Chiara Pagliarani

Anthocyanins Double the Shelf Life of Tomatoes by Delaying Overripening and Reducing Susceptibility to Gray Mold

Summary Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.

Recovery from water stress affects grape leaf petiole transcriptome

Fast and efficient recovery from water stress is a key determinant of plant adaptation to changing meteorological conditions modulating transpiration, i.e. air temperature and humidity. We analysed transcriptomic responses during rehydration after water stress in grapevine leaf petioles, where embolism formation and repair commonly take place, and where metabolic changes related to embolism recovery are expected to be particularly important. We compared gene expression of recovering plants with irrigated controls, upon high and low transpiration conditions, using cDNA microarrays. In parallel, we assessed the daily dynamics of water relations, embolism formation and repair, and leaf abscisic acid concentration. In recovering plants, the most affected gene categories were secondary metabolism, including genes linked to flavonoid biosynthesis; sugar metabolism and transport, and several aquaporin genes. The physiological dynamics of recovery were lower and the number of differentially expressed probes was much lower upon low transpiration than found in actively transpiring grapevines, suggesting the existence of a more intense metabolic reorganization upon high transpiration conditions and of a signal eliciting these responses. In plants recovering under high transpiration, abscisic acid concentrations significantly increased, and, in parallel, transcripts linked to abscisic acid metabolism and signalling (ABA-8′-hydroxylase, serine-threonine kinases, RD22 proteins) were upregulated; a trend that was not observed upon low transpiration. Our results show that recovery from water stress elicits complex transcriptomic responses in grapevine. The increase observed in abscisic acid cellular levels could represent a signal triggering the activation of responses to rehydration after stress.

The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange under Well-Watered Conditions But Not under Water Stress

We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape ‘Brachetto’, and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Insights on the Impact of Arbuscular Mycorrhizal Symbiosis on Tomato Tolerance to Water Stress

Arbuscular mycorrhizal symbiosis can improve tolerance to severe water stress conditions in tomato plants. Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS. To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved.

Gene expression in vessel-associated cells upon xylem embolism repair in Vitis vinifera L. petioles

In this work, the involvement of vessel-associated cells in embolism recovery was investigated by studying leaf petiole hydraulics and expression profiles of aquaporins and genes related to sugar metabolism. Two different stress treatments were imposed onto grapevines to induce xylem embolism: one involved a pressure collar applied to the stems, while the other consisted of water deprivation (drought). Embolism formation and repair were monitored during stress application and release (recovery). At the same time, stomatal conductance (gs), leaf water potential (Ψleaf) and leaf abscisic acid (ABA) concentration were measured. For each treatment, gene transcript levels were assessed on vessel-associated cells (isolated from leaf petioles by laser microdissection technique) and whole petioles. Both treatments induced severe xylem embolism formation and drops in gs and Ψleaf at a lesser degree and with faster recovery in the case of application of the pressure collar. Leaf ABA concentration only increased upon drought and subsequent recovery. Transcripts linked to sugar mobilisation (encoding a β-amylase and a glucose-6-P transporter) were over-expressed upon stress or recovery, both in vessel-associated cells and whole petioles. However, two aquaporin genes (VvPIP2;1 and VvPIP2;4N) were activated upon stress or recovery only in vessel-associated cells, suggesting a specific effect on embolism refilling. Furthermore, the latter gene was only activated upon drought and subsequent recovery, suggesting that either severe water stress or ABA is required for its regulation.

The functional role of xylem parenchyma cells and aquaporins during recovery from severe water stress

Xylem parenchyma cells [vessel associated cells (VACs)] constitute a significant fraction of the xylem in woody plants. These cells are often closely connected with xylem vessels or tracheids via simple pores (remnants of plasmodesmata fields). The close contact and biological activity of VACs during times of severe water stress and recovery from stress suggest that they are involved in the maintenance of xylem transport capacity and responsible for the restoration of vessel/tracheid functionality following embolism events. As recovery from embolism requires the transport of water across xylem parenchyma cell membranes, an understanding of stem-specific aquaporin expression patterns, localization and activity is a crucial part of any biological model dealing with embolism recovery processes in woody plants. In this review, we provide a short overview of xylem parenchyma cell biology with a special focus on aquaporins. In particular we address their distributions and activity during the development of drought stress, during the formation of embolism and the subsequent recovery from stress that may result in refilling. Complemented by the current biological model of parenchyma cell function during recovery from stress, this overview highlights recent breakthroughs on the unique ability of long-lived perennial plants to undergo cycles of embolism-recovery related to drought/rewetting or freeze/thaw events.

