immutans does not act as a stress-induced safety valve in the protection of the photosynthetic...

IMMUTANS Does Not Act as a Stress-Induced SafetyValve in the Protection of the Photosynthetic Apparatusof Arabidopsis during Steady-State Photosynthesis1

Dominic Rosso, Alexander G. Ivanov, Aigen Fu, Jane Geisler-Lee, Luke Hendrickson, Matt Geisler,Gregory Stewart, Marianna Krol, Vaughan Hurry, Steven R. Rodermel, Denis P. Maxwell,and Norman P.A. Huner*

Department of Biology and The Biotron, University of Western Ontario, London, Ontario, Canada,N6A 5B7 (D.R., A.G.I., J.G.-L., L.H., M.G., G.S., M.K., D.P.M., N.P.A.H.); Department of Genetics,Development and Cell Biology, Iowa State University, Ames, Iowa 50011 (A.F., S.R.R.); and Umea PlantScience Centre, Department of Plant Physiology, Umea University, Umea S–901 87, Sweden (L.H., V.H.)

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couplesthe reduction of O2 to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IMshares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the proteinencoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined invivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P700 to assess the capacity of IM tocompete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana).Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had beenoverexpressed six (OE-6 3) and 16 (OE-16 3) times. Immunoblots indicated that IM abundance was the only major variant thatwe could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pooloxidized compared to either the wild type or im grown under control conditions (25�C and photosynthetic photon flux densityof 150 mmol photons m22 s21). Similar results were observed either after 3-d cold stress at 5�C or after full-leaf expansion at 5�Cand photosynthetic photon flux density of 150 mmol photons m22 s21. Furthermore, IM abundance did not enhance protection ofeither photosystem II or photosystem I from photoinhibition at either 25�C or 5�C. Our in vivo data indicate that modulation ofIM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P700

1 during steady-statephotosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of publishedArabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses,which is consistent with our functional data. However, IM exhibited significant modulation in response to development inconcert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression linesnor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ poolduring stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Plants with a variegated phenotype display distinctcolor variation in their vegetative organs, the mostcommon of which being leaves with distinct green/white sectoring (Kirk and Tilney-Bassett, 1978; Rodermel,2002). Whereas cells in the green sectors contain es-sentially normal chloroplasts, cells in the white sectorshave plastids that are deficient in chlorophyll (Chl)and/or carotenoids (Rodermel, 2001, 2002). The varie-gated phenotype is most often the result of mutations

to distinct genes of either the nuclear or organellargenomes (Tilney-Bassett, 1975) and include the maize(Zea mays) nonchromosomal stripe (ncs) and iojap, as wellas Arabidopsis (Arabidopsis thaliana) mutants, chloro-plast mutator (chm), pale cress (pac), var1 and var2, cabunderexpressed (cue1), and immutans (im; for review, seeRodermel, 2002).

The im mutant was isolated and initially characterizednearly 40 years ago by Redei and coworkers, whoshowed that im is the result of a recessive mutation to anuclear gene and that the resulting variegated pheno-type is exacerbated by exposure to elevated tempera-tures and high light intensities (Redei, 1963, 1975;Robbelen, 1968; Wetzel et al., 1994). More recently,Wetzel et al. (1994) found that whereas the green sectorscontain the normal allotment of Chls and colored carot-enoids, the white sectors showed an accumulation of thecolorless carotenoid phytoene, the precursor of the ma-jor colored carotenoids. The white sectoring seen whenplants are grown under moderate to high irradiance isthus presumed to be the result of the photooxidation of

1 This work was supported by the Natural Sciences and Engi-neering Research Council of Canada (NSERC; grant to D.P.M., andN.P.A.H.) and by the U.S. Department of Energy (Energy Biosci-ences; grant no. DF–FG02–94ER20147 to S.R.R.). D.R. is the recipientof an NSERC postgraduate scholarship.

* Corresponding author; e-mail [email protected]; fax 519–661–3935.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Norman P.A. Huner ([email protected]).

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Chl due to the absence of protective carotenoids. Evi-dence suggests that the IM protein plays a critical role inthe desaturation reaction required to convert phytoeneinto photoprotective carotenoids (Wetzel et al., 1994).Briefly, the desaturation reaction is thought to requirethe donation of electrons to the plastoquinone (PQ)pool (Norris et al., 1995), a component of the photo-synthetic intersystem electron transport chain withinthe thylakoid membrane. Evidence suggests that sub-sequent oxidation of PQ involves IM, which acts as aplastid quinol oxidase (Carol et al., 1999; Wu et al.,1999). It follows that the lack of this oxidase in im plantsresults in the overreduction of PQ, leading to theinhibition of phytoene desaturation. This in turn wouldlead to phytoene accumulation, Chl photooxidation,and the appearance of white sectors (Wu et al., 1999).Consistent with the notion that white sectoring istriggered by photooxidation is that leaves of im plantsare almost indistinguishable from the wild type whenplants are grown under low light conditions (Redei 1963;Wetzel et al., 1994; Aluru and Rodermel, 2004).

In addition to its role in carotenoid biosynthesis,recent in vitro and in vivo evidence indicates that IMmay be the sought-after plastid terminal oxidaseinvolved in chlororespiration (Cournac et al., 2000b;Josse et al., 2000, 2003; Joet et al., 2002; Peltier andCournac, 2002; Fu et al., 2005). Bennoun (1982) pro-posed that, in the dark, reducing equivalents from astromal pool of NAD(P)H reduces PQ, which is me-diated by a NAD(P)H dehydrogenase. Subsequentoxidation of PQ is thought to require the action of IM.The recent identification of a chloroplastic NAD(P)Hdehydrogenase complex (Ohyama et al., 1986, 1988;Guedeney et al., 1996; Sazanov et al., 1996; Burrowset al., 1998; Field et al., 1998; Casano et al., 2000;Horvath et al., 2000) as well as immunological evi-dence that IM is localized in the stromal lamellae of thethylakoid membrane (Joet et al., 2002; Lennon et al.,2003) have supplied molecular evidence for the exis-tence of a chlororespiratory pathway (Carol et al.,1999; Wu et al., 1999; Cournac et al., 2000a, 2000b, 2002;Carol and Kuntz, 2001; Joet et al., 2002).

