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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.

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.

Sugars signal the plant cell’s energy status and trigger proper responses to stresses, also aiming to sustain growth and developmental processes. The ability to sense intracellular sugar levels is of utmost importance for the cell survival. The finely tuned levels of the nonreducing disaccharide trehalose (Tre) and its precursor, trehalose-6-P (T6P), are related to those of sucrose (Suc), whose accumulation is reflected in an increase in T6P levels. T6P in turn inhibits the regulatory protein Snf1-related protein kinase 1 (SnRK1), triggering changes in the energy metabolism. Alterations in the Tre/T6P ratios act as a signal leading to several effects on plant metabolic, physiological, and developmental processes. The present chapter will deal with the involvement of the Tre/T6P/SnRK1 system in response to abiotic stresses. Particular attention will be devoted to hypoxia and cold, in two model plants, that is, Arabidopsis and rice.

Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) 'Oro A'/melting flesh (MF) 'Springcrest' and 'Sanguinella'] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit.

The changes in endopolygalacturonase (endo-PG) levels and endo-PG expression in nonmelting flesh (NMF) and melting flesh (MF) peach fruits (Prunus persica) during softening were studied. The endo-PG gene was analysed to identify polymorphisms exploitable for early marker-assisted selection (MAS) of flesh texture. The role of endo-PG in softening was assessed by western and northern blotting and by biochemical analyses. Polymorphisms in the endo-PG gene were revealed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. An endo-PG protein was detected in both NMF and MF fruits. The levels of this endo-PG protein were higher and increased with softening in MF fruits, but remained lower and were constant in NMF fruits. The different levels of endo-PG appeared to be caused by the differential expression of an endo-PG gene, whose open-reading frame (ORF) showed five single nucleotide polymorphisms (SNPs) in NMF 'Oro A' compared with MF 'Bolero'. One of these SNPs allowed us to determine the allelic configuration at the melting flesh (M) locus and also seemed to be exploitable for early MAS in other NMF/MF phenotypes. The NMF phenotype does not seem to be caused by a large deletion of the endo-PG gene.

In peach fruit, it is desirable to individuate simple biochemical/physiological parameters, genetically determined and useful for marker-assisted selection to obtain improved genotypes for flesh firmness. The accumulation of proteins involved in cell wall re-organisation, such as endo-polygalacturonases (endo-PGs) and expansins, has been studied in fruits of two genotypes with either Non-Melting(NMF) or Melting Flesh (MF) texture, and in fruits of three 'Slow-Ripening' ('SR': altered ripening pattern) genotypes. In the flesh cell walls of MF fruits, the amounts of an endo-PG-like polypeptide were relevant and increased with ripening, while it was produced in extremely low amounts in NMF fruits. A similar pattern was observed for expansins. NMF fruits produced more ethylene than the MF ones. The Ca 2+ -dependent phosphorylation of a soluble polypeptide (Mw ≃ 52 kDa) decreased with ripening in NMF fruits and remained constant in the MF ones. In 'SR' genotypes, phosphorylation of this polypeptide was detectable only in ethylene-producing fruits, which softened their flesh and expressed the endo-PG-like polypeptide. The data suggest that phosphorylation of the 52-kDa polypeptide may be involved in the modulation of ethylene signalling and flesh softening.

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 toxicity of Cd2+in vivo during the early phases of radish (Raphanus sativus L.) seed germination and the in vitro Cd2+ effect on radish calmodulin (CaM) were studied. Cd2+ was taken up in the embryo axes of radish seeds; the increase in fresh weight of embryo axes after 24 h of incubation was inhibited significantly in the presence of 10 mmol m−3 Cd2+ in the external medium, when the Cd2+ content in the embryo axes was c. 1.1 μmol g−1 FW. The reabsorption of K+, which characterizes germination, was inhibited by Cd2+, suggesting that Cd2+ affected metabolic reactivation. The slight effect of Cd2+ on the transmembrane electric potential of the cortical cells of the embryo axes excluded a generalized toxicity of Cd2+ at the plasma membrane level. After 24 h of incubation, Cd2+ induced no increase in total acid-soluble thiols and Cd2+-binding peptides able to reduce Cd2+ toxicity. Ca2+ added to the incubation medium partially reversed the Cd2+-induced inhibition of the increase in fresh weight of embryo axes and concomitantly reduced Cd2+ uptake. Equilibrium dialysis experiments indicated that Cd2+ bound to CaM and competed with Ca2+ in this binding. Cd2+ inhibited the activation of Ca2+-CaM-dependent calf-brain phosphodiesterase, inhibiting the Ca2+-CaM active complex. Cd2+ reduced the binding of CaM to the Ca2+-CaM binding enzymes present in the soluble fraction of the embryo axes of radish seeds. The possibility that Cd2+ toxicity in radish seed germination is mediated by the action of Cd2+ on Ca2+-CaM is discussed in relation to the in vivo and in vitro effects of Cd2+.

