Alternative oxidase in resistance to biotic stresses: Nicotiana attenuata AOX contributes to resistance to a pathogen and a piercing-sucking insect but not Manduca sexta larvae

The role of the alternative respiratory pathway in the protection of plants against biotic stress was examined in transgenic tobacco ( Nicotiana attenuata ) plants (irAOX) silenced in the expression of ALTERNATIVE OXIDASE ( AOX ) gene. Wild-type (WT) and irAOX plants were independently challenged with: (i) chewing herbivores ( Manduca sexta ), (ii) piercing-sucking insects ( Empoasca spp.) and (iii) bacterial pathogens ( Pseudomonas syringae pv. tomato DC3000), showing that all these treatments can strongly elicit accumulation of AOX gene transcripts in WT plants. When N. attenuata chemical defenses and resistance were examined, irAOX plants showed WT levels of defense-related phytohormones, secondary metabolites and resistance to M. sexta . In contrast, piercing-sucking leafhoppers ( Empoasca spp.) caused more leaf damage and induced significantly higher salicylic acid (SA) levels in irAOX compared to WT plants in the field and/or glasshouse. Subsequently, irAOX plants accumulated lower levels of defense metabolites, 17-hydroxygeranyllinalool diterpene glycosides, caffeoylputrescine and nicotine compared to WT plants under prolonged attack of leafhoppers in the glasshouse. Finally, an accelerated cell death phenotype was observed in irAOX plants infected with P. syringae , which correlated with higher levels of SA and hydrogen peroxide levels in pathogen-infected irAOX compared to WT leaves. Overall, the AOX-associated changes in phytohormone and/or redox levels appear to support the resistance of N. attenuata plants against cell piercing-sucking insects and modulate the progression of cell death in pathogen-infected tissues but are not effective against rapidly feeding specialist herbivore M. sexta . These suggest that gene in mock and Pst DC3000-inoculated leaves of WT and two irAOX lines. No significant differences between genotypes at respective time points were determined by one-way ANOVA.


Introduction
Metabolic plasticity allows plants to adapt to variable environmental stress conditions. Plants rapidly reconfigure their primary metabolism during stress to cope with the increased metabolic demands of resistance responses, and mitochondria are known to play a central role in this reconfiguration (reviewed in Bolton, 2009;Millar et al., 2011). Under stress, electrons are frequently re-routed through an alternative respiratory pathway branching from the cytochrome (Cyt) pathway at the level of ubiquinone (UQ) pool to an enzyme known as the alternative oxidase (AOX; McDonald, 2008). In contrast to the Cyt pathway, the AOX pathway is resistant to cyanide and therefore known as cyanide-resistant respiration (Henry and Nyns, 1975).
The alternative pathway bypasses two important energy conservation sites (complex III and IV) and avoids oxidative phosphorylation and adenosine triphosphate (ATP) synthesis: energy is dissipated as heat after reduction of oxygen to water by AOX enzyme (Moore et al., 1978). While alternative respiration is widespread in plants, fungi and some prokaryotes (McDonald, 2008), its physiological function and seemingly energy-wasting character are not well understood.
One of the most investigated functions of AOX is heat production which, for example, can volatilize odoriferous compounds during flowering for the attraction of pollinators, for example in Arum lily flowers (Meeuse, 1975;Raskin et al., 1987).
Heat production is only associated with a limited number of thermogenic plant species (Seymour, 1997), and alternative functions of AOX have been extensively investigated. Subsequently, AOX has been established as one of the essential defense components in plant response to acute stress (Arnholdt-Schmitt et al., 2006). Abiotic stresses such as drought (Bartoli et al., 2005), high salt (Costa et al., 2007;Feng et al., 2010a), low temperature (Vanlerberghe and McIntosh, 1992;Popov et al., 2011;Wang et al., 2011) andwounding (Hiser andMcIntosh, 1990) are known to stimulate the activity of alternative respiratory pathway or at least increase AOX transcripts and/or protein levels. Phytochrome, phototropin and cryptochrome photoreceptors have been shown to mediate the light-responsiveness of the AOX1a gene in Arabidopsis 7 simulated herbivory treatment, and after direct feeding of the N. attenuata leaves by M. sexta neonates. The NaAOX transcripts strongly increased 1h after wounding of N. attenuata leaves with a pattern wheel and immediately applying either water to mechanical wounds (W+W) or M. sexta's oral secretions (OS) to simulate herbivory (W+OS) ( Figure 1A; Supplemental Figure S1B). Interestingly, NaAOX transcript and protein levels appeared higher in W+OS-compared to W+W-treated leaves at 1-3 h ( Figure 1A ; Supplemental Figure S2A). Similarly, direct M. sexta feeding significantly elevated basal NaAOX transcript and protein levels at 1 and 2 d after placing neonates on the leaves ( Fig. 1B; Supplemental Figure S2B). Because NaAOX transcript levels increased significantly more in response to M. sexta's salivary elicitors, we hypothesized that AOX may function in direct defense of N. attenuata against chewing herbivores. In addition to the herbivore-and wound-regulated patterns shown in Fig. 1, an apparent oscillation in NaAOX transcript abundance was observed in untreated control leaves, suggesting a possible circadian control of AOX transcription (Fig. 1A). In particular, AOX transcripts appeared to be higher during the light periods than during dark periods, which could be associated with a previously reported AOX function in buffering high photosynthetic and respiration rates in response to high light (Dinakar et al., 2010).

