The Effects of Salicylic Acid and Tobacco Mosaic Virus lnfection on the Alternative Oxidase of Tobacco’
Adrian M. Lennon’, Urs H. Neuenschwander, Miquel Ribas-Carbo, Larry Giles, John A. Ryals, and James N. Siedow*
DCMB Group (A.M.L., J.N.S.), and Phytotron (L.G.), Department of Botany, Duke University, Durham, North Carolina 27708; Biotechnology and Genomics Center, Novartis Crop Protection Inc., P.O. Box 12257, Research
Triangle Park, North Carolina 27709 (U.H.N., J.A.R.); and Departamento de Fisiologia Vegetal, Facultad de Ciencias, Universidad de Navarra, 31 008 Pamplona, Spain (M.R.-C.)
Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicofiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related l a mRNA (a marker for sys- temic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of ‘*O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahC tobacco p!ants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxi- dase protein, suggesting that steady-state alternative oxidase accu- mulates in an SA-independent manner. lnfection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahC plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.
One of the distinguishing features of plant mitochondria is the presence of a cyanide-resistant ”alternative” electron transfer pathway in addition to the standard cyanide- sensitive Cyt pathway (Moore and Siedow, 1991; Siedow and Umbach, 1995). The alternative pathway consists of a single ubiquinol oxidase, known as the alternative oxidase, that transfers electrons from reduced ubiquinone to molec-
This work was supported by U.S. Department of Agriculture National Research Initiative Competitive Grants Program grant no. 94-37360-0325 (J.N.S.), National Science Foundation (NSF) Di- vision of Environmental Biology grant no. 9112571 (Duke Univer- sity Phytotron), and a postdoctoral fellowship (5002-38012) from the Swiss NSF (U.H.N.).
Present Address: Department of Biochemistry, School of Med- ical Sciences, University of Bristol BS8 lTD, UK.
ular O,, producing water as the product (Moore and Siedow, 1991). The alternative oxidase does not pump pro- tons across the inner membrane and, because it bypasses the two sites of proton translocation at complexes 111 and IV, the free energy is released as heat (Whitehouse and Moore, 1995). The physiological role of this pathway re- mains unclear except in the specialized case of promoting thermogenesis during flowering in aroid spadices (Moore and Siedow, 1991). The pathway is present in a11 higher plants and also in a number of algae and fungi (Moore and Siedow, 1991).
SA has been demonstrated to induce expression of the alternative oxidase protein. This was first shown in voodoo lily (Sauromatum guttatum) appendix tissue (Raskin et al., 1987, 1989; Elthon et al., 1989a), where SA acts as an en- dogenous trigger of thermogenesis. Later, Rhoads and McIntosh (1992) demonstrated that in S. guttatum appendix tissue, SA induced a dramatic increase in the level of the alternative oxidase transcript, leading to an accumulation of the protein within the mitochondria. SA is also able to induce expression of the alternative oxidase in tobacco (Nicotiana tabacum L.) cell-suspension cultures (Rhoads and McIntosh, 1993), increasing the activity of the alternative pathway in the presence of inhibitors of the Cyt pathway (Kapulnik et al., 1992; Rhoads and McIntosh, 1993).
SA also plays a central role in the plant disease-resistance response (Delaney et al., 1994), including the activation of SAR, an inducible defense response that confers a long- lasting, enhanced resistance against a broad spectrum of pathogens (Chester, 1933; Ross, 1961; Kuc, 1982). A necro- tizing pathogen infection triggers the release of a phloem- mobile signal that travels throughout the plant and leads to activation of SAR in target tissues. Little is known about the signal cascade that triggers the activation of SAR, but elevated levels of SA have been found to be required for SAR induction (Neuenschwander et al., 1996; Ryals et al.,
Abbreviations: Aa, O, isotope fractionation by the alternative pathway; Ac, O, isotope fractionation by the Cyt pathway; An, O, isotope fractionation in the absence of inhibitors; PR, pathogenesis related; SA, salicylic acid; SAR, systemic acquired resistance; SHAM, salicylhydroxamic acid; Ta, the fraction of total electron flow partitioning to the alternative pathway; TMV, tobacco mosaic virus; V,,,, the rate of O, uptake by the alternative oxidase; V,,,, the rate of O, uptake in the presence of KCN.
