLV CP) gene including non-translatable and truncated forms were transferred via Agrobacterium-mediated transformation. Detection of transgenic sequences by PCR was positive in all lines. Southern blot analysis revealed that the number of inserted T-DNA copies ranged from 1 to 6. More than 46% of the tested transgenic lines contain one copy of the inserted T-DNA, qualifying them as interesting candidates for further breeding programs. Southern data of one line indicate the presence of an incomplete copy of the T-DNA, thus confirming previous PCR results. Since many putative transgenic lines shared identical hybridization patterns, they were clustered into 39 lines and considered as having originated from independent transformation events. The detection of the tetracycline (TET) resistance genes in 15% of the lines shows that an integration of plasmid backbone sequences beyond the T-DNA borders occurred. Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue did not show any accumulation of the GFLV CP in the 39 transgenic lines analyzed. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot were carried out; RT-PCR analyses showed that the GFLV CP mRNA was expressed at variable levels.

Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine.

Genetic transformation of fruit trees: current status and remaining challenges

Genetic transformation has emerged as a powerful tool for genetic improvement of fruit trees hindered by their reproductive biology and their high levels of heterozygosity. For years, genetic engineering of fruit trees has focussed principally on enhancing disease resistance (against viruses, fungi, and bacteria), although there are few examples of field cultivation and commercial application of these transgenic plants. In addition, over the years much work has been performed to enhance abiotic stress tolerance, to induce modifications of plant growth and habit, to produce marker-free transgenic plants and to improve fruit quality by modification of genes that are crucially important in the production of specific plant components. Recently, with the release of several genome sequences, studies of functional genomics are becoming increasingly important: by modification (overexpression or silencing) of genes involved in the production of specific plant components is possible to uncover regulatory mechanisms associated with the biosynthesis and catabolism of metabolites in plants. This review focuses on the main advances, in recent years, in genetic transformation of the most important species of fruit trees, devoting particular attention to functional genomics approaches and possible future challenges of genetic engineering for these species in the post-genomic era.

Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine ( Vitis spp.)

A novel method for initiating somatic embryogenesis in grapevine, based on immature whole flower culture, is presented. The embryogenic competence of flowers was compared to that of anthers and ovaries, the most widely used explant types, for five grapevine cultivars. Both the genotype and the explant source affected the differentiation of somatic embryos. The highest percentages of embryogenesis were obtained in ovary-derived calli from all cultivars tested with the exception of Brachetto a grappolo lungo. Whole flowers proved to be suitable material for initiating embryogenic cultures for most tested cultivars, and for 110 R, Chardonnay, and Grignolino they gave similar or better results than anthers. Collection of whole flowers from the inflorescence is easier and faster than excision of anthers and ovaries from the flower itself; it can be done without the use of a stereomicroscope and damage to the explant is unlikely. No morphological difference was noted among embryogenic cultures originated from ovaries, flowers, or anthers.

Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation

Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21–22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism.

Transcriptome analysis during berry development provides insights into co-regulated and altered gene expression between a seeded wine grape variety and its seedless somatic variant

BackgroundSeedless grapes are greatly appreciated for fresh and dry fruit consumption. Parthenocarpy and stenospermocarpy have been described as the main phenomena responsible for seedlessness in Vitis vinifera. However, the key genes underpinning molecular and cellular processes that play a significant role in seed development are not well characterized. To identify important regulators and mechanisms that may be altered in the seedless phenotype, we performed a comprehensive transcriptional analysis to compare the transcriptomes of a popular seeded wine cultivar (wild-type) and its seedless somatic variant (mutant) at three key developmental stages.ResultsThe transcriptomes revealed by Illumina mRNA-Seq technology had approximately 98% of grapevine annotated transcripts and about 80% of them were commonly expressed in the two lines. Differential gene expression analysis revealed a total of 1075 differentially expressed genes (DE) in the pairwise comparison of developmental stages, which included DE genes specific to the wild-type background, DE genes specific to the mutant background and DE genes commonly shared in both backgrounds. The analysis of differential expression patterns and functional category enrichment of wild-type and mutant DE genes highlighted significant coordination and enrichment of pollen and ovule developmental pathways. The expression of some selected DE genes was further confirmed by real-time RT-PCR analysis.ConclusionsThis study represents the most comprehensive attempt to characterize the genetic bases of seed formation in grapevine. With a high throughput method, we have shown that a seeded wine grape and its seedless somatic variant are similar in several biological processes. Nevertheless, we could identify an inventory of genes with altered expression in the mutant compared to the wild-type, which may be responsible for the seedless phenotype. The genes located within known genomic regions regulating seed content may be used for the development of molecular tools to assist table grape breeding. Therefore the data reported here have provided a rich genomic resource for practical use and functional characterization of the genes that potentially underpin seedlessness in grapevine.

Cultivar-specific gene modulation in Vitis vinifera : analysis of the promoters regulating the expression of WOX transcription factors

The family of Wuschel-related Homeobox (WOX) genes is a class of transcription factors involved in the early stages of embryogenesis and organ development in plants. Some of these genes have shown different transcription levels in embryogenic tissues and mature organs in two different cultivars of Vitis vinifera: ‘Chardonnay’ (CH) and ‘Cabernet Sauvignon’ (CS). Therefore, we investigated the genetic basis responsible for these differences by cloning and sequencing in both the cultivars the promoter regions (~2000 bp) proximal to the transcription start site of five VvWOX genes. We then introduced these promoters into Arabidopsis thaliana for expression pattern characterisation using the GUS reporter gene. In the transgenic Arabidopsis, two promoters isolated from CS (pVvWOX13C_CS and pVvWOX6_CS) induced increased expression compared to the sequence isolated in CH, confirming the data obtained in grapevine tissues. These results were corroborated by transient expression assays using the agroinfiltration approach in grapevine somatic embryos. Truncated versions of pVvWOX13C demonstrated that few nucleotide differences between the sequences isolated from CH and CS are pivotal for the transcriptional regulation of VvWOX13C. Analysis of promoters using heterologous and homologous systems appear to be effective for exploring gene modulation linked with intervarietal sequence variation in grapevine.

Field Assessment and Diagnostic Methods for Detection of Grapevine Viruses

The diagnosis of grapevine viral diseases and their associated viruses is usually performed by visual observation of symptoms or by using laboratory tests. The observation of symptoms can be carried out directly in the field, in case of manifest pathologies, or by means of biological indexing with susceptible indicators, in case of latent diseases. The laboratory assays include various serological and biomolecular tests, based on the detection of viral capsid and nucleic acid, respectively. Serological assays are largely used, also thanks to the availability of commercial kits. Biomolecular assays underwent a dramatic evolution in the last years, from dot-blot hybridisation to various PCR-based techniques and finally to microarrays, improving their sensitivity and reliability.