Maize pentatricopeptide repeat protein DEK41 affects cis-splicing of mitochondrial nad4 intron 3 and is required for normal seed development.

The splicing of organelle-encoded mRNA in plants requires proteins encoded in the nucleus. The mechanism of splicing and the factors involved are not well understood. Pentatricopeptide repeat (PPR) proteins are known to participate in such RNA-protein interactions. Maize defective kernel 41 (dek41) is a seedling-lethal mutant that causes developmental defects. In this study, the Dek41 gene was cloned by Mutator tag isolation and allelic confirmation, and was found to encode a P-type PPR protein that targets mitochondria. Analysis of the mitochondrial RNA transcript profile revealed that dek41 mutations cause reduced splicing efficiency of mitochondrial nad4 intron 3. Immature dek41 kernels exhibited severe reductions in complex I assembly and NADH dehydrogenase activity. Up-regulated expression of alternative oxidase genes and deformed inner cristae of mitochondria in dek41, as revealed by TEM, indicated that proper splicing of nad4 is essential for correct mitochondrial functioning and morphology. Consistent with this finding, differentially expressed genes in the dek41 endosperm included those related to mitochondrial function and activity. Our results indicate that DEK41 is a PPR protein that affects cis-splicing of mitochondrial nad4 intron 3 and is required for correct mitochondrial functioning and maize kernel development.

Introduction

Maize (Zea mays) is widely used for genetic research due to its easily observable phenotypes, and the broad range of defective kernel (dek) mutants provide good tools for investigating kernel development (Neuffer and Sheridan, 1980). Many maize dek mutants have been identified, for example dek1 exhibits severe growth and development defects (Lid ), reas1 causes a mild developmental delay (Qi ), and mutants of pentatricopeptide repeat (PPR) proteins exhibit reduced kernel size and arrested development of the embryo and endosperm (Liu ; Sun ; Cai ; Dai ). Thus, dek mutants offer opportunities to investigate many basic biological processes during kernel development.

In higher plants, the nucleus-encoded PPR proteins primarily localize to chloroplasts and mitochondria, and play critical roles in RNA editing, cis- and trans-splicing, cleavage, and maturation (Barkan and Small, 2014). PPR proteins are defined by tandem repeats of a degenerate 35-amino-acid motif and are classified into two major subgroups: P-type members possess only tandem repeats of the 35-amino-acid PPR motif, whilst PLS-type members include sequential repeats of P, short (S), and long (L) PPR motifs that often carry an E or E–DYW domain extension at the C terminus (Lurin ; Cheng ). In plants, the PPR family comprises more than 450 members, and a number of mutants associated with severe growth and development defects have been characterized (Fujii and Small, 2011; Barkan and Small, 2014; Hammani and Giegé, 2014; Møller, 2016; Gorchs Rovira and Smith, 2019). However, the regulatory functions of many PPR proteins have not yet been characterized.

Many P-type PPR proteins function in RNA processes including stabilization, cleavage, translational activation, and splicing. RNA splicing in mitochondria requires the coordination of PPR proteins because of the loss of the ability to self-splice (Bonen, 2008; Brown ). Group I and group II introns have distinct splicing patterns. More than 20 group II introns have been identified in the mitochondrial genomes of flowering plants, including 15 cis-spliced introns in nad1, nad2, nad4, nad5, nad7, rps3, cox2, and ccmF, and seven trans-spliced introns in nad1, nad2, and nad5 (Burger ; Berrisford and Sazanov, 2009). In the mitochondrial genome, most introns are in genes that encode subunits of complex I (NADH dehydrogenase), which is located in the inner membrane and transfers electrons from NADH to the primary acceptor FMN followed by the ubiquinone-binding site (Sazanov and Hinchliffe, 2006; Berrisford and Sazanov, 2009). Five P-type PPR proteins are required for group II intron splicing of mitochondrial transcripts in Arabidopsis, including transcripts of nad1 (de Longevialle ), nad2 (Liu ), nad5 (Colas des Francs-Small ), and nad7 (Hsieh ). In maize, EMP10, EMP16, and DEK37 are involved in cis-splicing of nad2 intron 4 and intron 1 (Xiu ; Cai ; Dai ), DEK2 is required for the trans-splicing of nad1 intron 1 (Qi ), and DEK35 affects cis-splicing of nad4 intron 1 (Chen ). Studies of maize kernel mutants provide an opportunity to reveal the important roles of other P-type PPR proteins in cis- and trans-splicing of mitochondrial RNA.

Among the large family of PPR proteins in flowering plants, only a small subset has been studied at the molecular level, particularly for mitochondrial PPRs, in which mutations are frequently embryo-lethal. Determining the molecular functions of mitochondrial PPR proteins may shed light on RNA-processing mechanisms as well as on the assembly of the oxidative phosphorylation machinery. In the present study, a maize seed mutant dek41 corresponding to a defective and seedling-lethal phenotype was characterized. Dek41 encodes a P-type PPR protein that targets to the mitochondria. The dek41 mutant shows reduced splicing efficiency of mitochondrial nad4 intron 3 in developing seeds. Lack of this splicing process affects the accumulation of complex I and the activity of NADH dehydrogenase, leading to the arrest of mitochondrial functioning and kernel development.

Materials and methods

Plant materials

The Zea mays MuDR stock (330I) and UFMu01110 mutant line were obtained from Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu). The MuDR stock was crossed into the B73 inbred line as the male parent to generate F1 population seeds, and then self-crossed to generate F2 population ears. Phenotypical screening for seed mutants was carried out on these F2 ears, which resulted in a new dek mutant being identified, and we designated it as dek41-ref. The dek41-ref mutant seeds on the F2 ear were white and during their development they were smaller than the wild-type (WT) seeds. The F2 ears of dek41-ref displayed a 1:3 segregation of mutant and WT seeds.

Mature seeds were used for genomic DNA extraction. Immature seeds were sampled for RNA and protein extraction, mitochondria isolation, preparation of paraffin and resin sections, TEM, and RNA-seq. Other plant tissues were harvested from one inbred B73 plant for RNA and protein extraction. All plants were cultivated in an experimental field in Shanghai University, Shanghai, China.