IM shows clear sequence similarity to the alternativeoxidase (AOX) of the respiratory chain of plant mito-chondria, with both proteins being nonheme di-ironcarboxylate proteins (Carol et al., 1999; Wu et al., 1999).AOX constitutes the alternative pathway of mitochon-drial electron transport, which can be differentiatedfrom the ubiquitous cytochrome pathway by the factthat it is insensitive to cyanide, but rather is inhibitedby salicylhydroxamic acid and n-propyl gallate. Duringalternative pathway respiration, AOX accepts elec-trons directly from reduced ubiquinone, bypassingthe respiratory complexes involved in proton translo-cation. Because of this, alternative pathway respirationdoes not contribute to the transmembrane pH gradientused to synthesize ATP. Instead, evidence suggeststhat, by providing a second pathway of electron flow,the alternative pathway and AOX may serve to preventthe overreduction of electron transport components,

which may occur in response to environmental stress(Vanlerberghe and McIntosh, 1997; Maxwell et al.,1999) and which would exacerbate the formation ofdamaging reactive oxygen species (Møller, 2001; Mooreet al., 2002). Similarly to AOX, IM has also been shownto be inhibited by n-propyl gallate (Cournac et al.,2000b; Josse et al., 2000), and it has been hypothesizedthat IM may be functionally analogous to AOX andserve to keep the photosynthetic electron transportchain relatively oxidized. Exposure of plants to excessirradiation may result in overreduction of electrontransport components and may lead to photoinhibitionof PSII, which, if not prevented, would result in de-creased energy transformation and ultimately a de-crease in plant biomass. Increased IM activity underconditions of high light thus may act as a safety valvefor excess electrons, preventing the overreduction ofthe photosynthetic electron transport chain (Niyogi,2000) and thereby minimizing the aberrant formationof potentially destructive reactive oxygen specieswithin the chloroplast (Melis, 1999; Niyogi, 2000).

Recently, Streb et al. (2005) reported that, comparedwith other alpine plant species, Ranunculus glacialisacclimated to high light and low temperature exhibi-ted increased levels of IM, which was correlated with amore oxidized electron transport chain as reflected bya lower measure of excitation pressure (EP). Whereasthis finding lends support to the hypothesis that IMmay serve to keep the photosynthetic electron trans-port chain more oxidized under environmental stress,direct experimental evidence by measuring the redoxstate of intersystem electron transport and the redoxstate of PSI are required to confirm such a role. Usingwild-type and im plants as well as transgenics inwhich IM has been overexpressed six (OE-6 3) and 16(OE-16 3) times, we have directly tested this hypoth-esis by making measurements of Chl a fluorescence aswell as measurements of PSI reoxidation kinetics dur-ing steady-state photosynthesis using fully expandedall-green leaves of Arabidopsis grown under strictlycontrolled conditions. We examined the extent to whichIM could compete with PSI for photosyntheticallygenerated electrons under controlled growth condi-tions as well as following a 3-d cold stress period. Wehypothesized that plants in which IM has been over-expressed should exhibit increased alternative elec-tron sink capacity, directly competing with PSI forintersystem electrons in comparison to the wild type,and be more resistant to high light-induced photo-inhibition. Conversely, plants lacking IM (im) shouldshow greater sensitivity to photoinhibition due tohaving a more reduced intersystem PQ pool and alower capacity to keep P700 oxidized.

RESULTS

Plant Phenotype

Previous reports investigating the consequence ofmutations to IM have compared all green wild-type

IMMUTANS as a Terminal Oxidase

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plants with im plants that display the variegated phe-notype (Aluru and Rodermel, 2004; Baerr et al., 2005).However, this approach is problematic because varie-gated leaves represent a heterogeneous population ofphotosynthetically competent cells with photooxidizedtissue displaying vastly different physiological prop-erties than green tissue. To determine accurately thephysiological role of IM requires plants to be grownunder conditions where the leaves are all green, re-gardless of genotype. We found that suppression of thevariegated phenotype was possible by first allowingthe plants to germinate and grow at 25�C and photo-synthetic photon flux density (PPFD) of 5 mmol photonsm22 s21 (25/5) for 7 d followed by growth at 50 mmolphotons m22 s21 (25/50) for 35 d. Subsequently, plantswere allowed to acclimate fully to the experimentalgrowth condition of 25�C and PPFD of 150 mmol pho-tons m22 s21 (25/150) for 40 d prior to exposure to coldstress by shifting plants to 5�C and PPFD of 150 mmolphotons m22 s21 (5/150) for an additional 3 d (Fig. 1). Itshould be noted that im plants did develop the varie-gated phenotype if they were shifted directly to PPFDof 150 mmol photons m22 s21 after the initial 7 d at 5mmol photons m22 s21 (Fig. 1).

When grown under the same conditions, whichsuppressed the variegation of im plants, minimal dif-ferences where observed in overall morphology (Fig. 2).Plants were similar in size and exhibited minimaldifferences in total Chl per leaf area compared with thewild type (Fig. 2). Separation of Chl-protein complexesby nondenaturing SDS-PAGE indicated no significantdifferences in the complement of thylakoid pigment-protein complexes (data not shown) among thefour genotypes, which is consistent with the fact thatall lines exhibited similar ratios of Chl a/b (Fig. 2; 25/150).

