Effect of Lead and Copper on Photosynthetic Apparatus in Citrus (Citrus aurantium L.) Plants. The Role of Antioxidants in Oxidative Damage as a Response to Heavy Metal Stress.

Photosynthetic changes and antioxidant activity to oxidative stress were evaluated in sour orange (Citrus aurantium L.) leaves subjected to lead (Pb), copper (Cu) and also Pb + Cu toxicity treatments, in order to elucidate the mechanisms involved in heavy metal tolerance. The simultaneous effect of Pb- and Cu on growth, concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2), chlorophylls, flavonoids, carotenoids, phenolics, chlorophyll fluorescence and photosynthetic parameters were examined in leaves of Citrus aurantium L. plants. Exogenous application of Pb and Cu resulted in an increase in leaf H2O2 and lipid peroxidation (MDA). Toxicity symptoms of both Pb and Cu treated plants were stunted growth and decreased pigments concentration. Furthermore, photosynthetic activity of treated plants exhibited a significant decline. The inhibition of growth in Pb and Cu-treated plants was accompanied by oxidative stress, as indicated by the enhanced lipid peroxidation and the high H2O2 concentration. Furthermore, antioxidants in citrus plants after exposure to high Pb and Cu concentrations were significantly increased compared to control and low Pb and Cu treatments. In conclusion, this study indicates that Pb and Cu promote lipid peroxidation, disrupt membrane integrity, reduces growth and photosynthesis and inhibit mineral nutrition. Considering the potential for adverse human health effects associated with high concentrations of Pb and Cu contained in edible parts of citrus plants the study signals that it is important to conduct further research into the accessibility and uptake of the tested heavy metals in the soil and whether they pose risks to humans.

Melatonin and resveratrol reverse the toxic effect of high boron (B) and modulate biochemical parameters in pepper plants (Capsicum annuum L.).

The objectives of this research were to test a possible involvement of melatonin (MEL) and resveratrol (RES) in restoring growth and to control boron (B) toxicity in peppers. The plants were subjected to four different nutrient solution treatments as following: 1) half-strength Hoagland's nutrient solution (Control), 2) half-strength Hoagland's nutrient solution+100 µM B (100 µMB), 3) half-strength Hoagland's nutrient solution+100 µM boron+100 µMresveratrol (100 µMRES), and 4) half-strength Hoagland's nutrient solution+100 µM B+1 µMmelatonin (1 µM MEL). Pepper plants subjected to B excess (100 µM) for 68 days (d) exhibited visible B toxicity symptoms, reduced rate of photosynthesis (Pn) and reduced dry weight (DW), while their leaf and fruit had the greatest increase of B concentration. The reduction of photosynthesis was restored, the reduction of DW was prevented, while the B leaf and fruit accumulation was moderated with the application of both 100 µMresveratrol (RES) and 1 µMmelatonin (MEL). Moreover, plants exposed to MEL and/or RES displayed no visible B toxicity symptoms. The present study revealed a novel role of MEL and/or RES in the adaptation of pepper plants to B excess based on plant growth, physiological and biochemical criteria.

Melatonin combined with ascorbic acid provides salt adaptation in Citrus aurantium L. seedlings.

Ascorbic acid (AsA) and melatonin (Mel) are known molecules participating in stress resistance, however, their combined role in counteracting the impact of salinity in plants is still unknown. In this work the effect of exogenous application of 0.50 mΜ AsA, 1 µΜ Mel and their combination (AsA + Mel) on various stress responses in leaves and roots of Citrus aurantium L. seedlings grown under 100 mΜ NaCl for 30 days was investigated. Application of AsA, Mel or AsA + Mel to saline solution decreased NaCl-induced electrolyte leakage and lipid peroxidation and prevented NaCl-associated toxicity symptoms and pigments degradation. Also, leaves exposed to combined AsA + Mel treatment displayed lower Cl(-) accumulation. Treatments with AsA and/or Mel modulated differently carbohydrates, proline, phenols, glutathione and the total antioxidant power of tissues as well as the activities of SOD, APX, POD, GR and PPO compared to NaCl alone treatment. Exposure of leaves and roots to chemical treatments and especially to combined AsA and Mel application was able to regulate CaMIPS, CaSLAH1 and CaMYB73 expression, indicating that sugar metabolism, ion homeostasis and transcription regulation were triggered by AsA and Mel. These results provide evidence that the activation of the metabolic pathways associated with combined AsA and Mel application are linked with salt adaptation in citrus plants.

