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. 2020 Sep 16;18(4):499–509. doi: 10.1080/15476286.2020.1817267

EMP32 is required for the cis-splicing of nad7 intron 2 and seed development in maize

Yan-Zhuo Yang 1, Shuo Ding 1, Xin-Yuan Liu 1, Jiao-Jiao Tang 1, Yong Wang 1, Feng Sun 1, Chunhui Xu 1, Bao-Cai Tan 1,✉
PMCID: PMC7971235  PMID: 32936708

ABSTRACT

Pentatricopeptide repeat (PPR) proteins play an important role in post-transcriptional regulation of mitochondrial gene expression. Functions of many PPR proteins and their roles in plant growth and development remain unknown. Through characterization of an empty pericarp32 (emp32) mutant, we identified the function of Emp32 in mitochondrial intron splicing and seed development in maize. The loss-of-function mutant emp32 shows embryo lethality with severely arrested embryo and endosperm development, and over-expression of Emp32 rescues the embryo-lethality. EMP32 is a P-type PPR protein targeted to mitochondria. Loss of function in Emp32 dramatically decreases the splicing efficiency of nad7 intron 2, while complementation of Emp32 restores the splicing efficiency. Although nad7 intron 2 is partially spliced in the wild type, over-expression of Emp32 does not increase the splicing efficiency. The splicing deficiency of nad7 intron 2 blocks the assembly of mitochondrial complex I and dramatically reduces its activity, which may explain the embryo-lethality in emp32. In addition to the one copy of nad7 in the maize mitochondrial genome, we identified one to six copies of nad7 in the nuclear genomes in different maize inbred lines. These copies appear not to be expressed. Together, our results revealed that the P-type PPR protein EMP32 is required for the cis-splicing of nad7 intron 2 and seed development in maize.

KEYWORDS: Maize (Zea mays), mitochondrion, nad7, intron splicing, seed development

Introduction

Mitochondrion, the powerhouse of the cell, is derived from an α-proteobacterium-like ancestor [1]. During evolution, many of mitochondrial genes are lost or transferred to the nuclear and chloroplast genome. Plant mitochondria contain 2000 to 3000 proteins [2], but less than 50 proteins are mitochondrial genome encoded [3]. The mitochondrion-encoded proteins include subunits of electron transport chain complexes, ribosomal proteins, proteins involved in cytochrome c maturation, a transporter and a maturase [3].

Intron splicing is an important post-transcriptional process and essential for gene expression in plant mitochondria [4]. A total of 26 mitochondrial introns have been identified in flower plants, and the vast majority of them are group II introns [5]. Group II introns are found within diverse genes in protozoa, fungi and plants and have ability to self-splice in vitro [6]. However, plant mitochondrial introns lost the ability to self-splice [5]. The splicing of plant mitochondrial introns is facilitated by a large number of nucleus-encoded accessory factors. These factors are RNA binding proteins, including pentatricopeptide repeat (PPR) protein [7], RNA helicase [8,9], chloroplast RNA splicing and ribosome maturation (CRM) protein [10,11], plant organelle RNA recognition (PORR) protein [12,13], mitochondrial transcription termination factors (mTERFs) [14], RCC protein [15] and maturase [16,17].

PPR proteins contain tandem arrays of a degenerate 31–36 amino acid motif [18,19], which binds to target RNA sites in a one-nucleotide one motif manner [20–22]. PPR protein family is expanded in terrestrial plants and contains more than 450 members in Arabidopsis [19]. PPR proteins are roughly classified into P and PLS subfamily, based on the characteristics of the PPR motifs [19,23]. P-type PPR proteins have been shown to participate in a wide range of organellar RNA processing, including RNA splicing, RNA transcription and stability and RNA maturation [7]. More than half of PPR proteins belongs to P subfamily [19], while the functions of many of them are unknown.

The nad7 gene, encoding the NADH dehydrogenase subunit7, resides in the nuclear genome in animals [24] and some fungi [25] and in the mitochondrial genome in angiosperms [26]. The maturation of angiosperm nad7 requires the cis-splicing of four group II introns, which involves a multitude of accessory proteins. For example, PPR proteins SLO3 [27] and MTL1 [28], CRM proteins mCSF1 [10] and CFM9 [11] and maturases MatR [17] and nMAT2 [16] are responsible for the splicing of nad7 intron 2 in Arabidopsis, and PPR protein PPR-SMR1 is necessary for the splicing of nad7 intron 2 in maize [29]. These proteins possibly play different roles in the splicing process. Maturases (MatR and nMAT2), CRM proteins (mCSF1 and CFM9) and PPR-SMR1 are required for the splicing of a large set of introns [10,11,16,17,29], whereas SLO3 and MTL1 are specific for a single introns [27,28]. However, the precise functions of these proteins in the splicing are unknown. Nad7 is a core subunit in mitochondrial complex I, and defect in the splicing of nad7 delays plant growth [27,28,30].

