Silvia Morgutti

Analysis of grape berry cell wall proteome: A comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23 °C and 3 °C. I. Biochemical and physiological parameters

SummaryIn the last 30 d of potato (Solanum tuberosum L.) tuber growth metabolic activity decreased. Levels of glucose-6-P and sucrose in whole tuber tissues declined and in tuber slices there was a decrease in the uptake from the medium and in the incorporation into macromolecules of [U-14C]sucrose. During storage at 23°C only the uptake of [U-14C]sucrose increased concomitant with tuber sprouting, indicating a possible involvement of the transport mechanisms in dormancy breaking. At 3°C, levels of reducing sugars and sucrose increased in response to the low temperature and increased release of K+ and malondialdehyde levels indicated cell membrane damage. The cell membrane functionality was restored at sprouting. The sprouting potential of the tubers was evaluated using the sprouting ability of single-bud explants (“seedcores”) in response to water, GA3 or ABA dips. This sprouting potential of tubers changed with stage of tuber growth and storage duration and temperature, indicating that the tissue hormonal state changed strongly throughout tuber life, probably in relation with the “sink” to “source” transition.

Evaluation of borage extracts as potential biostimulant using a phenomic, agronomic, physiological and biochemical approach

Biostimulants are substances able to improve water and nutrient use efficiency and counteract stress factors by enhancing primary and secondary metabolism. Premise of the work was to exploit raw extracts from leaves (LE) or flowers (FE) of Borago officinalis L., to enhance yield and quality of Lactuca sativa ‘Longifolia,’ and to set up a protocol to assess their effects. To this aim, an integrated study on agronomic, physiological and biochemical aspects, including also a phenomic approach, has been adopted. Extracts were diluted to 1 or 10 mL L–1, sprayed onto lettuce plants at the middle of the growing cycle and 1 day before harvest. Control plants were treated with water. Non-destructive analyses were conducted to assess the effect of extracts on biomass with an innovative imaging technique, and on leaf photosynthetic efficiency (chlorophyll a fluorescence and leaf gas exchanges). At harvest, the levels of ethylene, photosynthetic pigments, nitrate, and primary (sucrose and total sugars) and secondary (total phenols and flavonoids) metabolites, including the activity and levels of phenylalanine ammonia lyase (PAL) were assessed. Moreover, a preliminary study of the effects during postharvest was performed. Borage extracts enhanced the primary metabolism by increasing leaf pigments and photosynthetic activity. Plant fresh weight increased upon treatments with 10 mL L–1 doses, as correctly estimated by multi-view angles images. Chlorophyll a fluorescence data showed that FEs were able to increase the number of active reaction centers per cross section; a similar trend was observed for the performance index. Ethylene was three-fold lower in FEs treatments. Nitrate and sugar levels did not change in response to the different treatments. Total flavonoids and phenols, as well as the total protein levels, the in vitro PAL specific activity, and the levels of PAL-like polypeptides were increased by all borage extracts, with particular regard to FEs. FEs also proved efficient in preventing degradation and inducing an increase in photosynthetic pigments during storage. In conclusion, borage extracts, with particular regard to the flower ones, appear to indeed exert biostimulant effects on lettuce; future work will be required to further investigate on their efficacy in different conditions and/or species.

Effects of ophiobolin A on potassium permeability, transmembrane electrical potential and proton extrusion in maize roots

Abstract In maize seedling roots ophiobolin (OPH) A at higher concentration (5 × 10−5 M) promoted a depolarization of the transmembrane electrical potential (PD) and increased the depolarizing effect of K+; at lower concentration (7.5 × 10−6 M) it promoted a hyperpolarization of PD completely counteracted by K+ and still detectable in the presence of the uncoupler (p-trifluoromethoxy)-carbonyl cyanide-phenyl-hydrazone (FCCP). OPH A also increased both the leakage and the efflux of K+; moreover, all the concentrations of OPH A promoted the alkalinization of the medium; in the absence of K+, the capacity to acidify the incubation medium was restored by adding K+ to the medium. These data suggest that the lack of proton extrusion is not linked to a direct action of OPH A on the proton pump, but is linked to an effect of OPH A on the plasma-membrane permeability to K+. The above data are also discussed in relation to the regulation of proton extrusion by a chemical coupling with K+ or by an electrical coupling with PD.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23°C and 3°C. II. Evaluation of protein patterns

SummaryThe changes in polypeptide profiles (2D-PAGE) occurring in the soluble and microsomal fractions of parenchymatic tissue of potato (Solanum tuberosum L.) tubers were studied during the last 30 d of maturation and during storage at 23°C and 3°C. The major changes were observed in the last period of tuber maturation, when several polypeptides disappeared and new ones appeared. At both 23°C and 3°C specific polypeptides disappeared in dormant tubers and new polypeptides appeared during storage. At 3°C specific changes in protein composition occurred, particularly in the microsomal fraction. The changes in polypeptide profiles are discussed in relation to the transition from “sink” to “source” of the tuber, the onset of dormancy and of sprouting ability and the activation of cold acclimation responses. The results are also discussed on the basis of the physiological and biochemical changes that occur in the parenchymatic tissue.

