Transcriptional reprogramming in sound-treated Micro-Tom plants inoculated with Pseudomonas syringae pv. tomato DC3000.

Sound vibrations (SV) are known to influence molecular and physiological processes that can improve crop performance and yield. In this study, the effects of three audible frequencies (100, 500 and 1000 Hz) at constant amplitude (90 dB) on tomato Micro-Tom physiological responses were evaluated 1 and 3 days post-treatment. Moreover, the potential use of SV treatment as priming agent for improved Micro-Tom resistance to Pseudomonas syringae pv. tomato DC3000 was tested by microarray. Results showed that the SV-induced physiological changes were frequency- and time-dependent, with the largest changes registered at 1000 Hz at day 3. SV treatments tended to alter the foliar content of photosynthetic pigments, soluble proteins, sugars, phenolic composition, and the enzymatic activity of polyphenol oxidase, peroxidase, superoxide dismutase and catalase. Microarray data revealed that 1000 Hz treatment is effective in eliciting transcriptional reprogramming in tomato plants grown under normal conditions, but particularly after the infection with Pst DC3000. Broadly, in plants challenged with Pst DC3000, the 1000 Hz pretreatment provoked the up-regulation of unique differentially expressed genes (DEGs) involved in cell wall reinforcement, phenylpropanoid pathway and defensive proteins. In addition, in those plants, DEGs associated with enhancing plant basal immunity, such as proteinase inhibitors, pathogenesis-related proteins, and carbonic anhydrase 3, were notably up-regulated in comparison with non-SV pretreated, infected plants. These findings provide new insights into the modulation of Pst DC3000-tomato interaction by sound and open up prospects for further development of strategies for plant disease management through the reinforcement of defense mechanisms in Micro-Tom plants.

Microwave Radiation as an Inducer of Secondary Metabolite Production in Drosera rotundifolia In Vitro Plantlets.

In this study, low-intensity microwave irradiation (frequency of 2.45 GHz; 26.3 kW m-3 power density) for 0, 5, and 30 s was tested for the first time on Drosera rotundifolia in vitro plantlets to explore its effect on the production of highly valued phenolic compounds. Analysis of the extracts obtained from irradiated plantlets revealed time-dependent increases in the levels of photosynthetic pigments, particularly the carotenoids, whereas symptoms of growth decline were not observed. Similarly, the highest total antioxidant capacity and total phenolic and flavonoid contents were detected in 30-s-irradiated plantlets. High-performance liquid chromatography analysis revealed that the content of the bioactive phenolics 5-hydroxy-7-methylnaphthalene-1,4-dione (1), 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (2), and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one (3), which are considered to be responsible for the pharmacological properties of this species, was around twofold higher in 30-s-irradiated plantlets than in controls. Nevertheless, the accumulation of 5-hydroxy-2-methylnaphthalene-1,4-dione (4), which was present only in trace amounts in the plant roots, decreased by 30% upon microwave irradiation. The results indicate that microwave treatment acts as an effective inducer of the production of phenylpropanoid compounds, which opens up new opportunities for its use in biotechnological applications.

Differential Response of Phenol Metabolism Associated with Antioxidative Network in Elicited Grapevine Suspension Cultured Cells under Saline Conditions.

Highly productive trans-resveratrol (t-R) grapevine suspension cultured cells (SCC) and two effective elicitors, methyl jasmonate (MJ) and randomly methylated ß-cyclodextrins (CDs), were used to analyze the extent to which salt treatments alter the production of bioactive phenolic compounds. The expression/activity profile of the enzymes involved in phenol metabolism and antioxidant networks were also studied. A marked extracellular accumulation of phenolic compounds, especially t-R, was found in SCC elicited with CDs and/or MJ under saline conditions. However, the treatments with MJ alone and all those combined with salt favored the intracellular accumulation of catechin and ferulic acid. The salt-induced accumulation of phenolics was correlated with the higher total antioxidant capacity values found in cells, suggesting that cellular redox homeostasis under saline conditions was largely maintained by increasing phenolic compound production. These higher levels of phenolics found in elicited cells under saline conditions fit well with the highest activity of phenylalanine ammonia-lyase. Moreover, antioxidant enzyme activities were boosted in treatments with MJ and/or in those combined with salt and decreased in those treated with CDs. These results suggest a differential response of the antioxidative network to the presence of elicitors under saline conditions.

OPDA and ABA accumulation in Pb-stressed Zygophyllum fabago can be primed by salicylic acid and coincides with organ-specific differences in accumulation of phenolics.