Resveratrol and its combination with α-tocopherol mediate salt adaptation in citrus seedlings.

Resveratrol, a phytoalexin found in red wine, has the potential to impact a variety of human diseases but its function in plants exposed to stressful conditions is still unknown. In the present study the effect of exogenous application of resveratrol (Res), α-tocopherol (α-Toc) and their combination (Res+α-Toc) in salt adaptation of citrus seedlings was investigated. It was found that Res, α-Toc or Res+α-Toc treatments reduced NaCl-derived membrane permeability (EL), lipid peroxidation (MDA) and pigments degradation, whereas companied Res and α-Toc application also reduced H2O2 accumulation in leaves and restored the reduction of photosynthesis induced by NaCl. Application of Res under salinity retained Cl- in roots while Res+α-Toc reduced the translocation of Na+ and Cl- to leaves. Carbohydrates and proline, phenols, total ascorbic acid and glutathione were remarkably affected by NaCl as well as by chemical treatments in leaves and roots of citrus. NaCl treatment increased the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), glutathione reductase (GR), polyphenol oxidase (PPO) in leaves while SOD and POD activities were decreased in roots by this treatment. Also, Res, α-Toc or Res+α-Toc treatments displayed tissue specific activation or deactivation of the antioxidant enzymes. Overall, this work revealed a new functional role of Res in plants and provided evidence that the interplay of between Res and α-Toc is involved in salinity adaptation.

Volatile constituents and antioxidant activity of peel, flowers and leaf oils of Citrus aurantium L. growing in Greece.

The volatile constituents of the essential oils of the peel, flower (neroli) and leaves (petitgrain) of bitter orange (Citrus aurantium L.) growing in Greece were studied by GC-MS. The analytical procedures enabled the quantitative determination of 31 components. More specifically, the components of the essential oils identified were: twelve in the peel, twenty-six in the flowers, and twenty and sixteen in old and young leaves, respectively. The major constituents of the different parts of Citrus aurantium L. essential oils were: ß-pinene (0.62%-19.08%), limonene (0.53%-94.67%), trans-ß-ocimene (3.11%-6.06%), linalool (0.76%-58.21%), and α-terpineol (0.13%-12.89%). The DPPH test demonstrated that the essential oils in the old leaves had the maximum antioxidant activity, followed by the flowers, young leaves and the peel in that order. This study updates the data in the literature on the essential oils of bitter orange, and provides information on the composition of the oils for a further evaluation of this product.

Globular body production, their anatomy, DNase gel analysis and NDP kinase activity in root tips of Poncirus trifoliata L.

Green globular bodies were developed from Poncirus trifoliata L. root tip explants as a response to addition in the substrate of different growth regulators. From the globular bodies, shoots initiated and grew. Median section of the globular bodies reveals that they are composed of parenchyma cells and originate from the pericycle. The activity of DNases during shoot formation from globular bodies was influenced by the type and concentration of plant growth regulators that were added in the nutrient substrate. Peptide bands formation was also influenced by the increase of BA concentration. Consequently, BA, NAA and IAA combination influenced 5'-triphosphonucleosides (NTPs) appearance and activity in the presence of metal. Peptide bands resulted from the electrophoretic analysis of endogenous protein phosphorylation, proved to be catalytic subunits of NDP kinases, as they all phosphorylate diphosphonucleosides. The enzymes DNases and NDP kinases could be used as a scientific tool for the study of shoot formation from P. trifoliata L. green globular bodies.

Melatonin enhances root regeneration, photosynthetic pigments, biomass, total carbohydrates and proline content in the cherry rootstock PHL-C (Prunus avium × Prunus cerasus).

