Requirement of the C3HC4 zinc RING finger of theArabidopsis PEX10 for photorespiration and leafperoxisome contact with chloroplastsUwe Schumann*†, Jakob Prestele*, Henriette O’Geen‡, Robert Brueggeman§, Gerhard Wanner¶, and Christine Gietl*�

*Lehrstuhl fur Botanik, Technische Universitat Munchen, Am Hochanger 4, D-85350 Freising, Germany; ‡Genome Center, University of California,Davis, CA 95616; §Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164; and ¶Lehrstuhl fur Botanik,Ludwig-Maximilian-Universitat, Menzinger Strasse 67, D-80638 Munchen, Germany

Communicated by Diter von Wettstein, Washington State University, Pullman, WA, November 23, 2006 (received for review October 17, 2006)

Plant peroxisomes perform multiple vital metabolic processes in-cluding lipid mobilization in oil-storing seeds, photorespiration,and hormone biosynthesis. Peroxisome biogenesis requires thefunction of peroxin (PEX) proteins, including PEX10, a C3HC4 ZnRING finger peroxisomal membrane protein. Loss of function ofPEX10 causes embryo lethality at the heart stage. We investigatedthe function of PEX10 with conditional sublethal mutants. FourT-DNA insertion lines expressing pex10 with a dysfunctional RINGfinger were created in an Arabidopsis WT background (�Zn plants).They could be normalized by growth in an atmosphere of highCO2 partial pressure, indicating a defect in photorespiration. �-Oxidation in mutant glyoxysomes was not affected. However,an abnormal accumulation of the photorespiratory metaboliteglyoxylate, a lowered content of carotenoids and chlorophyll a andb, and a decreased quantum yield of photosystem II were detectedunder normal atmosphere, suggesting impaired leaf peroxisomes.Light and transmission electron microscopy demonstrated leafperoxisomes of the �Zn plants to be more numerous, multilobed,clustered, and not appressed to the chloroplast envelope as in WT.We suggest that inactivation of the RING finger domain in PEX10has eliminated protein interaction required for attachment ofperoxisomes to chloroplasts and movement of metabolites be-tween peroxisomes and chloroplasts.

�-oxidation � biogenesis � glyoxysome

Eukaryotic peroxisomes perform multiple metabolic pro-cesses, including fatty acid �-oxidation and H2O2 inactiva-

tion by catalase (1). In plants, leaf peroxisomes interact withchloroplasts and mitochondria in photorespiration, a metabolicpathway in which two molecules of glycolate are converted in aseries of enzymatic reactions through glyoxylate, glycine, serine,and hydroxypyruvate into CO2 and phosphoglycerate (2–4). Theadvantage of the photorespiratory cycle is twofold. When CO2in the plant canopy becomes limited in supply (which is frequentat midday), ribulose-bisphosphate carboxylase/oxygenase func-tions as an oxygenase and protects the photosynthetic machineryfrom photodamage. It does so by using energy for respiration,producing CO2, and regenerating the substrate to be used in CO2fixation. Mutants lacking enzymes of the photorespiratory cycleare incapable of surviving in ambient air but are able to grownormally in atmosphere enriched in CO2 because ribulose-bisphosphate oxygenase is suppressed (2). Plant peroxisomes arenecessary for jasmonic acid biosynthesis (5) and are implicatedin conversion of indole-3-butyric acid (IBA) into indole-3-aceticacid (IAA) (6–8). Specialized peroxisomes called glyoxysomescontain glyoxylate cycle enzymes for lipid mobilization in ger-minating oil seedlings and senescing leaves (1).

The peroxins (PEX proteins) are a set of cytosolic andmembrane proteins involved in peroxisome biogenesis. Muta-tions of PEX genes leading to impaired peroxisome biogenesisresult in severe metabolic and developmental disturbances inyeasts, humans (Zellweger syndrome), and plants (9–11).

