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.

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.

Different mechanisms of the metalliferous Zygophyllum fabago shoots and roots to cope with Pb toxicity.

Lead (Pb) remains classified as a priority pollutant. Zygophyllum fabago is considered an early colonizer of heavy metal-polluted soils under semiarid conditions, but physiological mechanisms underlying this colonizing capacity remain unclear. In order to characterize Z. fabago plants' performance on Pb-contaminated soils, we evaluated how Pb influenced root and shoot growth, carbon metabolism, and oxidative status. For that, 30-day-old seedlings from one population colonizing a mine tailing ("Mercader") at Murcia (southeast Spain) were exposed to 500-µM Pb(NO3)2 for 1 week. Results showed that this high dose of Pb induced no plant mortality nor senescence, though promoting plant nanism. Besides the efficiency of roots to accumulate Pb, shoots also demonstrate a high efficiency to translocate and accumulate this metal. Pb exposure decreased Zn uptake to the aerial part and reduced net photosynthetic rate (A), RuBisCO activity, chlorophyll, and soluble sugar contents in shoots. Moreover, in shoots, Pb exposure increased the levels of O2- and decreased antioxidant capacity, culminating with a loss of cell membrane integrity (electrolyte leakage) and increased protein oxidation. Compared to controls, exposed roots had less Mn and Zn levels, and despite the rise in H2O2 levels, they were able to modulate non-protein thiols presenting a robust defense capacity. This capacity may support the roots' ability to maintain cell membrane integrity (electrolyte leakage) with regard to control. Principal component analysis (PCA) contributed to elucidate how this species adjusts physiological mechanisms to cope with Pb toxicity, showing that roots and shoots evolved different antioxidant defenses, which demonstrates the importance of organ specificity in the response of Z. fabago to heavy metals.

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.

Induced plant-defenses suppress herbivore reproduction but also constrain predation of their offspring.

Inducible anti-herbivore defenses in plants are predominantly regulated by jasmonic acid (JA). On tomato plants, most genotypes of the herbivorous generalist spider mite Tetranychus urticae induce JA defenses and perform poorly on it, whereas the Solanaceae specialist Tetranychus evansi, who suppresses JA defenses, performs well on it. We asked to which extent these spider mites and the predatory mite Phytoseiulus longipes preying on these spider mites eggs are affected by induced JA-defenses. By artificially inducing the JA-response of the tomato JA-biosynthesis mutant def-1 using exogenous JA and isoleucine (Ile), we first established the relationship between endogenous JA-Ile-levels and the reproductive performance of spider mites. For both mite species we observed that they produced more eggs when levels of JA-Ile were low. Subsequently, we allowed predatory mites to prey on spider mite-eggs derived from wild-type tomato plants, def-1 and JA-Ile-treated def-1 and observed that they preferred, and consumed more, eggs produced on tomato plants with weak JA defenses. However, predatory mite oviposition was similar across treatments. Our results show that induced JA-responses negatively affect spider mite performance, but positively affect the survival of their offspring by constraining egg-predation.

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.