Contrasting Responses of Two Grapevine Cultivars to Drought: The Role of Non-structural Carbohydrates in Xylem Hydraulic Recovery.

Xylem embolism is one of the possible outcomes of decreasing xylem pressure when plants face drought. Recent studies have proposed a role for non-structural carbohydrates (NSCs) in osmotic pressure generation, required for refilling embolized conduits. Potted cuttings of grapevine Grenache and Barbera, selected for their adaptation to different climatic conditions, were subjected to a drought stress followed by re-irrigation. Stem embolism rate and its recovery were monitored in vivo by X-ray micro-computed tomography (micro-CT). The same plants were further analyzed for xylem conduit dimension and NSC content. Both cultivars significantly decreased Ψpd in response to drought and recovered from xylem embolism after re-irrigation. However, although the mean vessel diameter was similar between the cultivars, Barbera was more prone to embolism. Surprisingly, vessel diameter was apparently reduced during recovery in this cultivar. Hydraulic recovery was linked to sugar content in both cultivars, showing a positive relationship between soluble NSCs and the degree of xylem embolism. However, when starch and sucrose concentrations were considered separately, the relationships showed cultivar-specific and contrasting trends. We showed that the two cultivars adopted different NSC-use strategies in response to drought, suggesting two possible scenarios driving conduit refilling. In Grenache, sucrose accumulation seems to be directly linked to embolism formation and possibly sustains refilling. In Barbera, maltose/maltodextrins could be involved in a conduit recovery strategy via the formation of cell-wall hydrogels, likely responsible for the reduction of conduit lumen detected by micro-CT.

Relationships between population traits, nonstructural carbohydrates, and elevation in alpine stands of Vaccinium myrtillus.

PREMISE: Despite great attention given to the relationship between plant growth and carbon balance in alpine tree species, little is known about shrubs at the treeline. We hypothesized that the pattern of main nonstructural carbohydrates (NSCs) across elevations depends on the interplay between phenotypic trait plasticity, plant-plant interaction, and elevation. METHODS: We studied the pattern of NSCs (i.e., glucose, fructose, sucrose, and starch) in alpine stands of Vaccinium myrtillus (above treeline) across an elevational gradient. In the same plots, we measured key growth traits (i.e., anatomical stem features) and shrub cover, evaluating putative relationships with NSCs. RESULTS: Glucose content was positively related with altitude, but negatively related with shrub cover. Sucrose decreased at high altitude and in older populations and increased with higher percentage of vascular tissue. Starch content increased at middle and high elevations and in stands with high shrub cover. Moreover, starch content was negatively related with the number of xylem rings and the percentage of phloem tissue, but positively correlated with the percentage of xylem tissue. CONCLUSIONS: We found that the increase in carbon reserves across elevations was uncoupled from plant growth, supporting the growth limitation hypothesis, which postulates NSCs accumulate at high elevation as a consequence of low temperature. Moreover, the response of NSC content to the environmental stress caused by elevation was buffered by phenotypic plasticity of plant traits, suggesting that, under climate warming conditions, shrub expansion due to enhanced plant growth would be pronounced in old but sparse stands.

Analysis of Non-Structural Carbohydrates and Xylem Anatomy of Leaf Petioles Offers New Insights in the Drought Response of Two Grapevine Cultivars.

In grapevine, the anatomy of xylem conduits and the non-structural carbohydrates (NSCs) content of the associated living parenchyma are expected to influence water transport under water limitation. In fact, both NSC and xylem features play a role in plant recovery from drought stress. We evaluated these traits in petioles of Cabernet Sauvignon (CS) and Syrah (SY) cultivars during water stress (WS) and recovery. In CS, the stress response was associated to NSC consumption, supporting the hypothesis that starch mobilization is related to an increased supply of maltose and sucrose, putatively involved in drought stress responses at the xylem level. In contrast, in SY, the WS-induced increase in the latter soluble NSCs was maintained even 2 days after re-watering, suggesting a different pattern of utilization of NSC resources. Interestingly, the anatomical analysis revealed that conduits are constitutively wider in SY in well-watered (WW) plants, and that water stress led to the production of narrower conduits only in this cultivar.