1996). In tobacco and Arabidopsis exogenous application of SA activates both SAR and SAR gene expression (White, 1979; Ward et al., 1991; Uknes et al., 1992). Moreover, transgenic NahG plants, which are unable to accumulate elevated levels of SA due to expression of a bacterial salic- ylate hydroxylase, were found to be incapable of mounting an SAR response (Gaffney et al., 1993). Grafting experi- ments using wild-type and NahG plants have shown that SA is most likely not the phloem-mobile signal in SAR, but the signal requires SA to mount a systemic SAR response in tissues distant from the infection site (Vernooij et al., 1994).
Recently, Van Der Straeten et al. (1995) studied the re- sponse of tobacco leaves to exogenous application of SA. Using a high-resolution IR camera the authors recorded a 0.5 to 1.0"C rise in the surface temperature of the tobacco leaves following SA treatment. The temperature increase was found to be partially repressed by the exogenous application of SHAM, a specific inhibitor of the alternative oxidase. In addition, polarographic measurements of respi- ratory activity demonstrated an increase in the overall respiration rate, as well as that of the alternative pathway (i.e. following the addition of cyanide), after SA treatment. The authors suggested that the temperature increase seen in the tobacco leaves was due to an increase in alternative pathway activity stimulated by the application of SA.
In recent years it has been demonstrated that the alter- native oxidase can compete for electrons with an unsatur- ated Cyt pathway (Hoefnagel et al., 1995; Ribas-Carbo et al., 1995; Siedow and Umbach, 1995), and that the use of inhibitors to quantify the activity of the alternative oxidase is unsound (Millar et al., 1995; Ribas-Carbo et al., 1995; Day et al., 1996). Currently, the only available method to mea- sure the partitioning of electrons between the alternative and Cyt pathways in the absence of inhibitors is that of O,-isotope fractionation, which can be performed on intact tissue (Robinson et al., 1992, 1995) and on isolated mito- chondria (Guy et al., 1989; Ribas-Carbo et al., 1995, 1997).
Here we report the use of the O,-isotope fractionation technique to study the effects of salicylate on the alterna- tive oxidase of tobacco mitochondria. The results are dis- cussed in relation to a possible role for the alternative oxidase in SAR.
MATERIALS AND METHODS
Plant Material and Treatments
Tobacco (Nicotiana tabacum L.) plants were either cv Xan- thi. nc (referred to as cv Xanthi) or cv Samsun, which are both local lesion hosts for TMV. Salicylate hydroxylase- expressing lines were either NahG (also called NahG-10; Friedrich et al., 1995), a transgenic cv Xanthi line, or SH-L, a transgenic cv Samsun line. Both lines catabolize SA to catechol due to the activity of salicylate hydroxylase from Pseudomonas putida (Bi et al., 1995; Friedrich et al., 1995).
Plants were grown in a greenhouse under a 16-h/8-h light / dark regimen and were used for experiments at an age of 6 to 8 weeks. For SA treatments, 1.0 mM SA was infiltrated into the apoplast to fully saturate the leaves (Ward et al., 1991). Inoculation with TMV and control
"mock infections were performed as described by Ward et al. (1991). At least three individual plants and three leaves per plant were treated for each measurement. For mito- chondrial isolations and RNA extractions, tissues from sim- ilarly treated leaves were pooled upon harvesting, whereas for O,-isotope analysis, tissue of individual leaves was analyzed separately.
Mitochondrial lsolation
Mitochondrial mini-preparations were performed ac- cording to the method of Boutry et al. (1984), with some modifications. Five grams of leaf tissue was ground with glass beads in a mortar and pestle in 40 mL of mitochon- drial grinding buffer (Umbach and Siedow, 1993). The homogenate was filtered through four layers of cheesecloth and the filtrate was centrifuged for 2 min at 3,OOOg. The resulting supernatant was centrifuged for 10 min at 17,OOOg and the pellet was resuspended in 1 mL of wash medium (Umbach and Siedow, 1993). The resuspended pellet was then purified on a discontinuous Percoll gradient by cen- trifugation at 27,OOOg for 30 min. The gradient layers con- sisted of 1 mL of 45% Percoll, 2 mL of 21% Percoll, and 2 mL of 13.5% Percoll-containing wash medium. The mito- chondrial fraction at the 21/45% interface was removed and washed twice in wash medium at 27,OOOg for 10 min. The final pellet was resuspended in 100 pL of wash me- dium. Protein concentrations were estimated by the method of Lowry et al. (1951).