The present study, investigates the effects of melatonin (0, 0.05, 0.1, 0.5, 1, 5 and 10 µM) on the morphogenic and biochemical responses in the cherry rootstock PHL-C (Prunus avium L. × Prunus cerasus L.), from shoot tip explants. The incorporation of melatonin (0-10 µM) in the Murashige and Skoog (MS) medium, greatly influenced rooting either positively or negatively. Melatonin, irrespective of its concentration, had a negative effect concerning the number of roots. However, application of 0.5 µM melatonin significantly increased the root length; while 1 µM melatonin increased the root length by 2.5 times, and the fresh weight of the roots by 4 times, in comparison to the control. Although 0.05 µM melatonin increased rooting by 11.11%, 5 µM melatonin had a significant reduction on the number, the fresh weight of roots, and the rooting percentage. Melatonin concentration of 0.1 µM resulted in the greatest chlorophyll (a + b) content, and 5-10 µM reduced the chlorophyll concentration by 2 times, compared to the control. The high melatonin concentrations (5 and 10 µM), increased the levels of proline and carbohydrates in leaves by 3-4 times. In the roots, 0.5 µM of melatonin concentration increased the carbohydrate levels by 1.5 times, while 0.05, 0.1 and 1 µM melatonin concentration significantly reduced the proline content.

Comparative transcriptome analysis of two olive cultivars in response to NaCl-stress.

BACKGROUND: Olive (Olea europaea L.) cultivation is rapidly expanding and low quality saline water is often used for irrigation. The molecular basis of salt tolerance in olive, though, has not yet been investigated at a system level. In this study a comparative transcriptomics approach was used as a tool to unravel gene regulatory networks underlying salinity response in olive trees by simulating as much as possible olive growing conditions in the field. Specifically, we investigated the genotype-dependent differences in the transcriptome response of two olive cultivars, a salt-tolerant and a salt-sensitive one. METHODOLOGY/PRINCIPAL FINDINGS: A 135-day long salinity experiment was conducted using one-year old trees exposed to NaCl stress for 90 days followed by 45 days of post-stress period during the summer. A cDNA library made of olive seedling mRNAs was sequenced and an olive microarray was constructed. Total RNA was extracted from root samples after 15, 45 and 90 days of NaCl-treatment as well as after 15 and 45 days of post-treatment period and used for microarray hybridizations. SAM analysis between the NaCl-stress and the post-stress time course resulted in the identification of 209 and 36 differentially expressed transcripts in the salt-tolerant and salt-sensitive cultivar, respectively. Hierarchical clustering revealed two major, distinct clusters for each cultivar. Despite the limited number of probe sets, transcriptional regulatory networks were constructed for both cultivars while several hierarchically-clustered interacting transcription factor regulators such as JERF and bZIP homologues were identified. CONCLUSIONS/SIGNIFICANCE: A systems biology approach was used and differentially expressed transcripts as well as regulatory interactions were identified. The comparison of the interactions among transcription factors in olive with those reported for Arabidopsis might indicate similarities in the response of a tree species with Arabidopsis at the transcriptional level under salinity stress.

Effect of water deficit on leaf phenolic composition, gas exchange, oxidative damage and antioxidant activity of four Greek olive (Olea europaea L.) cultivars.

The olive tree (Olea europaea L.) is often exposed to severe water stress during the summer season. In this study, we determined the changes in total phenol content, oleuropein and hydroxytyrosol in the leaves of four olive cultivars ('Gaidourelia', 'Kalamon', 'Koroneiki' and 'Megaritiki') grown under water deficit conditions for two months. Furthermore, we investigated the photosynthetic performance in terms of gas exchange and chlorophyll a fluorescence, as well as malondialdehyde content and antioxidant activity. One-year-old self-rooted plants were subjected to three irrigation treatments that received a water amount equivalent to 100% (Control, C), 66% (Field Capacity 66%, FC(66)) and 33% (Field Capacity 33%, FC(33)) of field capacity. Measurements were conducted 30 and 60 days after the initiation of the experiment. Net CO(2) assimilation rate, stomatal conductance and F(v)/F(m) ratio decreased only in FC(33) plants. Photosynthetic rate was reduced mainly due to stomatal closure, but damage to PSII also contributed to this decrease. Water stress induced the accumulation of phenolic compounds, especially oleuropein, suggesting their role as antioxidants. Total phenol content increased in FC(33) treatment and oleuropein presented a slight increase in FC(66) and a sharper one in FC(33) treatment. Hydroxytyrosol showed a gradual decrease as water stress progressed. Malondialdehyde (MDA) content increased due to water stress, mostly after 60 days, while antioxidant activity increased for all cultivars in the FC(33) treatment. 'Gaidourelia' could be considered as the most tolerant among the tested cultivars, showing higher phenolic concentration and antioxidant activity and lower lipid peroxidation and photochemical damage after two months of water stress. The results indicated that water stress affected olive tree physiological and biochemical parameters and magnitude of this effect depended on genotype, the degree of water limitation and duration of treatment. However, the severity as well as the duration of water stress might exceed antioxidant capacity, since MDA levels and subsequent oxidative damage increased after two months of water deficit.