Plant Genotype

Expression of IM transcript and polypeptide abun-dance in the four different genotypes was confirmedusing RNA-blot analysis and immunoblotting utiliz-ing a polyclonal antibody raised against IM (Rizhskyet al., 2002). Compared to the wild type, the twooverexpressing lines had greater IM transcript abun-dance (Fig. 3A), which correlated with a 6- and 16-foldincrease in protein abundance (Fig. 3B). Furthermore,neither the IM transcripts (Fig. 3A) nor polypeptides(Fig. 3B) were detected in the all-green leaves of implants. In addition, because IM is a thylakoid mem-brane protein, the relative abundance of specific poly-peptides associated with PSI (PsaA), the NAD(P)Hdehydrogenase complex (H subunit), PSII (PsbA), thecytochrome b6 f (Cyt f) complex, and the major light-harvesting polypeptides associated with PSII (Lhcb2)and PSI (Lhca1) were examined to determine whetherthe various genotypes exhibited any other major dif-ferences in the stoichiometry of these photosyntheticcomponents (Fig. 3C). Minimal differences were ob-served in the relative abundance of these componentsof the photosynthetic apparatus in OE-6 3 and OE-16 3, as well as im compared to the wild type (Fig. 3C).

In vivo Chl a fluorescence was used to assess PSIIfunction. All genotypes grown at 25/150 possessed asimilar maximal PSII photochemical efficiency (Fv/Fm)of approximately 0.784 (Table I). A second indepen-dent measure of PSII photochemistry, thermolumines-cence, indicated no significant differences in the peakemission temperatures for either the B-band (29.3�C 61.0�C) or the Q-band (13.3�C 6 0.8�C) in all genotypestested. These measurements show that alterations toIM through mutation or overexpression had littleeffect on PSII photochemistry.

Figure 1. Experimental design for the suppression of the variegated phenotype. Seeds from im were germinated and allowedto grow at 25�C with an irradiance of 5 mmol photons m22 s21 (25/5) for 7 d. Plants were then shifted from an irradiance of 5 to50 mmol photons m22 s21 (25/50) for 4 weeks. Once the first rosette appeared, the plants were then shifted to 150 mmol photonsm22 s21 (25/150) for another 4 weeks until the second and third rosettes had completely developed. It is at this stage that fullyexpanded leaves were used for experimental analysis. Plants were then shifted from 25�C to 5�C for 3 d at an irradiance of150 mmol photons m22 s21 (25/150) to cold stress the plants. Alternatively, when plants were shifted directly from 5 to 150 mmolphotons m22 s21 after 7 d, im knockout plants exhibited a variegated phenotype when fully developed. All genotypes were grownwith an 8/16-h day/night cycle.

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EP, measured as 1 2 photochemical quenchingusing the puddle model (qP), is a measure of therelative reduction state of quinone A (QA), the firststable quinone acceptor in PSII reaction centers (Dietzet al., 1985; Huner et al., 1998, 2003; Kramer et al.,2004), and reflects the overall reduction state of theelectron transport chain. Whereas both overexpressinglines showed a 23% (OE-6 3) and a 53% (OE-16 3)higher EP compared to the wild type (Table I), it wasinteresting to find that im plants exhibited an EP thatwas approximately 26% lower than wild-type plants(Table I). However, none of the differences in EPbetween the genotypes tested was statistically signif-icant. Similar trends were observed when EP wascalculated using the more recently derived parameter,1 2 photochemical quenching using the lake model(qL; Table I).

To assess potential functional differences in photo-synthetic intersystem electron transport among thegenotypes, the oxidation-reduction of P700 was moni-tored by measuring the absorbance change at 820 nm(DA820/A820), which was normalized on a total Chl perleaf area basis (Mi et al., 1992a; Asada et al., 1993;Morgan-Kiss et al., 2001). Upon exposure to far-red(FR) light, wild-type leaves exhibited a rapid changein DA820/A820, indicating oxidation of P700 (Fig. 4A).P1

700 was transiently reduced with either a saturating,single-turnover (ST) flash or a saturating, multiple-turnover (MT) flash of white light in the presence ofbackground FR light (Fig. 4A). When the FR light wasturned off, P1

700 was completely reduced to P700 (Fig.4A). In the presence of 3-(3#, 4#-dichlorophenyl)-1,1-dimethylurea (DCMU; Fig. 4B), which inhibits PSIIat the quinone B (QB) binding site, the ST and MTflashes did not cause any transient reduction of P1

700even though the extent of the DA820/A820 signal wasunchanged. However, in the presence of DCMU, stro-mal electron donation to the PQ pool could stillcontribute to the reduction of P1

700. 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) inhibitsintersystem electron transport after the PQ pool at theCyt f complex. Thus, the maximal DA820 /A820 signalunder our measuring conditions would be expected inthe presence of DBMIB. Figure 4C illustrates that, as

expected, DBMIB inhibited the transient reductionof P700

1 by the ST and MT flashes. However, the extentof the DA820/A820 signal in the presence of DBMIB(Fig. 4C) was 24% greater than either that of the con-trol (Fig. 4A) or that observed in the presence of DCMU(Fig. 4B). This confirms that monitoring the redox stateof P700 is a valid measure of intersystem electron

Figure 2. Morphology, Chl per leaf area (mg Chlcm22), and Chl a/b ratios of different plant genotypesof Arabidopsis ecotype Columbia. Wild type (WT),OE-6 3, OE-16 3, and an all-green sectored knockoutmutant (im) are shown. All plant genotypes weregrown at 25�C at an irradiance of 150 mmol photonsm22 s21 (25/150). Cold-stressed plants were shiftedfrom 25�C to 5�C for an additional 3 d at the sameirradiance (5/150). All plants were grown under an 8/16-h day/night cycle to prevent flowering. Photo-graphs illustrate plants that were grown at 25�C. Let-ters represent significance between means for leavesgrown at 25�C and symbols represent significancebetween means for leaves that were cold stressed atthe 95% confidence interval.