Salicylic acid (SA) is a well-known priming agent that is widely used to protect plants against stressing agents, including heavy metals as Pb. A better understanding of the mechanisms that enable plants to counteract Pb toxicity would help to select strategies for land reclamation programs. Here we used a metallicolous population of Zygophyllum fabago to assess the extent to which SA pretreatment modulates Pb-induced changes in phenol metabolism and stress-related phytohormone levels in roots and leaves. Our data revealed that accumulation of different phytohormones, lignin, soluble and wall-bound phenolics as well as peroxidase (PRX) activity in Pb-stressed plants differed after SA-pretreatment. Exposure to Pb led to the induction of soluble and cell wall-bound PRX activities, particularly those involved in the oxidation of coniferyl alcohol and ferulic acid, while pretreatment with SA reduced the Pb-induced stimulation of PRX activities in roots but increased them in leaves. SA-treatment by itself induced accumulation of ABA and the JA-precursor 12-oxo-phytodienoic acid (OPDA) in the roots. Pb in turn inhibited these SA-induced effects with the exception of OPDA accumulation that was primed by the pretreatment. The SA treatment also induced accumulation of OPDA in leaves but suppressed the accumulation of JA-Ile although with relatively small absolute changes. Notably, Pb-induced accumulation of ABA was primed in the leaves of SA-pretreated plants. Together our data suggest that priming of OPDA accumulation in the roots and of ABA in the leaves by SA-pretreatment may play important regulatory roles, possibly via regulating PRX activities, for Pb stress in plants.

Subtle changes in light intensity affect in vitro responses but not ex vitro performance of Limonium sinuatum.

Environmental conditions during the in vitro stages of a micropropagation process can further affect the ex vitro plant performance. In this work, the behaviour of four blue cultivars of statice (Limonium sinuatum) was studied with respect to four distances (18.0, 12.5, 8.0, or 2.0 cm) between culture tubes and the light source during their in vitro multiplication stage. In vitro shoots of statice were cultured on MS medium supplemented with 4% sucrose, 0.9% agar and 0.2 mg L-1 BA and incubated under four slightly different light intensity treatments (PPFD of 96.9, 99.6, 101.2, or 102.7 µmol m-2 s-1) for 35 days at 23 °C with a daily photoperiod of 16 h. The light treatments assayed resulted in no significant differences in in vitro propagation ratio and ex vitro survival and crop production and quality (as judged by the number of inflorescence stems per plant, length of inflorescence stems and number of both spikes and branches per stem produced). However, in vitro growth (estimated as the length of the longest leaf in a shoot at the end of the culture period) and levels of pigments (chlorophylls and carotenoids) and antioxidants (as estimated by the DPPH test) showed variations among cultivars. This indicates that statice cultivars were able to adjust their metabolism in response to the small differences in light intensity of the treatments applied. Results point to the importance of optimization of growth room use to increase profitability of micropropagation processes.

Seasonal ionomic and metabolic changes in Aleppo pines growing on mine tailings under Mediterranean semi-arid climate.

Aleppo pine is the most abundant conifer species in Mediterranean basin. Knowledge of adaptive mechanisms to cope with different environmental stresses simultaneously is necessary to improve its resilience to the predicted climatic changes and anthropogenic stressors, such as heavy metal/metal(loid)s (HMMs) pollution. Here, one year-old needles and rhizosphere soil samples from five mining and non-mining (NM) populations of Aleppo pines grown spontaneously in SE Spain were sampled in two consecutive years during spring and summer. Quantitative determination of a wide suite of edaphic, biochemical, and physiological parameters was performed, including soil physicochemical properties, ionome profile, foliar redox components, primary and secondary metabolites. Mining rhizosphere soils were characterized by elevated contents of HMMs, particularly lead and zinc, and low carbon, nitrogen and potassium levels. Multivariate data analysis based on needle ionome and antioxidative/oxidative parameters revealed a clear distinction between seasons irrespective of the population considered. Spring needles were characterized by higher levels of HMMs, sulfur, glutathione (GSH), proanthocyanidins (PAs), and soluble phenols (TPC), whereas reduced chlorophylls and increased levels of carotenoids, relative water content and K+, Na+ and Cl- typified summer needles. In general mining populations had higher levels of ascorbate, and TPC, and exhibited higher antioxidant activities than the NM population. This could contribute to prevent oxidative injury induced by HMMs. Taken together, results suggest that seasonal factors have a more marked effect on the metabolism of the Aleppo pine populations studied than that exerted by soil conditions. This effect could be mediated by water availability in surface soil layers. If this conclusion is right, predicted rainfall reduction and temperature increase in the Mediterranean basin associated to global climate change would lead to pine needle metabolism to express the summer pattern for more prolonged periods. This, in turn, could negatively affect the performance of Aleppo pine populations.