In this study, we identified the function of a previously uncharacterized PPR protein, EMP32, in the mitochondrial intron splicing. EMP32 is specifically required for the cis-splicing of nad7 intron 2. Defect in the splicing impairs the assembly of mitochondrial complex I and decreases its activity, resulting in the embryo-lethality in the emp32 mutant. Over-expression of Emp32 in the emp32 mutant rescues the embryo-lethality and splicing deficiency, while in wild type does not increase the splicing efficiency.

Results

Seed development is arrested in the emp32 mutant

We ordered UniformMu stock UFMu-01098 for the study of a mitochondrial processing peptidase gene. However, we observed the segregation of empty pericarp (emp) phenotype which was not linked to the target gene. This unknown gene was named Emp32. The self-pollinated ear of emp32/+ heterozygote segregated normal to emp kernels at a ratio of 3:1 (Fig. 1A), indicating emp32 is a single recessive nuclear mutant. The developmental defect in the emp32 kernels is visible at early stages. At maturity, the emp32 kernels become white and shrunken (Fig. 1B). Longitudinal sectioning of mature kernels showed that the mutant kernels collapsed and the development of embryo and endosperm was severely arrested in the mutants (Fig. 1C, D, E). The emp32 mutant is embryo-lethal, as all attempts to geminate the mutants in soil or on MS medium were not successful.

Figure 1.

Figure 1.

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Seed development is arrested in the emp32 mutant. (A) An ear segregating emp32 mutants. Arrows indicate the mutants. (B) Germinal face of the wild type and emp32 mutant. (C) Sagittal cut of mature wild-type and emp32 mutant kernels. (D-E) Enlarged views of the wild-type (D) and emp32 mutant (E) embryos in (C). e, embryo. Bars = 0.5 cm in [48] and 0.1 cm in (E)

To further assess the impact of Emp32 mutation on embryogenesis and endosperm development, the emp32 kernels were sectioned and compared with wild-type sibling kernels in the same ear. At 9 days after pollination (DAP), the wild-type embryo reached the coleoptilar stage (Fig. 2A), whereas the mutant embryo only reached the transition stage (Fig. 2D). The wild-type endosperm completely filled pericarp (Fig. 2A), whereas the development of the mutant endosperm was severely retarded, producing a large cavity between endosperm and pericarp (Fig. 2D). At 13 DAP, the mutant embryo was blocked at the transition stage and the size of mutant endosperm remained small (Fig. 2E). At 16 DAP, the wild-type embryo developed a scutellum, shoot apical meristem, root apical meristem, coleorhiza and leaf primordial (Fig. 2C, G), but the mutant developed a comma-shaped embryo with a bloated embryo proper and an elongated suspensor (Fig. 2F, H). The wild-type endosperm was filled with starchy cells (Fig. 2C), whereas the starch level in the mutant endosperm was significantly reduced and the cavity between endosperm and pericarp still existed (Fig. 2F). These results indicate that the embryogenesis and endosperm development are significantly arrested in the emp32 mutant.

Figure 2.

Figure 2.

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Embryogenesis and endosperm development are arrested in the emp32 mutant. (A-H) Cytological sections of the wild type and the emp32 mutants. The wild-type kernels at 9 (A), 13 (B) and 16 DAP (C). The emp32 kernels at 9 (D), 13 (E) and 16 DAP (F). (G-H) Enlarged views of the wild type (H) and the emp32 mutant (G) embryos in (C) and (F), respectively. ep, embryo proper; su, suspensor; LP, leaf primordia; RAM, root apical meristem; SAM, shoot apical meristem; sc, scutellum; cor, coleorhiza. Bars = 1 mm in (A-F), 500 μm in (G) and 200 μm in (H)

Emp32 encodes a P-type PPR protein targeted to mitochondria

Eighteen Mu insertions identified in the stock UFMu-01098 were deposited in MaizeGDB database (https://www.maizegdb.org/data_centre/stock?id=2365693). To identify which insertion co-segregates with the emp32 phenotype, a PCR-based genotyping was performed with an insertion-specific primer and a Mu-specific primer TIR8. An insertion mu1017071 was identified to co-segregate with the emp32 phenotype. Sequencing result showed that the insertion is located at 40 nt downstream of the start codon (ATG) in GRMZM2G089959 (Fig. 3A). To confirm the linkage between the emp32 phenotype and the Mu insertion in GRMZM2G089959, a total 64 normal kernels from self-pollinated emp32/+ ears were geminated and the plants were genotyped by primers TIR8 and Emp32-R1. Forty-two plants harboured the Mu insertion in GRMZM2G089959, producing ears segregating for emp32 kernels (Fig. S1). Twenty-two plants did not harbour the insertion, producing normal ears (Fig. S1). These results indicate the Mu insertion in GRMZM2G089959 is tightly linked to the emp32 phenotype.