Measurement of protein and starch levels

For protein measurements, endosperms of mature WT and dek41-ref seeds were separated from the embryo and pericarp by dissection after soaking the seeds in water. The samples were dried to constant weight, ground with a mortar and pestle in liquid nitrogen. Proteins were extracted from 50 mg of three pooled endosperm powder samples from same ear, and then the protein content was measured according to a previously described protocol (Qi ). All the measurements were conducted on three biological replicates. For the starch measurements, mature seeds of the WT and dek41 mutants were ground in liquid nitrogen and the resulting powders were dried to a constant weight.100 mg of three pooled powders from same ear were used for the total starch measurements using an amyloglucosidase/α-amylase starch assay kit (Megazyme) according to a previously described protocol (Qi ). All the measurements were conducted on three biological replicates.

Light microscopy and TEM

For light microscopy, immature WT and dek41-ref seeds at 12 d after pollination (DAP), 15 DAP, and 18 DAP were collected from the same ear and were cut along the longitudinal axis for preparation of paraffin sections. The sections were fixed in a formalin–acetic acid–alcohol mixture and were dehydrated in an ethanol gradient series of 50, 60, 70, 85, 95, and 100%. After replacement of acetone and infiltration with paraffin, the sections were embedded and cut using a RM2265 microtome (Leica). The sections were stained with Basic Fuchsin Dye and were observed using a Leica DFC500 digital camera system.

For TEM analysis, immature WT and dek41-ref seeds at 15 DAP were collected from the same ear and were cut along the horizontal axis into small pieces and fixed in paraformaldehyde. The samples were further processed and stained by osmic acid, and were observed by TEM (Tecnai G2 Spirit BioTWIN, FEI Company).

Mutator tag isolation

The Mutator tag isolation was performed according to Williams-Carrier , with some modifications. Genomic DNA was prepared for mechanical shearing by the Majorbio Bio-Pharm Technology Company, Ltd (Shanghai, China). Fragments (200–500 bp) were ligated to modified Illumina adapters to mark samples from different individuals. Mu-containing DNA fragments were enriched by hybridization to a biotinylated oligonucleotide corresponding to the end of the Mu terminal inverted repeat. Two successive hybrid enrichment steps were performed to ensure that most of the sequenced DNA fragments harbored Mu sequences. Using primers that bind to the ends of the adapters, 15 and 18 cycles of PCR were used to bulk-up the recovered DNA after the first and second selection rounds, respectively. The PCR products were cloned into the vector pMD18-T (Takara). The effectiveness of the enrichment was tested by calculating the percentage of clones containing the Mu fragment. Enrichment rates above 30% were selected for Mu Illumina sequencing, which was carried out at BerryGenomics (Beijing, China).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from the samples using TRIzol reagent (Tiangen) according to previous Feng , and DNA was removed by treatment with RNase-Free DNase I (Takara). Using ReverTra Ace reverse transcriptase (Toyobo), RNA was reverse-transcribed to complementary DNA using the random primers provided in the kit (ReverTra Ace qPCR RT Master Mix, Toyobo). Amplification of mitochondrial transcripts was performed using primers as described previously (Chen ). Quantitative RT-PCR was performed using three independent sets of RNA with Ubiquitin as the reference gene. Primers for the mitochondrial introns were designed as described previously with some modifications (Qi ) (Supplementary Table S2 at JXB online). Quantitative RT-PCR was performed with SYBR Green Real-Time PCR Master Mix (Toyobo) using a Mastercycler ep realplex 2 (Eppendorf) according to the manufacturer’s protocol.

Phylogenetic analysis of DEK41 and its homologs

Sequences were compared with NCBI GenBank entries (http://www.ncbi.nlm.nih.gov/) using the protein–protein BLAST. To align the sequences of DEK41 and its homologs, the software Clustal X 1.81 was used (Thompson ). A phylogenetic tree was constructed using the neighbour-joining method of the MEGA 4.0 program (Tamura ) with the bootstrap test based on 2000 replicates.

Subcellular localization of DEK41

The full-length DEK41 coding region was cloned into pENTR/D-TOPO (Invitrogen), and transferred into the binary vector pB7WGY2 to produce the DEK41::EYFP fusion construct through LR site-specific recombination. The DEK41::EYFP fusion was placed under a Cauliflower mosaic virus 35S promoter. The construct was introduced into Agrobacterium tumefaciens strain Gv3101 and infiltrated into Nicotiana tabacum leaf epidermal cells, as described by van Herpen . The fluorescence signals were detected by laser-scanning confocal microscopy using a Zeiss LSM710 30 h after infiltration. The mCherry marker pBIN20-MT-RK (Nelson ) was a commercial vector purchased from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org). This vector was transferred into Agrobacterium, and the fluorescent signal observed after infecting tobacco mesophyll cells was used as a marker signal for mitochondria.

Preparation of mitochondria

Mitochondria were extracted from immature seeds of inbred B73 at 18 DAP as described previously (Chen ), with some modifications. Approximately 15 g of immature seeds was harvested from an individual plant at 18 DAP and ground into powder in liquid nitrogen with a mortar and pestle. Then, 20 ml of precooled (4 °C) extraction buffer (100 mM tricine, 300 mM sucrose, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA-K, 0.1% BSA, 5 mM DTT, pH 7.4) and 60 μl of plant protease inhibitor cocktail (Sigma-Aldrich) were added to the ground tissue. All the subsequent steps were performed at 4 °C. After filtration through a Miracloth membrane (Calbiochem, San Diego, CA), the samples were centrifuged twice at 2600 g for 15 min, and the supernatant was then centrifuged at 12 000 g for 25 min to pellet the crude mitochondria. The pellet was resuspended in wash buffer (100 mM tricine, 300 mM sucrose, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA-K, 0.1% BSA, pH 7.4) and loaded onto sucrose density gradients of 1.5, 2.5, 2.5, 2, and 2 ml respectively containing 1.8, 1.45, 1.2, 0.9, and 0.6 M sucrose diluted in wash buffer, as previously described (Clayton and Shadel, 2014). After 90 min of centrifugation at 62 000 g at 4 °C, the mitochondria were collected from the 1.2 M/1.45 M interface and washed four times in wash buffer. The purified mitochondria were collected after 20 min of centrifugation at 16 800 g at 4 °C.

