Plastidial and cytosolic thiol reductases participate in the control of stomatal functioning.

Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to post-translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol-reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry-based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH-thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2-CysPRXs or type-II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning.

Ectopic expression of human apolipoprotein D in Arabidopsis plants lacking chloroplastic lipocalin partially rescues sensitivity to drought and oxidative stress.

The chloroplastic lipocalin (LCNP) is induced in response to various abiotic stresses including high light, dehydration and low temperature. It contributes to protection against oxidative damage promoted by adverse conditions by preventing accumulation of fatty acid hydroperoxides and lipid peroxidation. In contrast to animal lipocalins, LCNP is poorly characterized and the molecular mechanism by which it exerts protective effects during oxidative stress is largely unknown. LCNP is considered the ortholog of human apolipoprotein D (APOD), a protein whose lipid antioxidant function has been characterized. Here, we investigated whether APOD could functionally replace LCNP in Arabidopsis thaliana. We introduced APOD cDNA fused to a chloroplast transit peptide encoding sequence in an Arabidopsis LCNP KO mutant line and challenged the transgenic plants with different abiotic stresses. We demonstrated that expression of human APOD in Arabidopsis can partially compensate for the lack of the plastid lipocalin. The results are consistent with a conserved function of APOD and LCNP under stressful conditions. However, if the results obtained with the drought and oxidative stresses point to the protective effect of constitutive expression of APOD in plants lacking LCNP, this effect is not as effective as that conferred by LCNP overexpression. Moreover, when investigating APOD function in thylakoids after high light stress at low temperature, it appeared that APOD could not contribute to qH, a slowly reversible form of non-photochemical chlorophyll fluorescence quenching, as described for LCNP. This work provides a base of understanding the molecular mechanism underlying LCNP protective function.

Variability in the redox status of plant 2-Cys peroxiredoxins in relation to species and light cycle.

Plant 2-Cys peroxiredoxins (2-CysPRXs) are abundant plastidial thiol-peroxidases involved in key signaling processes such as photosynthesis deactivation at night. Their functions rely on the redox status of their two cysteines and on the enzyme quaternary structure, knowledge of which remains poor in plant cells. Using ex vivo and biochemical approaches, we thoroughly characterized the 2-CysPRX dimer/monomer distribution, hyperoxidation level, and thiol content in Arabidopsis, barley, and potato in relation to the light cycle. Our data reveal that the enzyme hyperoxidization level and its distribution as a dimer and monomer vary through the light cycle in a species-dependent manner. A differential susceptibility to hyperoxidation was observed for the two Arabidopsis 2-CysPRX isoforms and among the proteins of the three species, and was associated to sequence variation in hyperoxidation resistance motifs. Alkylation experiments indicate that only a minor fraction of the 2-CysPRX pool carries one free thiol in the three species, and that this content does not change during the light period. We conclude that most plastidial 2-CysPRX forms are oxidized and propose that there is a species-dependent variability in their functions since dimer and hyperoxidized forms fulfill distinct roles regarding direct oxidation of partners and signal transmission.

A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety.

Barley displays a great genetic diversity, constituting a valuable source to delineate the responses of contrasted genotypes to environmental constraints. Here, we investigated the level of oxidative stress and the participation of antioxidant systems in two barley genotypes: Express, a variety known to be sensitive to drought, and Saïda, an Algerian landrace selected for its tolerance to water deficit. Soil-grown 15-day-old plants were subjected to water deficit for 8 days and then rewatered. We observed that upon water stress Express exhibits compared to Saïda accelerated wilting and a higher level of oxidative stress evaluated by HPLC measurements of lipid peroxidation and by imaging techniques. In parallel, Express plants also display lower levels of catalase and superoxide dismutase activity. No great difference was observed regarding peroxiredoxins and methionine sulfoxide reductases, enzymes detoxifying peroxides and repairing oxidized proteins, respectively. In contrast, upon water stress and recovery, much higher contents and oxidation ratios of glutathione and ascorbate were measured in Express compared to Saïda. Express also shows during water deficit greater increases in the pools of lipophilic antioxidants like xantophyll carotenoids and α-tocopherol. Altogether, these data show that the differential behavior of the two genotypes involves distinct responses regarding antioxidant mechanisms. Indeed, the drought sensitivity of Express compared with Saïda is associated with oxidative damage and a lower enzymatic ROS-scavenging capacity, but in parallel with a much stronger enhancement of most mechanisms involving low-molecular weight antioxidant compounds.

Differential responses to salinity of two Atriplex halimus populations in relation to organic solutes and antioxidant systems involving thiol reductases.

Atriplex halimus L. is a xero-halophyte species widespread in the Mediterranean basin. The tolerance to water stress and high salinity of two Atriplex populations from semi-arid (Djelfa) and arid saline (Laghouat) Algerian regions has been investigated in relation with organic solutes and antioxidant systems. Whereas no noticeable difference was observed between the two populations under water stress resulting from withholding watering or PEG treatment, Laghouat plants display significantly higher fresh and dry weights than Djelfa plants when exposed to high salinity. At 300mM NaCl, Laghouat plants exhibit higher concentrations in Na(+), proline and quaternary ammonium compounds, and a higher catalase activity than Djelfa plants. We then analysed the involvement of recently characterized plastidial thiol reductases, peroxiredoxins (Prxs) and methionine sulphoxide reductases (MSRs), key enzymes scavenging organic peroxides and repairing oxidized proteins, respectively. Upon salt treatment (300mM NaCl), we observed higher amounts of PrxQ and over-oxidized 2-Cys Prx in Laghouat than in Djelfa. An increased abundance of plastidial MSRA and a higher total MSR activity were also noticed in Laghouat plants treated with 300mM NaCl compared to Djelfa ones. We propose that mechanisms based on organic solutes and antioxidant enzymes like catalases, peroxiredoxins and MSRs party underlie the better tolerance of the Laghouat population to high salt.