Figure 3. IM gene expression and protein abundance. mRNA expres-sion of IM (A) relative abundance and of IM protein (B) and immuno-blots of polypeptides of the major photosynthetic complexes of isolatedthylakoid membranes (C) were performed on leaves obtained from thewild type (WT), OE-6 3 and OE-16 3, and knockout mutant (im) ofArabidopsis. The RNA gel was stained with ethidium bromide to showrRNA and demonstrate equal loading (A). Solubilized thylakoid mem-branes were loaded equally with 5 mg Chl/lane. Immunoblots wereprobed using polyclonal antibodies raised against PsaA, H subunitof the NAD(P)H complex, D1, Cyt f, and Lhcb2 and Lhca1. All plantgenotypes were grown at 25�C with an irradiance of 150 mmolphotons m22 s21.






transport and that PSII contributed minimally to theDA820/A820 signal in leaves of Arabidopsis.

Traces for the oxidation of P700 obtained for OE-6 3and OE-16 3 as well as im were qualitatively similarto those obtained for the wild type (data not shown).Overexpression of IM, which was postulated to com-pete for electrons with PSI, did result in significantdifferences in the extent of the DA820/A820 signal com-pared to the wild type, where OE-16 3 exhibited a14% increase in the extent of the oxidation of P700

1,when plants were grown at 25�C and PPFD of150 mmol photons m22 s21 (Fig. 4D). However,OE-6 3 was not statistically different compared withthe wild type in the extent of the DA820/A820 signal (Fig.4D). Unexpectedly, the DA820/A820 signal in im was 7%more oxidized than the wild type; considering thatthese plants lack this oxidase, there should have beena more reduced P700 pool (Fig. 4D). Furthermore, thenumber of electrons stored in the intersystem electrontransport chain (e2/P700) was measured as the ratio ofMT to ST. Calculated e2/P700 ranged from 8.6 to 11.1,with no statistical differences between all genotypestested. These data for e2/P700 were supported by in-dependent measurements of the transient rise in Fofluorescence after a transition from light to dark dur-ing steady-state photosynthesis. No differences in theextent of the transient rise in Fo fluorescence was ob-served between any of the genotypes tested (data notshown), indicating a comparable reduction state of theintersystem PQ pool (Mills et al., 1979; Endo et al.,1997; Corneille et al., 1998).

Photoinhibition and Recovery of PSII and PSI

To assess the sensitivity of the four genotypes tophotoinhibition of PSII, Fv/Fm was measured in plantsexposed to the photoinhibitory treatment of 1,200mmol photons m22 s21 at 5�C (Fig. 5). Prior to photo-inhibition, the Fv/Fm was approximately 0.80 for allgenotypes examined (Fig. 5) and, after 4 h, all geno-types were photoinhibited approximately to the sameextent (Fig. 5). Subsequent recovery from photoinhi-

bition by exposure of plants for 8 h at 25�C and lowlight (PPFD of 20 mmol photons m22 s21; Fig. 5) re-sulted in nearly 95% recovery of PSII photochemistryin all genotypes. Therefore, we conclude that either thepresence or absence of IM has minimal effects on thesensitivity of Arabidopsis to photoinhibition of PSII.

Whereas PSII is considered the main target ofphotoinhibition, PSI has also been shown to be sus-ceptible to photoinactivation (Ivanov et al., 1998;Terashima et al., 1998; Scheller and Haldrup, 2005).Thus, we also assessed the sensitivity of PSI to photo-inhibition in im versus wild-type plants (Fig. 6) underthe same conditions used for the photoinhibition andrecovery of PSII. The extent of the DA820/A820 signalprior to photoinhibition of the wild type (Fig. 6A)decreased by 56% after 4 h of photoinhibition (Fig. 6B),indicating that PSI in wild-type Arabidopsis is sensi-tive to photoinhibition. A comparison of the kineticsfor photoinhibition and recovery of PSI in the wildtype and im indicated that PSI in the wild type wasmore sensitive to photoinhibition than PSI in im(Fig. 6C), contrary to expectations. Both the wild typeand im exhibited the capacity to recover from photo-inhibition of PSI (Fig. 6C).

Cold Stress

It has been reported that cold acclimation of R.glacialis enhances the accumulation of IM, which mayprovide protection of these alpine plants from photo-inhibition (Streb et al., 2005). To test this in Arabidop-sis, all genotypes were shifted to the cold at 5�C andPPFD of 150 mmol photons m22 s21 (5/150) for 3 d(Fig. 1). No phenotypic differences between genotypesshifted to 5/150 were observed as reflected in mini-mal differences in Chl per leaf area and Chl a/b ra-tios (Fig. 2). Upon exposure to cold stress, the wildtype and OE-16 3 exhibited no statistical differencesin Fv/Fm, whereas the knockout mutant exhibiteda lower Fv/Fm (0.71; Table I). As shown in Table I,OE-6X 3 and OE-16 3 plants grown at 5/150 exhibi-ted higher EP to that of the same plants grown at

Table I. In vivo Chl a fluorescence measurements performed on wild type, OE-6 3 , OE-16 3 , and knockout mutant (im) of Arabidopsis

Fluorescence parameters measured include Fv/Fm, the relative reduction state of QA measured as 1 2 qP and 1 2 qL. Detached leaves weremeasured at their respective growth temperatures of 25�C (25/150) and 5�C (5/150) for 3-d cold-stressed leaves at an irradiance of 150 mmol photonsm22 s21. a and b, Significance between means for leaves grown at 25�C; *, #, and ^, significance between means for leaves that were cold stressed at aconfidence interval of 95%, where n 5 2 with three replicate measurements per experiment 6SE.