BN-PAGE and NADH dehydrogenase activity

The isolated mitochondria were resuspended in 50 μl B25G20 solution (25 mM Bis-Tris, 20% glycerin, pH 7.0), with 20% n-Dodecyl β-D-maltoside (DDM) added to a final concentration of 1%, and were gently mixed on ice for 1.5 h. After 15 min of centrifugation at 15 800 g at 4 °C, the supernatant was collected and added to the loading buffer for blue native polyacrylamide electrophoresis (BN-PAGE). The concentration of the separation gel was from 5–13.5%. The electrophoresis was initially run at 50 V, and increased by 25 V every 20 min to a final level of 150 V until the loading dye migrated to the edge of the gel. The gel was stained with Coomassie brilliant blue. Measurement of NADH dehydrogenase activity was performed as described previously (Meyer ). Briefly, the gel was washed three times for 5 min with distilled water and incubated in the reaction solution (0.14 mM NADH, 1.22 mM NBT, 0.1M Tris-HCl, pH 7.4). When the dark blue stain was strong enough, the reaction was stopped by transferring the gel to 40% (v/v) methanol /10% (v/v) acetic acid.

Immunoblot analysis

Total proteins were extracted from immature seeds of the WT and dek41 mutants at 18 DAP, and were then separated by SDS-PAGE. Separated protein samples were transferred to a nitrocellulose membrane (0.45 μm; Millipore). The membrane with the protein sample attached was incubated with primary and secondary antibodies. The signal was visualized using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) according to the manufacturer’s instructions. To produce polyclonal antibodies against NAD4, the 55–71 amino acid sequence (PRIQFDPSTANSQFVESC) of NAD4 was synthesized. This peptide was synthesized and dissolved in PBS solution (1.37M NaCl, 0.1M Na2HPO4, 0.018M KH2PO4, KCl 0.027M) containing 8 M urea, and was then coupled to KLH (keyhole limpet hemocyanin) protein for immunization in rabbits by the Shanghai Ying Ji Biotechnology Co., Ltd, according to standard protocols. The polyclonal antibodies against NAD4 were used at a dilution of 1:1000. The antibodies raised for NAD4 recognized a protein of an apparent molecular weight of 70 kDa (see Supplementary Fig. S1). Antibodies against cytochrome c (Agrisera) and α-tubulin (Sigma-Aldrich) were used at a dilution of 1:5000.

RNA-seq analysis

Total RNA was extracted from developing WT and dek41-ref seeds at 12 DAP. Library construction was performed according to Illumina standard protocols. Reads were aligned to the maize B73 genome using Bowtie 2 v2.1.0 (Langmead and Salzberg, 2012). Data were normalized as reads per kilobase of exon model per million mapped reads (RPKM), as the sensitivity of RNA-seq depends on both molar concentration and transcript length (Mortazavi ). Significant differentially expressed genes (DEGs) were identified as those with a fold-change >2.0 and P<0.001.

Data availability

The RNA-seq data are available from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the series entry GSE117091. Sequence data from this paper can be found in the GenBank/EMBL data libraries under the following accession numbers: DEK41, ONM56221, GRMZM2G127015; NAD4, AAR91196.

Results

dek41 mutants display defective seed development

dek41 is a defective kernel (dek) mutant that was isolated from F2 populations containing active MuDR (see Methods). F2 ears that displayed a 3:1 segregation (589:207, from three ears, P<0.05) of WT to dek phenotypes were termed dek41-ref. The dek41-ref mutant had white seeds that were smaller than WT during seed development (12 DAP) (Fig. 1A). Mature homozygous kernels of the dek41 mutants were small and flat, and had empty pericarps at the top of kernels (Fig. 1B, C). The 100-kernel weight of mutant seeds was only 13% that of the WT (Fig. 1G) and the total percentage starch content by weight was reduced by 20% (Fig. 1H). The zein protein and total protein contents of mature endosperm were also lower in mutant kernels than in the WT (Supplementary Fig. S2).

Fig. 1.

Phenotypic features of the maize dek41 mutant. (A) Ear of an F2 plant of dek41/wild-type (WT) at 12 d after pollination (DAP). The arrows indicate examples of mutant seeds. Scale bar is 1 cm. (B) A mature F2 ear of dek41/WT. The arrows indicate examples of mutant seeds. Scale bar is 1 cm. (C) Examples of mature WT and dek41 seeds from the segregated F2 population. Scale bar is 1 cm. (D) Paraffin sections of WT and dek41 seeds at 12 DAP and 18 DAP. Scale bar is1.5 mm. (E) Microstructure of developing endosperms of WT and dek41 at 12 DAP and 15 DAP. Scale bars are 100 μm. (F) Paraffin sections of the basal endosperm transfer layer of WT and dek41 at 12 DAP and 15 DAP. Scale bars are 50 μm. (G) Comparison of 100-seed weight of randomly selected mature WT and dek41 seeds in the segregated F2 population. Data means (±SE) of n=3 individual plants. (H) Comparison of total starch content in mature WT and dek41 endosperm, expressed as percentage dry weight. Data are means (±SE) of n=3 individual plants. Significant differences in (G, H) were determined using Student’s t-test: ***P<0.001.

Detailed phenotypic examination of WT and dek41-ref seeds at different developmental stages showed that embryo and endosperm development in the dek41-ref mutant was delayed at least 6 d compared to the WT (Fig. 1D). The endosperm cells of seeds of the dek41 mutants at 12 DAP and 15 DAP were less cytoplasmically dense with fewer starch granules compared to the WT (Fig. 1E). Analysis of paraffin sections at 12 DAP and 15 DAP showed that the development of the basal endosperm transfer layer was arrested in dek41 kernels (Fig. 1F). The dek41 seeds showed very low germination rates, and no seedlings survived after germination. Together, these results indicated that the growth and development of dek41 seeds were severely affected and that the homozygous dek41 mutation was seedling-lethal.