PEX10, PEX12, and PEX2 encode integral membrane proteinsdistinct in their primary sequence except for a shared C3HC4-type RING finger motif in the C-terminal domain. Loss offunction of any one of these three genes in Arabidopsis causesembryo lethality at the heart stage, supporting the notion thatthey act together during peroxisome biogenesis (12–15). Glyoxy-somes and leaf peroxisomes originate de novo, presumably withan involvement of the ER, and multiply by division (9–11). Theirdevelopmental transition with replacements of enzyme contentis induced by light (16–18).

The seed lethal T-DNA disruption phenotype of PEX10implicates this membrane PEX in multiple cell biological func-tions, but nothing is yet known about the physiological role ofPEX10 in plants. We therefore tried to elucidate its function withconditional sublethal mutants. Four different T-DNA insertionlines expressing pex10 with a dysfunctional C3HC4 RING fingerwere created in an Arabidopsis WT background. They could benormalized by growth in an atmosphere of high CO2 partialpressure, indicating a defect in photorespiration. Gene expres-sion, biochemical analyses, and light and electron microscopy of

Author contributions: U.S., J.P., G.W., and C.G. designed research; U.S., J.P., H.O., R.B., andG.W. performed research; U.S., J.P., H.O., R.B., and G.W. analyzed data; and C.G. wrote thepaper.

The authors declare no conflict of interest.

Abbreviations: TAIL-PCR, thermal asymmetric interlaced PCR; IBA, indole-3-butyric acid;IAA, indole-3-acetic acid; PEX, peroxin.

†Present address: Section of Molecular Biology, Division of Biological Sciences, University ofCalifornia at San Diego, La Jolla, CA 92093.

�To whom correspondence should be addressed. E-mail: christine.gietl@wzw.tum.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610402104/DC1.

© 2007 by The National Academy of Sciences of the USA

Fig. 1. The dysfunctional Zn finger motif in AtPex10p. The amino acidchanges resulting in a loss of Zn coordination sites are shown.

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these conditional mutants were undertaken to identify the roleof PEX10 in peroxisome proliferation, attachment to chloro-plasts, matrix protein import, and photorespiration.

ResultsGeneration of Conditional Sublethal Mutants Expressing pex10 with aDysfunctional Zn Finger (�Zn Lines). The motif C2GLG2C2, unableto coordinate Zn ions in the C3HC4 Zn finger of pex10 cDNA,was created by mutating C3, C4, and C5 to G and H1 to L (Fig.1). The mutated cDNA was cloned under control of the 35Spromoter into the Agrobacterium tumefaciens vector pBI121conferring kanamycin resistance and transformed into Arabi-dopsis Columbia WT plants. Screening of T2 transformants fornoticeable phenotypes such as dwarfism and atrophy in normalair (360 ppm CO2) and under 5-fold elevated CO2 partialpressure (1,800 ppm CO2) identified four dwarfs that could benormalized by high CO2 [for details of progeny analysis seesupporting information (SI) Text]. After verifying the pheno-types in T3 progenies, four lines (referred to as �Zn1–4) with thefollowing characteristics were established: in normal atmo-sphere, the plants grew as dwarfs with a 2- to 3-week-delayeddevelopment and retarded silique maturation, but under ele-vated CO2, the plants developed normally (Fig. 2 A and B), whichis typical for mutants impaired in photorespiration (2). Althoughseed yield was normal, �Zn seeds were often smaller, shrunken,and less tightly filled with storage material (Fig. 2C). The weightof 1,000 seeds was 26.9 � 0.4 mg for WT and 21.0 � 1.6 mg for�Zn1–4 grown in normal air, compared with 22.4 � 0.1 mg forWT and 23.0 � 2.5 mg for �Zn1–4 grown under high CO2.Transmission electron microscopy of maturing embryos revealedno conspicuous structural difference between WT and �Zn1–4(data not shown).

