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 and was 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 mendelian ratio (Fig. 1a, Additional file 1: Table S1). These results suggested that dek55-1 as a recessive phenotype is caused by a monogenic mutation, which was confirmed in other populations generated from dek55-1/+ heterozygotes crossed with the inbred lines C733 or S162 (Additional file 1: Table S1).
The dek55-1 kernels were of smaller size with whitish pericarp and can be distinguished from the wild-type (WT) kernel at 15 days after pollination (DAP) (Fig. 1a). At the maturity, dek55-1 kernels became even much smaller and shriveled (Fig. 1b, c). To further dissect the mutant phenotype, both WT and dek55-1 kernels were longitudinally sliced at 15 DAP. As compared to the WT, the mutant kernel has a tiny sized soft endosperm. (Fig. 1d, e). Furthermore, dek55-1 exhibited a smaller mature embryo and a decreased proportion of hard endosperm compared to WT (Fig. 1f, g). In addition, the kernel weight of dek55-1 was reduced by approximately 70% compared to that of WT kernels (Fig. 1h). No seed germinated (0/100) was recorded in the field conditions implying that embryo arrest is lethal in the dek55-1 mutants.
To further investigate the developmental structure of dek55-1 kernels, we examined the kernel tissue structure of WT and dek55-1 mutants at 12 and 18 DAP (Fig. 1i−l). At 12 DAP, the dek55-1 embryo had only a small scutellum arrested at the coleoptile stage and a large interspace between the endosperm the seed coat. In contrast, the WT embryo contained visible coleoptile, shoot apical meristem, scutellum, two leaf primordia, and the kernel was filled with endosperm cells (Fig. 1i, j). At 18 DAP, the WT embryo had developed into complete structures containing four-leaf primordia, shoot apical meristem, and a clearly seen root apical meristem (Fig. 1k), while dek55-1 embryos only generated one leaf primordium (Fig. 1l). Fewer 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 the dek55-1 endosperm (Fig. 1l). These results indicate that developmental defects in the 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 a self-pollinated filial 1 (F1) hybrid ear. Four genomic DNA pools (10 mutant kernels per pool), and both of the parents were used to identify the chromosome location of the DEK55 gene. The six simple sequence repeat (SSR) markers on chromosome 5 were highly correlated with defective kernel phenotypes, implying that the candidate gene may be on chromosome 5. Further analysis showed that the DEK55 gene is located between umc1705 and umc2302 on chromosome 5 (Fig. 2a). One thousand eight hundred and sixty eight mutant kernels from the F2 population were genotyped to narrow down the gene location, using six polymorphic molecular markers. Finally, the DEK55 gene was located on an approximately 1.29 Mb region between the molecular labels 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, the genomic DNA of 25 candidate genes was amplified and sequenced. Sequence alignment identified a single nucleotide polymorphism in the E-subgroup PPR protein gene (Zm00001d014471). In the dek55-1 mutant, nucleotide C was replaced by the nucleotide T at +449 bp, resulting in the substitution of amino acid Ser with Phe. However, no change in the mRNA expression level was observed. (Fig. 2a-d). To validate our results, we obtained a new mutant, dek55-2, from the maize ethyl methane sulfonate-induced mutant database [34]. The dek55-2 mutant showed 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 the dek55-2 heterozygote and WT were normal (Fig. 2e). These results indicate that the mutation in PPR gene Zm00001d014471 was responsible for the defective kernel phenotype, so the annotated gene was designated DEK55.
DEK55 is a mitochondrial E-subgroup PPR protein
Sequence alignment demonstrated that the DEK55 gene is 1893 bp long ORF with no introns. DEK55 encodes a 630 amino acid residue protein containing 13 PPR motifs and an E domain at 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 missing the last eight PPR motifs and the 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 (mitochondria-specific dye) (Fig. 3a), suggesting that in maize, DEK55 is a mitochondrial PPR protein (Fig. 3a). In addition, DEK55 expression analysis in various maize tissues demonstrated that DEK55 is relatively highly expressed in root, anther, and ear, with relatively low expression in stem, leaf, silk, tassel, and kernel (Fig. 3b).