Cloning of Dek41

We used F2 seeds of dek41-ref exhibiting a 3:1 segregation ratio (589:207, P<0.05) for genetic mapping. After characterizing 170 individual seeds, Dek41 was mapped between the markers InDel-6 and InDel-7, a region at ~9 Mb on chromosome 7 (maize B73 RefGen_v3) (Fig. 2A). As dek41-ref was isolated from an active Mutator line, Mu tags were isolated and sequenced (Williams-Carrier ). Only one unique Mu insertion was found in the mapped interval, which was located 632 bp downstream of the start codon of GRMZM2G127015 (Fig. 2B). Thus, GRMZM2G127015 was the candidate gene for dek41.

Fig. 2.

Cloning and identification of the maize Dek41 gene. (A) The Dek41 locus was mapped to an interval of ~9 Mb between molecular markers InDel-6 and InDel-7 on chromosome 7. The triangle indicates the site of Mu insertion in the dek41-ref mutant. (B) Schematic structure of GRMZM2G127015 and the allelic mutant. The triangles indicate the sites of Mu insertions. The matching positions of the primer pair Dek41-F1/Dek41-R1 that was used for detecting the full-length Dek41 transcript are indicated. (C) The dek41-ref and dek41-mu lines were used in allelic tests. The arrows indicate random examples of dek (dek41-ref/ dek41-mu) seeds. (D) Detection of the full-length transcript of Dek41 in wild-type (WT) and dek41-ref seeds at 12 d after pollination by reverse-transcription PCR using the primer pair Dek41-F1/R1, which was mapped to the 5´- and 3´-UTR sequences of Dek41 mRNA. 18S rRNA was used as the internal control.

In addition, a UniformMu insertion line (UFMu01110), carrying a Mu insertion at 1047 bp downstream of the start codon in GRMZM2G127015, was obtained from Maize Genetic Stock Center and named dek41-mu in this study (Fig. 2B). To determine whether the mutation in GRMZM2G127015 was indeed the cause of dek41, an allelic test was performed with the mutant allele dek41-mu. The test yielded the predicted 3:1 segregation ratio of WT to dek seed phenotypes (188:66 for dek41-ref × dek41-mu and 165:55 for dek41-mu × dek41-ref, P<0.05) (Fig. 2C). Twenty randomly selected seeds (WT and dek, 10 for each) from a test-cross ear (dek41-ref × dek41-mu) were analysed to detect the Mu insertion sites by co-segregation (Supplementary Fig. S3). Using the 1s and Mutir primers, three types of fragments (394 bps, 809 bps, and no fragment; Supplementary Fig. S3D) were amplified from WT seeds and identical bands (two bands per seed, 394 bps and 809 bps) were amplified from dek seeds. Using 1a and Mutir primers, three types of fragments (342 bps, 757 bps and no fragment; Supplementary Fig. S3E) were amplified from WT seeds and identical bands (two bands per seed, 757 bps and 342 bps) were amplified from the dek seeds. Both the phenotypes of ears in the allelic test and the detection of Mu insertion sites by co-segregation confirmed that dek41-mu was allelic to dek41-ref. Therefore, it was concluded that GRMZM2G127015 was the causal gene for dek41.

Dek41 encodes a P-type PPR protein

Sequence analysis of Dek41 revealed that it consisted of a single exon (without any introns) with a 1371-bp coding sequence encoding a putative PPR protein of 456 amino acids (Fig. 2B). Based on annotation in FLAGdb (http://tools.ips2.u-psud.fr/projects/FLAGdb++/HTML/index.shtml), DEK41 was classified as a P-type PPR protein with seven P repeat domains (Fig. 2B).

Phylogenetic analysis was conducted based on the full-length maize DEK41 protein sequence and homologous protein sequences from other organisms. The results showed that DEK41 homologs were highly conserved in angiosperms (Supplementary Fig. S4A). DEK41 was closely related to Sb02g029280 from Sorghum bicolor, to Os05g11700 from Oryza sativa japonica, and to homologs from Setaria italica (XP_004960609) and Brachypodium distachyon (XP_003566429). A detailed sequence alignment with homologs from these species indicated that DEK41 shared highly conserved P repeat domains with homologs in other monocotyledons (Supplementary Fig. S4B).

Dek41 is constitutively expressed in various tissues

To examine Dek41 expression, quantitative RT-PCR was performed using total RNA extracted from immature kernels of the WT and dek41 mutants at 12, , 15, and 18 DAP. Ubiquitin was used as an internal control. Surprisingly, increased transcript levels were detected at 12 DAP and 18 DAP in dek41 mutants relative to the WT (Supplementary Fig. S5). Full-length Dek41 transcripts from WT and dek41 seeds were also detected by reverse-transcription PCR using specific primers Dek41-F1/R1, matching the 5´- and 3´-UTR regional sequences of Dek41, respectively (Supplementary Table S2). Full-length (1650-bp) Dek41 transcripts were detected in WT seeds, but no target bands were amplified from either of the dek41 mutants (Fig. 2D). These results indicated that the expression of full-length Dek41 was completely arrested in the dek41 mutants.

The expression pattern of Dek41 in the WT was examined by quantitative RT-PCR. It was constitutively expressed in all tested tissues, with the highest expression level being observed in the ear (Fig. 3A). Dek41 was expressed throughout seed development, from 3–30 DAP, with the highest level recorded at 30 DAP.

Fig. 3.

Expression pattern of maize Dek41 and subcellular localization of DEK41. (A) RNA expression levels of Dek41 during seed development and in various tissues. DAP, days after pollination. The tissue samples were taken at the 12-leaf stage. The Ubiquitin gene was used as internal control. Data are means (±SE) from n=3 plants. Three technical replicates were performed for each RNA sample. (B) Subcellular localization in tobacco leaf epidermal cells. DEK41-EYFP represents the EYFP fusion protein at the C-terminus of the full-length DEK41 protein. The mitochondrial signals are marked by mCherry. DIC, differential interference contrast. Scale bar is 20 μm.

