Requirement of the C3HC4 Zinc RING Finger of the Arabidopsis PEX10 for Photorespiration and Leaf Peroxisome Contact with Chloroplasts

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Requirement of the C3HC4 zinc RING finger of the Arabidopsis PEX10 for photorespiration and leaf peroxisome contact with chloroplasts

Uwe Schumann*†, Jakob Prestele*, Henriette O’Geen‡, Robert Brueggeman§, Gerhard Wanner¶, and Christine Gietl*ʈ

*Lehrstuhl fu¨r Botanik, Technische Universita¨t Mu¨ nchen, 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 fu¨r Botanik, Ludwig-Maximilian-Universita¨t, Menzinger Strasse 67, D-80638 Mu¨nchen, 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 including lipid mobilization in oil-storing seeds, photorespiration, and hormone biosynthesis. Peroxisome biogenesis requires the function of peroxin (PEX) proteins, including PEX10, a C3HC4 Zn RING finger peroxisomal membrane protein. Loss of function of PEX10 causes embryo lethality at the heart stage. We investigated the function of PEX10 with conditional sublethal mutants. Four T-DNA insertion lines expressing pex10 with a dysfunctional RING finger were created in an Arabidopsis WT background (⌬Zn plants). They could be normalized by growth in an atmosphere of high CO2 partial pressure, indicating a defect in photorespiration. ␤- Oxidation in mutant glyoxysomes was not affected. However, an abnormal accumulation of the photorespiratory metabolite glyoxylate, a lowered content of carotenoids and chlorophyll a and PLANT BIOLOGY b, and a decreased quantum yield of photosystem II were detected under normal atmosphere, suggesting impaired leaf peroxisomes. Light and transmission electron microscopy demonstrated leaf Fig. 1. The dysfunctional Zn finger motif in AtPex10p. The amino acid peroxisomes of the ⌬Zn plants to be more numerous, multilobed, changes resulting in a loss of Zn coordination sites are shown. clustered, and not appressed to the chloroplast envelope as in WT. We suggest that inactivation of the RING finger domain in PEX10 has eliminated protein interaction required for attachment of PEX10, PEX12, and PEX2 encode integral membrane proteins peroxisomes to chloroplasts and movement of metabolites be- distinct in their primary sequence except for a shared C3HC4- tween peroxisomes and chloroplasts. type RING finger motif in the C-terminal domain. Loss of function of any one of these three genes in Arabidopsis causes ␤-oxidation ͉ biogenesis ͉ glyoxysome embryo lethality at the heart stage, supporting the notion that they act together during peroxisome biogenesis (12–15). Glyoxy- ukaryotic peroxisomes perform multiple metabolic pro- somes and leaf peroxisomes originate de novo, presumably with ␤ an involvement of the ER, and multiply by division (9–11). Their Ecesses, including fatty acid -oxidation and H2O2 inactivation by catalase (1). In plants, leaf peroxisomes interact with developmental transition with replacements of enzyme content chloroplasts and mitochondria in photorespiration, a metabolic is induced by light (16–18). pathway in which two molecules of glycolate are converted in a The seed lethal T-DNA disruption phenotype of PEX10 series of enzymatic reactions through glyoxylate, glycine, serine, implicates this membrane PEX in multiple cell biological functions, but nothing is yet known about the physiological role of and hydroxypyruvate into CO2 and phosphoglycerate (2–4). The PEX10 in plants. We therefore tried to elucidate its function with advantage of the photorespiratory cycle is twofold. When CO2 in the plant canopy becomes limited in supply (which is frequent conditional sublethal mutants. Four different T-DNA insertion at midday), ribulose-bisphosphate carboxylase/oxygenase func- lines expressing pex10 with a dysfunctional C3HC4 RING finger tions as an oxygenase and protects the photosynthetic machinery were created in an Arabidopsis WT background. They could be from photodamage. It does so by using energy for respiration, normalized by growth in an atmosphere of high CO2 