Condition Genotypes Fv/Fm 1 2 qP % 1 2 qL %

25/150 Wild type 0.784 6 0.01a 0.106 6 0.01a 100 0.266 6 0.01ab 100OE-6 3 0.758 6 0.01a 0.130 6 0.01a 123 0.301 6 0.01a 113OE-16 3 0.790 6 0.01a 0.162 6 0.04a 153 0.294 6 0.03a 111im 0.763 6 0.01a 0.078 6 0.02a 74 0.208 6 0.01b 78

5/150 Wild type 0.801 6 0.02* 0.244 6 0.02* 100 0.450 6 0.02* 100OE-6 3 0.755 6 0.01# 0.300 6 0.03* 123 0.496 6 0.04* 110OE-16 3 0.774 6 0.01*# 0.274 6 0.03* 112 0.452 6 0.03* 100im 0.712 6 0.02^ 0.275 6 0.02* 113 0.451 6 0.01* 100

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25/150, and the values were even higher than cold-stressed wild-type plants. Similar trends were observedwhen EP was calculated as 1 2 qL (Table I). Contraryto expectations, the EP of cold-stressed im plants wasnot significantly different from those of wild-typeplants (Table I).

Upon exposure to cold stress, both OE-6 3 andOE-16 3 exhibited a similar DA820/A820 signal as thewild type (Fig. 4E). The DA820/A820 of cold-stressed implants was also similar to both overexpression lines aswell as the wild type (Fig. 4E). Furthermore, based onthe ratio of MT to ST flashes, e2/P700 increased in allgenotypes tested upon cold acclimation and variedfrom 15.0 to 19.4, which was not statistically differentat the 95% confidence interval and no obvious trendswere observed between any of the genotypes. In ad-dition to cold stress, leaves of the wild type, OE-6 3 ,OE-16 3 , and im plants, fully expanded at 5/150,exhibited no significant differences with respect to EP,DA820/A820 signal, or e2/P700 (data not shown).

Expression of AOX1a and IM

It has been reported that IM displays sequencesimilarity with AOX, a family of nuclear genes thatencode mitochondrial AOX (Carol et al., 1999; Wuet al., 1999). Because AOX transcript, AOX protein,and associated alternative pathway respiration haveall been shown to be induced by a wide range ofenvironmental stresses (see Vanlerberghe and McIntosh1997; Maxwell et al., 1999), it has been proposedthat IM functions in a similar capacity within thephotosynthetic electron transport chain (Aluru andRodermel, 2004; Aluru et al., 2006). To compare theextent to which AOX genes and IM are transcription-ally coregulated, expression levels of the major AOXgene from Arabidopsis, AOX1a, were compared to IMusing previously published microarray data wherewild-type Arabidopsis plants were subjected to vari-ous abiotic and hormone stresses. From the datapresented in Figure 7A, the expression of IM doesnot correlate with the expression of AOX1a. WhereasAOX1a transcription abundance increased signifi-cantly under various abiotic stresses, such as H2O2,ozone, UV light, cold, and mannitol, as well as hor-mones such as abscisic acid and salicylic acid (Fig. 7A),the transcript abundance of IM remained at or nearcontrol levels. Expression levels of AOX1a and IMwere also compared under various stages of Arabi-dopsis development. In contrast to abiotic stress(Fig. 7A), there did appear to be a correlation betweenAOX1a and IM expression during development ofArabidopsis (Fig. 7B). Both AOX1a and IM exhibited

Figure 4. In vivo measurements of the relative redox state of P700.Detached plant leaves from wild type both untreated (A) and treatedwith the inhibitor DCMU (B) and treated with the inhibitor DBMIB (C)in Arabidopsis were dark adapted for 20 min prior to the measurementof the oxidation of P700. The steady-state oxidation of P700 (DA820/A820)was estimated for plants grown at 25�C (D) and 5�C (E) after the FR light

was turned on (FR ON) and the P700 transients were followed afterapplication of the ST and MT flashes of white light. Letters representstatistically significant differences between means at the 95% confi-dence interval.






increased gene expression in cauline leaves and sepals,as well as during leaf senescence (Fig. 7B).

DISCUSSION

A large body of literature proposes models in whichIM acts as a plastid terminal oxidase involved in thechlororespiratory pathway (Carol et al., 1999; Wu et al.,1999; Cournac et al., 2000a, 2000b, 2002; Josse et al.,2000, 2003; Joet et al., 2002; Peltier and Cournac, 2002;Fu et al., 2005). By analogy to the AOX of plantmitochondria, the role of the plastid terminal oxidasehas been extended to photoprotection of PSII duringsteady-state photosynthesis by keeping the intersys-tem PQ pool oxidized under excess excitation (Niyogi,2000; Streb et al. 2005). If the proposed role of IM inphotoprotection is correct, then its overexpression intransgenic plants should keep the PQ pool more ox-idized than in the wild type, and, conversely, im plantsshould exhibit a greater reduction of the PQ pool thanthe wild type. A corollary to this hypothesis is thatthe overexpression of IM should increase the compe-tition for intersystem electrons and thereby enhancethe capacity to keep PSI more oxidized. Our resultsindicate that modulation of IM expression and IMpolypeptide accumulation does not affect the flux ofelectrons through the photosynthetic intersystem elec-tron transport chain during steady-state photosynthesisfor the following reasons. First, both overexpressinglines exhibited higher excitation pressure than thewild type (Table I). More important, im plants failedto exhibit the expected higher EP than the wild type,but instead exhibited a 23% lower EP as comparedwith the wild type (Table I). Second, no significantdifferences were observed in either the intersystemelectron pool size (e2/P700) or the reduction state of

the PQ pool estimated either as the transient rise in Foafter a light-to-dark transition or as EP (Table I). Third,either a 6- or a 16-fold increase in IM abundance(Fig. 3B) failed to enhance the capacity to keep P700in the oxidized state as compared with the knockout(Fig. 4, D and E). Conversely, im plants that lack thisprotein (Fig. 3B) failed to exhibit the expected decreasein capacity to keep P700 oxidized (Fig. 4, D and E).Thus, we conclude that IM lacks the ability to competewith PSI for photosynthetically generated electronsduring steady-state photosynthesis, which is consis-tent with our data indicating that the levels of IM arenot correlated with the photoprotection of either PSIIor PSI from photoinhibition (Figs. 6 and 7). Theseresults for the sensitivity of PSII to photoinhibition areconsistent with those of Joet et al. (2002) and Baerr et al.(2005).