lmmunoblotting
SDS-PAGE was performed using 10% polyacrylamide gels, essentially according to Laemmli (1970). Proteins were transferred to nitrocellulose according to Towbin et al. (1979), and immunoblotting was performed by an initial incubation in 3% BSA and 2% milk powder in PBS solution for 2 h. The filters were then washed three times with PBS and subsequently probed with the alternative oxidase A monoclonal antibody against the alternative oxidase pro- tein (Elthon et al., 198913) or a polyclonal antibody against Cyt c oxidase subunit I1 (a generous gift of Dr. C.S. Levings, 111, North Carolina State University, Raleigh) at a dilution of 1:500 for 1 h. Following three washes in PBS solution, the filters were incubated with horseradish peroxidase- conjugated secondary antibody at a dilution of 1:20,000 for 1 h. The filters were then washed three times in blot rinse buffer (10 mM Tris, pH 7.4,150 mM NaCl, 1 mM EDTA, and 0.1% [v/v] Tween 20) and the bound antibodies were de- tected using the ECL detection system (Amersham), ac- cording to the manufacturer's instructions. To quantify the protein levels, densitometry was performed as described by Umbach and Siedow (1993). In a11 immunoblots of the alternative oxidase, only a single species running with an apparent molecular mass of 35 kD was ever observed.
RNA Analysis
RNA was isolated from frozen tissue samples by subse- quent phenol-chloroform extraction and lithium chloride
Salicylic Acid Effects on the Alternative Oxidase 785
precipitation (Lagrimini et al., 1987). Total RNA (10 pg) was separated by electrophoresis through formaldehyde- agarose gels and blotted to nylon-based membranes (GeneScreen Plus, NEN). Equal loading of samples was confirmed by including ethidium bromide in the sample- loading buffer, allowing visualization of RNA by photog- raphy under UV light (Ward et al., 1991). Blotted gels were hybridized with a 32P-labeled cDNA probe for PR-la, as described (Ward et al., 1991).
O,-lsotope Analysis
Three tobacco leaf discs (3.5 cm diameter) taken from separate plants were weighed and placed into a closed cuvette that was maintained under total darkness at room temperature. During inhibitor treatments either 1.0 mM KCN or 10 mM SHAM was applied by first sandwiching tissues between medica1 wipes saturated with the corre- sponding inhibitor for 10 min, and then surface-drying the tissues and transferring them to the cuvette. There was no apparent recovery from inhibitor treatments during the course of the experiments, as the Y' values for a11 linear regressions of the fractionation values reported were greater than 0.995.
O,-isotope analysis was performed as described in Rob- inson et al. (1995). Calculations of O,-isotope fractionation followed Robinson et al. (1995), and electron partitioning between the two pathways in the absence of inhibitors (Ta) was calculated as described by Guy et al. (1989). Linear regressions used to calculate the fractionation factor (D) were discarded if the resulting Y' was less than 0.995. The fractionation factor was converted to A, as in Guy et al. (1989).
RESULTS
Effects of SA on the Alternative Oxidase
O,-isotope fractionation by the Aa and Ac pathways was measured in the presence of 1.0 mM KCN and 10 mM SHAM, respectively, as described in "Materials and Meth- ods." The value of Aa was 31.4% 5 0.2 and that of Ac was 20.1% 5 0.3. These values are similar to those reported previously in green soybean cotyledons and leaf discs (Robinson et al., 1995; Ribas-Carbo et al., 1997). These values were subsequently used to calculate electron parti- tioning between the Cyt and alternative pathways in the absence of inhibitors.
Initial experiments, performed to optimize SA treatment of tobacco, revealed that infiltration of SA into the apoplast was preferable to spray treatment because infiltration led to more consistent results. A concentration of 1 mM SA was found to strongly induce PR-la gene expression with no observable phytotoxicity (data not shown). To determine the effect of SA on alternative oxidase electron partitioning in tobacco, leaves of cv Xanthi and cv Samsun were infil- trated with 1 mM SA, and O, fractionation by the tissue was measured over a time period from 1 h to 3 d. Electron partitioning (23.2%) between the two pathways in the ab- sence of inhibitors (An) did not change throughout the
experimental time course (Fig. 1). This value of An corre- sponds to a Ta of 0.27, indicating that 27% of the total electron flow is partitioned to the alternative pathway dur- ing respiration.