Figure 3.

Figure 3.

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Emp32 encodes a mitochondrion-targeted P-type PPR protein. (A) Schematic representation of the Emp32 gene and EMP32 protein structure. The Mu insertion is indicated by a triangle and the number indicates Mu insertion site. (B) Phenotype of wild type and the emp32 mutant complemented by over-expressing Emp32. (C) Molecular identification of the complemented emp32 mutants harbouring transgene Emp32 expression and mu insertion in the endogenous Emp32. (D) Subcellular localization of EMP32: GFP in tobacco leaf epidermis cells. Bars = 10 μm. DIC: differential interference contrast

To confirm that GRMZM2G089959 is the causal gene, we constructed transgenic maize lines over-expressing Emp32 under the maize ubiquitin promoter in inbred line KN5585. Eleven transgenic lines were generated and three with high Emp32 expression were used for further analysis (Fig. 6A). The emp32/+ heterozygotes were pollinated with pollens from the transgenic plants. T1 plants were genotyped to identify the ones harbouring both the transgene and the Mu insertion in GRMZM2G089959. Self-pollinated ears from these plants (emp32/+, grmzm2g089959/+) displayed approximately 6.25% (1/16) emp32 kernels, not 25%, indicating that the emp32 mutant phenotype is complemented by the transgene. The complemented kernels were viable and the seedlings appeared normal in growth and morphology (Fig. 3B). Genotyping by PCR indicates that the complemented seedlings carry both the transgene and homozygous for the Mu insertion in GRMZM2G089959 (Fig. 3C). These results indicate that GRMZM2G089959 is the causal gene for the emp32 phenotype, and thus named Emp32.

Figure 6.

Figure 6.

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Splicing efficiency of nad7 intron 2 is not increased in Emp32 over-expression plants. (A) The transcription level of Emp32 in the wild type and Emp32 over-expression plants. (B) The transcription level of spliced and unspliced nad7 intron 2 in the wild type and Emp32 over-expression plants. Values are the mean ± SE of three biological repeats

Emp32 encodes a P-type PPR protein containing 23 P-type PPR motifs and no other motifs (Fig. 3A). To determine the subcellular localization of EMP32, the full-length EMP32 was fused with green fluorescent protein (GFP) and the resulting fusion protein EMP32: GFP was transiently expressed in tobacco (Nicotiana benthamiana) leaf epidermis cells. Mitochondria were stained with MitoTracker red as a control. Confocal laser scanning microscopy analysis showed that the green fluorescence of EMP32: GFP was detected as dot-shaped granules which were merged with the red fluorescence of MitoTracker but not with the red auto-fluorescence of chlorophylls (Fig. 3D), indicating that EMP32 is a mitochondrion-localized protein.

The cis-splicing of nad7 intron 2 is defective in the emp32 mutant

Many studies showed that P-type PPR proteins are involved in organellar RNA metabolism, such as RNA splicing, stabilization and translation regulation [7]. As EMP32 is a mitochondrion-localized P-type PPR protein, we expected that it plays roles in mitochondrial RNA metabolism. Thus, the mitochondrial transcript profile was compared between the wild type and the emp32 mutant by RT-PCR. Maize mitochondrial genome encodes 33 protein-encoding transcripts [31]. As shown in Fig. 4A, except for nad7, all transcripts displayed approximately equal levels in the wild type and the emp32 mutant. In the emp32 mutant, the mature size nad7 transcript almost disappeared, whereas a large size transcript was accumulated (Fig. 4A). Estimated by the length of the large transcript (approximately 2600 bp, Fig. 4A), it appears to contain an unspliced intron 2. To prove this, four pairs of primers were designed to detect each splicing event (Fig. 4B). Four cis-introns are required to be removed from pre-mRNA to form the mature nad7 transcript (Fig. 4B). RT-PCR analysis showed that the fragments containing the unspliced intron 2 are sharply accumulated in the emp32 mutant and the fragments joining exons 2 and 3 are significantly reduced (Fig. 4C), indicating that the splicing of nad7 intron 2 is defective in the mutant. Sequencing analysis of the unspliced fragments confirmed that they contained the unspliced nad7 intron 2.

Figure 4.

Figure 4.

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The splicing of nad7 intron 2 is impaired in the emp32 mutant. (A) RT-PCR analysis of 33 mitochondrion-encoded transcripts in the wild type and emp32 mutant. Nearly full-length transcripts were amplified for each gene. ZmActin was used as a control for RNA normalization. (B) Schematic representation of maize mitochondrial nad7 gene. (C) RT-PCR analysis of the splicing of four cis-introns in nad7 pre-mRNA in the wild type and emp32 mutant

To verify the splicing specificity of EMP32, the splicing efficiency of all 22 maize mitochondrial group II introns was quantified by qRT-PCR. Splicing efficiency is calculated as a ratio of spliced form to unspliced form in the emp32 mutant normalized against the same ratio in the wild type. The results showed that only the splicing efficiency of nad7 intron 2 was dramatically decreased in the emp32 mutant, and the other introns were not significantly affected (Fig. 5).