Our functional data from in vivo experiments usingtwo overexpressing lines and an IM mutant (im) inArabidopsis support the conclusion of Ort and Baker(2002) that IM lacks the capacity to alter electron fluxthrough the intersystem electron transport chain sig-nificantly during steady-state photosynthesis. As aconsequence, IM cannot provide any significant pho-toprotection from excess light even when it is overex-pressed 16-fold in plants grown either under control(25/150) or cold stress by a sudden shift from 25/150to 5/150 or after cold acclimation.

Recently, Streb et al. (2005), investigating a high-mountain plant species, R. glacialis, acclimated to highlight and low temperature, found that compared withother alpine species it had greater IM protein abun-dance, which was correlated with having lower EP.The authors suggested that IM provides enhancedphotoprotection through its capacity to keep the PQpool oxidized. Data from our in vivo experimentswhere we genetically manipulated IM levels (Fig. 3)from complete absence (im) to 16-fold higher (OE-16 3) than the wild type do not support this conclu-sion. We believe that this discrepancy can best beexplained by the fact that acclimation to low temper-ature can significantly enhance the photosyntheticcapacity in many cold-tolerant herbaceous plants,such as rye (Secale cereale), wheat (Triticum aestivum;Gray et al., 1996; Savitch et al., 2002), Arabidopsis(Savitch et al., 2001), and Brassica napus (Savitch et al.,2005), which would prevent overreduction of theelectron transport chain independent of IM abun-dance. Furthermore, acclimation to high light hasbeen shown to enhance the Mehler reaction in wheat(Savitch et al., 2000), an alternative method of keepingthe electron transport chain oxidized. Thus, given thewide array of acclimatory strategies that prevent over-reduction of electron transport, the higher levels ofIM and the lower EP reported for cold-acclimatedR. glacialis may not reflect a cause-and-effect relation-ship, but rather a simple correlation.

How can im plants exhibit the tendency to keep PSIImore oxidized than either the wild type or the twooverexpressors (Table I)? Because im plants grown

Figure 5. Fv/Fm of all plant genotypes of Arabidopsis exposed to highlight (1,200 mmol photons m22 s21) at 5�C and allowed to recover at25�C with an irradiance of 20 mmol photons m22 s21 for up to 8 h. Fv/Fm

was measured in detached plant leaves.

Rosso et al.





under the specific conditions described here exhibit noobvious phenotype (Fig. 2), we suggest that this maybe due to the fact that inactivating IM has induced theexpression and biosynthesis of yet another plastoqui-nol oxidase in the chloroplast, which is more effectivethan IM. Several authors have concluded that a cya-nide-sensitive chloroplast oxidase (Buchel and Garab,1995; Lajko et al., 1997; Joet et al., 2002) and a thylakoidhydroquinone peroxidase may be involved in theoxidation of the PQ pool (Casano et al., 2000). Alter-natively, im may be able to keep PSII more oxidized ascompared to the wild type due to an up-regulation ofthe metabolic electron sink capacity at the acceptorside of PSI. Upon investigating the rates of photosyn-thesis in green-leaf sectors compared with white-leafsectors in im variegated leaves, Aluru et al. (2001)reported that the green-leaf sectors have increasedrates of photosynthesis relative to the wild type tocompensate for a lack of photosynthesis in the white-leaf sectors. Therefore, the lack of phenotype in implants may be caused by functional redundancy due tothe ability of networks to buffer the effects of pertur-bations by related pathways (Cutler and McCourt,2005). Our analyses indicate that extreme caution mustbe exercised in the interpretation of experimentalresults for the role of IM based solely on either ex-pression levels or immunoblotting without concomi-tant functional measurements. We show that evenlarge changes in either gene expression or proteinaccumulation are not necessarily associated with anyenhancement in the proposed function of IM as aplastid terminal oxidase during steady-state photo-synthesis.

Because IM shows sequence homology with AOX(Carol et al., 1999; Wu et al., 1999), it has beensuggested that IM is a stress-induced protein relatedto oxidative stress (Aluru et al., 2001, 2006; Aluru andRodermel, 2004; Mittler et al., 2004). AOX1a hasbeen previously reported as a stress-induced gene(Vanlerberghe and McIntosh, 1997; Maxwell et al.,1999; Molen et al., 2006). However, we have shownthrough a direct comparison of IM to AOX1a that thereis no correlation in gene expression between these twogenes due to stress, including oxidative stress (Fig. 7A).In contrast to environmental stress, IM does appear tobe correlated to AOX1a expression during develop-ment, especially in cauline leaves and sepals (Fig. 7B)and during senescence, which has also been reportedelsewhere (Aluru et al., 2001). We, therefore, concludethat IM expression is modulated minimally by abioticstress, which is consistent with our in vivo functionalmeasurements. However, IM expression is modulatedin concert with AOX1a in response to development(Fig. 7B). Consistent with previously published data(Wu et al., 1999; Aluru et al., 2001, 2006), as well aspreliminary results indicating the regulation of vari-egation by excitation pressure during the early stagesof leaf development in im (D. Rosso, S.R. Rodermel,and N.P.A. Huner, unpublished data), we suggest thatIM plays an important role in keeping the PQ pool

Figure 6. Photoinhibition of PSI. Photooxidation of P700 measured asDA820/A820 from detached plant leaves from wild-type control plantsgrown at 25/150 (A) and after 4 h of photoinhibition at 5�C with PPFDof 1,200 mmol photons m22 s21 (B). The steady-state oxidation of P700

was measured as DA820/A820 and normalized as a percentage of control.All genotypes of Arabidopsis were exposed to high light (1,200 mmolphotons m22 s21) at 5�C and allowed to recover at 25�C with anirradiance of 20 mmol photons m22 s21 (C). P700 was measured indetached plant leaves. Data presented are of one experimental treat-ment with three replicate measurements.






oxidized during chloroplast biogenesis and the assem-bly of the photosynthetic apparatus. However, oncechloroplast development is complete and photosyn-thetic competence is attained, IM has a minimal im-pact on the flux of electrons between PSII and PSI.

