A Mitochondrial Transcription Termination Factor, ZmSmk3, Is Required for nad1 Intron4 and nad4 Intron1 Splicing and Kernel Development in Maize

Abstract

The expression systems of the mitochondrial genes are derived from their bacterial ancestors, but have evolved many new features in their eukaryotic hosts. Mitochondrial RNA splicing is a complex process regulated by families of nucleus-encoded RNA-binding proteins, few of which have been characterized in maize (Zea mays L.). Here, we identified the Zea mays small kernel 3 (Zmsmk3) candidate gene, which encodes a mitochondrial transcription termination factor (mTERF) containing two mTERF motifs, which is conserved in monocotyledon; and the target introns were also quite conserved during evolution between monocotyledons and dicotyledons. The mutations of Zmsmk3 led to arrested embryo and endosperm development, resulting in small kernels. A transcriptome of 12 days after pollination endosperm analysis revealed that the starch biosynthetic pathway and the zein gene family were down-regulated in the Zmsmk3 mutant kernels. ZmSMK3 is localized in mitochondria. The reduced expression of ZmSmk3 in the mutant resulted in the splicing deficiency of mitochondrial nad4 intron1 and nad1 intron4, causing a reduction in complex I assembly and activity, impairing mitochondria structure and activating the alternative respiratory pathway. So, the results suggest that ZmSMK3 is required for the splicing of nad4 intron 1 and nad1 intron 4, complex I assembly and kernel development in maize.

Keywords: Zea mays; embryo; endosperm; intron splicing; mitochondrial.

Figure 1

The Zmsmk3 mutants are arrested in embryogenesis and endosperm development. (A) A mature segregating ear of maize. Arrows indicate the Zmsmk3 mutant kernels. (B) A size comparison between the wild-type (WT) and Zmsmk3 mutant kernels. (C) Dissection of mature WT (left) and Zmsmk3 (right) kernels. RAM, root apical meristem; SAM, shoot apical meristem; sc, scutellum, (D) Comparison of WT and Zmsmk3 kernel weights. Sections of developing kernels at 6 days after pollination (DAP; E-F), 12 DAP (G-H), and 21 DAP (I-J), and the basal endosperm transfer layer (BETL) at 21 DAP (K-L). (E, G, I, K) Wild-type (WT) kernels, (F, H, J, L) Zmsmk3 kernels. col, coleoptile; em, embryo; en, endosperm; LP1, first leaf primordium; SAM, shoot apical meristem; sc, scutellum. Arrows in K and L indicate the BETL. Values are the means with SE; n = 3 individuals. Scale bar = 1 cm in (A, B), 1 mm in (C, E, F, G, H, I, J) and 0.2 mm in (K–L).

Figure 2

Storage substances is reduced in the Zmsmk3 kernels. Starch accumulation in (A, B) 12 DAP kernels, (C, D) 25-DAP kernels, (A, C) wild-type kernels, and (B, D) Zmsmk3 kernels. Scale bar = 50 µm. (E) GO analysis of differentially expressed genes (DEGs) between the wild-type and Zmsmk3 kernels.

Figure 3

Identification of the candidate gene for Zmsmk3 . (A) Gene structure of the candidate gene for Zmsmk3. The location of the insertion in the candidate gene Zmsmk3 is indicated by the triangle. (B) Phylogenetic analysis and mTERF motifs alignment of candidate gene ZmSMK3 with its orthologs. (Left) Phylogenetic analysis of the candidate gene ZmSMK3. ZmSMK3 and identified homologous proteins in Sorghum bicolor, Setaria italica, Oryza sativa Japonica Group, Brachypodium distachyon, Populus trichocarpa, Prunus persica, Medicago truncatula, Vitis vinifera, Arabidopsis and Brassica napus were aligned by ClustalW. The phylogenetic tree was constructed using MEGA7 by Neighbor-joining method. The numbers at the nodes represent the percentage of 1000 bootstraps. The numbers at the lines represent the branch lengths. (Right) The alignment of the ZmSMK3 protein with its orthologs. mTERF motifs are indicated with different colors.

Figure 4

The expression of ZmSmk3 (A) Expression profiles of ZmSmk3 in various tissues. (B) Comparison of ZmSmk3 expression in the wild-type (WT) and Zmsmk3 mutant kernels. RNA was extracted from kernels at 12 days after pollination (DAP) following the removal of the pericarp. For each RNA sample in (A) and (B), three technical replicates were performed. Values are means with SE; n = 3 individuals. (C–H) mRNA in situ hybridization analysis of ZmSmk3 in 12-DAP WT kernels using an antisense probe (C–E) and a sense probe (F–H). (I) Subcellular localization of ZmSMK3. The ZmSMK3:GFP fusion protein was transiently expressed in maize leaf protoplasts. Fluorescent signals from ZmSMK3:GFP are displayed in green and mitochondria stained with MitoTracker are red. Scale bar = 100 µm in (C–H) and 10 µm in (I).

Figure 5

The expression of nad1 and nad4 is dramatically decreased due to the Splicing deficiency of mitochondrial nad4 intron 1 and nad1 intron 4. (A) RT-PCR analysis of 35 mitochondria-encoded transcripts in WT (left) and Zmsmk3 (right) seeds. (B) Schematic structure of the maize mitochondrial nad1 (up) and the splicing efficiency of the four introns by RT-PCR (down). (C) Schematic structure of the maize mitochondrial nad4 (up) and the splicing efficiency of the three introns by RT-PCR (down). (D) Quantitative RT-PCR analysis of 22 intron-containing mitochondrial transcripts. The RNA was isolated from the same ear segregating for WT and Zmsmk3 seeds at 12 DAP. ZmActin (GRMZM2G126010) was served as internal control.

Figure 6

The deficiency of complex I assembly and disordered mitochondrial structure in Zmsmk3. (A) Comparison of the accumulation of complex I in the wild type (WT) and Zmsmk3 mutant. Mitochondrial complexes of 12-DAP maize kernels without pericarps were separated using 3–12.5% blue native polyacrylamide gel electrophoresis (BN-PAGE). Complexes I, III, and IV are indicated. (B) Comparison of the NADH dehydrogenase activity of complex I in the WT and Zmsmk3. Darker staining indicates higher levels of activity. Dihydrolipoamide dehydrogenase (DLDH) was used as a loading control. (C) Comparison of the abundance of respiratory enzymes in the WT and Zmsmk3. Crude mitochondrial extracts from 12-DAP kernels with the pericarp removed were used for a western blot analysis using antibodies against Nad6 (subunit of complex I), Cytc (subunit of complex III), COX2 (subunit of complex IV), AtpB (subunit of complex V) and α-tubulin as a sample loading control. (D) RT-PCR analysis of AOX1, AOX2, and AOX3 expression in the WT and Zmsmk3 mutant. Total RNA was extracted from 12-DAP kernels with the pericarp removed. The expression level was normalized against ZmActin. (E) Western blot analysis using antibodies against AOX (alternative oxidase) and α-tubulin as a sample loading control. (F-G) Morphological analysis of mitochondria in wild-type (F) and Zmsmk3 (G) endosperms at 10-DAP. Mitochondria are indicated by the arrows. Scale bar = 0.5 µm.