Figure 5.

Figure 5.

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Splicing efficiency of nad7 intron 2 is decreased in the emp32 mutant. The splicing efficiency of 22 introns of mitochondrial genes was detected by Quantitative RT-PCR. Values are the mean ± SE of three biological repeats

Over-expression of Emp32 restores the splicing in the emp32 mutant, but does not increase the splicing efficiency in wild type

The splicing of nad7 intron 2 was detected in the complemented transgenic plants. RT-PCR analysis showed that the intron 2 was spliced equally in the wild-type and the complemented plants (Fig. S2), indicating that Emp32 is required for the splicing of nad7 intron 2.

Then we detected whether over-expression of Emp32 increases the splicing efficiency of nad7 intron 2 in over-expression plants. The transgenic plants showed 236, 31, 87-fold increase, respectively, in Emp32 expression (Fig. 6A). Although two of three transgenic plants (OE-2 and OE-3) showed slight increase in the splicing of nad7 intron 2, there was no correlation between the expression level of Emp32 and splicing efficiency (Fig. 6). As antibody against EMP32 was unavailable, we could not determine the level of EMP32 in these transgenic lines. Thus, it appears that an elevated level of Emp32 mRNA does not lead to an increase in the splicing efficiency of nad7 intron 2 in these transgenic plants.

EMP32 is required for the biogenesis of complex I

Nad7 is a core component in plant mitochondrial complex I. The defect in the splicing of nad7 intron 2 is expected to impair the synthesis of Nad7, and thus affect the assembly and activity of complex I. To test this, crude mitochondria were isolated from 13 DAP wild-type and emp32 mutant kernels. Mitochondrial complexes were solubilized with dodecyl maltoside and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). Comparing with the wild type, the bands corresponding to complex I and supercomplex I+ III2 were dramatically reduced in the emp32 mutant (Fig. 7A), indicating that the assembly of complex I is defective. To test the activity of complex I, BN-PAGE gel strip containing separated complexes was stained with an assay buffer containing NADH-NBT that can be reduced by complex I to form blue sediment. In the emp32 mutant, the activity corresponding to complex I and supercomplex I+ III2 bands was almost undetectable (Fig. 7B), confirming that the activity of complex I is defective.

Figure 7.

Figure 7.

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Loss-of-function mutation in Emp32 impairs the assembly and activity of mitochondrial complex I. (A) BN-PAGE analysis of mitochondrial complex I assembly. (B) In-gel staining of the mitochondrial complex I activity. The activity of dihydrolipoamide dehydrogenase (DLDH) is used as a loading control. C-I, complex I; C-I+ III2, supercomplex I+ III2. (C) Western blotting analysis of the accumulation of mitochondrial proteins

Furthermore, the accumulation of mitochondrial proteins from OXPHOS system was detected by Western blot. The protein level of Nad9, which is a core subunit of the complex I matrix arm, was largely decreased in the emp32 mutant, whereas the protein level of Cytc1 and Cox2 was dramatically increased (Fig. 7C). The protein level of ATPase-A was about equal in the wild type and the emp32 mutant (Fig. 7C). Alternative oxidase (AOX), which is activated by mitochondrial OXPHOS deficiency, was strongly induced in the emp32 mutant (Fig. 7C). Taken together, these results indicate that the function of mitochondrial complex I is defective in the emp32 mutant.

The maize nuclear genome contains one to six copies of nad7

To detect whether maize nuclear genome contains the nad7 copy, the maize B73 genome (B73-REFERENCE-NAM-5.0) was searched by BLAST. Five copies of nad7 mtDNA were found (Fig. 8A). One copy is located on chromosome 1 (hereafter named nad7-chr1) at position 340,041–357548 (17,508 bp) and other four copies on chromosome 9 (hereafter named nad7-chr9) at position 77192244–78127802 (935,558 bp, Fig. 8A). All copies contain the whole coding region of nad7 mtDNA. The nad7-chr1 is interrupted by two fragments in introns 1 and 2 of original nad7 mtDNA (Fig. 8A). The insertion in intron 1 is the 5ʹ LTR region of the retrotransposons RLC_ji and the insertion in intron 2 lacks recognizable sequence features. BLAST analysis revealed that the EST fragments found in maize EST databases were exon sequences of mitochondrial nad7 and edited, indicating that the nuclear nad7 copies are not expressed and probably pseudogenes.

Figure 8.

Figure 8.