Thus, neither our in vivo functional measurementsof photosynthetic intersystem electron transport norour meta-analyses of Arabidopsis microarrays sup-port the proposed analogous roles for IM and AOXas stress-induced safety valves during steady-state

Figure 7. A, Expression ratio of AOX1a(At3g22370) and IM (At4g22260) tran-scripts (log base 2) under different abi-otic stresses and hormone treatments.H2O2, 100 mM hydrogen peroxide for3 h; ozone, 200 ppb of ozone for 1 h;UVB, 15-min damaging UVB irradia-tion; salt, 150 mM NaCl; cold, 4�C shiftfrom room temperature; mannitol,300 mM mannitol; heat, 38�C shiftfrom room temperature (0.25–3 h)and recovery after 3 h (4–24 h);ACC, 10 mM 1-aminocyclopropane-1-carboxylic acid (ethylene precursor);MeJas, 10 mM methyl jasmonate; ABA,10 mM abscisic acid; SA, 10 mM sali-cylic acid. Times indicate hours aftertreatment initiation. Note significantup-regulation (1.5-fold stringent cutoff)of AOX1a in H2O2, ozone, 3- and 6-hUVB treatments, 12- and 24-h coldtreatments, 6-, 12-, and 24-h mannitoltreatments, and 3-h treatments in ABAand SA. IM showed no significant up-regulation or down-regulation. B, Dif-ferential expression of AOX1a and IMtranscripts in different tissues. Expres-sion level based on MAS 5.0 scaling byNASC (see ‘‘Materials and Methods’’)to give relative expression level (100units 5 genomic average). Roots,leaves, shoot apices and stems, flow-ers, siliques, and seeds in differentdevelopmental stages according toBoyes et al. (2001) and Schmid et al.(2005). L, Leaf number (L1 5 firstappearing leaf); d, days after germina-tion; SA, shoot apex; YL, young leaves;*, significant tissue-specific expressionespecially in senescing and caulineleaves (AOX1a) and sepals (IM andAOX1a) with more than 2-fold in-crease in average. Expression was notsignificantly above background inseeds for IM (stages 8–10) and AOX1a(stages 6–10), and in shoot apices(AOX1a). Roots were collected fromsoil-grown (Soil) and one-half-strengthMurashige and Skoog agar media (MS);note significant increase in AOX1a ex-pression on MS media.

Rosso et al.





photosynthesis. Therefore, care must be exercised inthe putative assignment of function based solely onDNA sequence analyses.

MATERIALS AND METHODS

Plant Growth

Arabidopsis (Arabidopsis thaliana ecotype Columbia) wild type, OE-6 3,

and OE-16 3, as well as the im mutant of IM, were germinated and grown

under controlled environmental conditions at 25�C with a PPFD of 5 mmol

photons m22 s21 for 1 week. Plants were thinned to one plant per pot and

grown under controlled environmental conditions at 25�C and with a PPFD of

50 mmol photons m22 s21 for an additional 4 weeks until the first rosette was

fully developed (see Fig. 1). After the first rosette had developed, plants were

shifted to 25�C with a PPFD of 150 mmol photons m22 s21 for an additional 40 d

(Fig. 1). All measurements were made on fully expanded leaves from the

second rosette and all plants were kept under an 8-h photoperiod to prevent

flowering. Plants were cold stressed by shifting them from 25/150 to 5/150 for

3 d prior to making measurements (Fig. 1).

Total Chl per Leaf Area

Chl was extracted with buffered 80% (v/v) aqueous acetone containing

2.5 mM sodium phosphate buffer, pH 7.8, and measured by the method of

Porra et al. (1989). The absorbance was measured at 663.6 nm and 646.6 nm

and corrected to 750 nm for light scattering in a Beckman DU-640 spectro-

photometer (Beckman Coulter). Leaf area was measured with a LI-COR area

meter (LI-3100C; LI-COR Biosciences).

Plasmid Constructs and Transformations

To generate IM overexpression plants, a full-length IM cDNA (Wu et al.,

1999) was cloned in the Xho1 and Sst1 sites of the binary vector pBI121; the

gene was driven by the cauliflower mosaic virus 35S promoter. The IM

construct was transferred into Agrobacterium tumefaciens, and wild-type

Arabidopsis (ecotype Columbia) plants were then transformed by the floral-

dip method (Clough and Bent, 1998). Kanamycin-resistant plants were se-

lected at the T1 generation on plates containing 1 3 MS salts, 1% (v/v) Suc,

0.8% (v/v) agar, pH 5.7, with 50 mg/mL kanamycin. PCR and Southern

blotting were performed to verify that the plants were transformed. RNA and

protein analyses were performed using T2 generation plants.

RNA Extraction and Gel-Blot Analysis

Total RNA was isolated from all genotypes of Arabidopsis using hot

phenol followed by LiCl precipitation (Maxwell et al., 1999). RNA was

separated on 1.2% (w/v) agarose gels, with equal amounts of RNA per gel

lane, containing formaldehyde and transferred to Hybond N membrane

(Amersham-Pharmacia Biotech) as previously described (Maxwell et al.,

1999). Radiolabeled DNA probes were made using the high-prime labeling

kit (Roche). The blots were probed with the full-length IM cDNA (Wu et al.,

1999).