partial pressure, indicating a defect in photorespiration. Gene expres- producing CO2, and regenerating the substrate to be used in CO2 fixation. Mutants lacking enzymes of the photorespiratory cycle sion, biochemical analyses, and light and electron microscopy of are incapable of surviving in ambient air but are able to grow normally in atmosphere enriched in CO2 because ribulose- Author contributions: U.S., J.P., G.W., and C.G. designed research; U.S., J.P., H.O., R.B., and bisphosphate oxygenase is suppressed (2). Plant peroxisomes are G.W. performed research; U.S., J.P., H.O., R.B., and G.W. analyzed data; and C.G. wrote the necessary for jasmonic acid biosynthesis (5) and are implicated paper. in conversion of indole-3-butyric acid (IBA) into indole-3-acetic The authors declare no conflict of interest. acid (IAA) (6–8). Specialized peroxisomes called glyoxysomes Abbreviations: TAIL-PCR, thermal asymmetric interlaced PCR; IBA, indole-3-butyric acid; contain glyoxylate cycle enzymes for lipid mobilization in ger- IAA, indole-3-acetic acid; PEX, peroxin. minating oil seedlings and senescing leaves (1). †Present address: Section of Molecular Biology, Division of Biological Sciences, University of The peroxins (PEX proteins) are a set of cytosolic and California at San Diego, La Jolla, CA 92093. membrane proteins involved in peroxisome biogenesis. Muta- ʈTo whom correspondence should be addressed. E-mail: [email protected]. tions of PEX genes leading to impaired peroxisome biogenesis This article contains supporting information online at www.pnas.org/cgi/content/full/ result in severe metabolic and developmental disturbances in 0610402104/DC1. yeasts, humans (Zellweger syndrome), and plants (9–11). © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610402104 PNAS ͉ January 16, 2007 ͉ vol. 104 ͉ no. 3 ͉ 1069–1074 Downloaded by guest on September 25, 2021 these conditional mutants were undertaken to identify the role of PEX10 in peroxisome proliferation, attachment to chloroplasts, matrix protein import, and photorespiration. Results Generation of Conditional Sublethal Mutants Expressing pex10 with a Dysfunctional Zn Finger (⌬Zn Lines). The motif C2GLG2C2, unable to 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 35S promoter into the Agrobacterium tumefaciens vector pBI121 conferring kanamycin resistance and transformed into Arabi- dopsis Columbia WT plants. Screening of T2 transformants for noticeable phenotypes such as dwarfism and atrophy in normal air (360 ppm CO2) and under 5-fold elevated CO2 partial pressure (1,800 ppm CO2) identified four dwarfs that could be normalized by high CO2 [for details of progeny analysis see supporting information (SI) Text]. After verifying the pheno- typesinT3 progenies, four lines (referred to as ⌬Zn1–4) with the following characteristics were established: in normal atmosphere, the plants grew as dwarfs with a 2- to 3-week-delayed development and retarded silique maturation, but under elevated CO2, the plants developed normally (Fig. 2 A and B), which is typical for mutants impaired in photorespiration (2). Although seed yield was normal, ⌬Zn seeds were often smaller, shrunken, and less tightly filled with storage material (Fig. 2C). The weight of 1,000 seeds was 26.9 Ϯ 0.4 mg for WT and 21.0 Ϯ 1.6 mg for ⌬Zn1–4 grown in normal air, compared with 22.4 Ϯ 0.1 mg for WT and 23.0 Ϯ 2.5 mg for ⌬Zn1–4 grown under high CO2. Transmission electron microscopy of maturing embryos revealed no 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 pex10 gene carrying additionally a MbiI restriction site marker for recognition were examined by RT-PCR. A 248-bp fragment spanning the WT or the mutated Zn finger motif was amplified in approximately equal amounts, indicating that the authentic and mutant PEX10 genes exhibited similar transcript levels in all ⌬Zn lines. Furthermore, all ⌬Zn lines exhibited approximately equal transcript amounts versus WT and vector control plants (Fig. 3). A T-DNA insertion disrupting the fourth exon of the AtPEX10 