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Transfer of mitochondrial nad7 to the nuclear genome in maize. (A) Maize B73 genome contains five copies of nad7 mitochondrial DNA. The copy on chromosome 1 is inserted by two fragments and other four copies on chromosome 9 are located at position 77192244–78127802. Green boxes present exons, green lines introns and yellow lines insertions. (B) The nad7 nuclear copy in multiple maize lines. Red triangles present the insertion in intron 1 and blue triangles in intron 2. Dotted lines indicate that two copies are far away from each other on the same chromosome

Then, we detected the nuclear nad7 copies in other 29 maize lines. Except for teosinte, all other lines contain the copy on chromosome 1. The copies (copy) in lines Ki11, M162W, NC358, CML228, CML247, CML322, Mo17, Tzi8, PH207 and CML322 do not contain the insertion; in lines W22, B97, CML52, F7, IL14H, K0326Y, Ki3, M37W, Ms71, NC350, PE0075, DK105, EP1, CML103, Oh43 and CML333 contain one insertion in intron 2; in P39 contains one insertion in intron 2; in B104 contains one insertion in introns 1 and 2, respectively (Fig. 8B). Oh43, CML103 and HP301 lines contain another copy on chromosome 1 (Fig. 8B). HP301 contains other 4 copies on chromosome 9, and CML333 contains other 4 copies on chromosome 1 (Fig. 8B). Teosinte nuclear genome does not contain full-length nad7 mtDNA (Fig. 8B). These results indicate that the nad7 mtDNA has been transferred to multiple maize nuclear genomes.

Discussion

Multiple factors are required for the splicing of nad7 intron 2

In this study, we have demonstrated that a previously uncharacterized P-type PPR protein EMP32 is required for the cis-splicing of nad7 intron 2 in mitochondria. The maize emp32 is an embryo-lethal mutant caused by Mu transposon insertion (Fig. 3). In the emp32 mutant, the splicing efficiency of nad7 intron 2 was dramatically decreased and the transcripts containing the unspliced intron 2 were accumulated (Fig. 4A, C and Fig. 5). Complementation of Emp32 in the emp32 mutants rescued the intron splicing deficiency and embryo-lethality (Fig. 3B and S2). These results indicate that EMP32 is essential for the cis-splicing of nad7 intron 2. It is also noted that several proteins have been identified to participate in the splicing of nad7 intron 2. These include PPR proteins SLO3 [27] and MTL1 [28], maturases AtnMat2 [16] and MatR [17], CRM-domain proteins mCSF1 [10] and CFM9 [11] in Arabidopsis. The likely ortholog of EMP32 in Arabidopsis is At5G57250 with a 33% identity, and the putative orthologs of SLO3, MTL1, AtnMat2, MatR, mCSF1 and CFM9 in maize are GRMZM2G177845, GRMZM2G030263, GRMZM2G154119, ZeamMp095, Zm-mCSF1 [29] and GRMZM2G054040, respectively. Thus, EMP32 is a previously unidentified splicing factor involved in the cis-splicing of nad7 intron 2.

Although CRM-domain proteins, maturases and PPR proteins belong to RNA binding protein, they are likely play different roles in the splicing processing. CRM-domain proteins and maturases, which are general splicing factors, have relatively broad RNA substrate binding activities, while PPR proteins, which are specific splicing factors, recognize and bind to specific RNA substrate. CRM-domain proteins may act as chaperons which help correct folding of RNA molecules by inducing secondary structure rearrangement of diverse RNA substrates with low sequence specificities [32]. Maturases act specifically on the introns or closely introns in which they are encoded in bacteria and yeast mitochondria [33]. However, they act on multiple introns in plant mitochondria [17]. Biochemical analyses show that the binding of maturases and their targets facilitates intron folding and enhances intron splicing activity [33]. The unspecific binding characteristics of CRM-domain proteins and maturases suggest that they need to be assisted by sequence-specific binding proteins, such as PPR proteins. Although CRM-domain proteins, maturases and PPR proteins are found in multiple protein and/or RNA complex [34–36], there is no evidence of the physical interaction between each other, and whether they are present in the same protein complex is unknown. Further analyses need to identify the exact functions of those proteins and the components of the splicing complex.

Over-expression of Emp32 does not increase the splicing efficiency

Restoration of splicing deficiency by over-expression of Emp32 in the emp32 mutant promotes us to test whether the over-expression can increase the splicing efficiency in wild-type background. However, there is no correlation between Emp32 expression level and the splicing efficiency of nad7 intron 2 in three independent overexpression lines (Fig. 6), indicating that over-expression of Emp32 does not increase the splicing efficiency. This result arises a question: why cannot increase the splicing efficiency of nad7 intron 2 by over-expressing Emp32? We propose several possibilities. First, the expression level of many of PPR genes in plants is low [19,37]. Thus, it is possible that the low level of Emp32 in plant is sufficient for the intron splicing and the over-expression cannot increase the efficiency. Second, group II intron splicing requires the cooperation of multiple factors. For nad7 intron 2, the splicing requires at least eight factors, SLO3 [27], MTL1 [28], AtnMat2 [16], MatR [17], mCSF1 [10] and CFM9 [11] in Arabidopsis, and PPR-SMR1 [29] and EMP32 in maize. Over-expression of a single factor may not increase the splicing efficiency. Meanwhile, it also opens the question of how many factors are needed to be over-expressed to increase the splicing efficiency. Third, it is known that domain 3 (D3) stimulates the chemical rate constant of group II intron reactions and functions as a ‘catalytic effector’. It is possible that EMP32 does not function on D3, and thus may not increase the splicing efficiency by over-expression. At last, we also cannot rule out the possibility that the level of EMP32 protein does not increase in the transgenic lines although the mRNA level is increased dramatically.

