Genome-Wide Expression Analysis of Glyoxalase I Genes Under Hyperosmotic Stress and Existence of a Stress-Responsive Mitochondrial Glyoxalase I Activity in Durum Wheat (Triticum durum Desf.)

Glyoxalase I (GLYI) catalyzes the rate-limiting step of the glyoxalase pathway that, in the presence of GSH, detoxifies the cytotoxic molecule methylglyoxal (MG) into the non-toxic D-lactate. In plants, MG levels rise under various abiotic stresses, so GLYI may play a crucial role in providing stress tolerance. In this study, a comprehensive genome database analysis was performed in durum wheat (Triticum durum Desf.), identifying 27 candidate GLYI genes (TdGLYI). However, further analyses of phylogenetic relationships and conserved GLYI binding sites indicated that only nine genes encode for putative functionally active TdGLYI enzymes, whose distribution was predicted in three different subcellular compartments, namely cytoplasm, plastids and mitochondria. Expression profile by qRT-PCR analysis revealed that most of the putative active TdGLYI genes were up-regulated by salt and osmotic stress in roots and shoots from 4-day-old seedlings, although a different behavior was observed between the two types of stress and tissue. Accordingly, in the same tissues, hyperosmotic stress induced an increase (up to about 40%) of both GLYI activity and MG content as well as a decrease of GSH (up to about –60%) and an increase of GSSG content (up to about 7-fold) with a consequent strong decrease of the GSH/GSSG ratio (up to about –95%). Interestingly, in this study, we reported the first demonstration of the existence of GLYI activity in highly purified mitochondrial fraction. In particular, GLYI activity was measured in mitochondria from durum wheat (DWM), showing hyperbolic kinetics with Km and Vmax values equal to 92 ± 0.2 μM and 0.519 ± 0.004 μmol min(–1) mg(–1) of proteins, respectively. DWM–GLYI resulted inhibited in a competitive manner by GSH (Ki = 6.5 ± 0.7 mM), activated by Zn(2+) and increased, up to about 35 and 55%, under salt and osmotic stress, respectively. In the whole, this study provides basis about the physiological significance of GLYI in durum wheat, by highlighting the role of this enzyme in the early response of seedlings to hyperosmotic stress. Finally, our results strongly suggest the existence of a complete mitochondrial GLYI pathway in durum wheat actively involved in MG detoxification under hyperosmotic stress

Antioxidant Activity of Free and Bound Compounds in Quinoa (Chenopodium quinoa Willd.) Seeds in Comparison with Durum Wheat and Emmer

Antioxidant activity (AA) of quinoa (Chenopodium quinoa Willd.) seeds, as well as of durum wheat (Triticum turgidum L. ssp. durum Desf.) and of emmer (T. turgidum L. ssp. dicoccum Sch¨ubler) grains, was evaluated by studying hydrophilic (H), lipophilic (L), free-soluble (FSP) and insoluble-bound (IBP) phenolic extracts using the new lipoxygenase/4-nitroso-N,N-dimethylaniline (LOX/RNO) method, able to simultaneously detect different antioxidant mechanisms, as well as using the Oxygen Radical Absorbance Capacity (ORAC) and the Trolox Equivalent Antioxidant Capacity (TEAC) assays, which measure the scavenging activity against peroxyl and ABTS [2,2-azino-bis-(3- ethylbenzothiazoline-6-sulfonate)] radicals, respectively. The species under study were compared with respect to the sum of AA values of H, L and FSP extracts (AAH+L+FSP), containing freely solvent-soluble antioxidants, and AA values of IBP extracts (AAIBP), representing the phenolic fraction ester-linked to insoluble cell wall polymers. The LOX/RNO and ORAC methods measured in quinoa flour a remarkable AAH+L+FSP higher than durum wheat, although lower than emmer; according to the same assays, the IBP component of quinoa resulted less active than the durum wheat and emmer ones. The TEAC protocol also revealed a high AAH+L+FSP for quinoa. Interestingly, the ratio AAH+L+FSP/AAH+L+FSP+IBP, as evaluated by the LOX/RNO and ORAC assays, resulted in quinoa higher than that of both durum wheat and emmer, and much higher than durum wheat, according to the TEAC protocol. This may suggest that antioxidants from quinoa seeds may be more readily accessible with respect to that of both the examined wheat species

ATP-sensitive cation-channel in wheat (triticum durum Desf.): Identification and characterization of a plant mitochondrial channel by patch-clamp

