Regulator of Chromosome Condensation 1-Domain Protein DEK47 Functions on the Intron Splicing of Mitochondrial Nad2 and Seed Development in Maize

Abstract

In flowering plants, mitochondrial genes contain approximately 20-26 introns. Splicing of these introns is essential for mitochondrial gene expression and function. Recent studies have revealed that both nucleus- and mitochondrion-encoded factors are required for intron splicing, but the mechanism of splicing remains largely unknown. Elucidation of the mechanism necessitates a complete understanding of the splicing factors. Here, we report the identification of a regulator of chromosome condensation 1 (RCC1)-domain protein DEK47 that is required for mitochondrial intron splicing and seed development in maize. Loss of function in Dek47 severely arrests embryo and endosperm development, resulting in a defective kernel (dek) phenotype. DEK47 harbors seven RCC1 domains and is targeted to mitochondria. Null mutation of DEK47 causes a deficiency in the splicing of all four nad2 introns, abolishing the production of mature nad2 transcript and resulting in the disassembly and severely reduced activity of mitochondrial complex I. In response, the expression of the alternative oxidase AOX2 is sharply increased in dek47. These results indicate that Dek47 is required for the splicing of all the nad2 introns in mitochondria, and essential for complex I assembly, and kernel development in maize.

Keywords: complex I; intron splicing; maize; mitochondria; nad2; regulator of chromosome condensation 1-domain protein; seed development.

Figure 1

Mutant dek47-1 arrests early in maize seed development. (A) The recessive dek47-1 mutant segregated in a self-pollinated ear at 17 days after pollination (DAP). Arrows point to dek47-1 mutant kernels. (B) Maturity ear of self-pollinated dek47-1 heterozygotes. Arrows point to dek47-1 mutant kernels. (C,D) Embryo side of mature kernels of wild-type (C) and dek47-1 (D) kernels at 30 DAP. (E,F) Dissection of mature wild-type (E) and dek47-1 (F) kernels at 30 DAP. (G-M) Paraffin sections of the wild-type (WT) and dek47-1 kernels at 8 and 14 DAP. Wild-type kernels at 8 DAP (G,K) and 14 DAP (I); the dek47-1 kernels at 8 DAP (H,L) and 14 DAP (J,M). Arrows indicate the embryo. en, endosperm; em, embryo; ram, root apical meristem; sam, shoot apical meristem; and sc, scutellum. Scale bars = 1 mm.

Figure 2

Dek47 encodes a mitochondrion-targeted RCC1-domain protein. (A) Genomic structure of Dek47 and the motifs of the encoded protein. The top panel represents the gene structure of Dek47 (blocks, exons; lines, introns) and the below panel represents the protein structures of DEK47. dek47-1: locations of Mu insertion are marked with triangle. dek47-2: the TGG (W286) codon is mutated to TGA (stop). (B) The subcellular localization of DEK47 in tobacco leaves. MitoTracker Red was used as a mitochondrial marker. Scale bars = 20 μm.

Figure 3

Blue native-PAGE analyses of mitochondrial complexes. (A) Assembly of mitochondrial complex I. Mitochondrial complexes of the embryo and endosperm of maize kernels were subjected to a 3–12.5% BN-PAGE. The BN gels were stained with Coomassie brilliant blue (CBB). The position of respiratory complexes is indicated. C-I, complex I; C-III, complex III; C-I+III₂, supercomplex I +III₂; and C-V, complex V. (B) Detection of NADH dehydrogenase activity of complex I. Dihydrolipoamide dehydrogenase (DLDH) was used as a loading control. (C) Analysis of mitochondrial proteins abundance. Western blot analysis with antibodies against Nad9, Cyt_C, Cyt_C1, Cox2, ATPase, and AOX. CBB-stained gel was used as reference of loading quantity.

Figure 4

The Dek47 mutant is deficient for mitochondrial nad2 mature transcript. (A) RT-PCR analysis of transcript levels of 35 mitochondrion-encoded genes in WT and dek47-1. RNA was isolated from the same ear segregating for WT and dek47-1. Normalization was performed against ZmActin. (B) Quantitative RT-PCR analysis of 35 mitochondrion-encoded transcripts in WT and dek47-1. RNA samples were normalized against ZmActin. Values represent the mean and standard deviation of three biological replicates, ±SD. The comparison groups of the Student’s t-test are wild type and dek47 mutant. Asterisks indicate significant differences between means calculated with Student’s t-test. ^*p < 0.05; ^**p < 0.01.

Figure 5

The intron splicing of nad2 transcript is impaired in dek47. (A) Schematic representation of nad2 gene. Intron 1, intron 3, and intron 4 of nad2 are cis-splicing introns. The expected amplification products and primers using are indicated. F and R present the primers used to detect splicing events in RT-PCR analysis. E1-E5, exon1-exon5. (B) RT-PCR analysis of the splicing of nad2 introns in WT and dek47. Amplifications are marked as in (A). Normalization was performed against ZmActin. S and U indicate the spliced and unspliced PCR products, respectively. (C) Quantitative RT-PCR analysis of 22 introns splicing efficiency of mitochondrial genes in WT and dek47. RNA samples were normalized against ZmActin. Values represent the mean and standard deviation of three biological replicates, ±SD. The comparison groups of the Student’s t-test are wild type and dek47 mutant. Asterisks indicate significant differences between means calculated with Student’s t-test. ^*p < 0.05; ^**p < 0.01.

Figure 6

DEK47 can rescue the Arabidopsis rug3 mutant phenotypes. (A) Phenotype comparison of Col-0, rug3, Dek47/rug3-1 (Com1), and Dek47/rug3-2 (Com2). (B) Quantitative RT-PCR analysis of intron splicing efficiency of nad2 transcript in WT, rug3, Com1, and Com2. RNA samples were normalized against Actin gene ACT2 (AT3G18780). Values represent the mean and standard deviation of three biological replicates, ±SD. The comparison groups of the Student’s t-test are between rug3 mutant and wild type, and between complemented line and wild type, respectively. Asterisks indicate significant differences between means calculated with Student’s t-test. ^*p < 0.05; ^**p < 0.01.

Figure 7

Interaction assay of DEK47 and the related splicing factors. (A) Schematic representation of nad2 transcript and the related splicing factors of nad2 intron 3. E1-E5, exon1-exon5. (B) Yeast two-hybrid assay of DEK47 and the related splicing factors. These constructs were co-transfected into Y2H Gold strain and spotted onto DDO (SD/−Leu/−Trp) medium and QDO (SD/−Ade/-His/−Leu/−Trp) medium for 4 days at 30°C.