The transfer of mitochondrial nad7 to the nucleus does not produce a functional gene in maize

The transfer of nad7 from the mitochondrion to the nucleus is an ongoing process. Transfers cause two results: resulting in functional nuclear genes and resulting in pseudogenes. In animals [24], certain fungi [25] and Marchantia polymorpha [38], functional nad7 was transferred to the nucleus successfully. In maize, except for teosinte, the wild ancestor of modern maize, all other lines we examined contain at least one copy of nad7 mtDNA (Fig. 8). However, these copies are not expressed and likely pseudogenes. We speculate that the nuclear copies cannot be functional genes even though they can be expressed. This is because the transfer of mitochondrial nad7 mtDNA to the nucleus in maize is mediated by DNA. These nuclear nad7 need to be edited and spliced to become functional genes. However, the nucleus lacks the mechanisms of group II intron splicing and C-to-U editing. In addition, the nuclear nad7 lacks a signal peptide for correctly targeting. In contrast, gene transfer mediated by RNA, which is already edited and spliced correctly, if can be inserted into a pre-existing gene for a mitochondrial protein, will be expressed and translocated into mitochondria. So far, in angiosperm, all functional genes transferred from the mitochondrial genome to the nuclear, such as cox2 in soybean [39] and cowpea [40], rps12 in Oenothera [41] and rps10 [42] and rps11 [43] in Arabidopsis, are mediated by RNA.

Materials and methods

Plant materials and growth condition

The maize (Zea mays L.) emp32 mutant was generated by introgressing the Mu-active line into the inbred W22 genetic background [44]. The UniformMu stock UFMu-01098 was requested from the Maize Genetics COOP Stock Center. The maize plants were grown in the experimental field at the Qingdao campus of Shandong University under the natural conditions.

Light microscopy of cytological sections

Immature wild type and emp32 mutant kernels were harvested from ears of self-pollinated heterozygous plants at 9, 13 and 16 DAP and cut along the longitudinal axis. Tissue fixing, dehydration, embedding, de-paraffining and section staining with 1% safranin O were carried out as described previously [45].

Subcellular localization of EMP32

The full-length coding region of EMP32 without stop codon was amplified by PCR using B73 genomic DNA as a template. After cloning into pENTR/D-TOPO vector (Thermo Fisher Scientific, USA), the fusion construct EMP32: GFP was generated by using gateway site-specific recombination in the pGWB5 vector. The expression of EMP32: GFP was placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Agrobacterium tumefaciens strain EHA105, and the resulting strain was infiltrated into tobacco (Nicotiana tabacum) leaf epidermal cells as described [46]. The fluorescence signals were detected under an Olympus FluoView FV1000 confocal microscope. MitoTracker Red (Thermo Fisher Scientific, USA) was used as the mitochondrial marker.

RNA extraction and quantitative RT-PCR (qPCR)

Total RNA was extracted from fresh kernels using the RNeasy Plant Mini Kit (Qiagen, USA). Before reverse transcription, RNA was treated with DNase I (New England Biolabs, USA) to eliminate residue genomic DNA contamination. cDNAs were synthesized by the SuperScript III One-Step RT-PCR System (Thermo Fisher Scientific, USA) with Oligo [47] and random hexamer primers. Transcription expression analysis of 33 mitochondrial genes by RT-PCR was performed as described preciously [45]. The relative quantification of gene expression was performed by qRT-PCR with Light Cycler Fast Start DNA Master SYBR Green I Kit and calculated with the 2−ΔΔCt method. ZmActin was used as a control for RNA normalization in RT-PCR and qPCR analysis. Primer sequences were listed in Supplementary Table S1 or published as descried preciously [45].

Plant transformation for emp32 complementation

The full-length coding region of EMP32 with stop codon was obtained by RT-PCR with primers as described above. The construct used for EMP32 over-expression carrying the ubiquitin promoter was homemade based on the backbone vectors of pTF101 and pUN1301. After restriction enzyme digestion and fragment linkage, the final construct was transformed into maize inbred line KN5585. The complementation was performed by crossing transgenic lines with emp32/+ heterozygotes. The T1 plants containing both Mu insertion and transgene were self-pollinated and the T2 plants were genotyped to identify the complemented emp32 mutants. The transgenic insertion was detected by PCR with primers Ubi-F and EMP32-R1 and the emp32 homozygote was detected with primers EMP32-F1 and EMP32-R1.