The Possible Role of Non-Structural Carbohydrates in the Regulation of Tree Hydraulics.

The xylem is a complex system that includes a network of dead conduits ensuring long-distance water transport in plants. Under ongoing climate changes, xylem embolism is a major and recurrent cause of drought-induced tree mortality. Non-structural carbohydrates (NSC) play key roles in plant responses to drought and frost stress, and several studies putatively suggest their involvement in the regulation of xylem water transport. However, a clear picture on the roles of NSCs in plant hydraulics has not been drawn to date. We summarize the current knowledge on the involvement of NSCs during embolism formation and subsequent hydraulic recovery. Under drought, sugars are generally accumulated in xylem parenchyma and in xylem sap. At drought-relief, xylem functionality is putatively restored in an osmotically driven process involving wood parenchyma, xylem sap and phloem compartments. By analyzing the published data on stem hydraulics and NSC contents under drought/frost stress and subsequent stress relief, we found that embolism build-up positively correlated to stem NSC depletion, and that the magnitude of post-stress hydraulic recovery positively correlated to consumption of soluble sugars. These findings suggest a close relationship between hydraulics and carbohydrate dynamics. We call for more experiments on hydraulic and NSC dynamics in controlled and field conditions.

Bioactive Polyphenols Modulate Enzymes Involved in Grapevine Pathogenesis and Chitinase Activity at Increasing Complexity Levels.

The reduction of synthetic chemistry use in modern viticulture relies on either the biological control of microorganisms or the induction of pathogenesis-related proteins. In the present study, the effects of hydro-alcoholic plant extracts (PEs) (i.e., by-products of Vitis vinifera L., leaves of Olea europaea L. and Ailanthus altissima (Mill.) Swingle) were tested on purified enzymes activity involved in plant-pathogen interactions. The polyphenolic composition was assayed and analyzed to characterize the extract profiles. In addition, suspension cell cultures of grapevine were treated with PEs to study their modulation of chitinase activity. Application of grape marc's PE enhanced chitinase activity at 4 g L-1. Additionally, foliar treatment of grape marc's PE at two doses (4 g L-1 and 800 g L-1) on grapevine cuttings induced a concentration-dependent stimulation of chitinase activity. The obtained results showed that the application of bioactive compounds based on PEs, rich in phenolic compounds, was effective both at in vitro and ex/in vivo level. The overall effects of PEs on plant-pathogen interaction were further discussed by applying a multi-criteria decision analysis, showing that grape marc was the most effective extract.

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

BACKGROUND: Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. RESULTS: Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. CONCLUSIONS: Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity.

In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

Involvement of mammalian bilitranslocase-like protein(s) in chlorophyll catabolism of Pisum sativum L. tissues.

Putative pea bilin and cyclic tetrapyrrole transporter proteins were identified by means of an antibody raised against a bilirubin-interacting aminoacidic sequence of mammalian bilitranslocase (TC No. 2.A.65.1.1). The immunochemical approach showed the presence of several proteins mostly in leaf microsomal, chloroplast and tonoplast vesicles. In these membrane fractions, electrogenic bromosulfalein transport activity was also monitored, being specifically inhibited by anti-bilitranslocase sequence antibody. Moreover, the inhibition of transport activity in pea leaf chloroplast vesicles, by both the synthetic cyclic tetrapyrrole chlorophyllin and the heme catabolite biliverdin, supports the involvement of some of these proteins in the transport of linear/cyclic tetrapyrroles during chlorophyll metabolism. Immunochemical localization in chloroplast sub-compartments revealed that these putative bilitranslocase-like transporters are restricted to the thylakoids only, suggesting their preferential implication in the uptake of cyclic tetrapyrrolic intermediates from the stroma during chlorophyll biosynthesis. Finally, the presence of a conserved bilin-binding sequence in different proteins (enzymes and transporters) from divergent species is discussed in an evolutionary context.

