Search

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate–sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on

Grapevine rootstocks play a pivotal role in plant responses to water deficiency (WD), therefore, the selection of new genotypes is a promising strategy for future agricultural managements aimed to cope with climate changes. Recent studies reinforced the central role of the root system in modulating WD responses, as it not only controls water uptake and transport to the leaves, but it also participates in stress perception and stress signalling to the shoot. The present work evaluated the performance of the 101.14 and M4 rootstocks in graft combination with the cultivar Cabernet Sauvignon (Cab) by assessing some of the canonical molecular, biochemical and physiological responses induced by WD. The autograft Cab/Cab was also included in the experimental design as a control. Under WD, Cab/M4 showed a greater capacity to sustain CO2 assimilation rate (An) and stomatal conductance (gs), while limiting the decrease of leaf potential (Ψleaf) compared with the other graft combinations. The enhanced adaptability of Cab/M4 to WD was also supported by the higher uptake of water from the soil, estimated by measuring the daily water lost of plants, and by the reduced effect of the drought treatment on the total root biomass. Quantification of ABA in both root and leaf organs revealed a reduced accumulation in Cab/M4 plants, thus confirming the lower sensitivity of the Cab/M4 combination to water deficit. At the molecular level, the expression of selected stress-responsive ABA-related genes was investigated, including genes involved in ABA biosynthesis (VviNCED3), ABA signalling (VviPP2C9, VviPP2C4,VviSnRk2.6), regulation of gene expression (VviABF2) and stomatal opening (VviSIRK, VviMYB60). Results indicated a tight correlation between the level of gene expression and of ABA accumulation in roots and leaves, suggesting that ABA synthesis and signalling were attenuated in Cab/M4 as compared with Cab/101.14 and Cab/Cab. As a whole, our data demonstrated the capacity of M4 to satisfy the water demand of the scion under limited water availability, as revealed by delayed stomatal closure and higher photosynthetic activity. Importantly, these physiological adaptive traits related to attenuated ABA-mediated responses in roots and leaves.

Basil (Ocimum basilicum L.) comprises green and purple cultivars, worldwide cultivated and appreciated for high contents of rosmarinic acid and anthocyanins, respectively. Although nitrogen (N) fertilization is needed for high yields, in basil it could have detrimental effects on the accumulation of phenolic compounds. In this study, plants of the cultivars &lsquo, Italiano Classico&rsquo, (green) and &lsquo, Red Rubin&rsquo, (purple) were grown in hydroponics and subjected to different nutritional treatments, consisting in N starvation, and nitrate (NO3&minus, ) or ammonium (NH4+) nutrition. Plant growth and nutritional status, estimated by the contents of NO3&minus, NH4+, and amino acids in roots and leaves, were evaluated and put in relation with quality traits of basil leaves, such as chlorophyll content, antioxidant capacity, total phenols, the activity of phenylalanine ammonia-lyase, and the concentrations of individual (poly)phenolic acids and flavonoids. This study reveals that N starvation, as well as the availability of the two inorganic N forms, differently affect the phenolic composition in the two cultivars. Compared to plants grown in NO3&minus, availability, in NH4+ availability, green basil showed a higher content of (poly)phenolic acids, while in purple basil, an increase in the contents of anthocyanins was detected. Overall, the study suggests that the management of NH4+ supply could contribute to enhance crop quality in hydroponics, and provides new knowledge about the relationship between N nutrition and phenolic metabolism in basil.

A prolamin paralog generated upon maize whole-genome duplication has changed its polymerization and solubility properties, allowing a new function in the assembly of maize protein bodies., In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Keywords: NaCl, Vitis, LC-ESI-MS/MS, carbon and energy metabolism, oxidative stress

Oxidations in grape berries are gaining major interest as they affect grape characteristics and quality. Considering berries, Reactive Oxygen Species are involved in the responses to both ripening process and stresses, including photooxidative sunburn. Redox metabolism involves a multitude of chemical and enzymatic reactions. In this study, four white grape cultivars were examined for natural ripening and photooxidative sunburn effects (obtained in artificial conditions) on berry pigmentation, chemical composition and enzymatic activity. The measured parameters included reflectance spectra, pigmentation (including berry browning), content of photosynthetic pigments, organic acid profiles, antioxidant activity, concentrations of antioxidants (total phenolics, ascorbic acid and reduced glutathione), enzymatic activities (guaiacol peroxidases, ascorbate peroxidase and catalase). The effects of the treatment (natural ripening and artificial photooxidative sunburn) on each considered parameter are described in the paper. Photooxidative sunburn strongly affected the contents of antioxidants and chlorophylls, increased the browning index and modulated the enzymatic activities investigated. Samples clearly clustered depending on the oxidation status. Furthermore, the PCA highlighted the similarities and differences in the responses to oxidative stress during ripening and photooxidative sunburn. PCA produced five functions with eigenvalues higher than 1, representing 87.03% of the total variability. In particular, the scores of the function 1 discriminated the samples based on the oxidation status, while the function 2 separated the samples based on the sampling date, representing the physiological responses characteristic of ripening. Our work sheds light on this topic, and will allow a more conscious vineyard management, thus supporting the agricultural adaptation to climate changes.

