Genetic and phenotypic analysis of the dek55-1 mutant
A mutant with a defective kernel phenotype was isolated from an ethyl methane sulfonate-induced maize B73 background population, named defective kernel 55 − 1 (dek55-1). The dek55-1 kernels were segregated from self-pollinated progenies of dek55-1/+ heterozygotes in a 1:3 ratio (Fig. 1a, Additional file 1: Table S1). This mutant was confirmed in other populations generated from dek55-1/+ heterozygotes crossed with the inbred lines C733 or S162 (Additional file 1: Table S1). These results suggest that dek55-1 as a recessive phenotype is caused by a monogenic mutation.
The dek55-1 kernels could be distinguished from wild type (WT) kernels at 15 days after pollination (DAP) (Fig. 1a). The dek55 mutant kernels exhibited a whitened pericarp and were smaller than WT kernels, which exhibited a yellow color (Fig. 1a). At the maturity stage, dek55-1 kernels became smaller and more shriveled (Fig. 1b, c). To further dissect the mutant phenotype, both WT and dek55-1 kernels were longitudinally sliced at different developmental stages. At 15 DAP, the pericarp of WT kernels, but not dek55-1 mutant kernels, was filled with endosperm cells (Fig. 1d, e). Furthermore, dek55-1 exhibited a smaller mature embryo and a decreased proportion of hard endosperm than that in WT (Fig. 1f, g). dek55-1 kernels could not germinate in the experimental field (0/100), implying that the arrested embryo was lethal in mutants. In addition, the kernel weight of dek55-1 was reduced by approximately 70% compared to that of WT kernels (Fig. 1h).
To further investigate the developmental structure of dek55-1 kernels, we examined the tissue structure of WT and dek55-1 kernels at 12 and 18 DAP (Fig. 1i − l). At 12 DAP, WT embryos contained visible coleoptiles, shoot apical meristems, scutella, and two leaf primordia (Fig. 1i). In contrast, dek55-1 embryos only had a small scutellum that was arrested at the coleoptile stage (Fig. 1j). Moreover, WT kernels were filled with endosperm cells, whereas a large interspace between endosperm and seed coat in dek55-1 was observed (Fig. 1i, j). At 18 DAP, WT embryos had developed into relatively complete structures containing four leaf primordia, shoot apical meristems, and a clearly seen root apical meristem (Fig. 1k), while dek55-1 embryos only generated one leaf primordium (Fig. 1l). Less starch grains were accumulated in dek55-1 than in WT endosperm cells at this stage (Fig. 1k, l). In addition, a cavity was observed in dek55-1 endosperm (Fig. 1l). These results indicate that developmental defects in embryo and endosperm are present in dek55-1 mutants.
Map-based cloning of DEK55
To identify the DEK55 gene, we performed the classical map-based cloning strategy to detect F2 mutant kernels, which were segregated from self-pollinated filial 1 (F1) hybrid ear. Four genomic DNA pools (10 mutant kernels per pool) and both parents were used for correlation analysis with polymorphic simple sequence repeats. The six simple sequence repeats at chromosome 5 were highly correlated with defective kernel phenotypes, implying that the candidate gene may be at chromosome 5. Further analysis showed that the DEK55 gene is located between umc1705 and umc2302 on chromosome 5 (Fig. 2a). Six polymorphic molecular markers in this region were used to analyze 1868 mutant kernels from the F2 population. Finally, the DEK55 gene was located on an approximately 1.29 Mb region between molecular label M3 and M4 (Fig. 2a). There are 25 putative protein-coding genes in this region (http://ensembl.gramene.org/Zea_mays/Info/Index). To identify the mutated genes, genomic DNA of 25 candidate genes were amplified and sequenced. This revealed that the E-subgroup PPR protein gene (Zm00001d014471) has a single nucleotide change (C to T) at + 449 in dek55-1, which might result in an amino acid replacement (Ser to Phe) in the protein sequence but not in expression level of DEK55 change (Fig. 2a − d). To validate this result, we obtained a new mutant, dek55-2, from the maize ethyl methane sulfonate-induced mutant database . The dek55-2 mutant had a single nucleotide mutation (G to A) at + 729 bp (Fig. 2b), which leads to protein truncation (Fig. 2d). The mutant dek55-2 also exhibited defective kernels with small and white pericarps (Fig. 2e). The allelic test between dek55-1 and dek55-2 heterozygotes revealed that normal and mutant kernels were segregated with the expected 3:1 ratio (normal/mutant; 450/143; P = 0.62) in the F1 ear (Fig. 2e). As a control, all the kernels from the ear that were crossed between dek55-2 heterozygote and WT were normal (Fig. 2e). These results indicate that the PPR gene Zm00001d014471 mutation is responsible for defective kernel phenotype, and the annotated gene was designated DEK55.
Dek55 Is A Mitochondrial E-subgroup Ppr Protein
Sequence alignment demonstrated that the DEK55 gene has one exon containing an 1893 bp ORF, which encodes a 630 amino acid residue protein containing 13 PPR motifs and an E domain on the carboxy-terminal end (Fig. 2b − d and Additional file 1: Fig. S1). Mutated sites in dek55-1 and dek55-2 were located in the third and fifth PPR motifs, respectively (Fig. 2d). The mutation in dek55-2 resulted in a truncated DEK55 protein without the last eight PPR motifs or E domain.
To examine the subcellular localization of DEK55, the p35S:DEK55-EGFP vector was constructed and transformed into maize protoplasts. The fluorescence signal of DEK55-EGFP overlapped with Mito Tracker, which is a mitochondria-specific dye (Fig. 3a), suggesting that the DEK55 protein is a mitochondrial PPR protein in maize (Fig. 3a). In addition, DEK55 expression analysis in various maize tissues demonstrated that DEK55 is highly expressed in root, anther, and ear, but lowly expressed in stem, leaf, silk, tassel, and kernel (Fig. 3b).