New perspective for the upscaling of plant functional response to flooding stress in salt marshes using remote sensing.

Understanding the response of salt marshes to flooding is crucial to foresee the fate of these fragile ecosystems, requiring an upscaling approach. In this study we related plant species and community response to multispectral indices aiming at parsing the power of remote sensing to detect the environmental stress due to flooding in lagoon salt marshes. We studied the response of Salicornia fruticosa (L.) L. and associated plant community along a flooding and soil texture gradient in nine lagoon salt marshes in northern Italy. We considered community (i.e., species richness, dry biomass, plant height, dry matter content) and individual traits (i.e., annual growth, pigments, and secondary metabolites) to analyze the effect of flooding depth and its interplay with soil properties. We also carried out a drone multispectral survey, to obtain remote sensing-derived vegetation indices for the upscaling of plant responses to flooding. Plant diversity, biomass and growth all declined as inundation depth increased. The increase of soil clay content exacerbated flooding stress shaping S. fruticosa growth and physiological responses. Multispectral indices were negatively related with flooding depth. We found key species traits rather than other community traits to better explain the variance of multispectral indices. In particular stem length and pigment content (i.e., betacyanin, carotenoids) were more effective than other community traits to predict the spectral indices in an upscaling perspective of salt marsh response to flooding. We proved multispectral indices to potentially capture plant growth and plant eco-physiological responses to flooding at the large scale. These results represent a first fundamental step to establish long term spatial monitoring of marsh acclimation to sea level rise with remote sensing. We further stressed the importance to focus on key species traits as mediators of the entire ecosystem changes, in an ecological upscaling perspective.

The mitochondrial permeability transition pore (PTP) - an example of multiple molecular exaptation?

The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins. In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role. Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.

Plant flavonoids--biosynthesis, transport and involvement in stress responses.

This paper aims at analysing the synthesis of flavonoids, their import and export in plant cell compartments, as well as their involvement in the response to stress, with particular reference to grapevine (Vitis vinifera L.). A multidrug and toxic compound extrusion (MATE) as well as ABC transporters have been demonstrated in the tonoplast of grape berry, where they perform a flavonoid transport. The involvement of a glutathione S-transferase (GST) gene has also been inferred. Recently, a putative flavonoid carrier, similar to mammalian bilitranslocase (BTL), has been identified in both grape berry skin and pulp. In skin the pattern of BTL expression increases from véraison to harvest, while in the pulp its expression reaches the maximum at the early ripening stage. Moreover, the presence of BTL in vascular bundles suggests its participation in long distance transport of flavonoids. In addition, the presence of a vesicular trafficking in plants responsible for flavonoid transport is discussed. Finally, the involvement of flavonoids in the response to stress is described.

Antifungal activity of chili pepper extract with potential for the control of some major pathogens in grapevine.

BACKGROUND: In recent years, biofungicides have drawn increasing interest in vineyards for a more sustainable integrated and copper-limited pest management. Among alternatives, botanicals could represent valuable tools, being rich sources of biologically active compounds. Conversely to the well-known antioxidant and biological properties in relation to health benefits, investigation on bioactivity of hot pungent Capsicum sp. products against fungal phytopathogens in vineyards is still scarce. Therefore, the present study aimed at exploring the biologically active compounds profile of a chili pepper (Capsicum chinense Jacq.) pod extract and its antimicrobial properties against some of the major fungal and Oomycetes pathogens of grapevine, including Botrytis cinerea Pers., Guignardia bidwellii (Ellis) Viala & Ravaz and Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. RESULTS: The ethyl acetate-extracted oleoresin from the most pungent varieties was rich in capsaicinoids and polyphenols (371.09 and 268.5 µg mg-1 dry weight, respectively). Capsaicin and dihydrocapsaicin, hydroxycinnamic and hydroxybenzoic acids and quercetin derivatives were the most abundant, while carotenoids represented only a minor fraction. The oleoresin was efficient to inhibit all three pathogenic fungi and ED50 values were determined, evidencing that G. bidwellii was the more sensitive (0.233 ± 0.034 mg mL-1 ). CONCLUSION: The results suggested a potentiality of chili pepper extract for the control of some important grapevine pathogens, their possible application being helpful for the recommended limitation in extensive use of copper in vineyard. The complex mixture of high amounts of capsaicinoids, associated to specific phenolic acids and other minor bioactive components might contribute to the observed antimicrobial action of chili pepper extract. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterisation and functionalisation of chitosan nanoparticles as carriers for double-stranded RNA (dsRNA) molecules towards sustainable crop protection.