Basil (Ocimum basilicum L.) is a culinary, medicinal, and ornamental plant appreciated for its antioxidant properties, mainly attributed to high content of rosmarinic acid. This species also includes purple varieties, characterized by the accumulation of anthocyanins in leaves and flowers. In this work, we compared the main morphological characteristics, the antioxidant capacity and the chemical composition in leaves, flowers, and corollas of green (&lsquo, Italiano Classico&rsquo, ) and purple (&lsquo, Red Rubin&rsquo, and &lsquo, Dark Opal&rsquo, ) basil varieties. The LC-ESI-MS/MS analysis of individual compounds allowed quantifying 17 (poly)phenolic acids and 18 flavonoids, differently accumulated in leaves and flowers of the three varieties. The study revealed that in addition to rosmarinic acid, basil contains several members of the salvianolic acid family, only scarcely descripted in this species, as well as, especially in flowers, simple phenolic acids, such as 4-hydroxybenzoic acid and salvianic acid A. Moreover, the study revealed that purple leaves mainly contain highly acylated anthocyanins, while purple flowers accumulate anthocyanins with low degree of decoration. Overall, this study provides new biochemical information about the presence of not yet characterized bioactive compounds in basil that could contribute to boosting the use of this crop and to gaining new knowledge about the roles of these compounds in plant physiology.

The somaclonal variant HS, from sweet cherry (Prunus avium L.) 'Hedelfinger' (H), was previously selected for reduced tree vegetative vigor and lesser canopy density. In this work, we compared H and HS fruits at early unripe (green) and full ripe (dark red) stages by biochemical and proteomic approaches. The main biochemical parameters showed that fruit quality was not affected by somaclonal variation. The proteomic analysis identified 39 proteins differentially accumulated between H and HS fruits at the two ripening stages, embracing enzymes involved in several pathways, such as carbon metabolism, cell wall modification, stress response, and secondary metabolism. The evaluation of fruit phenolic composition by mass spectrometry showed that HS sweet cherries have higher levels of procyanidin, flavonol, and anthocyanin compounds. This work provides the first proteomic characterization of fruit ripening in sweet cherry, revealing new positive traits of the HS somaclonal variant.

The Petunia hybrida ANTHOCYANIN1 (AN1) gene encodes a transcription factor that regulates both the expression of genes involved in anthocyanin synthesis and the acidification of the vacuolar lumen in corolla epidermal cells. In this work, the comparison between the red flowers of the R27 line with the white flowers of the isogenic an1 mutant line W225 showed that the AN1 gene has further pleiotropic effects on flavonoid biosynthesis as well as on distant physiological traits. The proteomic profiling showed that the an1 mutation was associated to changes in accumulation of several proteins, affecting both anthocyanin synthesis and primary metabolism. The flavonoid composition study confirmed that the an1 mutation provoked a broad attenuation of the entire flavonoid pathway, probably by indirect biochemical events. Moreover, proteomic changes and variation of biochemical parameters revealed that the an1 mutation induced a delay in the onset of flower senescence in W225, as supported by the enhanced longevity of the W225 flowers in planta and the loss of sensitivity of cut flowers to sugar. This study suggests that AN1 is possibly involved in the perception and/or transduction of ethylene signal during flower senescence.

The pear (Pyrus communis L.)/quince (Cydonia oblonga Mill.) graft incompatibility is associated to biochemical and morphological alterations impairing the establishment of a functional union between the two partners, in which the callus at graft interface plays pivotal roles. This work was aimed to study the early effects evoked by factor(s) released by quince cells on the metabolism of pear callus. Using two-dimensional difference gel electrophoresis, the study analysed the changes between the proteome of pear callus co-cultured on suspensions of pear microcalli (homologous combination) or of incompatible quince microcalli (heterologous combination) by an in vitro system. This approach revealed changes in the abundance of proteins involved in several metabolic pathways and cell processes. In pear callus in heterologous combination, the less abundance of α-amylase and enolase and the increase of enzymes involved in amino acid and pyrimidine recycling indicate an alteration of the carbohydrate usage and a concomitant upsurge of salvage pathways. Moreover, the higher accumulation of a polyphenol oxidase suggests a participation of the flavonoid metabolism in the protection from the stress evoked by the proximity of quince cells. At the same time, this stress condition resulted associated with a reduction of proteins involved in the cell translation machinery and vesicle traffic, supporting that in heterologous combination the pear callus undergoes a physiological status less favourable for proliferation and differentiation. Overall, the work provides new information about the pear/quince incompatibility, highlighting as it involves a network of different metabolic responses.