Treatment with SA did not affect the overall rate of respiration (approximately 110 nmol O, min-l g-' fresh weight) during the 3-d experimental time course (Fig. 1B). As such, Valt remained essentially unchanged following SA treatment and equal to 30 to 35 nmol O, min-' 8-l
A
22 21 1 B
O 10 20 30 40 50 60 70 80
C
o 10 20 30 40 50 60 70 80 Hours After Salicylate Treatment
Figure 1. Effect of treatment with 1 mM SA on the respiration of tobacco leaf discs. A, '80-isotope fractionation (An) in the absence of respiratory inhibitors. B, Total respiration rate (VJ of leaf discs in nmol O, min-' g-' fresh weight (FW). C, The rate of O, uptake by V,,, in nmol O, min-' g-' fresh weight. V,,, is calculated from the values of V, and A, as described in "Materials and Methods." All presented values and SES are calculated from four to seven repeti- tions. SE bars Iie within the data points when not seen.
fresh weight (Fig. 1C). Similar respiration rates and Anvalues were obtained with the cv Samsun (data notshown). Preincubation of leaf discs for 60 min (after cut-ting) did not affect the observed respiration rates or Anvalues, and the respiration rate remained constant duringthe 90-min time course of the measurement. This suggeststhat the measured respiration rates and values of An donot reflect any complicating involvement of a woundresponse. The rate of residual O2 in the presence of bothSHAM and KCN, which can potentially complicate mea-surements of O2-isotope fractionation, was well below 5%of the uninhibited respiration rate in the leaf discs andwas unchanged following SA treatment. Taken together,these results suggest that SA treatment does not affecteither the overall respiration rate or the relative contribu-tions of the Cyt and alternative pathways to respiration.
Figure 2, top, demonstrates that treatment with SA didaffect the amount of alternative oxidase, as detected on animmunoblot with the alternative oxidase monoclonal anti-body against the alternative oxidase protein (Elthon et al.,1989b). There was a 2.5-fold increase in alternative oxidaseprotein 5 h after treatment with SA. The level of the alter-
PR-1
12 24 72 HoursFigure 3. PR-1 a gene expression following treatment with SA. RNAwas isolated from the same plants as used in Figure 2 and analyzedby gel-blot hybridization using PR-la as a probe. Representative datafrom one of three experiments are presented.
45kDa
12 24 72 Hours
51 75 100 92 79 V,KCN
29 kDa
0 12 24 72 HoursFigure 2. Effect of treatment with 1 mM SA on the levels of thealternative oxidase (top) and Cyt oxidase subunit II proteins (bottom)in mitochondria isolated from cv Xanthi leaves, and on the rates ofO2 uptake in the presence of KCN (VKCN) in leaf discs (A). Equalamounts (15 /j.g) of mitochondrial protein were loaded in each laneand blotted with either the alternative oxidase monoclonal antibody(A) or a Cyt c oxidase subunit II polyclonal antibody (B). Lanes 1,Untreated cv Xanthi plants; lanes 2 through 5, 5, 12, 24, and 72 hfollowing treatment with SA, respectively. VKCN was measured asnmol O2 min"' g"1 fresh weight, using comparable leaf discs inisotope-fractionation experiments. Representative data from one ofthree experiments are presented.