DEK41 is targeted to the mitochondria

PPR proteins are predominantly targeted to plastids or mitochondria (Colcombet ). To determine the subcellular localization, full-length DEK41 was fused to EYFP in the binary vector pB7WGY2. The fusion was transiently expressed in tobacco leaf epidermal cells by Agrobacterium-mediated transformation. Confocal laser-scanning microscopy revealed that the EYFP signals co-localized with the mitochondria-mCherry marker pBIN20-MT-RK (Nelson ), indicating that DEK41 is targeted to mitochondria (Fig. 3B).

The dek41 mutation affects cis-splicing of mitochondrial nad4 intron 3

As DEK41 is a mitochondria-targeting P-type PPR protein, the transcripts of all the mitochondrial protein-coding genes from immature seeds of the WT and dek41 mutants at 12 DAP were amplified by RT-PCR using specific primers (Supplementary Table S2). Among the 35 genes, only the level of mature nad4 transcript was reduced in dek41, whilst the levels of several other genes were increased (Fig. 4A, Supplementary Fig. S6). These results suggested that DEK41 was involved in regulating the expression of nad4 in developing maize kernels. To further examine the nad4 precursor transcript, three specific primers were designed to amplify fragments containing each of the three introns in the nad4 transcript in both dek41-ref and dek41-mu (Fig. 4B). The splicing efficiencies of nad4 intron 3 and intron 1 were reduced in dek41-ref and dek41-mu compared with the WT, whereas the splicing efficiency of nad4 intron 2 was not affected (Fig. 4C).

Fig. 4.

Decrease of nad4 mature transcripts and splicing deficiency of nad4 intron 3 in the maize dek41 mutants. (A) RT-PCR analysis of 35 mitochondria-encoded transcripts in seeds of the wild-type (WT; left) and dek41 (right). The RNA was isolated from the same ear segregating for WT and dek41 seeds at 12 d after pollination. 18S rRNA served as the internal control. The asterisk indicates that nad4 had decreased transcript abundance. (B) Structure of the maize mitochondrial nad4 gene. All introns are cis-spliced. The expected amplification products using different primer pairs are indicated. (C) RT-PCR analysis of nad4 intron-splicing efficiency in the WT and dek41. Solid arrows indicate unspliced fragments and open arrows indicate spliced fragments. The expected sizes of the amplification products using different primer pairs are indicated below each band. W, wild-type; M, mutant.

P-type PPR proteins are involved in the splicing of group II introns (Cheng ; Xiu ). Maize mitochondria contain 22 group II introns, including three introns from nad4 transcripts. To investigate the alterations in splicing in the dek41 mutants, specific primers were designed for qRT-PCR to inspect all the group II introns in both dek41-ref and dek41-mu. The quantitative differences in spliced exons between the dek41 mutants and the WT were compared by PCR using primers across adjacent exons. The results showed a distinct reduction of the nad4 spliced exon 3–4 fragment and of the splicing efficiency of nad4 intron 3 in both the mutants compared to the WT (Fig. 5A, B). A slight reduction in splicing efficiency of nad4 intron 1 was also observed in both the mutants. These results suggested that DEK41 is required for the cis-splicing of mitochondrial nad4 intron 3.

Fig. 5.

The maize dek41 mutation affects cis-splicing of mitochondrial nad4 intron 3. (A) Quantitative RT-PCR analysis of mature transcripts. Primers spanning adjacent exons were used for measuring differences in each spliced fragment. (B) Quantitative RT-PCR analysis of splicing efficiency of mitochondrial introns. The ratio of mature transcripts to unspliced fragments was used to measure differences in splicing efficiency. The Ubiquitin gene was used as an internal control. Data are means (±SE) of n=3 biological replicates.

Mutations in Dek41 affect mitochondrial complex I assembly and NADH dehydrogenase activity

NAD4 is a subunit of the mitochondrial complex I, NADH dehydrogenase. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to detect the potential impact on mitochondrial complexes in dek41 mutants. The mitochondria were isolated and enriched from immature WT and dek41 seeds at 18 DAP, and then equivalent amounts of mitochondrial proteins were separated by BN-PAGE. The levels of complex I, as well as super complex I+III2, were reduced in both dek41-ref and dek41-mu, compared to the WT (Fig. 6A). An in-gel NADH dehydrogenase activity test was conducted to examine the activity of complex I (Meyer ). The dek41 mutants showed significantly reduced complex I and super complex I+III2 NADH dehydrogenase activity, indicating that the assembly of these complexes was impaired in the mutants (Fig. 6B). A smaller complex I with reduced activity was detected in the mutants, suggesting that a partially assembled complex I maintained residual NADH dehydrogenase activity.

Fig. 6.

The maize dek41 mutation affects mitochondrial functioning in seeds. (A) BN-PAGE analysis of mitochondrial complexes. The positions of complex I, complex III, complex V, and super complex I+III2 (composed of complex I and dimeric complex III) are indicated. Each lane was loaded with 250 μg of mitochondrial protein. WT, wild-type. (B) In-gel NADH dehydrogenase activity test of complex I. The positions of complex I and super complex I+III2 are indicated. * Partially assembled complex I. The activity of dihydrolipoamide dehydrogenase (DLDH) was used as a sample loading control. Each lane was loaded with 250 μg of mitochondrial protein. (C) Western blot comparing accumulation of complex-related protein NAD4 (subunit of complex I) and cytochrome c (Cyt c) in total seed protein from immature WT and dek41 seeds at 18 d after pollination (DAP). α-Tubulin was used as a sample loading control. (D) Quantitative RT-PCR analysis of Aox1, Aox2, and Aox3 that are associated with the alternative respiratory pathway. The Ubiquitin gene served as an internal control. Data are means (±SE) of n=3 individuals. Significant differences were determined using Student’s t-test: ns, not significant; *P <0.05, **P<0.01. (E) Ultrastructure of developing endosperms from WT and dek41-ref seeds at 15 DAP. Scale bars are 1 μm. Nu, nucleus; Mt, mitochondrion; Pb, protein body; St, starch grain. (F) The most significantly enriched GO terms of the 1124 functional annotated differentially expressed genes.