The Accumulation of miRNAs Differentially Modulated by Drought Stress Is Affected by Grafting in Grapevine

The abundance of drought-regulated miRNAs is affected by grafting in grapevine. Grapevine (Vitis vinifera) is routinely grafted, and rootstocks inducing drought tolerance represent a source for adapting vineyards to climate change in temperate areas. Our goal was to investigate drought stress effects on microRNA (miRNA) abundance in a drought-resistant grapevine rootstock, M4 (Vitis vinifera × Vitis berlandieri), compared with a commercial cultivar, Cabernet Sauvignon, using their autografts and reciprocal grafts. RNA extracted from roots and leaves of droughted and irrigated plants of different graft combinations was used to prepare cDNA libraries for small RNA sequencing and to analyze miRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). Measurements of leaf water potential, leaf gas exchange, and root hydraulic conductance attested that, under irrigation, M4 reduced water loss in comparison with cultivar Cabernet Sauvignon mostly through nonhydraulic, root-specific mechanisms. Under drought, stomatal conductance decreased at similar levels in the two genotypes. Small RNA sequencing allowed the identification of 70 conserved miRNAs and the prediction of 28 novel miRNAs. Different accumulation trends of miRNAs, observed upon drought and in different genotypes and organs, were confirmed by RT-qPCR. Corresponding target transcripts, predicted in silico and validated by RT-qPCR, often showed opposite expression profiles than the related miRNAs. Drought effects on miRNA abundance differed between the two genotypes. Furthermore, the concentration of drought-responsive miRNAs in each genotype was affected by reciprocal grafting, suggesting either the movement of signals inducing miRNA expression in the graft partner or, possibly, miRNA transport between scion and rootstock. These results open new perspectives in the selection of rootstocks for improving grapevine adaptation to drought.

Improved fluorescence-based evaluation of flavonoid in red and white winegrape cultivars

Background and Aims Modern viticulture requires robust, fast, non-destructive methods to assess berry composition. We tested a chlorophyll fluorescence screening method to estimate berry phenolic substances. Methods and Results We focused on anthocyanin and flavonol in red and white cultivars. The ANTH_RG index was dependent on the cultivar anthocyanin profile. In Nebbiolo, in which dihydroxylated anthocyanins prevail, ANTH_RG was 2.4 times higher than in Barbera, in which trihydroxylated anthocyanins prevail. Considering the profiles of the two cultivars at similar anthocyanin concentration and their relative in vitro absorbance, a bathochromic shift of 10 nm emerged, which can explain the different screening effect exerted by anthocyanin on chlorophyll fluorescence. As to flavanols, we propose the calibration of a new spectroscopic index, the FLAV_UV, in coloured and white berries, finding good correlation with flavonol concentration determined analytically (R2 higher than 0.7). Conclusions Spectroscopic indices can estimate the concentration of anthocyanin and flavonol in grape berries. Significance of the Study A calibration curve for Nebbiolo, which has a distinctive anthocyanin profile, and the calibration of a new index, the FLAV_UV, able to estimate flavonol concentration in both red and white cultivars, are described for the first time. These indices can effectively be applied for non-destructive assessment of grape flavonoid.

Exogenous strigolactone interacts with abscisic acid-mediated accumulation of anthocyanins in grapevine berries

The strigolactone analogue GR24 reduces ABA-induced anthocyanin accumulation in Vitis vinifera berries. GR24 treatment does not affect ABA biosynthesis while it activates ABA degradation and possibly ABA membrane transport.

miRVIT: a novel miRNA database and its application to uncover Vitis responses to Flavescence dorée infection

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it. Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling.