Impact of Chitosan-Based Foliar Application on the Phytochemical Content and the Antioxidant Activity in Hemp (Cannabis sativa L.) Inflorescences.

In the present study, the phytochemical content and the antioxidant activity in the inflorescences of the monoecious hemp cultivar Codimono grown in southern Italy were assessed, and their elicitation was induced by foliar spray application of 50 mg/L and 250 mg/L of chitosan (CHT) at three different molecular weights (low, CHT L; medium, CHT M; high CHT H). The analysis of the phytochemical profile confirmed that cannabinoids were the most abundant class (54.2%), followed by flavonoids (40.3%), tocopherols (2.2%), phenolic acids (1.9%), and carotenoids (1.4%). Cannabinoids were represented almost exclusively by cannabidiol, whereas cannabigerol and Δ9-tetrahydrocannabinol were detected at very low levels (the latter was below the legal limit of 0.3%). The most abundant flavonoids were orientin and vitexin, whereas tocopherols were mainly represented by α-tocopherol. The antioxidant activity was found to be positively correlated with flavonoids and tocopherols. Statistical analysis revealed that the CHT treatments significantly affected the phytochemical content and the antioxidant activity of hemp inflorescences. Notably, a significant increase in the total phenolic content (from +36% to +69%), the α-tocopherol (from +45% to +75%) and ß+γ-tocopherol (from +35% to +82%) contents, and the ABTS radical scavenging activity (from +12% to +28%) was induced by all the CHT treatments. In addition, treatments with CHT 50 solutions induced an increase in the total flavonoid content (from +12% to +27%), as well as in the vitexin (from +17% to +20%) and orientin (from +20% to +30%) contents. Treatment with CHT 50 L almost always resulted in the greatest increases. Overall, our findings indicated that CHT could be used as a low-cost and environmentally safe elicitor to improve the health benefits and the economic value of hemp inflorescences, thus promoting their employment in the food, pharmaceutical, nutraceutical, and cosmetic supply chains.

Effect of Genotype, Year, and Their Interaction on the Accumulation of Bioactive Compounds and the Antioxidant Activity in Industrial Hemp (Cannabis sativa L.) Inflorescences.

The phytochemical content and the antioxidant activity in the inflorescences of six industrial hemp (Cannabis sativa L.) genotypes, four monoecious (Codimono, Carmaleonte, Futura 75, and Santhica 27), and two dioecious (Fibrante and Carmagnola Selezionata), were assessed for three consecutive years from 2018 to 2020. The total phenolic content, total flavonoid content, and antioxidant activity were determined by spectrophotometric measurements, whereas HPLC and GC/MS were used to identify and quantify the phenolic compounds, terpenes, cannabinoids, tocopherols, and phytosterols. All the measured traits were significantly affected by genotype (G), cropping year (Y), and their interaction (G × Y), although the Y effect prevailed as a source of variation, ranging from 50.1% to 88.5% for all the metabolites except cannabinoids, which were equally affected by G, Y, and G × Y interaction (33.9%, 36.5%, and 21.4%, respectively). The dioecious genotypes presented a more constant performance over the three years compared to the monoecious genotypes, with the highest and most stable phytochemical content observed in the inflorescences of Fibrante, which was characterized by the highest levels of cannabidiol, α-humulene and ß-caryophyllene, which may confer on the inflorescences of this genotype a great economic value due to the important pharmacological properties of these metabolites. Conversely, the inflorescences of Santhica 27 were characterized by the lowest accumulation of phytochemicals over the cropping years, with the notable exception of cannabigerol, a cannabinoid that exhibits a wide range of biological activities, which was found at its highest level in this genotype. Overall, these findings can be used by breeders in future programs aimed at the selection of new hemp genotypes with improved levels of phytochemicals in their inflorescences, which can provide better health and industrial benefits.

Candidate Genes Associated with Abiotic Stress Response in Plants as Tools to Engineer Tolerance to Drought, Salinity and Extreme Temperatures in Wheat: An Overview.

Wheat represents one of the most important staple food crops worldwide and its genetic improvement is fundamental to meeting the global demand of the growing population. However, the environmental stresses, worsened by climate change, and the increasing deterioration of arable land make it very difficult to fulfil this demand. In light of this, the tolerance of wheat to abiotic stresses has become a key objective of genetic improvement, as an effective strategy to ensure high yields without increasing the cultivated land. Genetic erosion related to modern agriculture, whereby elite, high-yielding wheat varieties are the product of high selection pressure, has reduced the overall genetic diversity, including the allelic diversity of genes that could be advantageous for adaptation to adverse environmental conditions. This makes traditional breeding a less effective or slower approach to generating new stress-tolerant wheat varieties. Either mining for the diversity of not-adapted large germplasm pools, or generating new diversity, are the mainstream approaches to be pursued. The advent of genetic engineering has opened the possibility to create new plant variability and its application has provided a strong complement to traditional breeding. Genetic engineering strategies such as transgenesis and genome editing have then provided the opportunity to improve environmental tolerance traits of agronomic importance in cultivated species. As for wheat, several laboratories worldwide have successfully produced transgenic wheat lines with enhanced tolerance to abiotic stresses, and, more recently, significant improvements in the CRISPR/Cas9 tools available for targeted variations within the wheat genome have been achieved. In light of this, the present review aims to provide successful examples of genetic engineering applications for the improvement of wheat adaptation to drought, salinity and extreme temperatures, which represent the most frequent and most severe events causing the greatest losses in wheat production worldwide.

Genome-Wide Identification, Characterization and Expression Pattern Analysis of the γ-Gliadin Gene Family in the Durum Wheat (Triticum durum Desf.) Cultivar Svevo.

Very recently, the genome of the modern durum wheat cv. Svevo was fully sequenced, and its assembly is publicly available. So, we exploited the opportunity to carry out an in-depth study for the systematic characterization of the γ-gliadin gene family in the cv. Svevo by combining a bioinformatic approach with transcript and protein analysis. We found that the γ-gliadin family consists of nine genes that include seven functional genes and two pseudogenes. Three genes, Gli-γ1a, Gli-γ3a and Gli-γ4a, and the pseudogene Gli-γ2a* mapped on the A genome, whereas the remaining four genes, Gli-γ1b, Gli-γ2b, Gli-γ3b and Gli-γ5b, and the pseudogene Gli-γ4b* mapped on the B genome. The functional γ-gliadins presented all six domains and eight-cysteine residues typical of γ-gliadins. The Gli-γ1b also presented an additional cysteine that could possibly have a role in the formation of the gluten network through binding to HMW glutenins. The γ-gliadins from the A and B genome differed in their celiac disease (CD) epitope content and composition, with the γ-gliadins from the B genome showing the highest frequency of CD epitopes. In all the cases, almost all the CD epitopes clustered in the central region of the γ-gliadin proteins. Transcript analysis during seed development revealed that all the functional γ-gliadin genes were expressed with a similar pattern, although significant differences in the transcript levels were observed among individual genes that were sometimes more than 60-fold. A progressive accumulation of the γ-gliadin fraction was observed in the ripening seeds that reached 34% of the total gliadin fraction at harvest maturity. We believe that the insights generated in the present study could aid further studies on gliadin protein functions and future breeding programs aimed at the selection of new healthier durum wheat genotypes.

Chitosan-Induced Activation of the Antioxidant Defense System Counteracts the Adverse Effects of Salinity in Durum Wheat.

The present study was carried out with the aim of (i) evaluating the effect of chitosan (CTS) on the growth of durum wheat under salinity and (ii) examining CTS-regulated mechanisms of salinity tolerance associated with the antioxidant defense system. To achieve these goals, durum wheat seedlings were treated with CTS at different molecular weight, low (L-CTS, 50-190 kDa), medium (M-CTS, 190-310 kDa) and high (H-CTS, 310-375 kDa). The results obtained show that exposure to 200 mM NaCl reduced the shoot and the root dried biomass by 38% and 59%, respectively. The growth impairment induced by salinity was strongly correlated with an increase in the superoxide anion production (5-fold), hydrogen peroxide content (2-fold) and malondialdehyde (MDA) content (4-fold). Seedlings responded to the oxidative stress triggered by salinity with an increase in the total phenolic content (TPC), total flavonoid content (TFC) and total antioxidant activity (TAA) by 67%, 51% and 32%, respectively. A salt-induced increase in the activity of the antioxidant enzymes superoxide dismutase and catalase (CAT) of 89% and 86%, respectively, was also observed. Treatment of salt-stressed seedlings with exogenous CTS significantly promoted seedling growth, with the strongest effects observed for L-CTS and M-CTS, which increased the shoot biomass of stressed seedlings by 32% and 44%, respectively, whereas the root dried biomass increased by 87% and 64%, respectively. L-CTS and M-CTS treatments also decreased the superoxide anion production (57% and 59%, respectively), the hydrogen peroxide content (35% and 38%, respectively) and the MDA content (48% and 56%, respectively) and increased the TPC (23% and 14%, respectively), the TFC (19% and 10%, respectively), the TAA (up to 10% and 7%, respectively) and the CAT activity (29% and 20%, respectively). Overall, our findings indicate that CTS exerts its protective role against the oxidative damages induced by salinity by enhancing the antioxidant defense system. L-CTS and M-CTS were the most effective in alleviating the adverse effect of NaCl, thus demonstrating that the CTS action is strictly related to its molecular weight.

The Molecular and Functional Characterization of the Durum Wheat Lipoxygenase TdLOX2 Suggests Its Role in Hyperosmotic Stress Response.

In plants, lipoxygenases (LOXs) are involved in various processes, such as growth, development, and response to stress cues. In the present study, the expression pattern of six durum wheat LOX-encoding genes (TdLpx-B1.1, TdLpx-B1.2, TdLpx-A2, TdLpx-B2, TdLpx-A3 and TdLpx-B3) under hyperosmotic stress was investigated. With osmotic (0.42 M mannitol) and salt (0.21 M NaCl) stress imposed at the early stages of seedling growth, a strong induction of the TdLpx-A2 gene expression in the shoots paralleled an equally strong increase in the LOX activity. Enhanced levels of malondialdehyde (MDA) and increased rates of superoxide anion generation were also observed as a result of the stress imposition. Sequence analysis of the TdLOX2 encoded by the TdLpx-A2 gene revealed that it belonged to the type-1 9-LOX group. When overexpressed in E. coli, TdLOX2 exhibited normal enzyme activity, high sensitivity to specific LOX inhibitors, with 76% and 99% inhibition by salicylhydroxamic and propyl gallate, respectively, and a preference for linoleic acid as substrate, which was converted exclusively to its corresponding 13-hydroperoxide. This unexpected positional specificity could be related to the unusual TV/K motif that in TdLOX2 replaces the canonical TV/R motif of 9-LOXs. Treatment of seedlings with propyl gallate strongly suppressed the increase in LOX activity induced by the hyperosmotic stress; the MDA accumulation was also reduced but less markedly, whereas the rate of superoxide anion generation was even more increased. Overall, our findings suggest that the up-regulation of the TdLpx-A2 gene is a component of the durum wheat response to hyperosmotic stress and that TdLOX2 may act by counteracting the excessive generation of harmful reactive oxygen species responsible for the oxidative damages that occur in plants under stress.

Carotenoids in Cereal Food Crops: Composition and Retention throughout Grain Storage and Food Processing.

Carotenoids are C40 isoprenoids synthesized by plants, as well as some bacteria, fungi and algae, that have been reported to be responsible for a number of benefits conferred on human health. The inability of animals and humans to synthesize de novo these compounds is the reason why they must be introduced from dietary sources. In cereal grains, carotenoids are important phytochemicals responsible for the characteristic yellow colour of the endosperm, which confers nutritional and aesthetic quality to cereal-based products. Cereals are staple foods for a large portion of the world population, and the biofortification of cereal grains with carotenoids may represent a simple way to prevent many human diseases and disorders. Unfortunately, evidence exists that the storage and processing of cereal grains into food products may negatively impact their carotenoid content; so, this loss should be taken into consideration when analysing the potential health benefits of the cereal-based products. Focusing on the recent updates, this review summarizes the chemical composition of the carotenoids in the grains of staple cereals, including wheat, maize, rice and sorghum, the main factors that affect their carotenoid content during storage and processing and the most fruitful strategies used improve the grain carotenoid content and limit the carotenoid post-harvest losses.

Analysis of metabolic and mineral changes in response to salt stress in durum wheat (Triticum turgidum ssp. durum) genotypes, which differ in salinity tolerance.

The key mechanisms of salinity tolerance (ST) in durum wheat were investigated, with five genotypes used to determine changes in morpho-physiological traits and mineral and metabolite contents after exposure to 50, 100 and 200 mM NaCl. Plant growth impairment was evident at the highest salt level. Under this condition, a wide range of shoot Na+ contents and ST were observed within genotypes. However, no significant correlation was seen between ST and Na+ exclusion from the shoots, which indicates that tissue tolerance also has a role. Consistent with this, there was significant correlation between ST and the Na+:K+ ratio in the shoots. Indeed, the maintenance of the shoot Na+ and K+ homeostasis was found to be essential to achieve osmotic adjustment, which relied substantially on inorganic osmolytes, and to avoid toxicity symptoms, such as chlorophyll loss, which appeared only at the highest salinity level. Consistently, the metabolite changes occurred mainly in the shoots, with a dual response to salinity: (i) a conserved response that was common to all the genotypes and resulted in the accumulation of proline and in the depletion of organic acids, including some intermediates of the Krebs cycle; and (ii) a genotype-specific response that involved the accumulation of GABA, threonine, leucine, glutamic acid, glycine, mannose and fructose and appeared related to the different tolerance of genotypes to salinity. The lower magnitude of response to salinity detected in the roots confirmed the major role of the shoots in the determination of ST of durum wheat.

Molecular Approaches to Genetically Improve the Accumulation of Health-Promoting Secondary Metabolites in Staple Crops-A Case Study: The Lipoxygenase-B1 Genes and Regulation of the Carotenoid Content in Pasta Products.

Secondary metabolites, also known as phytochemicals, represent a large subset of plant molecules that include compounds with health-promoting effects. Indeed, a number of epidemiological studies have shown that, when taken regularly and in adequate amounts, these molecules can have long-term beneficial effects on human health, through reduction of the incidence of degenerative diseases, such as cardiovascular diseases, obesity, diabetes, and cancer. As the dietary intake of these phytochemicals is often inadequate, various strategies are in use to improve their content in staple crops, and the end-products thereof. One of the most effective strategies is crop improvement through genetic approaches, as this is the only way to generate new cultivars in which the high accumulation of a given phytochemical is stably fixed. Efforts to genetically improve quality traits are rapidly evolving, from classical breeding to molecular-assisted approaches; these require sound understanding of the molecular bases underlying the traits, to identify the genes/alleles that control them. This can be achieved through global analysis of the metabolic pathway responsible for phytochemical accumulation, to identify the link between phytochemical content and the activities of key enzymes that regulate the metabolic pathway, and between the key enzymes and their encoding genes/alleles. Once these have been identified, they can be used as markers for selection of new improved genotypes through biotechnological approaches. This review provides an overview of the major health-promoting properties shown to be associated with the dietary intake of phytochemicals, and describes how molecular approaches provide means for improving the health quality of edible crops. Finally, a case study is illustrated, of the identification in durum wheat of the Lipoxygenase-B1 genes that control the final carotenoid content in semolina-based foods, such as pasta products.

Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria-An Amazing Defense Tool Against Hyperosmotic Stress.

In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K(+)/H(+) antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress (mannitol or NaCl), PmitoKATP was found to be activated by ROS, so inhibiting further large-scale ROS production according to a feedback mechanism; moreover, a stress-activated phospholipase A2 may generate FFAs, further activating the channel. In conclusion, a main property of PmitoKATP is the ability to keep in balance the control of harmful ROS with the mitochondrial/cellular bioenergetics, thus preserving ATP for energetic needs of cell defense under stress.

Cloning, expression analysis, and functional characterization of two secretory phospholipases A2 in durum wheat (Triticum durum Desf.).

We previously isolated four cDNAs in durum wheat, TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV, that encode proteins with homology to plant secretory phospholipases A2 (sPLA2s) (Verlotta et al., Int. J. Mol. Sci., 14, 2013, 5146-5169). In this study, we have further characterized TdsPLA2II and TdsPLA2III sequences that, on the basis of our previous findings, might encode sPLA2 isoforms with different features. Functional analysis revealed that, similarly to other known sPLA2s, TdsPLA2II and TdsPLA2III have an optimum at pH 9.0, require Ca(2+), are heat stable, and are inhibited by the disulfide-bond-reducing agent dithiothreitol. However, differences emerged between these TdsPLA2 isoforms. Transcript analysis revealed that the TdsPLA2III gene is highly up-regulated under different environmental stresses; conversely, the TdsPLA2II gene is expressed at constant levels under almost all of the stress conditions examined. Moreover, TdsPLA2II is saturated at micromolar substrate and Ca(2+) concentrations, whereas TdsPLA2III requires millimolar concentrations to reach maximal activity. This suggests that TdsPLA2II normally functions under optimal conditions in vivo, whereas TdsPLA2III is only partially activated, depending on the specific phospholipid and Ca(2+) levels. Altogether these data lead to the hypothesis that in vivo TdsPLA2II and TdsPLA2III are differently regulated at both molecular and biochemical level and that TdsPLA2III plays a major role in durum wheat response to adverse environmental conditions.

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.

Transport pathways--proton motive force interrelationship in durum wheat mitochondria.

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoK(ATP)) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoK(ATP) neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoK(ATP) to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoK(ATP) and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa.

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering.

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP(+)-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.

The uniqueness of the plant mitochondrial potassium channel.

The ATP-inhibited Plant Mitochondrial K(+) Channel (PmitoKATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K(+) uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoKATP and K(+)/H(+) antiporter allows such a great operation of a K(+) cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoKATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics.

Secretory Phospholipases A2 in Durum Wheat (Triticum durum Desf.): Gene Expression, Enzymatic Activity, and Relation to Drought Stress Adaptation.

Phospholipases A2 (PLA2s) are known to mediate signaling cascades during plant growth and development, as well as biotic and abiotic stress responses. In this context, the present study provides extensive characterization of specific PLA2s in durum wheat, and assesses their involvement in durum wheat response to drought stress. In durum wheat leaves, four full-length expressed sequences encoding putative PLA2s were isolated and characterized as belonging to the class of secretory PLA2s (sPLA2s): TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV. PLA2 activity was also detected, the characteristics of which resemble those of previously characterized plant sPLA2s: strong preference for phospholipids; requirement for millimolar Ca2+ concentrations; optimal activity at basic pH; heat stability; and inhibition by the reducing agent dithiothreitol. With drought stress imposed at both the vegetative and reproductive stages, accumulation of TdsPLA2I and TdsPLA2III transcripts, and to a lesser extent of TdsPLA2IV transcript, paralleled increased PLA2 activity; both transcript levels and enzymatic activity decreased as a consequence of stress recovery. Consistently, free fatty acid analysis of drought-stressed leaves revealed increased linoleate, linolenate and palmitate contents, which were reversed by plant re-watering. Overall, these findings strongly suggest that there are inducible sPLA2 isoforms in durum wheat that have roles in orchestrating the plant response to drought stress.

The existence of phospholipase A(2) activity in plant mitochondria and its activation by hyperosmotic stress in durum wheat (Triticum durum Desf.).

The activity of mitochondrial phospholipase A(2) (PLA(2)) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA(2)/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA(2) (DWM-PLA(2)) showed maximal activity at pH 9.0 and 1mM Ca(2+), hyperbolic kinetics (K(m)=90±6µM, V(max)=29±1nmolmin(-1)mg(-1) of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA(2), that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca(2+)-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA(2) is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141-154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress.

Potassium channel-oxidative phosphorylation relationship in durum wheat mitochondria from control and hyperosmotic-stressed seedlings.

Durum wheat mitochondria (DWM) possess an ATP-inhibited K(+) channel, the plant mitoK(ATP) (PmitoK(ATP) ), which is activated under environmental stress to control mitochondrial ROS production. To do this, PmitoK(ATP) collapses membrane potential (ΔΨ), thus suggesting mitochondrial uncoupling. We tested this point by studying oxidative phosphorylation (OXPHOS) in DWM purified from control seedlings and from seedlings subjected both to severe mannitol and NaCl stress. In severely-stressed DWM, the ATP synthesis via OXPHOS, continuously monitored by a spectrophotometric assay, was about 90% inhibited when the PmitoK(ATP) was activated by KCl. Contrarily, in control DWM, although PmitoK(ATP) collapsed ΔΨ, ATP synthesis, as well as coupling [respiratory control (RC) ratio and ratio between phosphorylated ADP and reduced oxygen (ADP/O)] checked by oxygen uptake experiments, were unaffected. We suggest that PmitoK(ATP) may play an important defensive role at the onset of the environmental/oxidative stress by preserving energy in a crucial moment for cell and mitochondrial bioenergetics. Consistently, under moderate mannitol stress, miming an early stress condition, the channel may efficiently control reactive oxygen species (ROS) generation (about 35-fold from fully open to closed state) without impairing ATP synthesis. Anyway, if the stress significantly proceeds, the PmitoK(ATP) becomes fully activated by decrease of ATP concentration (25-40%) and increase of activators [free fatty acids (FFAs) and superoxide anion], thus impairing ATP synthesis.

Activation of the plant mitochondrial potassium channel by free fatty acids and acyl-CoA esters: a possible defence mechanism in the response to hyperosmotic stress.

The effect of free fatty acids (FFAs) and acyl-CoA esters on K(+) uptake was studied in mitochondria isolated from durum wheat (Triticum durum Desf.), a species that has adapted well to the semi-arid Mediterranean area and possessing a highly active mitochondrial ATP-sensitive K(+) channel (PmitoK(ATP)), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoK(ATP) activity was monitored. Linoleate and other FFAs (laurate, palmitate, stearate, palmitoleate, oleate, arachidonate, and the non-physiological 1-undecanesulphonate and 5-phenylvalerate), used at a concentration (10 µM) unable to damage membranes of isolated mitochondria, stimulated K(+) uptake by about 2-4-fold. Acyl-CoAs also promoted K(+) transport to a much larger extent with respect to FFAs (about 5-12-fold). In a different experimental system based on safranin O fluorescence measurements, the dissipation of electrical membrane potential induced by K(+) uptake via PmitoK(ATP) was found to increase in the presence of 5-phenylvalerate and palmitoyl-CoA, both unable to elicit the activity of the Plant Uncoupling Protein. This result suggests a direct activation of PmitoK(ATP). Stimulation of K(+) transport by FFAs/acyl-CoAs resulted in a widespread phenomenon in plant mitochondria from different mono/dicotyledonous species (bread wheat, barley, triticale, maize, lentil, pea, and topinambur) and from different organs (root, tuber, leaf, and shoot). Finally, an increase in mitochondrial FFAs up to a content of 50 nmol mg(-1) protein, which was able to activate PmitoK(ATP) strongly, was observed under hyperosmotic stress conditions. Since PmitoK(ATP) may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.