DEK55 is involved in the C-to-U editing of rps13, atp1, nad6, and nad9 transcripts at multiple sites
Usually, PPR proteins take part in modifying organelle transcripts . It has been reported that E-subgroup PPRs participate in the C-to-U editing of mitochondrial pre-RNA [14, 32, 33]. To explore whether DEK55 is involved in this processing, the transcriptional levels of 35 maize mitochondrial genes encoding functional proteins were analyzed in WT and dek55-1. RNA editing of these transcripts in WT and dek55 was detected by amplification sequencing. Direct sequencing of the PCR products and monoclonal sequencing revealed that the C-to-U editing ratio of 15 editing sites in the four transcripts rps13, atp1, nad6, and nad9 were significantly reduced in dek55. The C-to-U editing at the rps13-56 site was about 78.2% in WT kernels, whereas the editing efficiency of rps13-56 was dramatically decreased in dek55-1 (4.5%) and dek55-2 (0%) mutants (Fig. 4). The editing efficiency at the atp1-1490 and nad6-159 sites was dramatically decreased in dek55 (Fig. 4). The editing efficiencies of the two editing sites in WT were 100% and 68.8%, respectively, whereas they were reduced to 43.3% and 16.8% in dek55, respectively. In WT, the C-to-U editing of atp1-1490 changed the Pro codon (CCU) to the Leu codon (CUU), and the editing of nad6-159 kept the same amino acids (Phe) at this position (Fig. 4). Interestingly, the C-to-U editing ratio at 12 nad9 editing sites (nad9-14, nad9-92, nad9-113, nad9-167, nad9-190, nad9-233, nad9-298, nad9-311, nad9-328, nad9-356, nad9-368, and nad9-398) was dramatically decreased in dek55 (Fig. 4). The above results indicate that DEK55 is necessary for editing at rps13-56, atp1-1490, nad6-159, and 12 nad9 editing sites.
DEK55 is essential for the trans-splicing of nad1 introns 1 and 4 and for the cis-splicing of nad4 intron 1
The transcript levels of 35 maize mitochondrial genes were examined, and nad1 and nad4 were significantly downregulated in the dek55 mutant (Fig. 5a). The genomic DNA of nad1 contains four group II introns; intron 2 is a cis-splicing intron and the others are trans-splicing introns (Fig. 5c). The genomic DNA of nad4 has three cis-splicing introns (Fig. 5d) [13, 35]. The full maturation of nad1 and nad4 transcripts requires complete intron splicing. We further detected the intron splicing efficiency of nad1, nad4, and other genes in WT and dek55-1 by qRT-PCR. Compared with that in WT, the splicing efficiency of the first and fourth introns of nad1 and the first intron of nad4 in dek55-1 mutant were decreased (Fig. 5b). Furthermore, we amplified each intron and full transcripts of nad1 and nad4 by RT-PCR (Fig. 5c, d). The transcriptional abundance of nad1 exon 1–2, exon 4–5, and full-length DNA fragments were significantly decreased (Fig. 5c). The unspliced DNA fragments (1F + 2R, 3F + 4R, 4F + 5R) were not amplified by RT-PCR in WT and dek55, as nad1 introns 1, 3, and 4 are too long (Fig. 5c). The unspliced intron 2 fragments of nad1 in dek55 mutants were similar to those in WT (Fig. 5c). The abundance of nad4 spliced exon 1–2 and full-length DNA transcript fragments were significantly decreased, and the abundance of nad4 unspliced intron 1 transcript was significantly increased (Fig. 5d). This suggests that the significant decrease in the nad4 and nad1 transcript abundance in dek55 mutants was caused by the abnormal splicing of nad4 intron 1, nad1 intron 1, and intron 4, respectively (Fig. 5a − d). Therefore, DEK55 is necessary for the trans-splicing of two nad1 introns (1 and 4) and cis-splicing of the first nad4 intron in maize.
dek55-1 mutant exhibits reduced complex I activity and increased alternative respiratory pathway activity
The four genes nad1, nad4, nad6, and nad9 encode the subunits of complex I NAD1, NAD4, NAD6, and NAD9, respectively . The rps13 gene encodes ribosomal protein, and atp1 encodes the ATP1 subunit of ATP synthase F1 . Defects in post-transcriptional processing of these genes may impair the biosynthesis of mitochondrial complexes [17, 36–38]. We performed blue native polyacrylamide gel electrophoresis (BN-PAGE) and the in-gel NADH dehydrogenase activity assay to investigate the accumulation level and activity of mitochondrial complexes in WT and dek55-1 endosperm. BN-PAGE showed that the abundance of complex I and super-complex I + III2 in dek55-1 mutants significantly decreased (Fig. 6a). However, no significant differences were observed in the abundance of complex V between WT and dek55-1 (Fig. 6a). Furthermore, dek55-1 deficiency the activities of complex I and I + III2 (Fig. 6b). These results indicate that defects in mitochondrial transcript splicing and editing might affect the abundance and activity of mitochondrial complex I.
The mitochondrial respiratory chain in plants contains the cytochrome c and alternative oxidase (AOX) pathways . When the main cytochrome c pathway is blocked, AOX activity can be increased to compensate respiration pathways . In dek55-1, the functions of complex I were abolished (Fig. 6a, b). Thus, we performed qRT-PCR to detect the expression levels of Aox genes in WT and dek55-1. The expression of the Aox2 gene was increased approximately 512-fold in the dek55-1 mutant (Fig. 6c). Taken together, our results indicate that the respiration pathway is severely blocked in dek55-1 mitochondria.