PPR9 is encoded by the Arabidopsis gene AT1G03560 and produces a 660-amino-acid protein containing 13 recognized PPR motifs and a divergent PPR-like region. Database predictions indicated that the protein should enter mitochondria, and fluorescence microscopy supported that localization: a fusion containing the first 82 amino acids of PPR9 fused to green fluorescent protein formed punctate signals that colocalized with a mitochondrial marker in moss protoplasts. No comparable signal was detected in chloroplasts, the nucleus or the cytoplasm.

Public transcriptomic data also indicated relatively elevated PPR9 expression in embryos, young leaves, shoot apices, root tips and flowers. The researchers began from a T-DNA insertion mutant and used molecular, biochemical and physiological comparisons with wild-type, heterozygous and gene-complemented plants to test the protein’s function. Loss of PPR9 is developmentally severe.

About 24% of the seeds produced by heterozygous plants were shrunken and failed to germinate under standard soil or agar conditions, while approximately 98% of wild-type seeds appeared normal. Microscopy showed that mutant embryos in these abnormal seeds stopped developing at the torpedo to walking-stick stage, whereas embryos in green seeds were fully formed. No homozygous mutant could be established normally.

The researchers recovered a limited number of homozygotes through an in-vitro rescue procedure involving immature seeds, enriched Murashige and Skoog medium, sucrose and vitamins. Across three experiments, 7 of 16, 5 of 19 and 3 of 12 white seeds germinated on the supplemented medium. Those seedlings grew slowly and displayed variable, often bushy morphology, but many died within two to four weeks, none survived in soil, and none completed a life cycle or produced viable seeds.

To determine whether the mutant phenotypes were specifically caused by loss of PPR9, researchers introduced the full coding sequence under a cauliflower mosaic virus 35S promoter into heterozygous plants and screened for homozygous mutants carrying the transgene. This complemented line regained substantial mitochondrial RNA processing and restored detectable assembly of complete complex I. Heterozygous plants were morphologically normal and showed transcript, splicing and complex-assembly measurements similar to wild type. The complemented line did not match wild type in every protein or respiratory-complex measurement, which the authors noted could reflect tissue composition, mitochondrial abundance or the non-native expression promoter.

Nevertheless, the reappearance of the full complex provided functional support for PPR9’s role. Reverse-transcription quantitative PCR linked loss of PPR9 to changes in mitochondrial gene expression. In mutant seedlings, the processed transcript spanning exons 3 and 4 of nad2 was about 250-fold lower than in wild type.

Processed regions of nad7 were also depleted, with levels approximately eightfold lower across exons 1–2 and 20-fold lower across exons 2–3. Because these genes contain introns, the researchers tested splicing efficiency by comparing mature messenger RNA with corresponding precursor RNA across all 23 mitochondrial introns. The procedure was designed to identify processing defects by requiring precursor accumulation alongside a corresponding loss of mature RNA, rather than treating altered transcript abundance alone as evidence of impaired splicing.

Measurements were normalized to GAPDH, ACTIN2, 18S rRNA and the mitochondrial 26S rRNA. The analysis revealed that PPR9 is needed for efficient excision of nad2 intron 3 and introns 1 and 2 of nad7 . Splicing efficiency for nad2 intron 3 was about 1,000-fold lower in the mutant relative to wild type, while the reductions for the two nad7 introns were approximately 30-fold and 60-fold.

Most of the other mitochondrial introns showed efficiencies broadly comparable to those in wild-type plants. RNase-protection assays independently detected accumulation of unspliced precursors and reduction of mature products for the same three targets, while control transcripts such as cox2 , rpl2 and intronless nad6 appeared unaffected. A precursor associated with the nad2 trans-splicing pathway also accumulated, suggesting that processing steps within this multipartite gene are interdependent.

The data therefore support a role for PPR9 in maturation rather than a complete inability to splice the three introns. The molecular consequences were visible in the mitochondrial respiratory system. Blue-native polyacrylamide gel electrophoresis followed by immunoblotting showed that complete complex I, an assembly of roughly 1,000 kilodaltons, fell below detection in mutant mitochondrial membrane fractions.