�Zn1–4 were similarly dwarfed with �Zn1 showing some-times a slightly more severe phenotype under strong light.Transcript levels of the parental WT gene and the mutated pex10gene carrying additionally a MbiI restriction site marker forrecognition were examined by RT-PCR. A 248-bp fragmentspanning the WT or the mutated Zn finger motif was amplifiedin approximately equal amounts, indicating that the authenticand mutant PEX10 genes exhibited similar transcript levels in all�Zn lines. Furthermore, all �Zn lines exhibited approximatelyequal transcript amounts versus WT and vector control plants(Fig. 3).

A T-DNA insertion disrupting the fourth exon of the AtPEX10gene leads to embryo lethality; the mutant can be rescued bytransformation with the WT AtPEX10 cDNA (12). To charac-terize the significance of the C3HC4 Zn finger motif for thefunction of PEX10, the heterozygous kanamycin-resistant pex10disruption line was transformed with �Zn-pex10 under controlof the 35S promoter, by using the Bar resistance gene as selectionmarker (12). Forty-eight double-resistant seedlings, carryingboth the insertion and the �Zn-pex10, were obtained. Threepairs of PCR primers diagnostic for the presence of at least oneintact genomic copy of PEX10, the transgenic �Zn-pex10, andthe insertion element permitted genotyping of all possible

Fig. 2. Analysis of the �Zn lines, WT, and pB1727 vector control (contr). (A)Dwarfish growth of 4-week-old �Zn plants under 360 ppm CO2 (Left) andnormalization under 1,800 ppm CO2 (Right). (B) Six-week-old dwarfish �Znplant compared with WT and control. (C) Seeds of the �Zn mutant are smallerthan the WT seeds. (D) Ten-day-old �Zn plants grown in atmospheric CO2 (atthe bottom) show dwarfism and chlorosis that are normalized under 5-fold

elevated CO2 partial pressure (at the top). (E) Increased glyoxylate accumula-tion in the peroxisomes of the �Zn. (F) �Zn plants regained WT chlorophylla�b content after transfer to high CO2. (G) Photosynthetic yield of photosys-tem II in light-adapted leaves after a saturating actinic light pulse. Yield in �Znis decreased in low CO2. (H) Photochemical active quenching (qP) reduced andnonactive quenching (qN) increased in �Zn in normal atmosphere. (I) In-creased hydroxypyruvate reductase activity in �Zn organellar pellets. (J) In-creased glyoxysomal malate dehydrogenase in �Zn. (K) Lack of sucrose causesreduced root growth from day 3 after germination in �Zn with beginningphotoautotrophy. (L) Auxin precursor IBA inhibits root growth of WT, �Zn,and control, but not of IBA-resistant pex4 and pex6 seedlings.

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segregants (see figure 1 D and G in ref. 12). None of the 48transgenic plants could survive despite knockout of the WT bythe overexpressed �Zn-pex10; siliques of the surviving plantsexhibited the same ratio of segregating seeds as the noncomple-mented insertion line, i.e., green maturing to white lethal seeds(data not shown). Thus, mutation of the C3HC4 Zn finger motifconferred the same lethality as the complete disruption ofPEX10, and the phenotypes of �Zn1–4 were caused by compe-tition between the endogenous PEX10 and the mutated pex10transcripts.

Leaf Peroxisomes in the �Zn Lines Are Impaired in Photorespiration.�Zn plants showed the dwarfish and chlorotic phenotype innormal air after 10 days (Fig. 2D) and were used for character-ization. We quantified the parameters that are associated withdefective photorespiration, such as glyoxylate level, the amountof chlorophyll a and b and carotenoids (xanthophylls and car-otenes), and the maximum quantum yield of photosystem II. Allfour �Zn lines exhibited the same phenotype to a similarquantitative degree (Fig. 2 E–H).