native oxidase protein reached a maximum 12 h after SAtreatment, with a 9-fold increase in protein over that inuntreated plants. The level of the alternative oxidase pro-tein decreased after 12 h, but 3 d after SA treatment it stillremained elevated 2-fold over that observed in untreatedcontrol plants. There was also an increase in VKCN thattracked the amount of alternative oxidase protein, with amaximal increased rate (2-fold) observed at 12 h (Fig. 2,top). Even though a 9-fold increase in alternative oxidaseprotein level was observed at 12 h, the resulting VKCN wasapproximately equal to the respiration rate seen in theabsence of added inhibitor, suggesting that this is the max-imal sustainable respiratory rate in these leaves. After 24 hVKCN decreased, as did the level of alternative oxidaseprotein. However, 3 d after SA treatment VKCN was stillelevated in comparison to untreated control plants, as werelevels of alternative oxidase protein. Figure 2, bottom,demonstrates that the level of the Cyt oxidase subunit IIprotein was not elevated upon treatment with SA, althougha decrease in the level of this protein was observed at 3 d.Concomitant with the induction of alternative oxidase bySA, there was a marked increase in mRNA levels of thePR-la protein (Fig. 3), indicating that the SA treatment hadinduced SAR gene expression. PR-la is the predominantmarker for the onset of SAR in tobacco (Ward et al., 1991).
In tobacco there are significant levels of alternative oxi-dase protein constitutively present, in contrast to otherSA-inducible proteins, such as PR-la, which are rare inunstressed, healthy plants (Ward et al., 1991). To determineif the level of constitutively present alternative oxidase wasdependent on the SA content of the plant, alternative oxi-dase was measured in transgenic NahG (Friedrich et al.,1995) and SH-L (Bi et al., 1995) plants, which contain verylow (NahG) or undetectable (SH-L) levels of SA because ofthe activity of the enzyme salicylate hydroxylase fromPseudomonas putida. Both NahG and SH-L (data not shown) www.plantphysiol.orgon May 17, 2020 - Published by Downloaded from
Salicylic Acid Effects on the Alternative Oxidase 787
plants showed a An of 23.1% and had levels of the alter-native oxidase protein similar to control plants (Fig. 4).Therefore, NahG plants contain the alternative oxidaseprotein, and its activity during respiration is the same asthat of control cv Xanthi wild-type plants. O2-isotope frac-tionation by the Aa (30.3% ± 0.3) and Ac (20.0% ± 0.2) ofNahG leaf tissue were similar to the wild type, and theoverall respiration rate of NahG and SH-L plants was115 ± 5 nmol O2 min"1 g"1 fresh weight, also identical tothat of wild-type plants. Addition of SA to the NahG orSH-L plants did not cause a change in the electron parti-tioning between the alternative and Cyt pathways (data notshown).
45kDa
23.2 23.6 23.4 23.3 A
The Effects of TMV Infection on the Alternative Oxidase
To test the effect of pathogen infection on the alternativeoxidase, O2 fractionation during respiration in tobacco leaftissue and the level of alternative oxidase protein followinginfection by TMV were measured. These measurementswere made in both infected, primary, and uninfected sys-temic leaves. Immunoblots (Fig. 5) indicated that 7 d afterinfection, the level of alternative oxidase protein is elevatedin both TMV-infected primary leaves (lane 4) and in unin-fected systemic leaves (lane 3) over that observed in mock-inoculated control plants (lane 1). The increase was approx-imately 2-fold in both infected and uninfected leaves. Tofurther characterize the induction of alternative oxidase ininfected tissue, we compared levels of alternative oxidaseprotein in two types of leaf tissue: leaf sectors that dis-played lesion formation due to TMV infection, and leafsectors 1 cm distant to TMV-induced lesions. Figure 6Bdemonstrates that the level of alternative oxidase proteinwas increased to a greater extent in TMV-infected leaf
45kDa
Xanthi NahG
23.210.3 23.110.2 A
110i8 115!5 VtFigure 4. Immunoblots of the alternative oxidase protein, O2-isotopefractionation (A), and respiration rates (V,) in mitochondria isolatedfrom cv Xanthi wild-type (lane 1) and NahC (lane 2) tobacco plants.Equal amounts of mitochondrial protein (15 fjig) were blotted with thealternative oxidase monoclonal antibody. Respiration rates (nmol O2
min~' g~' fresh weight), A values (%), and SES were calculated fromfour to seven repetitions.
