Knockdown of mitochondrial alternative oxidase induces the ‘stress state’ of signaling molecule pools in Nicotiana tabacum, with implications for stomatal function

Abstract

SummaryThe mitochondrial electron transport chain (ETC) includes an alternative oxidase (AOX) that may control the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS act as signaling intermediates in numerous plant processes, including stomatal movement.The role of AOX in controlling ROS and RNS concentrations under both steady-state and different stress conditions was evaluated using Nicotiana tabacum plants lacking AOX as a result of RNA interference. A potential functional implication of changes in ROS and RNS homeostasis was also evaluated by examining stomatal function.The leaves of nonstressed AOX knockdowns maintained concentrations of H 2 O 2 and nitric oxide (NO) normally seen in wildtype plants only under stress conditions. Further, guard cell NO amounts were much higher in knockdowns. These guard cells were altered in size and were less responsive to NO as a signal for stomatal closure. This, in turn, compromised the stomatal response to changing irradiance.The results reveal a role for AOX in stomata. A working model is that guard cell AOX respiration maintains NO homeostasis by preventing over-reduction of the ETC, particularly during periods when high concentrations of NO acting as a signal for stomatal closure may also be inhibiting cyt oxidase respiration.

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

“…On the other hand, no differences were seen between the wild type and knockdown lines during drought in total Chl, stomatal conductance (g s ), mesophyll conductance (g m ), C i , or the concentration of CO 2 at the Rubisco carboxylation site (C c ; Table I). However, g s , C i , and C c were slightly lower in RI29 than in the wild type in well-watered plants, which is consistent with a previously reported stomatal limitation (Cvetkovska et al, 2014).…”

“…1). This correlates well with our previous studies showing that RI29 is the stronger of the two knockdowns (Wang et al, 2011;Wang and Vanlerberghe, 2013;Cvetkovska et al, 2014). In RI29, the effects of drought on A n , ETR II , excitation pressure, F PSII , and NPQ were all strongly magnified relative to the wild type, whereas RI9 (the slightly weaker AOX knockdown) always showed an intermediate response between RI29 and the wild type (Figs.…”

“…RI29 (and RI9 to a lesser extent) did show a slight (10%-15%) reduction in the light-saturated rate of photosynthesis estimated from light response curves. As previously described, this reduction is attributable to a stomatal limitation relating to disruption of guard cell nitric oxide homeostasis in well-watered knockdowns (Cvetkovska et al, 2014). Consistent with it resulting from a stomatal limitation, this reduction was not evident when A n between lines was compared at equivalent C i ( Fig.…”

“…Transgenic plant lines with suppressed levels of AOX protein (RI9 and RI29) attributable to the presence of an AOX1a RNA interference construct, or elevated levels of AOX protein (B7) due to the presence of an AOX1a transgene driven by a constitutive promoter were previously described (Wang et al, 2011;Wang and Vanlerberghe, 2013;Cvetkovska et al, 2014). In particular, it has been shown that AOX protein levels are strongly suppressed in the knockdowns, even under the strongly AOX-inducing conditions of low temperature (Wang et al, 2011) or severe drought (Wang and Vanlerberghe, 2013).…”

“…Similar to these Arabidopsis studies, we recently reported that well-watered N. tabacum AOX knockdowns grown at moderate irradiance display a slight reduced rate of photosynthesis (approximately 10%-15%) when measured at high irradiance. However, we established that the lower photosynthetic rate was the result of a stomatal rather than biochemical limitation of photosynthesis, and provided evidence that this stomatal limitation resulted from disrupted nitric oxide homeostasis within the guard cells of AOX knockdown plants (Cvetkovska et al, 2014).…”