gene leads to embryo lethality; the mutant can be rescued by transformation with the WT AtPEX10 cDNA (12). To charac- terize the significance of the C3HC4 Zn finger motif for the function of PEX10, the heterozygous kanamycin-resistant pex10 disruption line was transformed with ⌬Zn-pex10 under control of the 35S promoter, by using the Bar resistance gene as selection marker (12). Forty-eight double-resistant seedlings, carrying both the insertion and the ⌬Zn-pex10, were obtained. Three pairs of PCR primers diagnostic for the presence of at least one intact genomic copy of PEX10, the transgenic ⌬Zn-pex10, and the insertion element permitted genotyping of all possible

elevated CO2 partial pressure (at the top). (E) Increased glyoxylate accumulation in the peroxisomes of the ⌬Zn. (F) ⌬Zn plants regained WT chlorophyll aϩb content after transfer to high CO2.(G) Photosynthetic yield of photosystem II in light-adapted leaves after a saturating actinic light pulse. Yield in ⌬Zn is decreased in low CO2.(H) Photochemical active quenching (qP) reduced and Fig. 2. Analysis of the ⌬Zn lines, WT, and pB1727 vector control (contr). (A) nonactive quenching (qN) increased in ⌬Zn in normal atmosphere. (I) In- Dwarfish growth of 4-week-old ⌬Zn plants under 360 ppm CO2 (Left) and creased hydroxypyruvate reductase activity in ⌬Zn organellar pellets. (J) In- normalization under 1,800 ppm CO2 (Right). (B) Six-week-old dwarfish ⌬Zn creased glyoxysomal malate dehydrogenase in ⌬Zn. (K) Lack of sucrose causes plant compared with WT and control. (C) Seeds of the ⌬Zn mutant are smaller reduced root growth from day 3 after germination in ⌬Zn with beginning than the WT seeds. (D) Ten-day-old ⌬Zn plants grown in atmospheric CO2 (at photoautotrophy. (L) Auxin precursor IBA inhibits root growth of WT, ⌬Zn, the bottom) show dwarfism and chlorosis that are normalized under 5-fold and control, but not of IBA-resistant pex4 and pex6 seedlings.

1070 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610402104 Schumann et al. Downloaded by guest on September 25, 2021 ⌬Zn plants, WT, and vector control plants (Fig. 2 I and J). The enzyme content in the ⌬Zn plants was significantly increased as compared with control plants.

Glyoxysomes in the ⌬Zn Lines Are Not Impaired in ␤-Oxidation During Germination and Seedling Establishment. To assess ␤-oxidation in glyoxysomes, we tested sugar dependence during germination and IBA response of the ⌬Zn lines. Peroxisomal mutant seedlings of oil seed species such as Arabidopsis grow poorly in the absence of exogenous sugar (19). The developmental delay of ⌬Zn plants was quantified by measuring the root growth on medium either with or without 1% sucrose in the light (Fig. 2K). Fig. 3. Transcript levels of PEX10 and pex10 after ampliﬁcation by RT-PCR Mutant plants developed normally without exogenous sugar and digestion with MbiI; plasmid DNA was used as control. Lanes 1 and 3, until days 3–4 of germination, so long as lipid mobilization was undigested; lanes 2 and 4–10, digested with MbiI. Arrows indicate the undi- the limiting factor. However, a lag in root growth was seen from gested 248-bp PCR product of WT PEX10 and the 176-bp and 72-bp fragments the beginning of photoautotrophy at day 4 on medium lacking ⌬ of the mutated pex10 after digestion with MbiI. Zn lines exhibit both sucrose, when peroxisomal transport of metabolized phospho- transcripts in approximately equal amounts. M, 100-bp ladder. glycolate starts to play a crucial role for the normal photosynthetic development of seedlings. ␤ segregants (see figure 1 D and G in ref. 12). None of the 48 Mutants defective in -oxidation are resistant to the inhibitory transgenic plants could survive despite knockout of the WT by effect of IBA on root elongation, because the glyoxysomes ⌬ cannot convert IBA into IAA (6). Root growth was determined the overexpressed Zn-pex10; siliques of the surviving plants ⌬ exhibited the same ratio of segregating seeds as the noncomple- in WT, vector control, and Zn seedlings on medium with 