Mitochondrial complex I separation and activity

Isolation of crude mitochondria from the embryo and endosperm of immature maize kernels was performed as described previously [45]. The mitochondrial protein complexes were separated in a gradient Native PAGE Bis-Tris gel (Thermo Fisher). The gel was stained with Coomassie Brilliant Blue (CBB) to observe mitochondrial complexes and immersed in the assay buffer (0.1 M Tris/HCl, pH 7.4, 0.14 mM NADH and 1.22 mM nitrotetrazolium blue) to analyse complex I activity.

Acknowledgments

We thank Dr. Tsuyoshi Nakagawa (Shimane University, Japan) for the pGWB vectors and the Maize Genetics Cooperation Stock Center for the seed stocks.

Funding Statement

National Natural Science Foundation of China [31630053]; National Natural Science Foundation of China [91735301]; Shandong Provincial Natural Science Foundation [ZR2019MC005]; China Postdoctoral Science Foundation [2018M640624]; China Postdoctoral Science Foundation [2019T120583].

Author contributions

Y.-Z.Y., S.D. and B.-C.T. designed the research. Y.-Z.Y. and S.D. performed most of the experiments; X.-Y.L., J.-J.T. and Y.W. performed complementation experiment; F.S. performed the BN gel analysis; Y.-Z.Y., S.D., C.X. and B.-C.T. analyzed the data and wrote the article.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary materials

Supplemental data for this article can be accessed here.