Coordinated role of soluble and cell wall bound phenols is a key feature of the metabolic adjustment in a mining woody fleabane (Dittrichia viscosa L.) population under semi-arid conditions.

Environmental contamination by hazardous heavy metals/metalloids (metal(loid)s) is growing worldwide. To restrict the migration of toxic contaminants, the establishment of a self-sustainable plant cover is required. Plant growth in multi-polluted soils is a challenging issue not only by metal(loid) toxicities, but also by the co-occurrence of other stressors. Dittrichia viscosa is a pioneer Mediterranean species able to thrive in metal(loid)-enriched tailings in semi-arid areas. The aim of the present work was to examine the metabolic adjustments involved in the acclimation responses of this plant to conditions prevailing in mine-tailings during Mediterranean spring and summer. For this purpose, fully-expanded leaves, and rhizosphere soil of both mining and non-mining populations of D. viscosa grown spontaneously in south-eastern Spain were sampled in two consecutive years. Quantitative analysis of >50 biochemical, physiological and edaphic parameters were performed, including nutrient status, metal(loid) contents, leaf redox components, primary and secondary metabolites, salicylic acid levels, and soil physicochemical properties. Results showed that mining plants exhibited high foliar Zn/Pb co-accumulation capacity, without substantially affecting their photosynthetic metabolism or nutritional status even in the driest summer period. The comparison of the antioxidative/oxidative profile between mining and non-mining D. viscosa populations revealed no major seasonal changes in the content of primary antioxidants (ascorbate and GSH), or in the levels of ROS. Multivariate analysis showed that phenylalanine ammonia-lyase (PAL) and peroxidase (PRX) activities and soluble and cell wall-bound phenols were potential biomarkers for discriminating between both populations. During the dry season, a marked enhancement in the activity of both PAL and soluble PRX resulted in both a drop in the accumulation of soluble phenols and an increase of the strong metal chelator caffeic acid in the cell-wall fraction, supporting the view that the plasticity of phenylpropanoid metabolism provide an effective way to counteract the effects of stress combinations.

Seasonal changes in antioxidative/oxidative profile of mining and non-mining populations of Syrian beancaper as determined by soil conditions.

Soil pollution by heavy metals/metalloids (HMMs) is a problem worldwide. To prevent dispersion of contaminated particles by erosion, the maintenance of a vegetative cover is needed. Successful plant establishment in multi-polluted soils can be hampered not only by HMM toxicities, but also by soil nutrient deficiencies and the co-occurrence of abiotic stresses. Some plant species are able to thrive under these multi-stress scenarios often linked to marked fluctuations in environmental factors. This study aimed to investigate the metabolic adjustments involved in Zygophyllum fabago acclimative responses to conditions prevailing in HMM-enriched mine-tailings piles, during Mediterranean spring and summer. To this end, fully expanded leaves, and rhizosphere soil, of three contrasting mining and non-mining populations of Z. fabago grown spontaneously in south-eastern Spain were sampled in two consecutive years. Approximately 50 biochemical, physiological and edaphic parameters were examined, including leaf redox components, primary and secondary metabolites, endogenous levels of salicylic acid, and physicochemical properties of soil (fertility parameters and total concentration of HMMs). Multivariate data analysis showed a clear distinction in antioxidative/oxidative profiles between and within the populations studied. Levels of chlorophylls, proteins and proline characterized control plants whereas antioxidant capacity and C- and S-based antioxidant compounds were biomarkers of mining plants. Seasonal variations were characterized by higher levels of alkaloids and PAL and soluble peroxidase activities in summer, and by soluble sugars and hydroxycinnamic acids in spring irrespective of the population considered. Although the antioxidant systems are subjected to seasonal variations, the way and the intensity with which every population changes its antioxidative/oxidative profile seem to be determined by soil conditions. In short, Z. fabago displays a high physiological plasticity that allow it to successfully shift its metabolism to withstand the multiple stresses that plants must cope with in mine tailings piles under Mediterranean climatic conditions.

Pb-induced responses in Zygophyllum fabago plants are organ-dependent and modulated by salicylic acid.