An abnormally high level of glyoxylate was detected photo-metrically after reaction with phenylhydrazine in the presence ofK3Fe(CN)6 (Fig. 2E). The mutant plants regained WT chloro-phyll a and b contents after transfer to high CO2 (Fig. 2F) buthad a lowered content of carotenoids compared with WT grownin normal air and transferred to high CO2 (data not shown).Photosynthetic parameters were evaluated by chlorophyll f luo-rescence kinetics with the portable fluorometer PAM-2000 (Fig.2 G and H). The �Zn plants revealed a decreased photosystemII quantum yield under normal atmosphere; under high CO2these values reached WT levels (Fig. 2G). As a parameter for theredox state of the primary electron acceptor in photosystem II,photochemical quenching was determined. Under atmosphericCO2 conditions, active quenching was decreased and nonactivequenching was increased in the �Zn lines, indicating an inef-fective photosynthesis system. Under 5-fold-elevated CO2 partialpressure these parameters did not differ from WT and vectorcontrols (Fig. 2H).

The high level of glyoxylate indicates a defect in peroxisomalmetabolism, possibly a deficiency in serine-glyoxylate amino-transferase activity. We determined the serine-glyoxylate ami-notransferase activity by measuring the consumption of glyoxy-late with serine as amino donor in protein extracts of 10-day-oldplants. We found equal levels in both WT and �Zn plants:consumption of 0.24 �mol of glyoxylate min�1�mg�1 of proteincompared with 0.22 �mol of glyoxylate min�1�mg�1 of protein.We compared hydroxypyruvate reductase and glyoxysomalmalate dehydrogenase amounts in crude organelle pellets of

�Zn plants, WT, and vector control plants (Fig. 2 I and J). Theenzyme content in the �Zn plants was significantly increased ascompared with control plants.

Glyoxysomes in the �Zn Lines Are Not Impaired in �-Oxidation DuringGermination and Seedling Establishment. To assess �-oxidation inglyoxysomes, we tested sugar dependence during germinationand IBA response of the �Zn lines. Peroxisomal mutant seed-lings of oil seed species such as Arabidopsis grow poorly in theabsence of exogenous sugar (19). The developmental delay of�Zn plants was quantified by measuring the root growth onmedium either with or without 1% sucrose in the light (Fig. 2K).Mutant plants developed normally without exogenous sugaruntil days 3–4 of germination, so long as lipid mobilization wasthe limiting factor. However, a lag in root growth was seen fromthe beginning of photoautotrophy at day 4 on medium lackingsucrose, when peroxisomal transport of metabolized phospho-glycolate starts to play a crucial role for the normal photosyn-thetic development of seedlings.

Mutants defective in �-oxidation are resistant to the inhibitoryeffect of IBA on root elongation, because the glyoxysomescannot convert IBA into IAA (6). Root growth was determinedin WT, vector control, and �Zn seedlings on medium with 1%sucrose with or without 10 �M IBA. pex6, which has photore-spiration defects similar to the �Zn lines (7), and pex4, whichappears to grow normally (8), are IBA-resistant as seedlings andwere used as positive controls (Fig. 2L). �Zn seedlings re-sponded to the inhibitory effect of IBA on root elongationsimilar to WT and vector control plants, whereas pex4 and pex6seedlings exhibited the expected resistance to IBA.

Early normal development of the �Zn seedlings implies thattheir glyoxysomes contain sufficient matrix enzymes to perform�-oxidation to mobilize stored lipids and to convert IBA intoIAA. Glyoxysomal function thus appears to be unaffected by theoverexpression of a dysfunctional PEX10 protein that impairsphotorespiratory function in leaf peroxisomes.

Leaf Type Peroxisomes in �Zn Lines Are Pleomorphic and RarelyAssociated with Chloroplasts. The morphology and association ofleaf peroxisomes with other organelles in �Zn seedlings wereexamined by quantitative transmission electron microscopy (Fig.4) and light microscopy (Fig. 5). Peroxisomes are identifiedunequivocally by cytochemical staining for catalase (20). Asexemplified in Figs. 4A and 5, WT image, the spherical or ovoidperoxisomes in the mesophyll cells of WT Arabidopsis thalianahad a diameter of 0.88 � 0.23 �m (n � 50), were appressed tothe envelope of chloroplasts, and were frequently in closephysical contact with mitochondria. The peroxisomes of the �Znplants exhibit catalase activity equal to those of WT (Figs. 4 Band C and 5, image �Zn1–4), but they varied considerably in sizeand shape and were rarely associated with chloroplasts. The �Znperoxisomes form chains, protuberances, and interconnectedclusters within the cytoplasm (Fig. 5, image �Zn1–4), possiblyindicating an inhibition of the proliferating peroxisomes in theirfission.

