The Phosphofructokinase Isoform AtPFK5 Is a Novel Target of Plastidic Thioredoxin-f-Dependent Redox Regulation

The chloroplast primary metabolism is of central importance for plant growth and performance. Therefore, it is tightly regulated in order to adequately respond to multiple environmental conditions. A major fluctuation that plants experience each day is the change between day and night, i.e., the change between assimilation and dissimilation. Among other mechanisms, thioredoxin-mediated redox regulation is an important component of the regulation of plastid-localized metabolic enzymes. While assimilatory processes such as the Calvin–Benson cycle are activated under illumination, i.e., under reducing conditions, carbohydrate degradation is switched off during the day. Previous analyses have identified enzymes of the oxidative pentose phosphate pathway to be inactivated by reduction through thioredoxins. In this work, we present evidence that an enzyme of the plastidic glycolysis, the phosphofructokinase isoform AtPFK5, is also inactivated through reduction by thioredoxins, namely by thioredoxin-f. With the help of chemical oxidation, mutant analyses and further experiments, the highly conserved motif CXDXXC in AtPFK5 was identified as the target sequence for this regulatory mechanism. However, knocking out this isoform in plants had only very mild effects on plant growth and performance, indicating that the complex primary metabolism in plants can overcome a lack in AtPFK5 activity.

Kandelia candel Thioredoxin f Confers Osmotic Stress Tolerance in Transgenic Tobacco

Water deficit caused by osmotic stress and drought limits crop yield and tree growth worldwide. Screening and identifying candidate genes from stress-resistant species are a genetic engineering strategy to increase drought resistance. In this study, an increased concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in the nonsecretor mangrove species Kandelia candel. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localizes to chloroplasts. Enzymatic activity characterization revealed that KcTrxf recombinant protein possesses the disulfide reductase function. KcTrxf overexpression contributes to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) production. KcTrxf was shown to reduce the stomatal aperture by enhancing K+ efflux in guard cells, which increased the water-retaining capacity in leaves under drought conditions. Notably, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits plants exposed to drought by reducing water loss by promoting stomatal closure. KcTrxf-transgenic plants limited drought-induced H2O2 in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Additionally, glutathione (GSH) contributing to reactive oxygen species (ROS) scavenging and transgenic plants are more efficient at regenerating GSH from oxidized glutathione (GSSG) under conditions of drought stress. Notably, KcTrxf-transgenic plants had increased glucose and fructose contents under drought stress conditions, presumably resulting from KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributes to drought tolerance by increasing the water status, by enhancing osmotic adjustment, and by maintaining ROS homeostasis in transgene plants.