DEK55 is involved in the C-to-U editing of 14 transcripts at multiple sites
Usually, PPR proteins take part in modifying organelle transcripts [10]. It has been reported that E-subgroup PPRs participate in the C-to-U editing of mitochondrial pre-mRNA [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 dek55 (dek55-1 and dek55-2) and WT (WT-1 and WT-2) were detected by the strand- and transcript-specific RNA-seq (STS-PCRseq) strategy [35]. The sequence reads were mapped to the 35 mitochondrial gene transcripts and examined 482 C-to-U RNA editing sites in WT and dek55 (Additional file 2: Table S1). Compared with the editing ratio of these RNA editing sites between WT and dek55 (Additional file 2: Table S2), the results revealed that the C-to-U editing ratio of 31 editing sites in the 14 transcripts (atp1, atp8, ccmFc, ccmFn, cob, mat-r, nad3, nad4, nad6, nad7, rps12-ct, rps12, rps13, and rps3) were significantly altered in dek55 (Fig. 4, Additional file 2: Tables S2-S4), the editing ratio of 24 sites was decreased (Fig. 4a) and that of seven sites was increased in dek55 mutants compared with WT (Fig. 4b, Additional file 2: Tables S2, S4). The editing efficiency at the atp1-1490, ccmFn-287, mat-r-1877, and rps13-56 sites was dramatically decreased in dek55-1 and dek55-2 kernels, and the editing ratio in the dek55 mutant was more than 50% lower than that in the WT (Fig. 4a, Additional file 2: Table S3). Directed sequencing of RT-PCR products to evaluate the editing efficiency of atp1-1490, ccmFn-287, mat-r-1877 and rps13-56 sites also indicated that this was significantly reduced in dek55 at these RNA editing sites (Fig. 4c). The deficient C-to-U RNA editing leads to altered amino acid residues in dek55 (Fig. 4c). Meanwhile, at the atp8-123 site, only the editing efficiency of dek55-2 (5%) was more than 50% lower than the WT, and at nad4-77 sites, only the editing efficiency of dek55-1 (24.2%) was more than 50% lower than the WT (Fig. 4a). The above results indicated that DEK55 is required for RNA editing at multiple editing sites, especially the atp1-1490, ccmFn-287, mat-r-1877, and rps13-56 sites.
DEK55 is essential for the trans-splicing of nad1 introns 1 and 4 and the cis-splicing of nad4 intron 1
The transcript levels of 35 maize mitochondrial genes were examined, and the results depicted that nad1 and nad4 were significantly downregulated in the dek55 mutant (Fig. 5a). The genomic DNA of nad1 contains four group II introns, and except the 2nd intron, all are trans-splicing introns. (Fig. 5c). The genomic DNA of nad4 has three cis-splicing introns (Fig. 5d) [13, 36]. The full maturation of nad1 and nad4 transcripts requires complete intron splicing. We further analyze the intron splicing efficiency of nad1, nad4, and other genes in dek55-1 and WT by qRT-PCR. Compared with that in the WT, the splicing efficiency of the first and fourth introns of nad1 and the first intron of nad4 in the 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 the full-length DNA fragments were significantly decreased (Fig. 5c). RT-PCR could not amplify the intronic DNA fragments (1F+2R, 3F+4R, 4F+5R) in dek55 and WT because the 1st, 3rd, and the 4th intron of nad1 were too long. (Fig. 5c). The unspliced 2nd intronic fragments of nad1 in the dek55 mutants were similar to those in WT (Fig. 5c). The abundance of nad4 spliced exon 1-2 and full-length DNA fragments were significantly decreased, and the abundance of the nad4 unspliced intron 1 transcript was significantly increased (Fig. 5d). Our findings suggest 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 the two nad1 introns (1st and 4th) 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, i.e., nad1, nad4, nad3, and nad6 encode the subunits of complex I NAD1, NAD4, NAD3, and NAD6, respectively [36]. The rps13 gene encodes a ribosomal protein, atp1 and atp8 encode the ATPase subunit 1 and subunit 8 subunit of ATP synthase F1, respectively [36]. Defects in the post-transcriptional processing of these genes may impair the biosynthesis of mitochondrial complexes [17, 37-39]. 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 was significantly decreased (Fig. 6a). However, no significant differences were observed for the complex V between WT and dek55-1 (Fig. 6a). Furthermore, the activity of the complex I and I+III2 was reduced in the dek55-1 mutant (Fig. 6b). These results indicate that defects in mitochondrial transcript splicing and/or 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 [40]. When the main cytochrome c pathway is blocked, AOX activity can be increased to compensate respiration pathways [41]. 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, and the results showed a 512-fold increase in the expression level of the Aox2 gene in the dek55-1 mutant as compared to the WT. (Fig. 6c). Collectively, our results indicate that the respiration pathway is severely blocked in dek55-1 mitochondria.
DEK55 can interact with ZmMORF1 and ZmMORF8 in yeast
Previous studies explained that the MORFs directly interact with PPR proteins and play a role in RNA editing at numerous editing sites [42, 43]. In this study, DEK55 is responsible for 31 RNA editing in maize, so we speculated that DEK55 might interact with MORFs to form an editing complex involved in RNA editing in maize. Thus, we used MORFs in Arabidopsis as baits to search for putative MORFs in maize. Seven putative MORFs were identified in maize (Fig.7a). A yeast two-hybrid assay was performed to screen for MORFs interacting with DEK55, and the results indicated that the DEK55 can interact with ZmMORF1 and ZmMORF8 in yeast (Fig. 7b).