Zygophyllum fabago is a promising species for restoring heavy metal (HM) polluted soils, although the mechanisms involved in HM tolerance in this non-model plant remain largely unknown. This paper analyses the extent to which redox-active compounds and enzymatic antioxidants in roots, stems and leaves are responsible for Pb tolerance in a metallicolous ecotype of Z. fabago and the possible influence of salicylic acid (SA) pretreatment (24 h, 0.5 mM SA) in the response to Pb stress. SA pretreatment reduced both the accumulation of Pb in roots and even more so the concentration of Pb in aerial parts of the plants, although a similar drop in the content of chlorophylls and in the maximum quantum yield of photosystem II was observed in both Pb- and SA-Pb-treated plants. Pb increased the endogenous free SA levels in all organs and this response was enhanced in root tissues upon SA pretreatment. Generally, Pb induced a reduction in catalase, ascorbate peroxidase and glutathione reductase specific activities, whereas dehydroascorbate reductase was increased in all organs of control plants. SA pretreatment enhanced the Pb-induced H2O2 accumulation in roots by up-regulating Fe-superoxide dismutase isoenzymes. Under Pb stress, the GSH redox ratio remained highly reduced in all organs while the ascorbic acid redox ratio dropped in leaf tissues where a rise in lipid peroxidation products and electrolyte leakage was observed. Finally, an organ-dependent accumulation of proline and ß-carboline alkaloids was found, suggesting these nitrogen-redox-active compounds could play a role in the adaptation strategies of this species to Pb stress.

Antioxidant capacity as a marker for assessing the in vitro performance of the endangered Cistus heterophyllus.

Cistus heterophyllus subsp. carthaginensis is an endemic and endangered species from the SE Mediterranean coastal region of Spain. Within the framework of the efforts aiming to species conservation, in vitro culture techniques could be of interest. The aim of this study was to evaluate the antioxidant capacity of C. heterophyllus shoot cultures as a possible marker of in vitro performance. The effects of five different basal salt formulations and cytokinin levels on in vitro performance and antioxidant capacity were examined. K(+)/Na(+) and Ca(2+)/Na(+) ratios initially present in culture media greatly affected the antioxidant capacity (the lower the ratios the higher the antioxidant capacity). Increasing concentrations of BA resulted in higher antioxidant capacity. The results obtained point to antioxidant capacity as being a marker of incidence of stress conditions in in vitro cultured C. heterophyllus. A good correlation was found between antioxidant capacity and total soluble phenolics present in Cistus extracts. Catechin was identified in all the extracts and its levels were found to change parallel to the antioxidant capacity, pointing to a prominent role played by this flavonoid in C. heterophyllus defence against oxidative stress, which in turn affects the in vitro performance of this species.

Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate.

Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and ß-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom.

Heavy metal stress reduces the deposition of calcium oxalate crystals in leaves of Phaseolus vulgaris.

Calcium oxalate (CaOx) crystals in plants may serve as a sink for the absorption of excess calcium, and they could play an important role in heavy metal detoxification. In this study, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of Phaseolus vulgaris was investigated. Different analytical techniques were used to determine the influence of exogenous lead and zinc on CaOx deposition and to detect a presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Energy dispersive X-ray spectrometry did not detect the inclusion of heavy metals inside the CaOx crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in P. vulgaris but do play an important role in bulk calcium regulation.

Changes in phenol content during strawberry (Fragariaxananassa, cv. Chandler) callus culture.

Total soluble phenols, soluble flavanols, (+)-catechin, ferulic acid and 1-O-feruloyl-beta-d-glucose were analyzed during the development of a strawberry (Fragariaxananassa, cv. Chandler) callus culture. The time-course changes of the different phenols assayed were well correlated with callus growth and morphology. The changes in polyphenol oxidase (EC 1.10.3.1-2) and beta-glucosidase (EC 3.2.1.21) activities in the callus were also examined. The total phenol, soluble flavanols and (+)-catechin contents were high during the preexponential and exponential phases of growth. The subsequent decrease in (+)-catechin concentration coincided with high levels of polyphenol oxidase activity. The 1-O-feruloyl-beta-d-glucose content was highest as callus growth ceased, and its subsequent decrease was accompanied by the increased production of ferulic acid. This increase in ferulic acid was accompanied by an increase in beta-glucosidase activity. The ferulic acid content decreased at the end of culture, when callus growth had stopped and showed clear symptoms of senescence. This decrease in the ferulic acid concentration was accompanied by an increase in the levels of ferulic acid bound to cell wall components.