A quantitative analysis of the peroxisomal profiles revealed3.32 � 1.5 per WT (n � 25) and 9.0 � 4.2 per �Zn1 (n � 25)cell section (t for difference � 4.11; ***, P � 0.001). In WT cells88 � 16% (n � 25) of peroxisomes were appressed to achloroplast, whereas in an equal number of �Zn1 cell sections31 � 21% were appressed to chloroplasts (t for difference � 27.7;***, P � 0.001). Measurements of the width of peroxisomalprofiles (n � 50) in both WT and �Zn1 did not reveal anysignificant difference with 0.88 � 0.23 �m for WT and 1.28 �0.78 �m for �Zn1 (t for comparison � 0.331; ***, P � 0.07).However, there was a large difference in length. There were twoperoxisomal populations in �Zn1 cells, one with a normal lengthof 0.68 � 0.15 �m (n � 29) and a second with a highly variable

Fig. 3. Transcript levels of PEX10 and pex10 after amplification by RT-PCRand digestion with MbiI; plasmid DNA was used as control. Lanes 1 and 3,undigested; lanes 2 and 4–10, digested with MbiI. Arrows indicate the undi-gested 248-bp PCR product of WT PEX10 and the 176-bp and 72-bp fragmentsof the mutated pex10 after digestion with MbiI. �Zn lines exhibit bothtranscripts in approximately equal amounts. M, 100-bp ladder.

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length of 3.51 � 1.93 �m (n � 21) (tk for difference � 6.227; ***,P � 0.001). The elongated structures could reach a length of 7�m. Plants overexpressing WT PEX10 have normal-shapedperoxisomes attached to the chloroplast envelope (Fig. 5, imageoverexpr.).

Isolation and Mapping of Genomic Sequences Flanking the T-DNAIntegration Sites in �Zn Lines 1–4. Southern blot analysis of the�Zn lines revealed the presence of at least one integration siteof the T-DNA carrying the pex10 cDNA (data not shown).Thermal asymmetric interlaced PCR (TAIL-PCR) was carriedout to identify genomic sequences flanking the T-DNA inser-tions (Fig. 6A). The two bands in the left lane for each plantDNA represent the products of the second cycle of the TAIL-PCR created with the arbitrary degenerate primer Nr.2 (AD-2)and the pBI right border primer (RB-2). The two bands in theright lane for the individual plant DNA amplificates (marked ‘‘1’’

and ‘‘2’’) are the specific products of the third TAIL-PCR cycle.These PCR fragments are shorter than the bands in the left lane,because nested T-DNA primers were used to demonstrate thespecificity of the PCR. WT DNA produced no PCR products.The bands marked ‘‘1’’ and ‘‘2’’ were sequenced and subjected toBLAST search. Bands marked with ‘‘2’’ were primarily T-DNAregions. The boxes in Fig. 6A mark the sequenced bands thatmatched different genomic sequences of A. thaliana. Thus,T-DNA integration into the different �Zn lines occurred atdifferent sites. In �Zn1, the insertion was 335 bp upstream of thestart codon within the 5� UTR of an ortholog to the MtN24 geneof Medicago trunculata encoding a sequence similar to a pro-karyotic membrane lipoprotein with a lipid attachment site(At3g55390). In �Zn2, the integration was found 140 bp down-stream of the stop codon within the 3� UTR of a gene forglycerol-phosphodiesterase involved in glycerol metabolism(At5g58050). Integration in �Zn4 occurred in the last intron ofa gene for an unknown but expressed protein (At5g24314).TAIL-PCR for �Zn3 did not reach genomic regions. Thus, thegene integrations, especially in the �Zn1 and �Zn2 lines, areunlikely to have interrupted expression of genes essential forphotorespiration, and the phenotype must be due to the expres-sion of the mutated PEX10 Zn finger protein. The integrationsites were confirmed by genomic PCR by using primers specific