To determine the influence of the dek41 mutation on oxidation respiratory chain complexes, the levels of mitochondrial complex-related proteins in dek41 and WT plants were analysed by western blotting using antibodies against NAD4 (subunit of complex I) and cytochrome c. The NAD4 protein was less abundant in the dek41 mutants, while the amount of cytochrome c was increased (Fig. 6C). The defect of NAD4 was in accordance with the changes observed using BN-PAGE. These results suggested that dek41 affected the abundance of NAD4 and the assembly of complex I, which probably also affected the accumulation of other proteins related to the electron transport chain (such as cytochrome c) in dek41.

dek41 mutants exhibit abnormal mitochondrial morphology

To determine the effects of the dek41 mutation on mitochondrial structure, the endosperms of the WT and dek41 mutants at 15 DAP were observed by TEM. Normal activation of the electron transport chain (ETC) is required for the proper formation of the inner envelope cristae in mitochondria (Logan, 2006). The mitochondria in the WT endosperm formed distinct inner-envelope cristae surrounded by a dense matrix (Fig. 6E). By contrast, the internal structure of mitochondria in the dek41 mutants was less distinct, without typical cristae and instead having a grainy appearance. Thus, morphological changes to the mitochondria occurred in dek41, suggesting that mitochondrial functioning was severely arrested in the dek41 during seed development.

The dek41 mutation affects the expression of genes related to mitochondrial function

In plants, when there is an electron transport defect in the cytochrome c pathway alternative oxidases (AOXs) are activated to maintain the tricarboxylic acid cycle and electron transport (Vanlerberghe and Ordog, 2002). The alternative pathway was examined by analysing the expression levels of AOX genes in dek41. Quantitative RT-PCR showed that both Aox2 and Aox3 were up-regulated in dek41 compared to the WT (Fig. 6D), indicating that the expression levels of genes in the alternative respiratory pathway were up-regulated as the result of inefficient mitochondrial oxidative phosphorylation in the mutant.

The transcript profiles of the endosperm of dek41 mutants and the WT at 12 DAP were compared using RNA-seq. Among the 41 406 gene transcripts detected, significant DEGs were identified as those with a threshold fold-change >2 and P<0.001. Based on these criteria, 1823 genes showed significant altered expression between dek41 and the WT. There were 1542 genes with increased transcription, while 281 genes showed decreased transcription (Supplementary Table S1). Of these DEGs, 1124 genes could be functionally annotated (BLAST analysis against the GenBank database). Gene ontology (GO; http://bioinfo.cau.edu.cn/agriGO) analysis indicated that the DEGs were mostly related to 10 terms. Among them, six GO terms were closely related to mitochondrial function: ‘mitochondrial membrane’, ‘protein targeting to mitochondria’, ‘GTP binding’, ‘NAD or NADH binding’, ‘purine nucleoside monophosphophate biosynthetic process’, and ‘hydrogen ion transmembrane transporter activity’ (Fig. 6F). In addition, DEGs involved in ‘tricarboxylic acid cycle’, ‘glucose catabolic process’, and ‘nucleosome assembly’ were also up-regulated in dek41. ‘Nutrient reservoir activity’, which is mainly attributed to storage proteins, was down-regulated in dek41 compared with the WT (Supplementary Table S1). This finding was in accordance with the reduced total protein content in mature dek41 seeds compared to the WT (Supplementary Fig. S2D). The RNA-seq data were further validated by quantitative RT-PCR of some of the most significant DEGs from each GO category (Supplementary Fig. S7), and four basal endosperm transfer layer ( BETL) marker genes (BAP-1A, BAP-1B, BETL3, and BETL9) were also detected using qPCR (Supplementary Fig. S8). These results were all in accordance with the RNA-seq data.

Discussion

DEK41 is a P-type PPR protein required for cis-splicing of mitochondrial nad4 intron 3 in maize

In this study, we identified a novel P-type PPR protein DEK41 that affects the cis-splicing of nad4 intron 3 in maize. Our characterization of DEK41 adds the list of P-type PPR proteins that function as splicing factors in organelle RNA metabolism. P-type PPR proteins with splicing effects have been studied in Arabidopsis mitochondria, and include OTP43, ABO5, OTP439, TANG2, SLO3, and MTL1 (Table 1). These PPRs are required for the splicing of mitochondrial nad1, nad2, nad5, and nad7 introns. In addition to PPRs, Arabidopsis mutants that affect complex I also include RNA maturase (nMAT1, nMAT2, nMAT4) mutants (Keren , 2012; Cohen ) and RNA helicase (PMH2 and ABO6) mutants (He ; Köhler ). Mutation of these genes (both PPR and non-PPR) in Arabidopsis usually causes a slow-growth phenotype of plants. In maize, the mutants affecting complex I are mostly PPR mutants. The P-type PPRs (EMP8, EMP10, EMP11, EMP16, DEK2, DEK35, and DEK37) are reported to affect the splicing of mitochondrial nad1, nad2, and nad4 introns (Table 1). These P-type PPRs together with the PLS-types (Smk1, EMP5, EMP7), which are required for the transcript editing of mitochondrial nad7, rpl16, and ccmF (Table 1), affect the assembly of mitochondrial complex I and the corresponding mutants all demonstrate abnormal morphology of mitochondria. The maize dek10 mutant affects complex IV, the Emp9 mutant affects complex I and III, and the corresponding genes of these mutants both encode mitochondrial E-subgroup PPRs (Qi ; Yang ). The mutants that affect complex I, III, and IV all demonstrate abnormal morphology of mitochondria, indicating that the mitochondrial structure and function are affected. In maize, mutation of these genes usually causes a lethal phenotype of plants (Liu ; Qi ; Yang ; Dai ; Sun ).