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.

Incubation of radish (Raphanus sativus L.) seeds in the presence of 1 or 5 mol m −3 Ca-EGTA, which increased Ca 2+ activity in the incubation medium (c. 0.24 or 0.37 mol m −3 at 24 h with respect to c. 0.13 mol m −3 in the control), did not affect germination, the restoration of K + net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m −3 Na-EGTA, which reduced Ca 2+ activity in the incubation medium (20 mmol m −3 at 24 h), decreased the total Ca 2+ level in embryo axes (−21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K + net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m −3 Na-EGTA (Ca 2+ activity in the incubation medium was 0.6 mmol m −3 ), the decrease in the total Ca 2+ level was greater (c.-27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca 2+ availability does not affect germination and suggest that the effect of Na-EGTA, at least up to 5 mol m −3 , is a result of an induction of Ca 2+ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000 g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5-fold and 7-fold, respectively). This increase was very similar in the Ca-EGTA-treated seeds but was inhibited (c.-38%) by 1 mol m −3 Na-EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m −3 Na-EGTA (c.-30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci & N. Negrini (1988) Plant Physiology 88, 910-914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca 2+ availability and that the CaM inhibitor has a role in controlling the amount of Ca-CaM available for the Ca-CaM-dependent enzymes

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the "unripe" to the "ripe" phase. The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; 'Oro A') and a melting flesh (MF; 'Bolero') cultivar, at "unripe" and "ripe" stages as defined by some parameters typical of ripening, were then analyzed. The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage ("unripe" versus "ripe") and/or the genetic background of the cultivar ('Oro A' versus 'Bolero'). Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress. Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric ("unripe") to the climacteric ("ripe") phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status. The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.

Germination in the dark and at 16°C of photoblastic and thermosensitive seeds of Phacelia tanacetifolia was inhibited when incubated with EGTA and the Ca2+-ionophore A 23187; A 23187 in the presence of Ca2+ still inhibited germination, but to a lesser extent. Treatments with EGTA or Ca2+ at different concentrations in the presence or in the absence of A 23187 did not remove light inhibition. The calmodulin (CaM) inhibitor, calmidazolium, strongly inhibited germination. The specificity of these inhibitors and their effects on seed germination are discussed. CaM from Phacelia tanacetifolia seeds has been purified and its characteristics (molecular weight, heat and acid stability, kinetics of phosphodiesterase [EC 3.1.4.17] activation) were very similar to those of other plant sources. More than 90% of total CaM was present in the soluble fraction (ca 41 μg g-1 fresh weight in ungerminated seeds). The CaM level greatly increased in the early phases of seed germination; this increase did not take place when germination was inhibited by light or high temperature. When fusicoccin, a toxin which promotes germination by activating membrane functions, relieved light or high temperature inhibition, CaM increased up to the control value in the dark at 16°C. The parallel increase in CaM and seed germination suggest that CaM plays an important role in the process. Fusicoccin in the dark at 16°C stimulated CaM and fresh weight increase, but not the metabolic reactivation measured as increase in DNA and total RNA levels; at 30°C fusicoccin stimulated the increase in fresh weight and in CaM level, but the increases in DNA and total RNA were very low. These results suggest that the activation of membrane functions with cell enlargement induced by fusicoccin is related to CaM increase.