1% sucrose with or without 10 ␮M IBA. pex6, which has photore- mented insertion line, i.e., green maturing to white lethal seeds ⌬ (data not shown). Thus, mutation of the C HC Zn finger motif spiration defects similar to the Zn lines (7), and pex4, which 3 4 appears to grow normally (8), are IBA-resistant as seedlings and conferred the same lethality as the complete disruption of ⌬ PEX10, and the phenotypes of ⌬Zn1–4 were caused by compe- were used as positive controls (Fig. 2L). Zn seedlings re- tition between the endogenous PEX10 and the mutated pex10 sponded to the inhibitory effect of IBA on root elongation PLANT BIOLOGY similar to WT and vector control plants, whereas pex4 and pex6 transcripts. seedlings exhibited the expected resistance to IBA. Early normal development of the ⌬Zn seedlings implies that Leaf Peroxisomes in the ⌬Zn Lines Are Impaired in Photorespiration. their glyoxysomes contain sufficient matrix enzymes to perform ⌬Zn plants showed the dwarfish and chlorotic phenotype in ␤-oxidation to mobilize stored lipids and to convert IBA into normal air after 10 days (Fig. 2D) and were used for character- IAA. Glyoxysomal function thus appears to be unaffected by the ization. We quantified the parameters that are associated with overexpression of a dysfunctional PEX10 protein that impairs defective photorespiration, such as glyoxylate level, the amount photorespiratory function in leaf peroxisomes. of chlorophyll a and b and carotenoids (xanthophylls and car- otenes), and the maximum quantum yield of photosystem II. All Leaf Type Peroxisomes in ⌬Zn Lines Are Pleomorphic and Rarely four ⌬Zn lines exhibited the same phenotype to a similar Associated with Chloroplasts. The morphology and association of quantitative degree (Fig. 2 E–H). leaf peroxisomes with other organelles in ⌬Zn seedlings were An abnormally high level of glyoxylate was detected photo- examined by quantitative transmission electron microscopy (Fig. metrically after reaction with phenylhydrazine in the presence of 4) and light microscopy (Fig. 5). Peroxisomes are identified K3Fe(CN)6 (Fig. 2E). The mutant plants regained WT chloro- unequivocally by cytochemical staining for catalase (20). As phyll a and b contents after transfer to high CO2 (Fig. 2F) but exemplified in Figs. 4A and 5, WT image, the spherical or ovoid had a lowered content of carotenoids compared with WT grown peroxisomes in the mesophyll cells of WT Arabidopsis thaliana in normal air and transferred to high CO2 (data not shown). had a diameter of 0.88 Ϯ 0.23 ␮m(n ϭ 50), were appressed to Photosynthetic parameters were evaluated by chlorophyll fluo- the envelope of chloroplasts, and were frequently in close rescence kinetics with the portable fluorometer PAM-2000 (Fig. physical contact with mitochondria. The peroxisomes of the ⌬Zn ⌬ 2 G and H). The Zn plants revealed a decreased photosystem plants exhibit catalase activity equal to those of WT (Figs. 4 B II quantum yield under normal atmosphere; under high CO2 and C and 5, image ⌬Zn1–4), but they varied considerably in size these values reached WT levels (Fig. 2G). As a parameter for the and shape and were rarely associated with chloroplasts. The ⌬Zn redox state of the primary electron acceptor in photosystem II, peroxisomes form chains, protuberances, and interconnected photochemical quenching was determined. Under atmospheric clusters within the cytoplasm (Fig. 5, image ⌬Zn1–4), possibly CO2 conditions, active quenching was decreased and nonactive indicating an inhibition of the proliferating peroxisomes in their quenching was increased in the ⌬Zn lines, indicating an inef- fission. fective photosynthesis system. Under 5-fold-elevated CO2 partial A quantitative analysis of the peroxisomal profiles revealed pressure these parameters did not differ from WT and vector 3.32 Ϯ 1.5 per WT (n ϭ 25) and 9.0 Ϯ 4.2 per ⌬Zn1 (n ϭ 25) controls (Fig. 2H). cell section (t for difference ϭ 4.11; ***, P Ͻ 0.001). In WT cells The high level of glyoxylate indicates a defect in peroxisomal 88 Ϯ 16% (n ϭ 