Table 1.

List of mitochondrial PPR proteins affecting mitochondrial genes in Arabidopsis and maize that are involved in transcript splicing, editing, maturation, stability, and translation

Function	Organism	Target transcript	Protein	Reference
Splicing	Arabidopsis	nad1	OTP43	de Longevialle et al. (2007)
Splicing	Arabidopsis	nad2	ABO5	Liu et al. (2010)
Splicing	Arabidopsis	nad5	OTP439	Colas des Francs-Small et al. (2014)
Splicing	Arabidopsis	nad5	TANG2	Colas des Francs-Small et al. (2014)
Splicing	Arabidopsis	nad7	SLO3	Hsieh et al. (2015)
Splicing	Maize	nad2	EMP16	Xiu et al. (2016)
Splicing	Maize	nad2	EMP10	Cai et al. (2017)
Splicing	Maize	nad4	DEK35	Chen et al. (2017)
Splicing	Maize	nad1	DEK2	Qi et al. (2017b)
Splicing	Maize	nad1	EMP11	Ren et al. (2017)
Splicing	Maize	nad2	DEK37	Dai et al. (2018)
Splicing	Maize	nad1, nad2, nad4	EMP8	Sun et al. (2018)
Splicing	Maize	nad4	DEK41	This study
Editing	Maize	rpl16, nad9, cox3, rps12	EMP5	Liu et al. (2013)
Editing	Maize	nad7	SMK1	Li et al. (2014)
Editing	Maize	ccmF N	EMP7	Sun et al. (2015)
Maturation	Arabidopsis	nad9, cox3	RPF2	Jonietz et al. (2010)
Maturation	Arabidopsis	nad4	RPF1	Hölzle et al. (2011)
Maturation	Arabidopsis	nad6, atp9	RPF5	Hauler et al. (2013)
Stability	Arabidopsis	nad4	MTSF1	Haïli et al. (2013)
Stability	Maize	nad5	PPR78	Zhang et al. (2017)
Translation and splicing	Arabidopsis	nad7	MTL1	Haïli et al. (2016)

The maize dek41-ref is a defective kernel mutant with arrested endosperm and embryo development (Fig. 1A, D). The full-length Dek41 transcript was completely repressed in the dek41-ref mutant (Fig. 2D), indicating that loss-of-function of DEK41 was the intrinsic cause of the arrested development in dek41 seed. The splicing efficiency of nad4 intron 3 was clearly reduced in both the dek41-ref and dek41-mu mutants, and this was unique across the 22 mitochondrial group II introns (Fig. 5B). Although splicing of nad4 intron 3 was not completely abolished in the dek41 mutants, the level of mature nad4 transcript was reduced (Fig. 4A), which was probably due to a loss of stabilization resulting from a splicing defect. We also noticed that the transcripts of several genes were found to be increased in the dek41 mutants (such as nad6, rps4, rps13, atp9, ccmF, mat-r, and rpl16; Fig. 4A). This could have been due to a feedback response to the mutation in Dek41. Similar feedback responses in the transcription of mitochondrial genes have also been observed in other P-type PPR gene mutations (Xiu ; Qi ). A slight reduction in splicing efficiency of nad4 intron 1 was also observed in both the dek41 mutants (Figs 4C, 5B). However, a reduction in the levels of spliced nad4 exon1-2 was not found (Fig. 5A), suggesting that nad4 intron 1 might not be the unique splicing target of DEK41. Our previous work showed that DEK35 also affects the splicing of nad4 intron 1 (Chen ), and that EMP8 affects the splicing of mitochondrial nad4 intron 1, nad2 intron 1, and nad1 intron 4 (Sun ), so it is possible that the splicing of nad4 intron 1 requires DEK41, DEK35, and EMP8. Further analysis of mitochondrial complexes showed significant deficiencies of complex I and super complex I + III2 (Fig. 6A), suggesting that mitochondrial function was severely defective in dek41 mutants due to reduced nad4 transcript levels.

NAD4 is one of nine mitochondria-encoded subunits (NAD1–7, NAD4L, and NAD9) that constitute NADH dehydrogenase (Clifton ). NADH dehydrogenase is the major entry point for transporting electrons in plants (Braun ). Previous studies have shown that different types of nuclear-encoded proteins are involved in the diverse RNA processing of nad4 transcripts: MTSF1 is essential for the 3´-processing of mitochondrial nad4 mRNA and its stability in Arabidopsis (Haïli ), RPF1 is required for the efficient generation of a 5´-end 228 nucleotides upstream of the mitochondrial nad4 gene in Arabidopsis (Hölzle ), AHG11 has potential roles in nad4 RNA editing of mitochondria in Arabidopsis (Murayama ), CSS1 is a splicing factor of the mitochondrial nad4 intron in Arabidopsis (Nakagawa and Sakurai, 2006), EMP8 is required for the splicing of mitochondrial nad4 intron 1 in maize (Sun ), and MS1 is involved in splicing of mitochondrial nad4 intron 1 in Nicotiana sylvestris (Brangeon ). These studies all report severe effects of faulty regulation of nad4 transcripts, indicating that NAD4 plays a crucial role in mitochondrial function. In the dek41 mutants, nad4 intron 3 exhibited reduced splicing processes, which resulted in severe deficiencies of mitochondrial complex I assembly and activity, and in impaired seed development. Our results demonstrate that splicing of the nad4 transcript is another crucial post-transcriptional process that is required to maintain the normal function of mitochondrial complex I.