Calli of Actinidia deliciosa were cultured on medium with 30 mM sucrose and 0.2 M 3-O-methyl-D-glucose (3-OMG) and their metabolic adaptation to the hyperosmotic condition was studied. 3-OMG concentration in the tissue reached that of the medium. Experiments with labelled 3-O-methyl-D-U-14C]glucose (3-OM[U-14C]G) showed that it was partially metabolised but not oxidised to CO2. In vivo 31P-NMR showed that in 3-OMG-treated calli a phosphorylated compound, with a chemical shift similar to that of monophosphoesters, was accumulated. The 3-OMG-treated calli showed more negative water (Ψw) and osmotic (Ψπ) potentials than the controls. In the following of subcultures an increase in organic and inorganic osmolytes, together with increase in O2 uptake and decrease in ATP/ADP ratio occurred. The data show that, even if 3-OMG can not be considered a classic osmotic agent due to its permeability, it induced metabolic changes similar to those described for osmotic adaptation, suggesting that the adaptations might be due to a physical or metabolic effect of 3-OMG not linked to a difference between Ψπ and Ψw agar

(1995). Effect of culture filtrates of Stemphylium vesicarium on pear leaves. Giornale botanico italiano: Vol. 129, No. 4, pp. 1070-1070.

(1995). Effect of ABA on radish (Raphanus sativus, L) seeds germinating at low calcium availability. Giornale botanico italiano: Vol. 129, No. 4, pp. 1030-1030.

An inhibitor of Ca(2+)-calmodulin (Cam)-dependent brain phosphodiesterase was present in the soluble fraction of embryo axes from ungerminated radish (Raphanus sativus L.) seeds. This inhibitor is a Ca(2+)-dependent, Cam-binding protein; in fact: (a) its effect was strongly reduced by treatment with proteases; (b) the inhibition was counteracted by Cam but not by Ca(2+); (c) on gel filtration in the presence of Ca(2+), Cam co-chromatographed with the inhibitor. The inhibitor is heat stable and positively charged at pH 7.5. During early phases of germination, the fresh weight and the levels of DNA and RNA of embryo axes increased, the level of the inhibitor decreased, and the level of Cam increased. Abscisic acid (ABA) inhibited germination, the decrease of inhibitor, and the increase of Cam. Fusicoccin (FC) stimulated the increase in fresh weight but not the increase in the RNA and DNA levels; in this condition, the inhibitor level decreased and the increase in Cam level was higher than in the control. In the presence of both ABA and FC, there was an increase in fresh weight not accompanied by an increase in DNA and RNA levels; Cam increased and, on a fresh weight basis, reached the value of the control. These results indicate that the Ca(2+)-Cam system was activated in early germination of radish seeds by an increase in Cam and a decrease in the inhibitor levels, that FC, probably through the activation of membrane functions, increased Cam level, and that the ABA inhibition on germination was not mediated by the Ca(2+)-Cam system.

‘Big Top’ nectarine [Prunus persica (L.) Batsch] has appreciable keeping quality because it resembles, at ripening, the stony hard (SH) peach (P. persica) in firmness and crispness but melts at a slow speed at full ripening. We have characterized the postharvest behavior of ‘Big Top’ fruit, treated or not with ethylene for 5 days after harvest (DAH), and compared it with that of a SH peach (‘Ghiaccio’). Pp-ACS1 expression, ethylene evolution, endo-polygalacturonase (endo-PG) production, and softening were evaluated and compared with those of the physiologically ripe melting flesh (M) cultivar Bolero. Like ‘Bolero’, ‘Big Top’ fruit expressed Pp-ACS1 and evolved ethylene but with a 5-day delay. Pp-endo-PG expression, production of an active endo-PG, and fruit melting showed a parallel behavior; ethylene treatment enhanced all these features. In SH ‘Ghiaccio’ Pp-ACS1 expression, ethylene evolution, endo-PG production, and softening were absent during the first 5 DAH in air. ‘Ghiaccio’ neither expressed Pp-ACS1 nor evolved ethylene even after ethylene treatment but responded by accumulating Pp-endo-PG transcripts and an active endo-PG protein, with flesh melting. A ‘Big Top’ Pp-endo-PG clone showed several single nucleotide (SNP) and insertion-deletion (InDel) polymorphisms in comparison with the M Pp-endo-PG clone of ‘Bolero’ and substantial similarity with the Pp-endo-PG clone of ‘Ghiaccio’. In ‘Big Top’, we identified a peculiar SNP (bp 348) and InDels shared with ‘Ghiaccio’, possibly suitable for discriminating among different genotypes. Overall, the data confirm the pivotal role of ethylene in the regulation of endo-PG production and in the determination of peach flesh texture and support the evidence that ‘Big Top’ could be classified as a melting (slow-melting) phenotype.