The need to minimise the impact of phytosanitary treatments for disease control boosted researchers to implement techniques with less environmental impact. The development of technologies using molecular mechanisms based on the modulation of metabolism by short dsRNA sequences appears promising. The intrinsic fragility of polynucleotides and the high cost of these techniques can be circumvented by nanocarriers that protect the bioactive molecule enabling high efficiency delivery to the leaf surface and extending its half-life. In this work, a specific protocol was developed aiming to assess the best methodological conditions for the synthesis of low-size chitosan nanoparticles (NPs) to be loaded with nucleotides. In particular, NPs have been functionalised with partially purified Green Fluorescent Protein dsRNAs (GFP dsRNA) and their size, surface charge and nucleotide retention capacity were analysed. Final NPs were also stained with FITC and sprayed on Nicotiana benthamiana leaves to assess, by confocal microscopy, both a distribution protocol and the fate of NPs up to 6 days after application. Finally, to confirm the ability of NPs to increase the efficacy of dsRNA interference, specific tests were performed: by means of GFP dsRNA-functionalised NPs, the nucleotide permanence during time was assessed both in vitro on detached wild-type N. benthamiana leaves and in planta; lastly, the inhibition of Botrytis cinerea on single leaves was also evaluated, using a specific fungal sequence (Bc dsRNA) as the NPs' functionalising agent. The encouraging results obtained are promising in the perspective of long-lasting application of innovative treatments based on gene silencing.

Bioavailability of flavonoids: a review of their membrane transport and the function of bilitranslocase in animal and plant organisms.

Fruits and vegetables are rich in flavonoids, and ample epidemiological data show that diets rich in fruits and vegetables confer protection against cardiovascular, neurodegenerative and inflammatory diseases, and cancer. However, flavonoid bioavailability is reportedly very low in mammals and the molecular mechanisms of their action are still poorly known. This review focuses on membrane transport of flavonoids, a critical determinant of their bioavailability. Cellular influx and efflux transporters are reviewed for their involvement in the absorption of flavonoids from the gastro-intestinal tract and their subsequent tissue distribution. A focus on the mammalian bilirubin transporter bilitranslocase (TCDB 2.A.65.1.1) provides further insight into flavonoid bioavailability and its relationship with plasma bilirubin (an endogenous antioxidant). The general function of bilitranslocase as a flavonoid membrane transporter is further demonstrated by the occurrence of a plant homologue in organs (petals, berries) where flavonoid biosynthesis is most active. Bilitranslocase appears associated with sub-cellular membrane compartments and operates as a flavonoid membrane transporter.

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of Abies alba.

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial K (ATP)(+) channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the K (ATP)(+) channel activity could induce an entry of K(+) into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.

Identification and localization of the bilitranslocase homologue in white grape berries (Vitis vinifera L.) during ripening.