Toxicity effects of olive-mill wastewater on growth, photosynthesis and pollen morphology of spinach plants.

Olive mill-wastewater (OMW), a by-product of the olive oil extraction process, represents a significant environmental problem in Mediterranean areas. We studied the impact of OMW dilutions (1:10 and 1:20) on growth, photosynthesis, proline and sugar accumulation as well as on pollen morphology of spinach (Spinacia oleracea L.) plants, to evaluate the application of OMW dilutions as pretreatment technique, prior to land disposal. Biomass, height, total chlorophyll and leaf area of spinach declined progressively with decreasing OMW dilution. Since fatty acids and phenolic compounds (present in the OMW) are considered precursors in the polymerization of sporopollenin, we suggest that under OMW treatment spinach plants seem to 'direct' the excess of these substances in the production and formation of increased pollen grains. Proline did not accumulate under OMW stress, but decreased possible due to transport to pollens in response to increased demand to over-production of pollens. Both OMW dilutions resulted in a decreased efficiency of PSII functioning and an increased excitation pressure (1-q(p)). It is concluded that, higher than 1:20 OMW dilutions should be used, and/or additional treatment should be applied before use of the OMW in the environment.

Melatonin promotes adventitious root regeneration in in vitro shoot tip explants of the commercial sweet cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and MxM 60 (P. avium × P. mahaleb).

The objectives of this study were to test the effects of melatonin (N-acetyl-5-methoxytryptamine), a natural compound of edible plants on the rooting of certain commercial sweet cherry rootstocks. Shoot tip explants from previous in vitro cultures of the cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and M × M 60 (P. avium × P. mahaleb) were included in the experiment. The effect of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) alone or in combination with melatonin was tested concerning their rooting potential. Seven concentrations of melatonin (0, 0.05, 0.1, 0.5, 1, 5, and 10 µM) alone or in combination with 5.71 µM of IAA or 4.92 µM of IBA were tested. For each rootstock, 21 treatments were included. The explants were grown in glass tubes containing 10 mL of substrate. The parameters measured include rooting percentage, number of roots per rooted explant, root length, and callus formation. The data presented in this study show that melatonin has a rooting promoting effect at a low concentration but a growth inhibitory effect at high concentrations. In the absence of auxin, 1 µM melatonin had auxinic response concerning the number and length of roots, but 10 µM melatonin was inhibitory to rooting in all the tested rootstocks. The final conclusion of this experiment is that exogenously applied melatonin acted as a rooting promoter and its action was similar to that of IAA.

Polyphenol oxidase activity of two olive (Olea europaea L.) cultivars as an early criterion of Mn toxicity.

BACKGROUND: The time course of polyphenol oxidase (PPO) activity in the leaves of two olive cultivars (Picual and FS-17) irrigated with nutrient solutions differing in Mn concentration (0, 2 and 1280 micromol L(-1)) was studied under hydroponic conditions to determine whether PPO activity could be used as an early criterion of Mn status of olive plants, and to elucidate whether genotypic differences exist between the two olive cultivars studied, concerning the effect of Mn concentration on PPO activity. RESULTS: In all the Mn treatments, PPO activity was greater in Picual than in FS-17. Under excess Mn (1280 micromol L(-1)), PPO activity gradually increased with time, starting from day 30 of the experiment in both cultivars, and this increase preceded the appearance of Mn toxicity symptoms. In contrast, in the other two Mn treatments (0 and 2 micromol L(-1)) PPO activity increased and afterwards decreased during the experiment, but the trend was not clear. In the 1280 micromol L(-1) treatment, PPO activity linearly increased (R = 0.8836 for Picual and 0.943 for FS-17) with the increase of Mn concentration in the leaves of both cultivars. In the 1280 micromol L(-1) Mn treatment, PPO activity was negatively related with Fe and Zn concentrations in the leaves, and positively in the 0 and 2 micromol L(-1) Mn treatments with the Ca, Mg and K concentrations. CONCLUSION: From the differential time course of PPO activity in the three Mn treatments (0, 2 and 1280 micromol L(-1)), it is concluded that periodic measurements of PPO activity in the leaves of the olive cultivars Picual and FS-17 can be used for the early detection of Mn toxicity (before the appearance of symptoms).

