Expression and kinetics of the mitochondrial alternative oxidase in nitrogen‐fixing nodules of soybean roots

Abstract

Mitochondria from nitrogen-fixing soybean nodules had previonsly been found to display very low rates of cyanideinsensitive O2 consumption and to contain little protein immunoreactive with alternative oxidase (AOX) antibodies (A. Kearns et al. 1992, Plant Physiology 99,, pp. 712-717). Following the recent identification of novel regulatory mechanisms for AOX, we have re-investigated AOX activity in nodule mitochondria. When AOX is fully activated by adding pyruvate and a reductant, significant AOX activity can be measured in nodule mitochondria when substrates that cause the ubiquinone (Q) pool to become > 90% reduced are provided. These nodule mitochondria contain =10% of the immunoreactive protein observed in soybean root or cotyledon mitochondria. Furthermore, transcripts from all three members of the soybean AOX multigene family were significantly less abundant in total RNA from nodules than from other tissues. The ratio of transcripts from the three AOX genes varied between the outer cortex and the inner infected region of the nodule. Measurements of Q redox state {QJQi) versus O2 consumption showed that much higher levels of Q reduction were required for AOX to be active in mitochondria from nodules than in mitochondria from roots and cotyledons, and that root and nodule mitochondria were more dependent on added pyruvate for maximum AOX activity. Kinetic modelling suggests that these differences were simply the result of differences in the relative amount of active AOX protein, rather than differences in Q content or the AOX isoform expressed, in the different tissues.

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation

“…When pyruvate was included in the reaction medium to activate AOX, activity was detectable at a lower Qr/Qt as more AOX protein became engaged and apparent Vmax increased [30,35]. These results are very similar to those observed with mitochondria isolated from soybean roots and cotyledons [7,15,36], and show that the soybean AOX interacts in a 'normal' manner with the Q pool when produced in tobacco.…”

“…3), showing that succinate was not acting as a substrate but rather as an activator of AOX. This effect of succinate on Qr and 02 uptake was very similar to the effect of pyruvate on soybean root mitochondria [35,36] in which G m A O X 3 is the sole A O X protein [10]. The relationship between Q and the different A O X proteins is analysed further in Fig.…”

“…The Qr response curve of AOX in these mitochondria was shifted slightly to the left compared to (activated) wild-type GmAOX3 activity, probably because of the higher expression of mutant protein (Fig. 1) which alters the relationship [35].…”

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate

“…This, however, does not yet explain the effect pyruvate has on the steadystate kinetics (Umbach et al, 1994;Hoefnagel and Wiskich, 1996), but some alternative explanations can be given. First, the steady-state Q-titration curve for soybean mitochondria can be converted to the pyruvate-activated curve by applying an increased V,,, to the model of Van den Bergen et al (1994), where both Q-oxidizing and Q-reducing reactions are considered (Millar et al, 1997). Second, pyruvate could have an effect on the redox potential of AOX, allowing a faster reaction rate at lower levels of reduction of the Q pool.…”

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

“…Studies on soybean AOX suggest that pyruvate action is due to its interaction with a Cys sulfhydryl to form a thiohemiacetal, since the activation is mimicked by iodoacetate (Umbach and Siedow, 1996). It has also been shown that pyruvate acts to increase the V max of AOX without any significant effect on its affinity for ubiquinol (Hoefnagel et al, 1997;Millar et al, 1997).…”