Grape skin is characterized by a very intense metabolism during ripening involving many biosynthetic activities such as those relating to secondary compounds like the anthocyanins. In order to improve our knowledge about the physiology of this tissue, we performed a comparison among the proteomes of the ripe skin of ?Riesling Italico?, ?Pinot Gris?, ?Pinot Noir? and ?Croatina?, genotypes that accumulate anthocyanins to different extents. In order to examine quantitative changes in proteomic maps, data underwent a two-way ANOVA test using cultivars and years as factors. The 297 significant variations underwent two different clustering procedures. By means of the two-way hierarchical clustering, it was possible to observe that the gels of the four cultivars were arranged in an order that clearly reflected anthocyanin levels and emphasized the similarities between the two Pinots, from one side, and the divergence of the proteome of ?Croatina?, from the other. It was then possible to group the 297 spots, according to their trends, in six clusters through the k-means procedure. One of the clusters included 85 spots showing a variation profile that could be associated with anthocyanin levels. Some were identified through LC-ESI-MS/MS and their role was discussed.

Although molecular markers such as SSRs are extensively used for the identification of different vines, they often fail to discriminate the clones of a given cultivar. For these reasons, many attempts are made to find devices able to achieve this purpose. In this view, we evaluated the potential of proteomics in this field comparing the skin 2-DE profiles of berries belonging to three clones of the Italian cultivar ?Aglianico? (Taurasi, Taburno and Vulture) grown in the same field and considered in different moments to reduce the impact of environmental and seasonal variability. After a preliminary and completely automated gel alignment, we tested some multivariate statistical techniques to distinguish the three clones and to isolate the variables (i.e., the spots on the gels) responsible for the clonal discrimination. Although further analyses will be necessary to refine such an approach, the results are encouraging in order to identify markers useful in discriminating clones.

Background and Aims: In the light of possible climate change, a crucial aspect for future Mediterranean Europe viticulture is the selection of new rootstocks exhibiting adaptability to drought and salt stress. In this context and considering recent literature, it is fundamental to increase our knowledge of the biochemical and physiological events that characterise stress responses in grapevine roots. In this study, leaf and root responses induced by water stress (WS) and sodium chloride (NaCl) exposure in a new selected genotype, named M4 (Vitis vinifera × V. berlandieri) × V. berlandieri cv. Resseguier n. 1), were compared with those of the commercial rootstock 101.14. Methods and Results: The effect of progressive WS and NaCl exposure was studied under controlled environmental conditions on ungrafted plants grown in pots. Shoot growth, plant water status and leaf photosynthetic parameters were measured. The concentration of sugars, amino acids and total proteins, as well as the concentration of the more abundant ions, was determined in both leaf and root organs. The M4 genotype showed greater capacity to maintain photosynthetic activity, to accumulate osmotic compounds as well as to counteract Na and Cl accumulation. Conclusion: The M4 genotype exhibited a greater capacity to tolerate both WS and exposure to an increasing concentration of Na and Cl, maintaining photosynthetic activity also under severe stress conditions. The root system appeared to play a central role in sustaining biochemical and physiological responses experienced under these adverse conditions. Significance of the Study: This study showed that the tolerance to abiotic stress conditions, such as WS and NaCl exposure, depends on root integrity/functionality, confirming that these aspects must be considered in further selection programs.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

