Andrea Carra

Drought-induced changes in development and function of grapevine (Vitis spp.) organs and in their hydraulic and non-hydraulic interactions at the whole-plant level: a physiological and molecular update

This review deals with grapevine responses to water stress by examining perturbations to physiological and molecular processes at the root, shoot, leaf and berry levels. Long-distance signalling among organs is also considered. Isohydric or anisohydric Vitis genotypes are described in relation to their response to drought, which is linked to stomatal behaviour. Stomatal regulation of grapevine under abscisic acid and hydraulic control (the latter being linked to embolism formation and recovery in water pathways upstream the stomata) is reviewed and linked to impairments of photosynthetic assimilation. We define three stages of photosynthesis regulation in grapevines that are subjected to progressive water stress on the basis of the main causes of assimilation decline. Early and late contributions of aquaporins, which play a fundamental role in water stress control, are discussed. Metabolic mechanisms of dehydration tolerance are rewieved, and variation linked to differences in transcript abundance of genes involved in osmoregulation, photosynthesis, photorespiration, detoxification of free radicals and coping with photoinhibition. Results of these defence strategies accumulated in berries are reviewed, together with perturbations of their molecular pathways. Features observed in different organs show that grapevine fits well as a complex model plant for molecular and physiological studies on plant drought avoidance/tolerance.

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.

Profiling of Hydroxycinnamoyl Tartrates and Acylated Anthocyanins in the Skin of 34 Vitis vinifera Genotypes

The diversity of berry skin flavonoids in grape genotypes has been previously widely investigated with regard to major compounds (nonacylated anthocyanins and flavonols), but much less with regard to acylated anthocyanins and hydroxycinnamoyl tartrates (HCTs). In this study, the composition of the phenolic fraction of the berry skin (free and acylated anthocyanins, flavonols, and HCTs) was assessed on 34 grapevine genotypes grown in a collection vineyard in northwestern Italy. The phenolic fraction was profiled on berries collected in the same vineyard, at the same ripening level across two successive vintages. The anthocyanin, HCT, and flavonol profiles were specific of each genotype, and the first two were relatively little affected by the vintage. A wide diversity in the polyphenolic fraction was shown among cultivars. Besides expected discriminatory effects of free anthocyanins and flavonol profiles, principal component analyses allowed a good discrimination of cultivars on the basis of coumaroylated anthocyanins and of the HCT profile. Anthocyanins were mostly acylated by aromatic acids, and acylation was independent from the anthocyanin substrate. HCTs were present mostly as coumaroyl and caffeoyl derivatives, and no correlation was observed between the same acylation patterns of tartrate and of anthocyanins. The results of this study are discussed in the light of new hypotheses on still unknown biosynthetic steps of phenolic substances and of the potential use of these substances in discrimination and identification of different grape cultivars in wines.

Expression Analysis of Aquaporins from Desert Truffle Mycorrhizal Symbiosis Reveals a Fine-Tuned Regulation Under Drought

We have performed the isolation, functional characterization, and expression analysis of aquaporins in roots and leaves of Helianthemum almeriense, in order to evaluate their roles in tolerance to water deficit. Five cDNAs, named HaPIP1;1, HaPIP1;2, HaPIP2;1, HaPIP2;2, and HaTIP1;1, were isolated from H. almeriense. A phylogenetic analysis of deduced proteins confirmed that they belong to the water channel proteins family. The HaPIP1;1, HaPIP2;1, and HaTIP1;1 genes encode functional water channel proteins, as indicated by expression assays in Saccharomyces cerevisiae, showing divergent roles in the transport of water, CO2, and NH3. The expression patterns of the genes isolated from H. almeriense and of a previously described gene from Terfezia claveryi (TcAQP1) were analyzed in mycorrhizal and nonmycorrhizal plants cultivated under well-watered or drought-stress conditions. Some of the studied aquaporins were subjected to fine-tuned expression only under drought-stress conditions. A beneficial effect on plant physiological parameters was observed in mycorrhizal plants with respect to nonmycorrhizal ones. Moreover, stress induced a change in the mycorrhizal type formed, which was more intracellular under drought stress. The combination of a high intracellular colonization, together with the fine-tuned expression of aquaporins could result in a morphophysiological adaptation of this symbiosis to drought conditions.

Non Coding RNAs and Gene Silencing in Grape

Grapevine (Vitis vinifera L.) is a worldwide fruit crop of primary economic interest for berry consumption and winemaking. The molecular basis of grape berry ripening has been partially elucidated with the isolation and functional characterization of transcription factors which regulate sugar accumulation and secondary metabolism. After the recent publication of the complete sequence draft of two grapevine genotypes, a set of small non coding RNAs has been isolated by Sanger and high-throughput sequencing of small RNA libraries. These include conserved and grapevine-specific microRNAs as well as other small RNAs potentially involved in berry ripening. Small non coding RNAs are effectors of silencing pathways that underlie transgene silencing phenomena observed in several experiments of Agrobacterium-mediated transformation of grapevine. The knowledge of the silencing mechanisms in grapevine promises to facilitate the development of transient systems for gene functional studies.

Identification of an Alternative rRNA Post-transcriptional Maturation of 26S rRNA in the Kingdom Fungi

Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap. rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.

Small RNA Extraction and Expression Analysis by Northern Blot

Small non-coding RNAs are important regulatory elements able to cause endogenous gene silencing at both the transcriptional and post-transcriptional levels. Here we describe in detail the procedure of small RNAs extraction from grape tissues, which was developed as a modification of an RNA extraction method initially used for pine tree tissues. Polyacrylamide gel electrophoresis in denaturing conditions (obtained with high concentrations of urea) is used to efficiently separate small RNAs. In hybridization of small RNA northern blot, a single stranded DNA or LNA oligonucleotide in antisense orientation to the target small RNA is used as probe. The technique reported here was used for the isolation and expression analysis of small RNAs from grape berries at different stages of maturation and allowed the identification of several non-conserved miRNAs and siRNAs. Some major constraints of the procedure are discussed, including the comparably high amounts of tissues needed for the isolation of small RNAs, especially when berries are used as samples. Some recently developed methods to address these problems are briefly discussed, which are based on Real-Time (or quantitative) PCR.