Mn-induced changes in leaf structure and chloroplast ultrastructure of Citrus volkameriana (L.) plants.

Seedlings of Citrus volkameriana (L.) were grown hydroponically for 43 days in order to study the effect of Mn concentration (0, 2, 14, 98 and 686 microM) in the nutrient solution on leaf anatomy and mesophyll chloroplast ultrastructure. Increasing Mn concentration stimulated leaf lamina thickness. The size of mesophyll chloroplasts decreased and increased under 0 and 686 microM Mn, respectively, compared to the intermediate Mn concentrations, similar with regard to the number of chloroplasts per mesophyll cell area. Thylakoid membranes of plants grown under 0 microM Mn were somewhat swelled, while those in other Mn treatments did not present any visible malformation. The relative volume of starch grains per chloroplast was significantly smaller under 0-98 microM Mn (12.8-16.0%) than in the treatment with 686 microM Mn (67.6%). Further, under 686 microM Mn, dark deposits were found in vacuoles. The existence of a cell adaptation mechanism to excessive Mn availability (686 microM Mn) by increasing the size of chloroplasts as well as their number per cellular area, is discussed.

Effects of 4-month Fe deficiency exposure on Fe reduction mechanism, photosynthetic gas exchange, chlorophyll fluorescence and antioxidant defense in two peach rootstocks differing in Fe deficiency tolerance.

Fe deficiency was imposed by omission of Fe (-Fe), or by inclusion of bicarbonate (supplied as 20 mM NaHCO3) in the nutrient solution in two contrasting peach rootstocks (GF-677; tolerant to Fe deficiency and Cadaman; sensitive to Fe deficiency) for 4 months. In the Fe-deprived leaves and roots, and especially in those treated with bicarbonate, a decrease in Fe concentrations was recorded. Omission of Fe resulted in an increase of the activity of root Fe(III)-chelate reductase (FCR) in both rootstocks, whereas FCR activity decreased in the bicarbonate-treated roots of Cadaman. The results obtained from the FCR assay were confirmed by an agarose-based staining technique used to localize FCR activity. Also, an agar-pH-test revealed that the roots of GF-677 exposed to (-Fe) treatment induced a strong H+ extrusion. In addition, Fe deficiency resulted in reduction of the total chlorophyll (CHL) content. Apart from the (-Fe)-treated leaves of GF-677, Fe deficiency caused a decline in the photosynthetic rate (P(n)) and stomatal conductance (g(s)), without changes of the intercellular CO2 concentration (C(i)), as well as a reduction in the maximum quantum yield of PSII (F(v)/F(m)) and the ratio between variable to initial fluorescence F(v)/F0. The above changes were particularly evident for the bicarbonate-treated leaves of Cadaman. On the other hand, Fe deficiency resulted in an increase of leaf superoxide dismutase (SOD) activity and a depression of catalase (CAT) activity in the leaves and roots, irrespective of the rootstock. Although the non-enzymatic antioxidant activity (FRAP values) was increased in the roots of both rootstocks exposed to -Fe treatment, however, FRAP values were stimulated in the (-Fe)-treated leaves of GF-677 and decreased in the bicarbonate-treated leaves of Cadaman. The H2O2 content was increased in Fe-deprived tissues except for the (-Fe)-treated leaves and roots of GF-677. As a result of Fe deficiency, peroxidase (POD) activity and isoform expression were diminished in the tissues of Cadaman. However, in the tissues of GF-677 subjected to -Fe treatment POD activity was increased whereas an additional POD isoform was detected in the roots suggesting that expression of POD isoforms might be an important attribute linked to the tolerance to Fe deficiency.