NaAOX silencing does not compromise defense of N. attenuata against M. sexta caterpillars
To examine the function of NaAOX in resistance of native tobacco against its specialist herbivore M. sexta caterpillars, we generated stably AOX-silenced N. attenuata plants (irAOX) using RNA interference and inverted repeat fragment of the was not surprising that caterpillar performance in feeding bioassays did not differ (Fig.   2B). These results suggest that despite increased transcript levels of NaAOX in response to M. sexta attack, NaAOX genes do not play any significant role in the direct defense of N. attenuata plants against chewing specialist herbivores. The induction of NaAOX transcripts and protein levels by insect feeding could be a consequence of/and defense against mechanical wounding, similar to previously described accumulation of AOX protein in mechanically wounded potato (Solanum tuberosum) tubers (Hiser and McIntosh, 1990).
We also considered an alternative hypothesis that increased AOX expression during hervivory could facilitate higher emissions of volatile organic compounds (VOCs), mediators of indirect defenses, from herbivore-attacked leaf surfaces of N.
attenuata. However, in our previous experiments we did not find any consistent differences in α-bergamotene or benzyl acetone emissions from WT and irAOX  phytohormone and defense metabolite profiles after Empoasca spp. attack.

irAOX plants contain higher levels of SA after leafhopper attack
Piercing-sucking insects such as aphids, mites and leafhoppers can activate both salicylate-and jasmonate-dependent defense signaling pathways in plants (Ament et al., 2004;Mozoruk et al., 2006;Kempema et al., 2007). less in irAOX compared to WT plants exposed for 8-12 d to leafhopper feeding ( Fig.   6), suggesting a positive role of AOX in efficient accumulation of these metabolites after prolonged Empoasca spp. feeding. The initial feeding of leafhoppers, however, is known to be independent of nicotine, HGL-DTGs and PIs in N. attenuata plants (Kallenbach et al., 2012). Consistently, after 4 d of Empoasca spp. feeding, defense metabolites were not yet elicited (Fig. 6).

NaAOX transcripts increase in response to pathogen infection
The treatment of tobacco cells with pathogen-associated elicitors is known to alter partitioning between Cyt-and AOX-dependent respiratory pathways, and transiently increase AOX expression (Vidal et al., 2007). We examined if  To characterize Pst-induced cell death at a molecular level, the expression of a tobacco hypersensitive response marker HAIRPIN-INDUCED 1 (HIN1) gene was examined. HIN1 is known to be highly expressed during incompatible plant-pathogen interactions in tobacco (Takahashi et al., 2004a;2004b). NaHIN 1 transcripts were significantly induced by Pst DC3000 in N. attenuata and they were significantly higher in two irAOX lines compared to WT plants 3 d after infection (Fig. 8C).

irAOX plants accumulate higher levels of hydrogen peroxide after Pst infection
Cellular ROS is an important signaling molecule in eukaryotic cells (Rhoads et al., 2006). One of the proposed functions of AOX in abiotic stress resistance is the prevention of over-reduction of mitochondrial ubiquinone (UQ) pool that can counteract the formation of mitochondrial ROS (Maxwell et al., 1999;Umbach et al., 2005).
We first examined the levels of hydrogen peroxide (H 2 O 2 ) by semi-quantitative histochemical DAB staining of WT and irAOX leaves after Pst DC3000 infection. A strong brown precipitate of oxidized DAB was observed in both WT and two irAOX lines at 1-3 d after inoculation but the staining intensity at 2 d appeared stronger in 1 3 irAOX compared to WT leaves (Fig. 9A). In order to determine H 2 O 2 contents more precisely, we used sensitive quantitative Amplex Red hydrogen peroxide assay kit and determined H 2 O 2 levels in the leaves. Pst DC3000 infiltration resulted in a strong increase in H 2 O 2 content at 2 DPI in both WT and irAOX plants, and these levels were significantly higher in irAOX leaves (Fig. 9B), coinciding with a more rapid development of cell death symptoms in these plants (Fig. 8A).
Previously, the Arabidopsis respiratory burst oxidase homolog D (RbohD) was shown to produce a majority of ROS found in pathogen-infected Arabidopsis leaves (Torres et al., 2002). We therefore examined the expression of a putative NaRbohD homolog in N. attenuata after Pst DC3000 inoculation. The transcripts of this NaRbohD gene increased both in WT and irAOX (lines 200 and 203) but appeared significantly higher in irAOX infected leaves (Fig. 9C). This suggests that this RbohD homolog may contribute to the higher ROS levels observed in irAOX lines, possibly in response to a retrograde mitochondrial signal and/or the accumulation of SA, as proposed below.

