The plasma membrane

Grapevine under deficit irrigation: hints from physiological and molecular data.

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°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. Copyright © 2008 John Wiley & Sons, Ltd.

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

The ongoing climate change is characterized by increased temperatures and altered precipitation patterns In addition, there has been an increase in both the frequency and intensity of extreme climatic events such as drought Episodes of drought induce a series of interconnected effects, all of which have the potential to alter the carbon balance of forest ecosystems profoundly at different scales of plant organization and ecosystem functioning During recent years, considerable progress has been made in the understanding of how aboveground parts of trees respond to drought and how these responses affect carbon assimilation In contrast, processes of belowground parts are relatively underrepresented in research on climate change In this review, we describe current knowledge about responses of tree roots to drought Tree roots are capable of responding to drought through a variety of strategies that enable them to avoid and tolerate stress Responses include root biomass adjustments, anatomical alterations, and physiological acclimations The molecular mechanisms underlying these responses are characterized to some extent, and involve stress signaling and the induction of numerous genes, leading to the activation of tolerance pathways In addition, mycorrhizas seem to play important protective roles The current knowledge compiled in this review supports the view that tree roots are well equipped to withstand drought situations and maintain morphological and physiological functions as long as possible Further, the reviewed literature demonstrates the important role of tree roots in the functioning of forest ecosystems and highlights the need for more research in this emerging field

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How tree roots respond to drought

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

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.

/pdf/drought-induced-changes-in-development-and-function-of-1x7v890ccr.pdf

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

Small non-coding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are effectors of regulatory pathways underlying plant development, metabolism, and responses to biotic and abiotic stresses. To address the nature and functions of these regulators in grapevine (Vitis vinifera L.), we have produced a small RNA library from mixed-stage grape berries. Thirteen conserved miRNAs belonging to nine miRNA families, a non-conserved miRNA, and four putative non-conserved miRNAs were isolated, and their expression and targets are described. Experimentally validated targets of non-conserved miRNAs and putative miRNAs included three genes encoding NB-LRR proteins and a gene encoding a heavy metal ion transport/detoxification protein. Of the endogenous and pathogen-derived siRNAs that were also isolated, four endogenous siRNAs mapped to genes encoding RD22-like proteins and two to a gene encoding a cytokinin synthase. The siRNA id65 targeted the cytokinin synthase gene transcript with antisense complementarity, and was specifically expressed in mature berries, in which, by contrast, expression of the cytokinin synthase gene was strongly repressed. 5' RACE revealed that the transcript of this gene was processed in 21 nucleotide increments from the id65 cleavage site, and that further cleavage was mediated by secondary siRNAs in cis. These results indicate that grapevine miRNA- and siRNA-mediated regulatory circuits have evolved to comprise processes associated with defence and fruit ripening, and broaden the range of small RNA-mediated regulation, which was previously associated with auxin, ABA, gibberellins and jasmonate, to encompass cytokinin metabolism.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2009.03906.x

Cloning and characterization of small non-coding RNAs from grape

A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues.

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.

Profiling of hydroxycinnamoyl tartrates and acylated anthocyanins in the skin of 34 Vitis vinifera genotypes.

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 p

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-07-12-0178-R

Andrea Carra

Papers

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

Cloning and characterization of small non-coding RNAs from grape

A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues.

Profiling of hydroxycinnamoyl tartrates and acylated anthocyanins in the skin of 34 Vitis vinifera genotypes.

Expression analysis of aquaporins from desert truffle mycorrhizal symbiosis reveals a fine-tuned regulation under drought.