Thylakoid Preparation and Immunoblotting

Thylakoid membranes were isolated according to the method of Harrison

and Melis (1992). Proteins were separated on 15% (w/v) SDS-PAGE in the

presence of 6 M urea according to the method of Laemmli (1970). Immuno-

blotting was performed by electrophoretically transferring the proteins from

SDS-PAGE gel to a nitrocellulose membrane (Bio-Rad Laboratories). Immu-

nodetection was performed using horseradish peroxidase-conjugated secondary

antibodies (Sigma) and enhanced chemiluminescence according to the man-

ufacturer (ECL; Amersham-Pharmacia Biotech). Proteins were immunode-

tected with specific polyclonal antibodies raised against the PsaA polypeptide

of the PSI reaction center (1:1,000 dilution), the H subunit of the NAD(P)H

dehydrogenase complex (1:500 dilution; D. Rumeau, unpublished data), D1

polypeptide of the PSII reaction center (1:1,000 dilution), Cyt f (1:1,000 dilu-

tion), Lhca1 and Lhcb2, respectively (1:1,000 dilution; Agri-sera), and IM

(1:1,000 dilution).

Thylakoid Isolation and Pigment Protein Analysis

Leaf material was ground in cold isolation buffer (50 mM Tricine, 0.4 M

sorbitol, 10 mM NaCl, 5 mM MgCl2 hexahydrate, pH 7.8) in a mortar and pestle

on ice, filtered through two layers of miracloth (typical pore size 22–25 mm;

Calbiochem), and centrifuged for 5 min (5,000g). The pellet was either

resuspended in isolation buffer for thermoluminescence measurements (as

described above) or in cold 50 mM Tricine, pH 8.0, wash buffer before Chl

determination in 80% (v/v) acetone according to Porra et al. (1989).

Thylakoids were centrifuged at 10,000g for 5 min, solubilized in an SDS/

dodecylmaltoside (DM) buffer with a 20:1 (w/w) DM 1 SDS:Chl ratio (0.1%

[w/v] SDS, 0.45% [w/v] DM, 0.3 M Tris, 13% [v/v] glycerol), and then

centrifuged to remove the insoluble material. Separation of Chl proteins was

undertaken by nondenaturing electrophoresis in a 5% to 10% (w/v) linear

gradient polyacrylamide gel according to Laemmli (1970), as modified by

Komenda (2000), where the gel contained no detergent and the SDS was

replaced by 0.2% (v/v) Deriphat 160 in electrophoretic buffer. To determine

relative band intensity, the green gels were scanned at 671 nm (DU-540;

Beckman).

P700 Photooxidation

The relative redox state of P700 was estimated in vivo as DA820/A820 using a

PAM-101 modulated fluorometer (Heinz Walz) equipped with an ED-800DT

emitter-detector and PAM-102 units, following the procedure of Schreiber et al.

(1988), as described by Ivanov et al. (1998). FR light (lmax 5 715 nm, 10 W

m22; Schott filter RG 715) was provided by the FL-101 light source. MT (50 ms)

and ST half-peak width of 14-ms saturating flashes were applied with XMT-

103 and XST-103 power/control units, respectively. Leaves were vacuum

infiltrated with 20 mM DCMU in darkness. One-way ANOVA was performed

to determine statistical significance between genotypes (P # 0.05) followed

by a Bonferroni test to test for differences between group means at a 95%

confidence interval (Microcal Origin 7.5; Origin Lab Corporation).

Chl a Fluorescence and Relative Redox State of QA

Steady-state fluorescence measurements were made using a PAM Chl

fluorometer (PAM-101, 103; Heinz Walz). Two Schott lamps (KL 1500) pro-

vided saturating flashes and actinic illumination for photosynthesis. Samples

were dark adapted for 20 min prior to all measurements. Fluorescence

parameters, such as the Fv/Fm, qP, qL, and quenching coefficient of the

nonphotochemical quenching (qN), were calculated (Bradbury and Baker,

1981; Schreiber et al., 1986; van Kooten and Snell, 1990; Kramer et al., 2004). A

post hoc test followed by a Student-Newman-Keuls test was performed to

determine statistical differences between means at a confidence interval of

95% (SPSS; Systat Software).

Photoinhibition and Recovery

Detached leaves were photoinhibited either at 25�C and PPFD of 500 mmol

photons m22 s21, or at 5�C and 1,200 mmol photons m22 s21. Recovery from

photoinhibition took place at 25�C with an irradiance of 20 mmol photons

m22 s21 for an additional 8 h. Photoinhibition was measured as the decrease in

Fv/Fm using a PAM fluorometer (PAM-103; Heinz Walz). A post hoc test

followed by a Student-Newman-Keuls test was performed to determine

statistical differences between means at a confidence interval of 95% (SPSS;

Systat Software).

Microarray Expression Analysis

The microarray analysis followed Geisler-Lee et al. (2006). Arabidopsis

oligo microarray data were downloaded from the Nottingham Arabi-

dopsis Stock Center (NASC; http://affymetrix.arabidopsis.info/narrays/

experimentbrowse.pl). Stress series and developmental series were provided

by the AtGene Express project (http://www.uni-frankfurt.de/fb15/botanik/

mcb/AFGN/atgenex.htm). The oligo microarrays were based on unique

oligonucleotides for the Arabidopsis genome, which did not cross-hybridize

closely related genes. We totaled the Microarray Suite, version 5.1, (http://

www.affymetrix.com/products/software/specific/mas.affx), scaled and nor-

malized signal values for AOX1a (At3g22370) and IM (At4g22260) genes, and

then determined relative expression in different Arabidopsis tissue. AFGN






and Affymetrix did all methods for microarray handling and normalization

and published them online within the NASC database.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AF098072 (IM) and AF370166 (AOX1a).

ACKNOWLEDGMENTS

We thank Dr. Maneesha Aluru for her help and expertise. We would also

like to thank Dr. Dominique Rumeau for the gift of antibody against the H

subunit of the NDH complex, and Dr. Jean-Marc Ducruet for providing and

assisting with the signal analysis software for thermoluminescence.

Received June 28, 2006; accepted July 27, 2006; published August 4, 2006.

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immutans does not act as a stress-induced safety valve in the protection of the photosynthetic...

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