SA levels increase significantly more in pathogen-infected irAOX leaves
Accumulation Interestingly, higher levels of SA in pathogen-infected irAOX plants negatively correlated with lower levels of induced JA in these leaves, suggesting a negative crosstalk between Pst DC3000-induced SA and JA accumulations (Fig. 10A).
In agreement with the higher SA levels, transcripts of two known

AOX-modulated responses to abiotic stress
In contrast to the well-known role of AOX in the production of heat in thermogenic plants (Meeuse, 1975)

Reported roles of AOX in plant resistance to biotic stress
To further complement the role of AOX in stress responses, the role of AOX in resistance against two types of herbivores and one pathogen was examined in native tobacco plants. Previously, the role of AOX in response to biotic stress was described in tobacco plants (Xanthi nn genotype) infected with tobacco mosaic virus (TMV).
While the pretreatment of tobacco leaf discs with SA suppressed TMV replication,

AOX and herbivory in N. attenuata
Here we show that AOX transcription is efficiently induced by the feeding of a chewing herbivore. However, the suppression of AOX in irAOX plants did accumulation, similar to that we found in Pst DC3000-infected irAOX N. attenuata plants.

Possible mechanisms of accelerated PCD in irAOX plants
Mitochondria represent one of the most important sources of ROS in plant cells.
Recently, it has been shown that mitochondrial complex II of the electron transport  interesting to see in the future if AOX could be directly involved in these interactions.

Conclusions
Although AOX expression is induced by numerous biotic stress factors, the induction of AOX and protection against stress may not always be in a positively  were imported under 07-341-101n.

Generation and characterization of NaAOX-silenced plants (ir-AOX)
A 510 bp fragment of the cDNA sequence of NaAOX1 gene (

Expression analysis by RT-qPCR
Total RNA was extracted from approximately 100 mg leaf tissue using TRIZOL

AOX protein determination by western blotting
Leaves were ground to a fine powder in liquid nitrogen and 100µL of 2x Laemmli The manufactures' instructions were followed except in the following modifications: membranes were blocked overnight at 4 °C and washing steps were prolonged to 10 min.

Measurement of respiration in N. attenuata leaves
The

Analysis of secondary metabolites
Secondary metabolites were analyzed by HPLC coupled to diode array detector (DAD) as described in Kaur et al. (2010). Approximately 100 mg of leaf material was extracted with 1 mL of extraction buffer containing 40% methanol and 0.5% acetic acid. After centrifugation, the supernatants were collected and injected into an Agilent

Determination of cell death
Trypan blue staining specific to dead cells with permeabilized cytoplasmic membranes was carried out as described in Koch et al. (1990). Electrolyte leakage was measured as described in Pike et al. (1998) using a conductivity meter (Mettler Toledo, Ohio; http://us.mt.com/). Eight discs (0.785 cm 2 each) were punched from the treated leaves using a cork-borer and placed in 15 mL Falcon tubes (BD Biosciences http://www.bdbiosciences.com) together with 10 mL of sterile distilled water.
Samples were incubated for 4 h under slow rotation at 20 ºC and the conductivity of resulting water solutions was measured by conductivity meter.

Hydrogen peroxide visualization and determinations
DAB staining was performed on the leaves inoculated with Pst DC3000 or mock solutions as described by Thordal-Christensen et al. (1997)

Supplemental tables
Supplemental Table 1 Real time PCR primers and probes. The primers and Taqman probe sequences used for Taqman qPCR, and primer sequences used for SYBR Green-based RT-qPCR assays.     Leaf NaAOX gene transcript levels increase in response to Empoasca spp. leafhopper attack. Means  NaAOX gene transcripts increase in response to infection with Pst DC3000 in N. attenuata leaves. Mean (±SE) NaAOX relative transcript abundances quantified with RT-qPCR in 3 independent mockand Pst pv. tomato DC3000-inoculated WT plants at indicated time points. Asterisks indicate significant differences between mock-and Pst DC3000-infected leaves at respective time points determined by unpaired Student's t-test (*P<0.05; **P<0.01; *** P<0.001; n=3).