25) of peroxisomes were appressed to a metabolism, possibly a deficiency in serine-glyoxylate amino- chloroplast, whereas in an equal number of ⌬Zn1 cell sections transferase activity. We determined the serine-glyoxylate ami- 31 Ϯ 21% were appressed to chloroplasts (t for difference ϭ 27.7; notransferase activity by measuring the consumption of glyoxy- ***, P Ͻ 0.001). Measurements of the width of peroxisomal late with serine as amino donor in protein extracts of 10-day-old profiles (n ϭ 50) in both WT and ⌬Zn1 did not reveal any plants. We found equal levels in both WT and ⌬Zn plants: significant difference with 0.88 Ϯ 0.23 ␮m for WT and 1.28 Ϯ Ϫ Ϫ consumption of 0.24 ␮mol of glyoxylate min 1⅐mg 1 of protein 0.78 ␮m for ⌬Zn1 (t for comparison ϭ 0.331; ***, P ϭ 0.07). compared with 0.22 ␮mol of glyoxylate minϪ1⅐mgϪ1 of protein. However, there was a large difference in length. There were two We compared hydroxypyruvate reductase and glyoxysomal peroxisomal populations in ⌬Zn1 cells, one with a normal length malate dehydrogenase amounts in crude organelle pellets of of 0.68 Ϯ 0.15 ␮m(n ϭ 29) and a second with a highly variable

Schumann et al. PNAS ͉ January 16, 2007 ͉ vol. 104 ͉ no. 3 ͉ 1071 Downloaded by guest on September 25, 2021 Fig. 5. Light microscopy of semithin sections of leaf tissue from WT and ⌬Zn1–4 plants and AtPEX10-overexpressing plants stained for catalase activity Fig. 4. Electron micrographs of leaf tissue of WT (A) and ⌬Zn1 (B and C) with diaminobenzidine. plants stained for catalase activity with diaminobenzidine. The leaf peroxisomes of WT are ovoid and in physical contact with chloroplasts (A). Mutant ⌬Zn plants exhibit peroxisomes with catalase activity, however rarely associ- and ‘‘2’’) are the specific products of the third TAIL-PCR cycle. ated with chloroplasts. Their shape is pleomorph, ranging from ovoid to These PCR fragments are shorter than the bands in the left lane, elongated with protrusions (B). Clusters of peroxisomes forming local net- because nested T-DNA primers were used to demonstrate the works are frequent (C). P, chloroplast; V, vacuole; MB, microbody (peroxi- specificity of the PCR. WT DNA produced no PCR products. some); M, mitochondrion. The bands marked ‘‘1’’ and ‘‘2’’ were sequenced and subjected to BLAST search. Bands marked with ‘‘2’’ were primarily T-DNA Ϯ ␮ ϭ ϭ regions. The boxes in Fig. 6A mark the sequenced bands that length of 3.51 1.93 m(n 21) (tk for difference 6.227; ***, P Ͻ 0.001). The elongated structures could reach a length of 7 matched different genomic sequences of A. thaliana. Thus, ␮ T-DNA integration into the different ⌬Zn lines occurred at m. Plants overexpressing WT PEX10 have normal-shaped ⌬ peroxisomes attached to the chloroplast envelope (Fig. 5, image different sites. In Zn1, the insertion was 335 bp upstream of the start codon within the 5Ј UTR of an ortholog to the MtN24 gene overexpr.). of Medicago trunculata encoding a sequence similar to a pro- karyotic membrane lipoprotein with a lipid attachment site Isolation and Mapping of Genomic Sequences Flanking the T-DNA (At3g55390). In ⌬Zn2, the integration was found 140 bp down- Integration Sites in ⌬Zn Lines 1–4. Southern blot analysis of the Ј ⌬ stream of the stop codon within the 3 UTR of a gene for Zn lines revealed the presence of at least one integration site glycerol-phosphodiesterase involved in glycerol metabolism of the T-DNA carrying the pex10 cDNA (data not shown). (At5g58050). Integration in ⌬Zn4 occurred in the last intron of Thermal asymmetric interlaced PCR (TAIL-PCR) was carried a gene for an unknown but expressed protein (At5g24314). out to identify genomic sequences flanking the T-DNA inser- TAIL-PCR for ⌬Zn3 did not reach genomic regions. Thus, the tions (Fig. 6A). The two bands in the left lane for each plant gene integrations, especially in the ⌬Zn1 and ⌬Zn2 lines, are DNA represent the products of the second cycle of the TAIL- unlikely to have interrupted expression of genes essential for PCR created with the arbitrary degenerate primer Nr.2 (AD-2) photorespiration, and the phenotype must be due to the expres- and the pBI right border primer (RB-2). The two bands in the sion of the mutated PEX10 Zn finger protein. The integration right lane for the individual plant DNA amplificates (marked ‘‘1’’ sites were confirmed by genomic PCR by using primers specific