110 105 120 122 VtFigure 5. Immunoblots of the alternative oxidase protein in isolatedmitochondria and O2-isotope fractionation (A) and respiration rates(V,) in leaf discs from cv Xanthi plants treated with SA or infectedwith TMV. Lane 1, Infiltration with H2O 3 d before analysis; lane 2,infiltration with 1 mM SA 3 d before analysis; lane 3, systemic leaf ofplant in which the primary leaf was infected with TMV 7 d beforeanalysis; and lane 4, primary leaf of plant infected with TMV 7 dbefore analysis. Equal amounts of mitochondrial protein (15 ;u,g) wereloaded in each lane and blotted with the alternative oxidase mono-clonal antibody. Respiration rates (nmol O2 min~' g~' fresh weight)and A values (%) were calculated from four to seven repetitions. SESfor the respiration rates were within ± 5% and for the A values werewithin ± 1%.
sectors (4-fold; lane 4B), compared with regions of the leafadjacent to infected sectors (1.5-fold; lane 3B). The parti-tioning of electrons between the Cyt and alternative path-ways in both the infected and uninfected leaves did notdiffer from that in uninfected control plants (An = 23%),and the total respiration rate remained comparable to thatof control plants (Fig. 5).
The induction of alternative oxidase in systemic leavesoccurred concomitantly with the accumulation of PR-lamRNA in the same leaves (Fig. 7, lanes 1 and 2), indicatingthat this process might be related to the onset of SAR inthese leaves. To test whether accumulation of the alterna-tive oxidase in systemic leaves of TMV-treated plants is SAdependent (as is the induction of SAR, Gaffney et al., 1993;Vernooij et al., 1994), the TMV infection experiments wererepeated with NahG and SH-L plants. In both NahG andSH-L plants (data not shown), TMV infection of the pri-mary leaves did not cause an increase in either PR-lamRNA accumulation (Fig. 7, lanes 3 and 4) or the level ofalternative oxidase protein (Fig. 6A, lanes 1 and 2) insystemic leaves.
DISCUSSION
It has been known for some time that SA modulates theinduction of SAR following pathogen attack (Neuen-schwander et al., 1996; Ryals et al., 1996) and that SA caninduce the expression of the alternative oxidase (Rhoadsand Mclntosh, 1992, 1993). In this study we have re-examined the effects of SA on the alternative oxidase usingO2-isotope fractionation, which allows measurement of the www.plantphysiol.orgon May 17, 2020 - Published by Downloaded from
A BFigure 6. Immunoblots of the alternative oxidase protein in mitochondria isolated from cv NahG (A) and cv Xanthi (B)tobacco 7 d after treatment with TMV. A, Lane 1, NahG systemic leaf of mock-inoculated control; lane 2, NahG systemicleaf of TMV-infected plant. B, Lane 1, cv Xanthi systemic leaf of mock-inoculated control; lane 2, cv Xanthi systemic leafof TMV-infected plant; lane 3, cv Xanthi primary leaf of TMV-treated plant (uninfected region); and lane 4, cv Xanthi primaryleaf of TMV-treated plant (infected region). Equal amounts of mitochondrial protein (A, 15 /xg; B, 10 /ng) were loaded in eachlane and blotted with the alternative oxidase monoclonal antibody. Representative data from one of three experiments arepresented.
partitioning of electrons between the alternative and Cytpathways in the absence of added inhibitors (Robinson etal., 1995).
Infiltration of 1.0 mM SA into leaves of tobacco caused amarked increase in the level of both the alternative oxidaseprotein and PR-la mRNA over a 3-d period. There was nochange in the partitioning of electrons between the twopathways at any point during the experimental timecourse. In all cases a ra of 0.27 to 0.30 was observed,indicating that SA does not act to modulate the activity ofthe alternative pathway. Although no change in electronpartitioning was observed, the increased alternative oxi-dase protein did lead to an increase in VKCN- There was no
PR-1
X XM T
NM
NT
Figure 7. PR-la gene expression in uninfected systemic leaves fol-lowing treatment of cv Xanthi or cv NahG plants with TMV. RNA wasisolated from uninfected systemic leaves of TMV or mock-inoculatedplants 7 d after treatment and analyzed by gel-blot hybridizationusing PR-1 a as a probe. The same plants used in Figure 6 (lanes 1A,2A, 1 B, and 2B) were analyzed. X, cv Xanthi; N, cv NahG; M, mockinoculation of primary leaf; and T, TMV inoculation of primary leaf.Representative data from one of three experiments are presented.