The dek41 mutation results in defective mitochondrial function and kernel development

Mitochondrial complex I is the primary entry point for electrons into the electron transport chain (ETC), disruption of which by inhibitors or by loss of subunits leads to problems with primary metabolism (Noctor ). Complex I is embedded in the inner membrane and mediates the transfer of electrons from NADH to ubiquinone (Lee ). BN-PAGE and in-gel NADH dehydrogenase activity analyses indicated that the assembly and activity of complex I and super-complex I + III2 were severely impaired in the dek41 mutants (Fig. 6A, B). A smaller complex I with partial activity was observed in the in-gel NADH dehydrogenase activity assays (Fig. 6B), suggesting that dek41 mutants can still synthesize proteins in mitochondria, probably at reduced capability and/or with increased errors. Such partially assembled complex Is have also been observed in other PPR mutants, including emp9, ppr78, and dek35, which exhibit arrested mitochondrial complex I activity and seed development (Chen ; Yang ; Zhang ). Similar to EMP16 (Xiu ) and Dek2 (Qi ), increased Aox2 and Aox3 level was also observed in dek41. AOXs can reduce the levels of reactive oxygen species in situations when ETC complexes are unable to function properly for the maintenance of electron flux. A rapid increase in transcriptional level of Aoxs has previously been reported in PPR mutants (Sun ; Xiu ; Chen ; Qi ). Cytochrome c is located in the mitochondrial intermembrane/intercristae spaces and functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (Garrido ). Therefore, in addition to the reduction of NAD4 and the increase of cytochrome c in the dek41 mutants (Fig. 6C), we suggest that the dek41 mutation affects the assembly and NADH dehydrogenase activity of mitochondrial complex I, which probably promotes the accumulation of other complex-related proteins (including cytochrome c).

ETC biogenesis is required for the proper morphology of the cristae in mitochondria (Logan, 2006). In maize PPR mutants, the formation of mitochondrial cristae by the inner membrane is strongly impaired, and these structurally altered mitochondria are likely less functional than those with a normal inner structure (Sosso ; Chen ; Qi ; Dai ). A voided internal structure with abnormal morphology of mitochondria was also observed in the dek41 mutants (Fig. 6E), suggesting that mitochondrial function was severely disordered. The loss of DEK41 function results in a defect of ETC biogenesis, which affects not only respiratory metabolism but also the inner structure of the mitochondria.

The defect in mitochondrial function in dek41 also led to changes in the expression levels of several important genes in GO categories associated with energy-consuming biological process (Fig. 6F). Among 10 GO classifications that were identified as being significantly affected, six that were highly related to mitochondrial structure and activity exhibited extensive up-regulated DEGs, which indicated that the oxidation respiratory chain was severely affected in the dek41 mutants. The critical effects on mitochondrial function were in accordance with increases identified by transcriptome analysis in ‘mitochondrial membrane’, ‘protein targeting to mitochondria’, ‘GTP binding’, ‘NAD or NADH binding’, ‘hydrogen ion transmembrane transporter activity’ and ‘purine nucleoside monophosphate biosynthetic process’. The ‘tricarboxylic acid cycle’ (TCA cycle) is a series of enzyme-catalysed chemical reactions that form a key part of aerobic respiration in mitochondria, and the ‘glucose catabolic process’ determines the reaction rate of the TCA cycle. ‘Nucleosome assembly’ is essential for a variety of biological processes, such as cell-cycle progression, development, and senescence (Gal ), which are also energy-consuming processes. The dek41 mutation caused abnormal mitochondrial function, which might have resulted in the feedback up-regulation of genes related to nucleosome assembly, leading in turn to developmental delay (Supplementary Table S1). Whilst GO classifications were up-regulated, only one, ‘nutrient reservoir activity’, showed decreased DEGs (Supplementary Table S1). Immature dek41 seeds showed delayed embryo and endosperm development (Fig. 1A, D) and mature seeds exhibited significantly decreased dry weight, and starch and total protein contents (Fig. 1G, H, Supplementary Fig. S2D). These phenotypic data were in accordance with the decreased DEGs identified in the transcriptome analysis (Supplementary Table S1). We postulate that the wide down-regulation of genes encoding zein proteins might explain the reduced total protein content in the seeds. Our results highlight the necessity of mitochondrial activity for nutrient generation and accumulation (Law ).

The basal endosperm transfer layer (BETL) develops extensive cell-wall ingrowths that support an enlarged plasma membrane surface, promoting primary nutrient uptake in the endosperm (Pate and Gunning, 1972; Thompson ; Offler ) that requires high metabolic rates. Therefore, transfer cells are typically rich in mitochondria. In our study, the development of the BETL was dramatically arrested in dek41 (Fig. 1F), and the transcriptional levels of BETL marker genes were decreased (Supplementary Fig. S8). The absence of a properly formed transfer cell layer is always correlated with reduced rates of grain filling and with seed abortion (Brink and Cooper, 1947; Charlton ), and mutants with arrested BETL development exhibit small and hollow kernels. Mutations of maize EMP4, EMP16, DEK35, and DEK37 that encode PPR proteins also result in a defective transfer cell layer (Gutierrez ; Xiu ; Chen ; Dai ). Therefore, the arrested development of the BETL in dek41 might be the consequence of reduced energy for nutrient transport. In summary, the dek41 mutation in maize affects the mitochondrial structure and function, resulting in arrested BETL formation and embryo morphogenesis, which ultimately affects kernel development.

Supplementary data

Supplementary data are available at JXB online.

Fig. S1. Original gels of western blots comparing accumulation of NAD4 in total proteins from immature wild-type and dek41 seeds at 18 DAP.

Fig. S2. Analysis of protein content in the maize dek41 mutant.

Fig. S3. Detection of Mu insertion sites by co-segregation for test-cross ears of dek41-ref × dek41-mu.

Fig. S4. Phylogenetic analysis and sequence alignment of DEK41 with its homologs.

Fig. S5. Quantitative RT-PCR comparing Dek41 transcript levels in the wild-type and dek41 at 12, 15, and 18 DAP.

Fig. S6. Original gels used for RT-PCR analysis of 35 mitochondria-encoded transcripts in immature wild-type and dek41 seeds at 12 DAP.

Fig. S7. Quantitative RT-PCR confirmation of the transcriptome data.

Fig. S8. Quantitative RT-PCR identification of BETL marker genes in immature dek41-ref and wild-type seeds at 15 DAP and 18 DAP.

Table S1. Gene ontology classifications of DEGs with functional annotations.

Table S2. Primers used in this study.

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