1072 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610402104 Schumann et al. Downloaded by guest on September 25, 2021 different regions of the Arabidopsis genome exhibited equal transcript levels in the four transformants supporting the rationale that the mutant protein lacking the RING finger metal ligands com- petes with the WT protein for interacting partners and will therefore interfere with the normal function of PEX10. By determining the sites of integration of three of the transgenes in the Arabidopsis genome and by analyses of knockout mutants of the genes targeted by the insertions, it was shown that they are not encoding proteins involved in photorespiration and thus the integrations were not responsible for the defective phenotype. The RING finger with the Zn metal ligands was required for normal-shaped peroxisome organelles that can physically associate through their single membrane with the outer membrane of the chloroplast envelope. This was correlated with proliferation of elongated peroxisomes that were unable to fission into normal-sized peroxisomes (Fig. 5). The dysfunctional RING finger did not affect the function of glyoxy- Fig. 6. Localization of T-DNA integrations in ⌬Zn lines. (A) TAIL-PCR of ⌬Zn somes that carry out ␤-oxidation and the glyoxylate cycle for lipid lines 1–4, WT, and pBI121 vector control (contr). PCR products marked ‘‘1’’ and mobilization during oil seed germination; it did not influence the ‘‘2’’ were sequenced and subjected to a BLAST search. PCR products ‘‘2’’ conversion of IBA into the plant growth hormone IAA. Further- represent T-DNA regions. PCR products ‘‘1’’ (boxes) indicate positive matches more, the dysfunctional RING finger did not impair the abundance in the Arabidopsis genome. (B) Genomic PCR of the ⌬Zn lines. The lack of DNA fragments obtained with ⌬Zn DNA from lines ⌬Zn1, ⌬Zn2, and ⌬Zn4 with the of catalase within leaf peroxisomes as demonstrated in situ by respective forward and reverse specific primers (sp1–4 fϩr), and the creation cytochemical staining. of bands with the specific primers in combination with a right border primer Functional photorespiration requires the localization of matrix (RB) for pBI121, confirm the T-DNA integrations within the genes proposed by enzymes within peroxisomes as well as the association of per- TAIL-PCR and BLAST search. The band from ⌬Zn4 DNA created by the specific oxisomes, chloroplasts, and mitochondria to enable the metab- reverse primer (sp4r) and the RB primer, additionally to the band generated olite flow between these organelles. Our results let us conclude by the specific forward primer (sp4f) and the RB primer, indicates a full or the necessity of the PEX10 Zn RING finger for the physical partial inverse tandem integration into chromosomal DNA in ⌬Zn4. M, 100-bp attachment between peroxisome membrane and chloroplast PLANT BIOLOGY ladder with the accentuated 500-bp fragment. envelope. PEX10 is already known as an essential component of the peroxisomal protein import machinery and is thought to for the affected genes. As expected, PCR using two gene-specific form a complex with the Zn RING finger proteins PEX2 and PEX12 in the peroxisomal membrane (9). We found equal or primers resulted in an absence of PCR products, whereas a ⌬ combination of either of the appropriate gene-specific primers even enhanced activity levels for Zn plants compared with WT with primers for the pBI121 right border yielded PCR products for the peroxisome marker enzymes serine-glyoxylate amino- confirming the results of the BLAST search (Fig. 6B). In ⌬Zn4, transferase in protein extracts and hydroxypyruvate reductase both specific primers in combination with the right border