increase in the level of the COXII protein following SAtreatment, indicating that the elevated alternative oxidasedid not result from an increase in all mitochondrial pro-teins. The increase in the level of the alternative oxidaseprotein and VKCN are consistent with the results of Rhoadsand Mclntosh (1993), who observed both an increase in thelevel of alternative oxidase protein and an increase in VKCNusing tobacco cell-suspension cultures. However, the re-sults of this study clearly indicate that an increase in thelevel of the alternative oxidase protein does not necessarilylead to an increase in Val, in the absence of inhibitors. Allother factors being constant, a doubling of the amount ofalternative oxidase protein would be expected to increasethe partitioning of electrons to the oxidase in the absenceof inhibitors. Whereas the specific alternative oxidaseisozyme induced by SA could conceivably have differentkinetic properties from that of the constitutively expressedoxidase, the alternative oxidase is also felt to be subject toa number of regulatory features (e.g. by a-keto acids andsulfhydryl/disulfide redox regulation) that can markedlymodulate its activity (Siedow and Umbach, 1995). The re-sults of the present study suggst that there is an apparentregulation of the alternative oxidase that compensates forthe higher amount of alternative oxidase protein present tokeep the partitioning of electrons to the oxidase constant ata given total respiration rate.
The respiratory data from this study differ from theresults obtained by Van Der Straeten et al. (1995). Eighthours after SA treatment of tobacco plants, these authorsobserved an increase in the total respiration rate, as well asan increase in the activity of the alternative pathway mea-sured as the difference between total respiration and therespiration after the addition of 25 mM SHAM. The tem-perature of the leaf surface was also shown to increase by0.5 to 1°C following treatment with SA. This temperatureincrease was proposed to be derived from the observedincrease in alternative pathway activity. Van der Straetenet al. (1995) reported that the overall increase in respirationupon treatment with SA was 18 nmol O2 min"1 g"1 freshweight. Assuming that the entire increase in the respiration www.plantphysiol.orgon May 17, 2020 - Published by Downloaded from
Salicylic Acid Effects on the Alternative Oxidase 789
rate occurred via the alternative pathway, application of the Stefan-Boltzmann equation (Nobel, 1983) indicates that the total heat that could potentially be produced is two orders of magnitude below that required to account for the 0.5 to 1°C temperature increase observed by Van Der Straeten et al. (1995). Further, the reported increase in alternative oxidase activity was demonstrated by the addi- tion of SHAM, which may not represent a true measure of Val, in vivo in the absence of inhibitors (Day et al., 1996).
Our results obtained in the absence of inhibitors indicate that following SA treatment there is no engagement of the alternative pathway above that observed in untreated con- trol plants. The rate of O, consumption for untreated plants obtained in this study is higher than that reported by Van Der Straeten et al. (1995), the difference being approximately 35 nmol O, min-' g-' fresh weight, perhaps due to varietal differences. The respiration rates reported by Van Der Straeten et al. (1995) following treatment with SA (approxi- mately 90 m o 1 O, min-' g-' fresh weight) are closer to the rates obtained in our experiments for both treated and un- treated plants. It is possible that the increase in respiration rate observed by Van Der Straeten et al. (1995) was due to the different method of application of SA to their plants. Van Der Straeten et al. (1995) sprayed 5 mM SA onto the leaf surface, whereas in our study 1 mM SA was infiltrated into the leaf (Friedrich et al., 1995). However, spraying 2.5, 5, or 10 mM SA did not result in an increase in either the parti- tioning of electrons to the alternative pathway or an increase in the total respiration rate with our plants (data not shown). Spraying tobacco leaves with 5 to 10 mM SA can lead to considerable toxicity (L. Friedrich, B. Vernooij, and J. Ryals, unpublished observations).