Changes in some physiological and biochemical parameters during two subcultures in kiwi (Actinidia deliciosa) callus

Abstract Kiwi ( Actinidia deliciosa ) calli were grown for two successive 45-day subcultures on agarised medium in order to evaluate the metabolic changes occurring during the growth of the callus. At different times (10, 35 and 45 days of each subculture), increase in fresh weight, oxygen uptake rate, levels of inorganic cations (K + , Ca 2+ , Mg 2+ and Na + ) and of some metabolites (sucrose, glucose-6-phosphate, malic acid, amino acids and adenosine phosphates) were measured. After an adaptive growth phase (0–6 days) and a phase of high growth (6–12 days), the callus growth rate decreased sharply and then remained essentially constant up to the 45th day. The parameter listed above changed with time following a similar pattern in the two analysed subcultures. In the first 10 days, most of them increased (in particular, oxygen uptake rate and the energetic status of the cells); the levels of Na + decreased. After the first 10 days, the levels of glucose-6-phosphate (glu-6-P) and of the adenylate pool and the availability of the high-energy phosphate bonds of ATP and ADP decreased and the Na + levels began to increase; in this period, the rate of oxygen uptake increased, but this corresponded to neither increased availability of the high-energy phosphate bonds of ATP and ADP nor increased growth rate. In the last period (35–45 days) the main metabolic parameters dropped, with a large increase in Na + levels. Transfer onto new medium restored, after the adaptive period, the maximum growth and the levels of the parameters listed above. The data show that during the subculture periods of Actinidia deliciosa calli important metabolic changes occur. They are probably linked to a different status of suffering (for reduction of nutrient availability, accumulation of catabolic products, and/or variations in oxygen and CO 2 diffusion) with the activation of metabolic mechanisms of adaptation and repair. These metabolic changes appear not severe enough to inhibit the growth of the calli. The results obtained with Actinidia deliciosa calli are discussed in relation to protocols of selection and micropropagation.

Effects of Ni2+ on proton extrusion and related transport processes and on the transmembrane electrical potential in maize roots

Abstract The presence of Ni 2+ in the incubation medium of maize roots induces an early and significant enhancement of the rate of proton extrusion, an inhibition of K + and leucine uptake and a slight stimulation of 3- O -methylglucose (3- O -MG) and phosphate uptake. The uptake of Ni 2+ is accompanied by a rapid depolarization of the trans-membrane electrical potential (PD). At the 1-mM concentration, both the uptake and the depolarizing effect of Ni 2+ are quantitatively similar to those of K + . These data suggest that the primary effect of Ni 2+ is the depolarization of PD, which would activate H + extrusion; the other transport phenomena would thus be differently influenced by the changes of the electrical and H + gradients. Some probable secondary consequences of Ni 2+ accumulation in the tissue are also discussed.

Light or high (30°C) temperature effects on l-[U-14C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-14C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-14C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-14C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-14C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.

A possible relationship between stalk necrosis and membrane transport in grapevine cultivars

Abstract The higher susceptibility to stalk necrosis of the grape (Vitis vinifera L.) cultivar ‘Bonarda’ (clusters with few berries) than the cultivar ‘Barbera’ (clusters with many berries) was correlated to higher K + Ca 2+ ratios in the tissue and to a different functioning of the transport mechanism. At the middle of the meristematic growth phase of the berries, segments excised from grapevine clusters of the more susceptible cultivars showed lower proton extrusion activity, lower K+ and Ca2+ leakage and similar uptake capacity both for K+ and for Ca2+. These results are discussed in regard to the onset of stalk necrosis in grapevine clusters.

Effect of nickel on proton extrusion in segments of maize seedling roots

Abstract Nickel affects the proton extrusion capacity in segments of maize seedling roots; as in the field condition, this effect is enhanced by phosphate and counteracted by Ca++ and Mg++. The effect of nickel on the proton‐extruding activity can explain either nickel toxicity and its stimulating effect on plant growth. Such hypothesis is discussed in this paper.