The Golgi-located phosphate exporter PHT4;6 has been described as involved in salt tolerance but further analysis on the physiological impact of PHT4;6 remained elusive. Here we show that PHT4;6-GFP is targeted to the trans-Golgi compartment and that loss of function of this carrier protein has a dramatic impact on plant growth and development. Knockout mutants of pht4;6 exhibit a dwarf phenotype that is complemented by the homologous gene from rice (Oryza sativa). Interestingly, pht4;6 mutants show altered characteristics of several Golgi-related functions, such as an altered abundance of certain N-glycosylated proteins, altered composition of cell-wall hemicelluose, and higher sensitivity to the Golgi α-mannosidase and the retrograde transport inhibitors kifunensine and brefeldin A, respectively. Moreover, pht4;6 mutants exhibit a 'mimic disease' phenotype accompanied by constitutively activated pathogen defense mechanisms and increased resistance against the virulent Pseudomonas syringae strain DC3000. Surprisingly, pht4;6 mutants also exhibit phosphate starvation symptoms, as revealed at the morphological and molecular level, although total Pi levels in wild-type and pht4;6 plants are similar. This suggested that subcellular Pi compartmentation was impaired. By use of nuclear magnetic resonance (NMR), increased Pi concentration was detected in acidic compartments of pht4;6 mutants. We propose that impaired Pi efflux from the trans-Golgi lumen results in accumulation of inorganic phosphate in other internal compartments, leading to low cytoplasmic phosphate levels with detrimental effects on plant performance.

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.

The role of grape berry skin as a protective barrier against damage by physical injuries and pathogen attacks requires a metabolism able to sustain biosynthetic activities such as those relating to secondary compounds (i.e., flavonoids). In order to draw the attention on these biochemical processes, a proteomic and metabolomic comparative analysis was performed among Riesling Italico, Pinot Gris, Pinot Noir, and Croatina cultivars, which are known to accumulate anthocyanins to a different extent. The application of multivariate statistics on the dataset pointed out that the cultivars were distinguishable from each other and the order in which they were grouped mainly reflected their relative anthocyanin contents. Sorting the spots according to their significance 100 proteins were characterized by LC-ESI-MS/MS. Through GC-MS, performed in Selected Ion Monitoring (SIM) mode, 57 primary metabolites were analyzed and the differences in abundance of 16 of them resulted statistically significant to ANOVA test. Considering the functional distribution, the identified proteins were involved in many physiological processes such as stress, defense, carbon metabolism, energy conversion and secondary metabolism. The trends of some metabolites were related to those of the protein data. Taken together, the results permitted to highlight the relationships between the secondary compound pathways and the main metabolism (e.g., glycolysis and TCA cycle). Moreover, the trend of accumulation of many proteins involved in stress responses, reinforced the idea that they could play a role in the cultivar specific developmental plan.

The development of methods for the analysis and comparison of the nucleic acids contained in single cells is an ambitious and challenging goal that may provide useful insights in many physiopathological processes. We review here some of the published protocols for the amplification of whole genomes (WGA). We focus on the reaction known as Multiple Displacement Amplification (MDA), which probably represents the most reliable and efficient WGA protocol developed to date. We discuss some recent advances and applications, as well as some modifications to the reaction, which should improve its use and enlarge its range of applicability possibly to degraded genomes, and also to RNA via complementary DNA.

The amount of DNA available for informative genomic studies is often limiting. Thus, several methods have been developed to achieve a whole genome amplification (WGA). Particu-larly promising is a variant, called multiple displacement amplification (MDA) (1,2), which exploits the high processivity of Φ29 phage DNA polymerase and suffers an amplifi-cation bias significantly lower than previous, PCR-based WGA (3).Even though MDA-DNA has been successfully used for many applica-tions (1,4–7), some problems have been reported. For example, sequences near the ends of linear chromosomes appear underrepresented after MDA (6,8,9). Both defective priming events and abortive chain terminations could contribute to this problem, which limits the applications of MDA and jeopardizes its use on short linear DNA (e.g., cDNA and degraded genomes from forensic, archeological, and fetal origin) (10). In these cases, the terminal underrepresentation would cause the loss of substantial information.We studied this problem using λ phage DNA (48.5 kb; Promega Biosciences, San Luis Obispo, CA, USA). MDA was performed using the Genomiphi

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.

Grapevine breeding has been actively conducted in Italy since the end of the nineteenth century, producing approximately 800 varieties (rootstocks, wine and table grapes). The major Italian rootstock, wine and table grape breeders are listed, including the main varieties they obtained, such as Pirovano (cv. Italia), Paulsen (1103P rootstock), Dalmasso (cv. Albarossa), Ruggeri (140Ru rootstock), Prosperi (cv. Clotilde Prosperi), Manzoni (cv. Manzoni bianco), Terzi (cv. Incrocio Terzi 1) and Rigotti (cv. Rebo). Current breeding programs to obtain new rootstocks, new disease-resistant varieties and crossings within Vitis vinifera cvs. to improve some technological traits are described.