Instead, the mutants accumulated smaller assembly particles, including a stable intermediate of approximately 800–850 kilodaltons and lower-mass species detected with an antibody against the complex I membrane protein CA2. A roughly 700-kilodalton species detected with an antibody against the soluble-arm protein NAD9 was consistent with disturbed assembly. These findings align with the established roles of NAD2 and NAD7, which contribute to the membrane and matrix-associated arms of complex I, respectively.

The study did not directly reconstruct the assembly pathway or resolve the molecular contacts between PPR9 and its RNA targets. Mutant mitochondria also showed remodeling of electron transport. Alternative oxidase AOX1a accumulated to about 20 times the wild-type level, and transcripts for several alternative oxidases and rotenone-insensitive NAD(P)H dehydrogenases increased, although changes in some genes were not statistically significant.

The Rieske iron-sulfur protein associated with complex III rose about threefold, while cytochrome c oxidase and ATP synthase proteins showed moderate increases. Oxygen-consumption rates did not simply collapse: two-month-old mutant seedlings consumed 129 ± 8 nanomoles of oxygen per minute per gram fresh weight, compared with 125 ± 9 in wild type. Rotenone, which inhibits complex I, suppressed wild-type respiration by 47% but mutant respiration by only about 20%.

Combined cyanide and salicylydroxamic acid reduced oxygen uptake by roughly 90% in wild type and 85% in mutants, showing that these inhibitors strongly suppressed the measured respiration under the experimental conditions. Together, the observations support a model in which nuclear-encoded PPR9 enters mitochondria and enables maturation of selected mitochondrial transcripts, thereby supplying NAD2 and NAD7 proteins required for assembly of complex I. When that pathway fails, the respiratory chain is remodeled, complex I falls below detection, alternative electron-flow components rise, and early development is disrupted. The study nevertheless has important limitations.

Its main mutant was a single T-DNA insertion, although complementation supported the assignment of the phenotype to PPR9. Most molecular and biochemical analyses used unusual in-vitro-rescued, approximately two-month-old homozygous seedlings rather than normally developing plants, and the rescued mutants were metabolically unstable and infertile. The predicted PPR recognition code generated more than 200 possible RNA-binding sites and did not isolate an unambiguous binding motif.

Genetic and biochemical measurements identified the three affected introns, but the study did not directly demonstrate the nucleotide contacts between PPR9 and those RNA sequences or establish why this P-class protein acts on these particular group II introns. The study was limited to a single gene in the model plant Arabidopsis thaliana , and its principal mutant was a T-DNA insertion whose homozygous descendants could be analyzed only after specialized embryo rescue. Most RNA, protein and respiratory measurements were therefore made on unusual, developmentally impaired plants cultured in liquid medium for about two months.

The complemented line used a constitutive 35S promoter rather than the native regulatory sequence and did not reproduce every wild-type measurement, although it restored detectable mature complex I and substantially improved transcript processing. The PPR-code analysis also produced more than 200 candidate mitochondrial RNA-binding sites, so the molecular basis for PPR9 recognition of the three validated intron targets remains unresolved. Even with those constraints, the combined genetic, transcript, assembly and physiological evidence identifies PPR9 as an essential nuclear regulator linking mitochondrial RNA maturation to respiratory complex I biogenesis and early plant development.

Subject of Research: PPR9-dependent mitochondrial RNA splicing, respiratory complex I biogenesis and early development in Arabidopsis.

Article Title: PPR9 mediates mitochondrial nad transcript maturation required for complex I biogenesis and early plant development in Arabidopsis

Article References: Kobaivanov, E., Maltz, T., Kittel, M., Matan, R., Mizrahi, R., Carmi, N., & Ostersetzer-Biran, O. (2026). PPR9 mediates mitochondrial nad transcript maturation required for complex I biogenesis and early plant development in Arabidopsis. Plant Molecular Biology, 116(6), Article 105. https://doi.org/10.1007/s11103-026-01769-9

Image Credits: AI Generated

DOI: 10.1007/s11103-026-01769-9

Keywords: Arabidopsis, PPR9, mitochondrial RNA splicing, respiratory complex I, plant mitochondria, group II introns, alternative respiration, embryo development, pentatricopeptide repeat proteins, oxidative phosphorylation, mediates, mitochondrial