Pentatricopeptide repeat 336 as the candidate gene for paternal sorting of mitochondria (Psm) in cucumber

Abstract

Pentatricopeptide repeat (PPR) 336 was identified as the candidate gene for Paternal Sorting of Mitochondria ( Psm ), a nuclear locus that affects the predominant mitochondria transmitted to progenies. Cucumber (Cucumis sativus L.) is a useful plant to study organellar-nuclear interactions because its organelles show differential transmission, maternal for chloroplasts and paternal for mitochondria. The mitochondrial DNA (mtDNA) of cucumber is relatively large due in part to accumulation of repetitive DNAs and recombination among these repetitive regions produces structurally polymorphic mtDNAs associated with paternally transmitted mosaic (MSC) phenotypes. The mitochondrial mutant MSC16 possesses an under-representation of ribosomal protein S7 (rps7), a key component of the small ribosomal subunit in the mitochondrion. A nuclear locus, Paternal Sorting of Mitochondria (Psm), affects the predominant mitochondria transmitted to progenies generated from crosses with MSC16 as the male parent. Using single nucleotide polymorphisms, Psm was mapped to a 170 kb region on chromosome 3 of cucumber and pentatricopeptide repeat (PPR) 336 was identified as the likely candidate gene. PPR336 stabilizes mitochondrial ribosomes in Arabidopsis thaliana and because MSC16 shows reduced transcription of rps7, the cucumber homolog of PPR336 (CsPPR336) as the candidate for Psm is consistent with a nuclear effect on ribosome assembly or stability in the mitochondrion. We used polymorphisms in CsPPR336 to genotype progenies segregating at Psm and recovered only one Psm -/- plant with the MSC phenotype, indicating that the combination of the Psm- allele with mitochondria from MSC16 is almost always lethal. This research illustrates the usefulness of the MSC mutants of cucumber to reveal and study unique interactions between the mitochondrion and nucleus.

Fig. 1

Families of wild-type (WT), mosaic (MSC), and segregating (WT + MSC) progenies from crosses of plants segregating for Paternal Sorting of Mitochondria (Psm) with MSC16 as the male parent. Each row is seed from one cross and the percentage of wild-type progenies was used for mapping of Psm

Fig. 2

Composite interval mapping of percentage of wild-type progenies from crosses of F₂ plants segregating for Paternal Sorting of Mitochondria (Psm) with MSC16 as the male parent. Single nucleotide polymorphisms (SNPs) along the x-axis are listed in Supplemental File 1. Numbers in parentheses following SNPs indicate kilobase positions. Horizontal line shows threshold LOD score from permutation analysis. Scaffolds from the sequence assembly of GY14 (Yang et al. 2012) are shown below SNPs

Fig. 3

Amino-acid alignment between predicted protein sequences of CsPPR336 from Gy14 and TMG1 (both Psm +/+) and 401734-3A (P3A; Psm −/−)

Fig. 4

BsaWI digestion of genomic amplicons of CsPPR336 from 401734-3A (P3A; Psm −/−), inbred B (Psm +/+), and an artificial heterozygote (AH) from mixture of equal amounts of DNAs from P3A and inbred B