The effect of growth conditions (medium and photoperiod) on in vitro tuberisation and microtuber dormancy of the cv. Desiree (Solanum tuberosum subspeciestuberosum) and Imilla Negra (Solanum tuberosum subspeciesandigena) was evaluated. The short photoperiod and presence of CCC in the medium reduced the dry matter of plants as well as the size of microtubers. The two cultivars showed a difference, in favour of Desiree, in the percentage of plants having more than one tuber and in the average time of sprouting after storage. The average time of sprouting was more rapidly obtained under long days and in a medium without CCC. The CCC and short days caused the same dormancy conditions and the same K+ quantity in the microtubers. The microtubers mineral composition is involved in the dormancy status.

Summary Ni2+- accumulation and the toxic effects of Nit+ on growth were investigated in pea seedlings. In roots, Ni2+ absorption increased as a function of metal supply, while in the shoots the accumulation of the metal was restricted, particularly with higher treatments. Ten μmol/L NiZ+ was found to be the lowest effective concentration to cause a significant reduction in root growth. NiZ+ did not induce changes in shoot length, even though biomass production was affected in both roots and shoots. The toxic effects of Ni2+ on K+ uptake and on water content were markedly higher in roots than in shoots. In the Ni-treated roots, these effects, and the concomitant increase in phenol content and extracellular peroxidase activity with affinity towards syringaldazine, were associated with an early induced cellular differentiation. The significance of this senescent-like state, together with other cellular defence mechanisms such as the stimulation of intracellular peroxidase activity, is discussed in relation to Ni-stress.

In 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.

The 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.

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.

The effects of Ni 2+ on Raphanus sativus seeds during the first 48 h of germination were investigated. Increasing concentrations of Ni 2+ (0, 80, 100, 200 and 400 μM) progressively inhibited the growth of radish seedlings. In parallel, Ni 2+ affected the restoration of reabsorption of K + and Mg 2+ and the release of Ca 2+ into the incubation medium. The transport mechanisms appeared to be well operative, as indicated by the negative values of the transmembrane electrical potential. Ni 2+ strongly affected the reactivation of oxygen uptake and the increase in energy charge, the mobilisation of sugar and phospho-organic compounds and inhibited the synthesis of DNA, RNA and proteins. The presence of Ni 2+ retarded the degradative and synthetic changes of protein of the soluble and microsomal fractions, occurring in seeds during germination; moreover, in both the soluble and microsomial fractions, two polypeptides from each were not expressed.

bye of Kplant cells involves a high-affinity system predominating atlow external concentration (< 1-0.3 mM) mediating the ca-tion influx against its electrochemical potential (Maathuisand Sanders 1993, Gassmann and Schroeder 1994), anda low affinity system predominating at higher external ca-tion concentrations. Several mechanisms have been pro-posed for the high-affinity system (Poole 1978, Rodriguez-Navarro et al. 1986, Kochian and Lucas 1989, Gibrat et al.1990) but the low-affinity system is largely accepted to be achannel mechanism (Kochian et al. 1985, Schroeder andFang 1991, Kourie and Goldsmith 1992, Gassman et al.1993, Kochian and Lucas 1993, Gassmann and Schroeder1994). It has also been proposed tha

To elucidate the inhibitory effect of light on the germination of the negatively photoblastic seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair), we have compared the changes that occur during the first 24 h of imbibition in the dark and in the light. Here. we give data on transcription products, translation activity, activities of some enzymes involved in the metabolic reactivation of seeds (glucose-6-phosphate dehydrogenase. pyruvate kinase, glucokinase and malate dehydrogenase), and the capability to absorb L-[U- 14 C]-leucine. During the first stages of imbibition, the changes in the protein pattern and in the mRNA populations that occurred were not related to the light/dark regimes. Differences due to the light treatment did not appear until after 24 h, when new microsomal proteins appeared in the dark and some mRNA populations encoding low molecular mass polypeptides were increased in the light. The translation activity, measured as L-[U- 14 C]-leucine incorporated into proteins and as the ratio polysomes/ monosomes, increased with time in both darkness and light, but to a much higher extent in the dark. The activities of the four enzymes tested were higher in the light than in the dark before 16 h, then decreased in the light and increased sharply in the dark. Together with the behaviour of the increase in the ratio polysomes/monosomes in the dark, this suggests an increased rate of protein degradation in the dark during the period 0-16 h. The absorption of L-[U- 14 C]-leucine was severely inhibited by light during the whole period studied, as expected since light inhibits the reactivation of the transport mechanisms at the plasma membrane. In seeds imbibed in the dark and then transferred to light, the inhibition by light of absorption and of incorporation into proteins of L-[U- 14 C]-leucine, as well as the inhibition of the increase in the ratio poly-somes/monosomes, was effective before the 22nd h; after which time the control exerted by light disappeared.