Fig. 4. Electron micrographs of leaf tissue of WT (A) and �Zn1 (B and C)plants stained for catalase activity with diaminobenzidine. The leaf peroxi-somes of WT are ovoid and in physical contact with chloroplasts (A). Mutant�Zn plants exhibit peroxisomes with catalase activity, however rarely associ-ated with chloroplasts. Their shape is pleomorph, ranging from ovoid toelongated with protrusions (B). Clusters of peroxisomes forming local net-works are frequent (C). P, chloroplast; V, vacuole; MB, microbody (peroxi-some); M, mitochondrion.

Fig. 5. Light microscopy of semithin sections of leaf tissue from WT and�Zn1–4 plants and AtPEX10-overexpressing plants stained for catalase activitywith diaminobenzidine.

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for the affected genes. As expected, PCR using two gene-specificprimers resulted in an absence of PCR products, whereas acombination of either of the appropriate gene-specific primerswith primers for the pBI121 right border yielded PCR productsconfirming the results of the BLAST search (Fig. 6B). In �Zn4,both specific primers in combination with the right borderprimers led to amplified DNA, indicating a full or partial tandemintegration. Furthermore, crosses between homozygous �Znlines resulted in F1 progeny that were homozygous for the pex10cDNA expression but heterozygous for the gene affected by theT-DNA insertion. These F1 progenies consistently exhibitedthe dwarf chlorotic �Zn phenotype, again confirming that theexpressed pex10 cDNA was responsible for the phenotype (datanot shown).

Knockout Lines with T-DNA Integrations in the Genes Containing theTransgenes in the �Zn Lines Do Not Exhibit Impaired Photorespiration.As a final confirmation that expression of pex10 is responsible forthe dwarf chlorotic phenotype of �Zn lines, knockout mutantswith T-DNA integrations close to the integration sites of thepex10 cDNA in the �Zn lines were examined. Homozygousknockout mutants were grown and genotyped to confirm thepresence and position of the T-DNA insertions: SALK line534800 and SAIL line 809777 with an insertion in the third exonof the gene At3g55390 affected in the �Zn1 line, and SALK line537722 with an insertion in the fifth exon of the gene At5g58050affected in the �Zn2 line. Their phenotype was similar to WTplants.

DiscussionGiven the lethal phenotype of the previously analyzed null mutantsof PEX10, we generated nonlethal partial loss-of-function mutants.By expressing a version of AtPEX10 cDNA with a dysfunctional Znfinger motif under the control of the 35S CaMV promoter, weobtained four dominant negative, conditional transformants withall landmarks of mutants compromised in photorespiration (Fig. 2).The parental WT gene and the mutated pex10 cDNA inserted in

different regions of the Arabidopsis genome exhibited equal tran-script levels in the four transformants supporting the rationale thatthe mutant protein lacking the RING finger metal ligands com-petes with the WT protein for interacting partners and will there-fore interfere with the normal function of PEX10. By determiningthe sites of integration of three of the transgenes in the Arabidopsisgenome and by analyses of knockout mutants of the genes targetedby the insertions, it was shown that they are not encoding proteinsinvolved in photorespiration and thus the integrations were notresponsible for the defective phenotype. The RING finger with theZn metal ligands was required for normal-shaped peroxisomeorganelles that can physically associate through their single mem-brane with the outer membrane of the chloroplast envelope. Thiswas correlated with proliferation of elongated peroxisomes thatwere unable to fission into normal-sized peroxisomes (Fig. 5). Thedysfunctional RING finger did not affect the function of glyoxy-somes that carry out �-oxidation and the glyoxylate cycle for lipidmobilization during oil seed germination; it did not influence theconversion of IBA into the plant growth hormone IAA. Further-more, the dysfunctional RING finger did not impair the abundanceof catalase within leaf peroxisomes as demonstrated in situ bycytochemical staining.