A homologue of the mammalian bilirubin transporter bilitranslocase (BTL) (TCDB 2.A.65.1.1), able to perform an apparent secondary active transport of flavonoids, has previously been found in carnation petals and red grape berries. In the present work, a BTL homologue was also shown in white berries from Vitis vinifera L. cv. Tocai/Friulano, using anti-sequence antibodies specific for rat liver BTL. This transporter, similarly to what found in red grape, was localized in the first layers of the epidermal tissue and in the vascular bundle cells of the mesocarp. In addition, a strong immunochemical reaction was detected in the placental tissue and particularly in peripheral integuments of the seed. The protein was expressed during the last maturation stages in both skin and pulp tissues and exhibited an apparent molecular mass of c. 31 kDa. Furthermore, the transport activity of such a carrier, measured as bromosulphophthalein (BSP) uptake, was detected in berry pulp microsomes, where it was inhibited by specific anti-BTL antibodies. The BTL homologue activity exhibited higher values, for both K(m) and V(max), than those found in the red cultivar. Moreover, two non-pigmented flavonoids, such as quercetin (a flavonol) and eriodictyol (a flavanone), inhibited the uptake of BSP in an uncompetitive manner. Such results strengthen the hypothesis that this BTL homologue acts as a carrier involved also in the membrane transport of colourless flavonoids and demonstrate the presence of such a carrier in different organs and tissues.

Caspase-3-like activity and proteasome degradation in grapevine suspension cell cultures undergoing silver-induced programmed cell death.

Toxic metal contamination is one of the major environmental concerns of the recent decade, due to the large application of metals in industrial, healthcare and commercial products, even in the form of nanostructures and nanomaterials. Nevertheless, the effects of silver (Ag+) on plants have not yet thoroughly elucidated. Therefore, suspension cell cultures of grapevine were used as a model for investigating silver toxicity. To do this, oxidative stress and programmed cell death (PCD), evaluated as reactive oxygen species production, caspase-3-like activity and ubiquitin-proteasome system, were investigated. As a result, the highest concentration (10 µM) of Ag+ caused a rapid (within 24 h) induction of PCD (approx. 80%), accompanied by generation of reactive oxygen species and activation of caspase-3-like activity. In the presence of specific inhibitor of this enzyme, a partial recovery of cell viability and a strong inhibition of caspase-3-like activity was observed. In addition, silver-induced PCD was accompanied either by increase of poly-ubiquitin conjugated proteins and degradation of subunit PBA1 of the proteasome 20S core, similarly to what found for metal-induced neurotoxicity in animals. The present study shows that silver could induce PCD in grapevine suspension cell cultures, mediated by caspase-3-like activity and oxidative stress. These effects were associated to accumulation of poly-ubiquitin conjugated proteins, suggesting the impairment of ubiquitin-proteasome complex, confirmed by the decrease of the PBA1 subunit. These findings indicate that animal and plant cells could share a common pathway in response to toxic metal, which involves PCD and disassembling of proteasome complex.

Properties of the Permeability Transition of Pea Stem Mitochondria.

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by Mg-ADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel.

Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: The possible link between carbon starvation and hydraulic failure.

Drought-induced tree decline is a complex event, and recent hypotheses suggest that hydraulic failure and carbon starvation are co-responsible for this process. We tested the possible role of non-structural carbohydrates (NSC) content on post-drought hydraulic recovery, to verify the hypothesis that embolism reversal represents a mechanistic link between carbon starvation and stem hydraulics. Measurements were performed in laurel plants subjected to similar water stress levels either over short or long term, to induce comparable embolism levels. Plants subjected to mild and prolonged water shortage (S) showed reduced growth, adjustment of turgor loss point driven by changes in both osmotic potential at full turgor and bulk modulus of elasticity, a lower content of soluble NSC and a higher content of starch with respect to control (C) plants. Moreover, S plants showed a lower ability to recover from xylem embolism than C plants, even after irrigation. Our data suggest that plant carbon status might indirectly influence plant performance during and after drought via effects on xylem hydraulic functioning, supporting the view of a possible mechanistic link between the two processes.

Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase.

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (K(M) = 5.3 microm), which is competitively inhibited by cyanidine 3-glucoside (Ki = 51.6 microm) and mainly noncompetitively by cyanidin (Ki = 88.3 microm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd = 1.7 microm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabeled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.