and glyoxysomal malate dehydrogenase in organellar pellets. Because the function of the different PEX10 subdomains in primers led to amplified DNA, indicating a full or partial tandem matrix protein import is not known, it could be that the complex integration. Furthermore, crosses between homozygous ⌬Zn between the mutated PEX10 and the PEX2 and PEX12 is lines resulted in F progeny that were homozygous for the pex10 1 stabilized by the intact Zn finger of PEX2 and PEX12, thus cDNA expression but heterozygous for the gene affected by the allowing enzyme import into the abnormal-shaped peroxisomes. T-DNA insertion. These F1 progenies consistently exhibited ⌬ This interpretation is supported by the intact glyoxysome import the dwarf chlorotic Zn phenotype, again confirming that the machinery that might be inherited during developmental tran- expressed pex10 cDNA was responsible for the phenotype (data sition to leaf peroxisomes as demonstrated by the cytochemical not shown). catalase staining. We cannot, however, completely exclude the mislocalization of peroxisome enzymes to the cytosol, thus Knockout Lines with T-DNA Integrations in the Genes Containing the contributing to the defective photorespiration phenotype. ⌬ Transgenes in the Zn Lines Do Not Exhibit Impaired Photorespiration. Efficient processing of glyoxylate into glycine for transport As a final confirmation that expression of pex10 is responsible for into mitochondria was severely curtailed. The elevated level of ⌬ the dwarf chlorotic phenotype of Zn lines, knockout mutants glyoxylate might be an inheritance of the glyoxysome metabo- with T-DNA integrations close to the integration sites of the lism rather than owing to the transfer from chloroplasts. Because ⌬ pex10 cDNA in the Zn lines were examined. Homozygous the activity of serine-glyoxylate aminotransferase is comparable knockout mutants were grown and genotyped to confirm the in WT and ⌬Zn plants, the reason for the disturbed metabolism presence and position of the T-DNA insertions: SALK line is either the mislocalization of the enzyme to the cytosol or the 534800 and SAIL line 809777 with an insertion in the third exon lack of serine that is thought to be on the return trip from of the gene At3g55390 affected in the ⌬Zn1 line, and SALK line mitochondria. 537722 with an insertion in the fifth exon of the gene At5g58050 It is of interest whether the RING finger domains of PEX12p affected in the ⌬Zn2 line. Their phenotype was similar to WT and PEX2p are also required for generating associations be- plants. tween peroxisomes, chloroplasts, and mitochondria. In a comparable strategy, RNA interference plants with reduction of the Discussion PEX12 transcript were used to obtain sublethal mutants (15). Given the lethal phenotype of the previously analyzed null mutants These plants exhibited impaired peroxisome biogenesis and of PEX10, we generated nonlethal partial loss-of-function mutants. function and inhibition of plant growth. Down-regulation of By expressing a version of AtPEX10 cDNA with a dysfunctional Zn PEX12 by RNA interference, however, yielded plants that were finger motif under the control of the 35S CaMV promoter, we also less responsive to IBA. obtained four dominant negative, conditional transformants with In conclusion, transformants with peroxins mutated in an all landmarks of mutants compromised in photorespiration (Fig. 2). individual protein domain can detect novel functions of the The parental WT gene and the mutated pex10 cDNA inserted in peroxins. In the present case, the requirement of PEX10 for