Indirect evidence points to the presence of K + channels in plant mitochondria. In the present study, we report the results of the first patch clamp experiments on plant mitochondria. Single-channel recordings in 150 mM potassium gluconate have allowed the biophysical characterization of a channel with a conductance of 150 pS in the inner mitochondrial membrane of mitoplasts obtained from wheat (Triticum durum Desf.). The channel displayed sharp voltage sensitivity, permeability to potassium and cation selectivity. ATP in the mM concentration range completely abolished the activity. We discuss the possible molecular identity of the channel and its possible role in the defence mechanisms against oxidative stress in plants

Evaluation of synergistic interactions of antioxidants from plant foods by a new method using soybean lipoxygenase

A proper evaluation of synergism among antioxidants remains so far rather difficult to obtain. We recently reported that a new method for antioxidant activity measurement, based on the secondary reaction of soybean lipoxygenase (LOX)-1 isoenzyme with 4-nitroso-N,N-dimethylaniline (RNO), so-called LOX/RNO method, may assess very well the synergism among antioxidants from durum wheat whole semolina. To evaluate whether this behaviour is generalizable to different food matrices, herein, antioxidants from other very different sources were analysed. For this purpose, antioxidant activity of food-grade preparations enriched in catechins, quercetin, resveratrol, tyrosol/hydroxytyrosol and lycopene was evaluated by the LOX/RNO method in comparison with the Trolox equivalent antioxidant ca pacity (TEAC) assay. The antioxidant activity values obtained by LOX/RNO method were 2–90-times higher than those obtained by the TEAC protocol, depending on the tested antioxidant. Synergism was evaluated by comparing antioxidant activity of the mixture of compounds (AAmix) with the sum of antioxidant activity values of individual compounds (AAsum). The LOX/RNO method revealed a strong synergism, being AAmix 5.69 ± 0.31 times higher than AAsum (statistically significant, p < 0.001), while the TEAC method showed a synergistic increase of only 0.31 ± 0.04 (statistically significant, p < 0.01). These findings suggest that the LOX/RNO method is able to assess very well the synergism in various food samples

In vitro antioxidant capacity of Opuntia spp. fruits measured by the LOX-FL method and its high sensitivity towards betalains

Current in vitro methodologies neglect or subestimate the contribution of betalains to antioxidant capacity in foods because they do not reflect their in vivo biological mechanisms. In this study, we assessed the sensibility of the lipoxygenase-fluorescein (LOX-FL) method towards betalains, phenolic compounds and ascorbic acid from Opuntia spp. fruits; and (ii) the antioxidant capacity of peel and pulp extracts from Opuntia ficus-indica L. Mill (var. Fresa, Colorada and Blanco) and Opuntia stricta var. Dillenii; by comparing the LOX-FL method to traditional antioxidant methods (ORAC and TEAC). The spectrophotometric monitoring of the LOX-FL reaction avoided interference caused by betalain pigments. Indicaxanthin and betanin showed high antiperoxidative and radical scavenging mechanisms in the LOX-FL assay. O. stricta var. Dillenii tissues the highest antioxidant capacity which correlated with betanin content. ORAC and TEAC antioxidant methods were less sensible towards betalain antioxidant activity. To our knowledge, this is the first time the LOX-FL antioxidant method has been used on betalains and betalain-rich foods.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.We thank the funding from Spanish Ministry of Science and Innovation (Spain), projects INIA RTA2015-00044-C02-02 and PID2020-118300RB-C21. We thank Gloria Lobo (ICIA) for the recollection and provision of prickly pear fruits (RTA2015-00044-C02-01). Andrea Gómez-Maqueo thanks CONACyT (Mexico) for her doctoral scholarship 692751.Peer reviewe

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K+ Channel (PmitoKATP)was discovered about fifteen years ago in Durum WheatMitochondria (DWM). PmitoKATP catalyses the electrophoreticK+ 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 tocollapse mitochondrial membrane potential (ΔΨ) and ΔpH, thusimpairing protonmotive force (Δp). A possible physiological roleof such ΔΨ control is the restriction of harmful reactive oxygenspecies (ROS) production under environmental/oxidative stressconditions. Interestingly, DWM lacking Δp were found to benevertheless fully coupled and able to regularly accomplish ATPsynthesis; this unexpected behaviour makes necessary to recastin some way the classical chemiosmotic model. In the whole,PmitoKATP may oppose to large scale ROS production bylowering ΔΨ under environmental/oxidative stress, but, whenstress is moderate, this occurs without impairing ATP synthesisin a crucial moment for cell and mitochondrial bioenergetics.[BMB Reports 2013; 46(8): 391-397

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoKATP) 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 PmitoKATP 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 PmitoKATP 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 PmitoKATP 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