Legume Alternative Oxidase Isoforms Show Differential Sensitivity to Pyruvate Activation

Alternative oxidase (AOX) is an important component of the plant respiratory pathway, enabling a route for electrons that bypasses the energy-conserving, ROS-producing complexes of the mitochondrial electron transport chain. Plants contain numerous isoforms of AOX, classified as either AOX1 or AOX2. AOX1 isoforms have received the most attention due to their importance in stress responses across a wide range of species. However, the propensity for at least one isoform of AOX2 to accumulate to very high levels in photosynthetic tissues of all legumes studied to date, suggests that this isoform has specialized roles, but we know little of its properties. Previous studies with sub-mitochondrial particles of soybean cotyledons and roots indicated that differential expression of GmAOX1, GmAOX2A, and GmAOX2D across tissues might confer different activation kinetics with pyruvate. We have investigated this using recombinantly expressed isoforms of soybean AOX in a previously described bacterial system (Selinski et al., 2016, Physiologia Plantarum 157, 264-279). Pyruvate activation kinetics were similar between the two GmAOX2 isoforms but differed substantially from those of GmAOX1, suggesting that selective expression of AOX1 and 2 could determine the level of AOX activity. However, this alone cannot completely explain the differences seen in sub-mitochondrial particles isolated from different legume tissues and possible reasons for this are discussed.

Different Metabolic Roles for Alternative Oxidase in Leaves of Palustrine and Terrestrial Species

The alternative oxidase pathway (AOP) is associated with excess energy dissipation in leaves of terrestrial plants. To address whether this association is less important in palustrine plants, we compared the role of AOP in balancing energy and carbon metabolism in palustrine and terrestrial environments by identifying metabolic relationships between primary carbon metabolites and AOP in each habitat. We measured oxygen isotope discrimination during respiration, gas exchange, and metabolite profiles in aerial leaves of ten fern and angiosperm species belonging to five families organized as pairs of palustrine and terrestrial species. We performed a partial least square model combined with variable importance for projection to reveal relationships between the electron partitioning to the AOP (τa) and metabolite levels. Terrestrial plants showed higher values of net photosynthesis (AN) and τa, together with stronger metabolic relationships between τa and sugars, important for water conservation. Palustrine plants showed relationships between τa and metabolites related to the shikimate pathway and the GABA shunt, to be important for heterophylly. Excess energy dissipation via AOX is less crucial in palustrine environments than on land. The basis of this difference resides in the contrasting photosynthetic performance observed in each environment, thus reinforcing the importance of AOP for photosynthesis.

Comparison of the Kinetic Parameters of Alternative Oxidases From Trypanosoma brucei and Arabidopsis thaliana—A Tale of Two Cavities

The alternative oxidase (AOX) is widespread in plants, fungi, and some protozoa. While the general structure of the AOX remains consistent, its overall activity, sources of kinetic activation and their sensitivity to inhibitors varies between species. In this study, the recombinant Trypanosoma brucei AOX (rTAO) and Arabidopsis thaliana AOX1A (rAtAOX1A) were expressed in the Escherichia coli ΔhemA mutant FN102, and the kinetic parameters of purified AOXs were compared. Results showed that rTAO possessed the highest Vmax and Km for quinol-1, while much lower Vmax and Km were observed in the rAtAOX1A. The catalytic efficiency (kcat/Km) of rTAO was higher than that of rAtAOX1A. The rTAO also displayed a higher oxygen affinity compared to rAtAOX1A. It should be noted that rAtAOX1a was sensitive to α-keto acids while rTAO was not. Nevertheless, only pyruvate and glyoxylate can fully activate Arabidopsis AOX. In addition, rTAO and rAtAOX1A showed different sensitivity to AOX inhibitors, with ascofuranone (AF) being the best inhibitor against rTAO, while colletochlorin B (CB) appeared to be the most effective inhibitor against rAtAOX1A. Octylgallate (OG) and salicylhydroxamic acid (SHAM) are less effective than the other inhibitors against protist and plant AOX. A Caver analysis indicated that the rTAO and rAtAOX1A differ with respect to the mixture of polar residues lining the hydrophobic cavity, which may account for the observed difference in kinetic and inhibitor sensitivities. The data obtained in this study are not only beneficial for our understanding of the variation in the kinetics of AOX within protozoa and plants but also contribute to the guidance for the future development of phytopathogenic fungicides.

Gasotransmitter H2S accelerates seed germination via activating AOX mediated cyanide-resistant respiration pathway

Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of enzymes (LCD and DES1) responsible for H2S production. Moreover, NaHS (the H2S donor) fumigation significantly accelerated seed germination, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. Further results indicated that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition, and the expression of AOX genes was provoked lag behind H2S production during germination. Additionally, exogenous H2S fumigation significantly reinforced imbibition induced enhancement of AOX1A expression, and mediated post-translational modification to keep AOX in its reduced and active state, which mainly involved H2S induced increase of the GSH/GSSG ratio and the cell reducing power. Consequently, H2S signaling acts as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide metabolism, which also participates in endogenous H2S generation, providing evidence for more extensive studies of H2S signaling.