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

Abstract

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 C(3)HC(4) 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 (DeltaZn plants). They could be normalized by growth in an atmosphere of high CO(2) partial pressure, indicating a defect in photorespiration. beta-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 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 peroxisomes of the DeltaZn 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 PEX10 has eliminated protein interaction required for attachment of peroxisomes to chloroplasts and movement of metabolites between peroxisomes and chloroplasts.

Fig. 1.

The dysfunctional Zn finger motif in AtPex10p. The amino acid changes resulting in a loss of Zn coordination sites are shown.

Fig. 2.

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

Fig. 3.

Transcript levels of PEX10 and pex10 after amplification by RT-PCR and 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 undigested 248-bp PCR product of WT PEX10 and the 176-bp and 72-bp fragments of the mutated pex10 after digestion with MbiI. ΔZn lines exhibit both transcripts in approximately equal amounts. M, 100-bp ladder.

Fig. 4.

Electron micrographs of leaf tissue of WT (A) and ΔZn1 (B and C) plants stained for catalase activity with diaminobenzidine. The leaf peroxisomes of WT are ovoid and in physical contact with chloroplasts (A). Mutant ΔZn plants exhibit peroxisomes with catalase activity, however rarely associated with chloroplasts. Their shape is pleomorph, ranging from ovoid to elongated with protrusions (B). Clusters of peroxisomes forming local networks are frequent (C). P, chloroplast; V, vacuole; MB, microbody (peroxisome); M, mitochondrion.

Fig. 5.

Light microscopy of semithin sections of leaf tissue from WT and ΔZn1–4 plants and AtPEX10-overexpressing plants stained for catalase activity with diaminobenzidine.

Fig. 6.

Localization of T-DNA integrations in ΔZn lines. (A) TAIL-PCR of ΔZn lines 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 matches in the Arabidopsis genome. (B) Genomic PCR of the ΔZn lines. The lack of DNA fragments obtained with ΔZn DNA from lines ΔZn1, ΔZn2, and ΔZn4 with the respective forward and reverse specific primers (sp1–4 f+r), and the creation of bands with the specific primers in combination with a right border primer (RB) for pBI121, confirm the T-DNA integrations within the genes proposed by TAIL-PCR and BLAST search. The band from ΔZn4 DNA created by the specific reverse primer (sp4r) and the RB primer, additionally to the band generated by the specific forward primer (sp4f) and the RB primer, indicates a full or partial inverse tandem integration into chromosomal DNA in ΔZn4. M, 100-bp ladder with the accentuated 500-bp fragment.