Schumann et al. PNAS ͉ January 16, 2007 ͉ vol. 104 ͉ no. 3 ͉ 1073 Downloaded by guest on September 25, 2021 establishing the physical attachment of the peroxisome mem- collected with the portable pulse amplitude modulator fluorom- brane to the chloroplast envelope has been demonstrated. It eter (PAM-2000; Walz, Effeltrich, Germany) at 25 Ϯ 1°C and opens the way to finding out how this attachment shown in ambient CO2 partial pressure (29). For details see SI Text. numerous published micrographs is established at the molecular level and which other proteins and possible transporters are Electron and Light Microscopy. Pieces of leaf tissue from 12-day-old involved. The attachment has been presaged in textbook car- light-grown control and ⌬Zn plants were fixed with 2.5% toons to explain the metabolite flow from chloroplasts via glutaraldehyde in 75 mM sodium cacodylate/2 mM MgCl2 (pH peroxisomes to mitochondria and back via peroxisomes into the 7.0) for1hat25°C, rinsed several times in fixative buffer, and chloroplast to describe the photorespiratory pathway. postfixed for2hwith1%osmium tetroxide in fixative buffer at Methods 25°C. After two washing steps in distilled water, the pieces were stained en bloc with 1% uranyl acetate in 20% acetone for 30 Generation of the Mutated C3HC4 Zn Finger. Four point mutations min. Dehydration and embedding in Spurr’s low-viscosity resin PEX10 were introduced into the Zn finger motif of the cDNA were essentially as in ref. 19. Ultrathin sections of 50–70 nm were with gene splicing by overlap extension (21, 22) to change the cut with a diamond knife and mounted on uncoated copper codons TGT or TGC for Cys into GGT for Gly and CAT for His into CTT for Leu (Fig. 1). The required primers are listed in SI grids. The sections were poststained with aqueous lead citrate Text. (100 mM, pH 13.0). Micrographs were taken with an EM 912 electron microscope (Zeiss, Oberkochen, Germany) equipped RT-PCR. Total RNA was isolated from 100 mg of 3-week-old with an integrated OMEGA energy filter operated in the seedlings grown on MS plates. After cDNA first-strand synthesis, zero-loss mode. Specific localization of catalase activity was PCR was carried out with the PEX10 sense and antisense primers performed as in ref. 20. For light microscopy, semithin sections spanning a 248-bp-long fragment of the C terminus including the with a thickness of 500 nm were cut with a diamond knife, Zn finger and part of the 3Ј UTR. The products of amplified mounted onto glass slides, and embedded in epoxy resin. PEX10/pex10 were purified, reamplified, digested with MbiI, and analyzed on a 3% agarose gel. The required primers are listed in Characterization of T-DNA Insertion Sites Within the Chromosomes SI Text. ⌬Zn Lines. TAIL-PCR (30) used a combination of primers recognizing the pBI121 right border (RB2 or RB3) and three Assays. Glyoxylate was measured according to ref. 23, leaf different arbitrary degenerate primers (AD primers) annealing pigments were measured as in ref. 24, serine-glyoxylate amino- randomly in the genome of A. thaliana. The AD-2 primer transferase was measured as in ref. 25, and hydroxypyruvate combined with the RB3 primer resulted in the specific PCR reductase activity was measured as in ref. 26. Organellar pellets products. Genomic PCR for verification of the integration sites were prepared as in ref. 27. For Western blotting, polyclonal identified by TAIL-PCR and BLAST research in ⌬Zn1, ⌬Zn2, antibodies generated against glyoxysomal malate dehydrogenase and ⌬Zn4 used a primer pair annealing Ϸ900 bp upstream and were used (28). Defects in ␤-oxidation or photorespiration were downstream of the putative integration sites. The primers used assayed by growth on MS plates with or without 1% sucrose and and their locations are listed in SI Text. measurement of root length (mean value of 20 plants) at days 3, 4, and 6. The inhibitory effect of IBA on root elongation (mean We thank Dr. Bonnie Bartel for the Arabidopsis pex4 and pex6 mutant value of 20 plants) was tested as in ref. 6. seeds and Dr. Markus Schmid for ideas on this project, especially for the design of the ⌬Zn-pex10. The skillful technical assistance of Ms. Silvia Photosynthetic Parameters. The photosynthetic yield and the Dobler is gratefully acknowledged. This work was supported by Deutsche active and nonactive quenching were calculated from data Forschungsgemeinschaft Grant Gi 154/12-1.

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