Functional photorespiration requires the localization of matrixenzymes within peroxisomes as well as the association of per-oxisomes, chloroplasts, and mitochondria to enable the metab-olite flow between these organelles. Our results let us concludethe necessity of the PEX10 Zn RING finger for the physicalattachment between peroxisome membrane and chloroplastenvelope. PEX10 is already known as an essential component ofthe peroxisomal protein import machinery and is thought toform a complex with the Zn RING finger proteins PEX2 andPEX12 in the peroxisomal membrane (9). We found equal oreven enhanced activity levels for �Zn plants compared with WTfor the peroxisome marker enzymes serine-glyoxylate amino-transferase in protein extracts and hydroxypyruvate reductaseand glyoxysomal malate dehydrogenase in organellar pellets.Because the function of the different PEX10 subdomains inmatrix protein import is not known, it could be that the complexbetween the mutated PEX10 and the PEX2 and PEX12 isstabilized by the intact Zn finger of PEX2 and PEX12, thusallowing enzyme import into the abnormal-shaped peroxisomes.This interpretation is supported by the intact glyoxysome importmachinery that might be inherited during developmental tran-sition to leaf peroxisomes as demonstrated by the cytochemicalcatalase staining. We cannot, however, completely exclude themislocalization of peroxisome enzymes to the cytosol, thuscontributing to the defective photorespiration phenotype.

Efficient processing of glyoxylate into glycine for transportinto mitochondria was severely curtailed. The elevated level ofglyoxylate might be an inheritance of the glyoxysome metabo-lism rather than owing to the transfer from chloroplasts. Becausethe activity of serine-glyoxylate aminotransferase is comparablein WT and �Zn plants, the reason for the disturbed metabolismis either the mislocalization of the enzyme to the cytosol or thelack of serine that is thought to be on the return trip frommitochondria.

It is of interest whether the RING finger domains of PEX12pand PEX2p are also required for generating associations be-tween peroxisomes, chloroplasts, and mitochondria. In a com-parable strategy, RNA interference plants with reduction of thePEX12 transcript were used to obtain sublethal mutants (15).These plants exhibited impaired peroxisome biogenesis andfunction and inhibition of plant growth. Down-regulation ofPEX12 by RNA interference, however, yielded plants that werealso less responsive to IBA.

In conclusion, transformants with peroxins mutated in anindividual protein domain can detect novel functions of theperoxins. In the present case, the requirement of PEX10 for

Fig. 6. Localization of T-DNA integrations in �Zn lines. (A) TAIL-PCR of �Znlines 1–4, WT, and pBI121 vector control (contr). PCR products marked ‘‘1’’ and‘‘2’’ were sequenced and subjected to a BLAST search. PCR products ‘‘2’’represent T-DNA regions. PCR products ‘‘1’’ (boxes) indicate positive matchesin the Arabidopsis genome. (B) Genomic PCR of the �Zn lines. The lack of DNAfragments obtained with �Zn DNA from lines �Zn1, �Zn2, and �Zn4 with therespective forward and reverse specific primers (sp1–4 f�r), and the creationof bands with the specific primers in combination with a right border primer(RB) for pBI121, confirm the T-DNA integrations within the genes proposed byTAIL-PCR and BLAST search. The band from �Zn4 DNA created by the specificreverse primer (sp4r) and the RB primer, additionally to the band generatedby the specific forward primer (sp4f) and the RB primer, indicates a full orpartial inverse tandem integration into chromosomal DNA in �Zn4. M, 100-bpladder with the accentuated 500-bp fragment.

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establishing the physical attachment of the peroxisome mem-brane to the chloroplast envelope has been demonstrated. Itopens the way to finding out how this attachment shown innumerous published micrographs is established at the molecularlevel and which other proteins and possible transporters areinvolved. The attachment has been presaged in textbook car-toons to explain the metabolite flow from chloroplasts viaperoxisomes to mitochondria and back via peroxisomes into thechloroplast to describe the photorespiratory pathway.

