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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 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 in- cluding 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 inactiva- tion 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 func- tions, 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 chloro- plasts, 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 atmo- sphere, the plants grew as dwarfs with a 2- to 3-week-delayed development and retarded silique maturation, but under ele- vated 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 accumula- tion in the peroxisomes of the ⌬Zn. (F) ⌬Zn plants regained WT chlorophyll aϩb 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 ⌬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.
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