The ability of SA to increase the level of alternative oxidase protein led to the question of whether SA-deficient plants would contain less alternative oxidase than wild- type tobacco. Alternative oxidase protein was measured in two transgenic lines (SH-L and NahG) that have very low levels of SA (Bi et al., 1995; Friedrich et al., 1995) due to expression of a bacterial salicylate hydroxylase. Both trans- genic lines exhibited comparable amounts of alternative oxidase protein and the same alternative pathway activity as wild-type plants. These results suggest that endogenous SA is not required for a constitutive level of expression of the alternative oxidase, but that the presence of enhanced levels of cellular SA does induce an increase in the level of the alternative oxidase protein. Rhoads and McIntosh (1992) have previously proposed that the developmental regulation of alternative oxidase expression in voodoo lily (Saurotum guttatum) may not be solely dependent on SA, and recent work by Vanlerberghe and McIntosh (1996) has shown, in tobacco suspension-cell cultures, that a number of compounds, including citrate, Cys, and H,O,, can in- duce an increase in both the level of alternative oxidase protein and V,,,. Our results are in agreement with those of Vanlerberghe and McIntosh (1996), who found that ex- pression of the alternative oxidase was not regulated solely by SA, which further supports the concept that there are a number of cellular species that can regulate expression of the alternative oxidase. Whelan et al. (1995) have reported the presence of two distinct alternative oxidase cDNAs in
tobacco, leading to the possibility that only one of these represents a gene product that is responsive to SA.
After infection of wild-type tobacco plants with TMV, we found an increase in the level of the alternative oxidase protein in the infected leaves after 7 d, but no change in activity of the alternative pathway was detected. However, this result may be misleading. Increased engagement of the alternative oxidase could be restricted to cells close to the edge of the lesion sites. As such, no significant increase in alternative oxidase activity would be observed, as the vast majority of the tissue would still have a fractionation of 23% and mask the larger fractionation in the small percent- age of tissue near the lesion site.
Similar results were obtained with tissue from unin- fected leaves of the same plants. Infection of wild-type tobacco plants with TMV also resulted in increased levels of the alternative oxidase protein in uninfected leaves after 7 d, whereas the partitioning of electrons between the alternative and Cyt pathways was not affected in these leaves. Moreover, TMV treatment of transgenic plants un- able to accumulate SA did not result in detectable changes in the uninfected leaves. In the same set of experiments the levels of PR-la mRNA in uninfected leaves were induced in wild-type, but not in SA-deficient, transgenic plants. These data suggest that induction of alternative oxidase protein in uninfected leaves is SA dependent and correlates with expression of PR-la, a primary marker for SAR in tobacco (Ward et al., 1991).
We speculate that elevated levels of alternative oxidase protein may be an element of the SAR response in tobacco. The systemic response may be poising the level of the alternative oxidase, along with other SAR proteins, such that they can react rapidly to subsequent pathogen infec- tion. This suggests that the alternative oxidase could play a role in the response of plants to pathogen infection. One possibility is that the alternative oxidase may be involved in maintaining the oxidative burst that occurs following infection with pathogens that trigger a hypersensitive re- sponse (Hammond-Kosack and Jones, 1996). High local levels of SA have been proposed to contribute to maintain- ing the oxidative burst by inhibition of oxidoreductases (Chen et al., 1993; Neuenschwander et al., 1995), leading to an increase in the half-life of reactive O, intermediates. Generation of the oxidative burst is believed to require NADPH (Hammond-Kosack and Jones, 1996). Production of large quantities of NADPH could occur via the pentose phosphate shunt, which would result in the oxidation of Glc-6-phosphate to pyruvate with the production of ATP and NADPH (Stryer, 1981). To maintain the pathway the resulting pyruvate would need to be oxidized to CO,. The alternative oxidase could aid in the oxidation of pyruvate by allowing turnover of the Krebs cycle under reduced adenylate control. As such, the increased level of alterna- tive oxidase protein produced during the SAR response would poise the tissue for mobilizing a rapid response to pathogen infection and maintenance of the oxidative burst. Therefore, treatment with SA alone does not result in an increase in the percentage of electrons partitioning to the alternative pathway, even though it does lead to an in- crease in the amount of alternative oxidase protein. In this
scenario the alternative oxidase will only show a higher leve1 of activity in the immediate aftermath of a subsequent pathogen attack. Future studies will be needed to verify this hypothesis.
ACKNOWLEDCMENTS
We wish to thank Dr. T.E. Elthon for his gift of the alternative oxidase monoclonal antibody, Dr. C.S. Levings, 111, for his gift of the Cyt c oxidase polyclonal antibody, Dr. Eric Ward for useful discussions, and Drs. Ann Umbach, Michelle Hunt, and Mike Willits for their critica1 reading of the manuscript.
Received March 24, 1997; accepted July 2, 1997. Copyright Clearance Center: 0032-0889 /97/ 115/0773/09.
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