The pentatricopeptide repeat gene OTP43 is required for trans-splicing of the mitochondrial nad1 Intron 1 in Arabidopsis thaliana

Abstract

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a large protein complex formed from both nuclearly and mitochondrially encoded subunits. Subunit ND1 is encoded by a mitochondrial gene comprising five exons, and the mature transcript requires four RNA splicing events, two of which involve trans-splicing independently transcribed RNAs. We have identified a nuclear gene (OTP43) absolutely required for trans-splicing of intron 1 (and only intron 1) of Arabidopsis thaliana nad1 transcripts. This gene encodes a previously uncharacterized pentatricopeptide repeat protein. Mutant Arabidopsis plants with a disrupted OTP43 gene do not present detectable mitochondrial Complex I activity and show severe defects in seed development, germination, and to a lesser extent in plant growth. The alternative respiratory pathway involving alternative oxidase is significantly induced in the mutant.

Figure 1.

The otp43 Mutant Line. (A) T-DNA insertion map of the Sail_867F07 line. (B) Mutant (left) and wild-type (right) seeds from a plant heterozygous for the Sail_867F07 insertion. (C) Rosette from a homozygous otp43 plant growing on soil in short-day conditions. (D) Rosettes growing on soil in short-day conditions, from left to right: wild-type (Columbia [Col-0]), otp43, and complemented otp43. Rosettes from otp43 are 4 weeks older than those of the wild type and the complemented otp43 to approximately match the developmental stages of the plants.

Figure 2.

A Splicing Defect in nad1 Intron 1 in otp43. (A) Schematic representation of the localization of the primers used to detect the four splicing events by RT-PCR on the nad1 pre-mRNA. 1, nad1exon1F/nad1exon2R; 2, nad1exon2F/nad1exon3R; 3, nad1exon3F/nad1exon4R; 4, nad1exon4F/nad1exon5R; 5, nad1exon1F/nad1exon5R. (B) RT-PCR on Col-0 cDNA, on cDNA from otp43, and on cDNA from complemented otp43 using primers designed to amplify across nad1 splice junctions. No amplification is detected from otp43 with primer pairs 1 and 5, showing that intron 1 is not spliced in the mutant. Molecular weight markers are shown on the right. (C) PCR on cDNA from otp43 leaves 48, 72, or 92 h after infiltration with Agrobacterium carrying a T-DNA containing the wild-type OTP43 gene.

Figure 3.

Secondary Structure Model of Domains V and VI of the Arabidopsis nad1 Intron 1. The C-to-U editing site found in nad 1 intron 1 domain VI in wheat, highlighted in gray, is not edited in Arabidopsis. The second nucleotide highlighted in gray in domain V is a novel C-to-U editing site that was found to be edited in the wild type and in otp43. The underlined adenosine in domain V is a uracil in almost all other group II introns, while the underlined uracil in domain VI lies approximately where the bulged adenosine involved in lariat formation would normally be located.

Figure 4.

Complex I Is Lacking from otp43 Mitochondria. (A) Blue-native gel (left) and NADH dehydrogenase activity staining of the same gel (right) of isolated mitochondrial proteins from Col-0 rosettes and from otp43 rosettes growing in short-day conditions. I and III+I indicate the positions of Complex I and the supercomplex of Complex I + Complex III. (B) Two-dimensional blue-native/SDS-PAGE of mitochondrial proteins from Col-0 wild-type plants (left) and otp43 plants (right). The gels are stained with Coomassie blue. Apparent molecular mass of subunits is shown in kilodaltons. Arrows indicate spots that have been identified by mass spectrometry (see Supplemental Table 2 online): 1, 76-kD subunit (At5g37510); 2, 51-kD subunit (At5g08530), ND5 (NP_085478/AtMg01460); 3, ND7 (NP_085511/AtMg00510); 4, 39-kD subunit (At2g20360), ND2 (NP_085584/AtMg01450); 5, CAL1 (At5g63510), 23-kD subunit (At1g79010), ND1 (NP_085565/AtMg01275); 6, ND9 (NP_085479/AtMg0070); 7, 16-kD subunit (At2g27730), PDSW subunit (At3g18410, At1g49140); 8, B14 subunit (At3g12260), B18 subunit (At2g02050); 9, B18 subunit (At2g02050). These proteins are all subunits of Complex I and none are visible in the gel of otp43 proteins. 10 and 19, ATP2 (At5g08670), ATP1 (NP_085571/AtMg01190); 11 and 20, ATP3 (At2g33040); 12 and 21, ATP FAD (At2g21870); 13 and 22, ATP8 (NP_085508/AtMg00480). These proteins, identified in both wild-type and otp43 plants, are all subunits of Complex V. 14 and 23, MPP b (At3g02090); 15 and 24, MPP a (At1g51980); 16 and 25, Cyt c1-1 (At3g27240), Cyt c1-2 (At5g40810), COB (NP_085492/AtMg00220); 17 and 26: URC1 (At5g13430); 18 and 27, QCR7 (At4g32470). These proteins, identified in both wild-type and otp43 plants, are all subunits of Complex III.

Figure 5.

Alternative Electron Transport Pathway Transcripts and Proteins Are Induced in otp43. (A) Quantitative RT-PCR on cDNA from three independent otp43 plants (otp43-a, otp43-b, and otp43-c). (B) Immunoblot analysis of AOX proteins. Coomassie blue–stained SDS-PAGE gel (left) of mitochondrial proteins extracted from wild-type and otp43 plants. This gel was used for a protein gel blot (right) using anti-AOX antibodies. Bound antibodies were revealed by chemiluminescence.