MethodsGeneration of the Mutated C3HC4 Zn Finger. Four point mutationswere introduced into the Zn finger motif of the PEX10 cDNAwith gene splicing by overlap extension (21, 22) to change thecodons TGT or TGC for Cys into GGT for Gly and CAT for Hisinto CTT for Leu (Fig. 1). The required primers are listed in SIText.

RT-PCR. Total RNA was isolated from 100 mg of 3-week-oldseedlings grown on MS plates. After cDNA first-strand synthesis,PCR was carried out with the PEX10 sense and antisense primersspanning a 248-bp-long fragment of the C terminus including theZn finger and part of the 3� UTR. The products of amplifiedPEX10/pex10 were purified, reamplified, digested with MbiI, andanalyzed on a 3% agarose gel. The required primers are listed inSI Text.

Assays. Glyoxylate was measured according to ref. 23, leafpigments were measured as in ref. 24, serine-glyoxylate amino-transferase was measured as in ref. 25, and hydroxypyruvatereductase activity was measured as in ref. 26. Organellar pelletswere prepared as in ref. 27. For Western blotting, polyclonalantibodies generated against glyoxysomal malate dehydrogenasewere used (28). Defects in �-oxidation or photorespiration wereassayed by growth on MS plates with or without 1% sucrose andmeasurement of root length (mean value of 20 plants) at days 3,4, and 6. The inhibitory effect of IBA on root elongation (meanvalue of 20 plants) was tested as in ref. 6.

Photosynthetic Parameters. The photosynthetic yield and theactive and nonactive quenching were calculated from data

collected with the portable pulse amplitude modulator fluorom-eter (PAM-2000; Walz, Effeltrich, Germany) at 25 � 1°C andambient CO2 partial pressure (29). For details see SI Text.

Electron and Light Microscopy. Pieces of leaf tissue from 12-day-oldlight-grown control and �Zn plants were fixed with 2.5%glutaraldehyde in 75 mM sodium cacodylate/2 mM MgCl2 (pH7.0) for 1 h at 25°C, rinsed several times in fixative buffer, andpostfixed for 2 h with 1% osmium tetroxide in fixative buffer at25°C. After two washing steps in distilled water, the pieces werestained en bloc with 1% uranyl acetate in 20% acetone for 30min. Dehydration and embedding in Spurr’s low-viscosity resinwere essentially as in ref. 19. Ultrathin sections of 50–70 nm werecut with a diamond knife and mounted on uncoated coppergrids. The sections were poststained with aqueous lead citrate(100 mM, pH 13.0). Micrographs were taken with an EM 912electron microscope (Zeiss, Oberkochen, Germany) equippedwith an integrated OMEGA energy filter operated in thezero-loss mode. Specific localization of catalase activity wasperformed as in ref. 20. For light microscopy, semithin sectionswith a thickness of 500 nm were cut with a diamond knife,mounted onto glass slides, and embedded in epoxy resin.

Characterization of T-DNA Insertion Sites Within the Chromosomes�Zn Lines. TAIL-PCR (30) used a combination of primersrecognizing the pBI121 right border (RB2 or RB3) and threedifferent arbitrary degenerate primers (AD primers) annealingrandomly in the genome of A. thaliana. The AD-2 primercombined with the RB3 primer resulted in the specific PCRproducts. Genomic PCR for verification of the integration sitesidentified by TAIL-PCR and BLAST research in �Zn1, �Zn2,and �Zn4 used a primer pair annealing �900 bp upstream anddownstream of the putative integration sites. The primers usedand their locations are listed in SI Text.

We thank Dr. Bonnie Bartel for the Arabidopsis pex4 and pex6 mutantseeds and Dr. Markus Schmid for ideas on this project, especially for thedesign of the �Zn-pex10. The skillful technical assistance of Ms. SilviaDobler is gratefully acknowledged. This work was supported by DeutscheForschungsgemeinschaft Grant Gi 154/12-1.

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