References

  • [1].Gray MW. Evolutionary biology: mitochondrial genes on the move. Nature. 2000;408(6810):302–305. [DOI] [PubMed] [Google Scholar]
  • [2].Millar AH, Eubel H, Jansch L, et al. Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits. Plant Mol Biol. 2004;56(1):77–90. . [DOI] [PubMed] [Google Scholar]
  • [3].Unseld M, Marienfeld JR, Brandt P, et al. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature Genet. 1997;15(1):57–61. [DOI] [PubMed] [Google Scholar]
  • [4].Binder S, Brennicke A.. Gene expression in plant mitochondria: transcriptional and post-transcriptional control. Phil Trans R Soc Lond B. 2003;358(1429):181–188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Bonen L. Cis- and trans-splicing of group II introns in plant mitochondria. Mitochondrion. 2008;8(1):26–34. [DOI] [PubMed] [Google Scholar]
  • [6].Jacquier A. Group II introns: elaborate ribozymes. Biochimie. 1996;78(6):474–487. [DOI] [PubMed] [Google Scholar]
  • [7].Barkan A, Small I. Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol. 2014;65(1):415–442. [DOI] [PubMed] [Google Scholar]
  • [8].Matthes A, Schmidt-Gattung S, Kohler D, et al. Two DEAD-box proteins may be part of RNA-dependent high-molecular-mass protein complexes in Arabidopsis mitochondria. Plant Physiol. 2007;145(4):1637–1646. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Kohler D, Schmidt-Gattung S, Binder S. The DEAD-box protein PMH2 is required for efficient group II intron splicing in mitochondria of Arabidopsis thaliana. Plant Mol Biol. 2010;72(4–5):459–467. [DOI] [PubMed] [Google Scholar]
  • [10].Zmudjak M, Colas Des Francs-Small C, Keren I, et al. mCSF1, a nucleus-encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis. New Phytol. 2013;199(2):379–394. . [DOI] [PubMed] [Google Scholar]
  • [11].Lee K, Park SJ, Park YI, et al. CFM9, a Mitochondrial CRM Protein, is crucial for mitochondrial intron splicing, mitochondria function and Arabidopsis growth and stress responses. Plant Cell Physiol. 2019;60(11):2538–2548. [DOI] [PubMed] [Google Scholar]
  • [12].Chettoor AM, Yi G, Gomez E, et al. A putative plant organelle RNA recognition protein gene is essential for maize kernel development. J Integr Plant Biol. 2015;57(3):236–246. . [DOI] [PubMed] [Google Scholar]
  • [13].Des Francs-Small CC, Kroeger T, Zmudjak M, et al. A PORR domain protein required for rpl2 and ccmFC intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria. Plant J. 2012;69(6):996–1005. . [DOI] [PubMed] [Google Scholar]
  • [14].Hsu YW, Wang HJ, Hsieh MH, et al. Arabidopsis mTERF15 is required for mitochondrial nad2 intron 3 splicing and functional complex I activity. PLoS One. 2014;9(11):e112360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Kuhn K, Carrie C, Giraud E, et al. The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. Plant J. 2011;67(6):1067–1080. . [DOI] [PubMed] [Google Scholar]
  • [16].Keren I, Bezawork-Geleta A, Kolton M, et al. AtnMat2, a nuclear-encoded maturase required for splicing of group II introns in Arabidopsis mitochondria. RNA. 2009;15(12):2299–2311. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Sultan LD, Mileshina D, Grewe F, et al. The reverse transcriptase/RNA maturase protein MatR is required for the splicing of various group II Introns in brassicaceae mitochondria. Plant Cell. 2016;28(11):2805–2829. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Small ID, Peeters N. The PPR motif - a TPR-related motif prevalent in plant organellar proteins. Trends Biochem Sci. 2000;25(2):46–47. [DOI] [PubMed] [Google Scholar]
  • [19].Lurin C, Andres C, Aubourg S, et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell. 2004;16(8):2089–2103. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Barkan A, Rojas M, Fujii S, et al. A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet. 2012;8(8):e1002910. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Takenaka M, Zehrmann A, Brennicke A, et al. Improved computational target site prediction for pentatricopeptide repeat RNA editing factors. PLoS One. 2013;8(6):e65343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Yagi Y, Hayashi S, Kobayashi K, et al. Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS One. 2013;8(3):e57286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Cheng SF, Gutmann B, Zhong X, et al. Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J. 2016;85(4):532–547. . [DOI] [PubMed] [Google Scholar]
  • [24].Fearnley IM, Runswick MJ, Walker JE. A homolog of the nuclear coded 49 Kd subunit of Bovine mitochondrial NADH-ubiquinone reductase is coded in chloroplast DNA. Embo J. 1989;8(3):665–672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Preis D, Vanderpas JC, Nehls U, et al. The 49 K subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria: primary structure of the gene and the protein. Current Genet. 1990;18(1):59–64. [DOI] [PubMed] [Google Scholar]
  • [26].Bonen L, Williams K, Bird S, et al. The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome. Mol Gen Genet. 1994;244(1):81–89. [DOI] [PubMed] [Google Scholar]
  • [27].Hsieh WY, Liao JC, Chang CY, et al. The SLOW GROWTH3 pentatricopeptide repeat protein is required for the splicing of mitochondrial NADH dehydrogenase subunit7 intron 2 in arabidopsis. Plant Physiol. 2015;168(2):490–501. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Haili N, Planchard N, Arnal N, et al. The MTL1 pentatricopeptide repeat protein is required for both translation and splicing of the mitochondrial NADH DEHYDROGENASE subunit 7 SUBUNIT7 mRNA in arabidopsis. Plant Physiol. 2016;170(1):354–366. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Chen Z, Wang HC, Shen J, et al. PPR-SMR1 is required for the splicing of multiple mitochondrial introns, interacts with Zm-mCSF1, and is essential for seed development in maize. J Exp Bot. 2019;70(19):5245–5258. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Koprivova A, Des Francs-Small CC, Calder G, et al. Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts. J Biol Chem. 2010;285(42):32192–32199. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Clifton SW, Minx P, Fauron CM, et al. Sequence and comparative analysis of the maize NB mitochondrial genome. Plant Physiol. 2004;136(3):3486–3503. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Rajkowitsch L, Chen D, Stampfl S, et al. RNA chaperones, RNA annealers and RNA helicases. RNA Biol. 2007;4(3):118–130. . [DOI] [PubMed] [Google Scholar]
  • [33].Blocker FJ, Mohr G, Conlan LH, et al. Domain structure and three-dimensional model of a group II intron encoded reverse transcriptase. RNA. 2005;11(1):14–28. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Asakura Y, Barkan A. A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts. Plant Cell. 2007;19(12):3864–3875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Zmudjak M, Shevtsov S, Sultan LD, et al. Analysis of the roles of the Arabidopsis nMAT2 and PMH2 proteins provided with new insights into the regulation of group II intron splicing in land plant mitochondria. Int J Mol Sci. 2017;18(11):2428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Sandoval R, Boyd RD, Kiszter AN, et al. Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2. Plant J. 2019;99(6):1116–1126. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Sosso D, Mbelo S, Vernoud V, et al. PPR2263, a DYW-subgroup pentatricopeptide repeat protein, is required for mitochondrial nad5 and cob transcript editing, mitochondrion biogenesis, and maize growth. Plant Cell. 2012;24(2):676–691. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Kobayashi Y, Knoop V, Fukuzawa H, et al. Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha. Mol Gen Genet. 1997;256(5):589–592. [DOI] [PubMed] [Google Scholar]
  • [39].Covello PS, Gray MW. Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome c oxidase (cox2) in soybean: evidence for RNA-mediated gene transfer. Embo J. 1992;11(11):3815–3820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Nugent JM, Palmer JD. RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution. Cell. 1991;66(3):473–481. [DOI] [PubMed] [Google Scholar]
  • [41].Grohmann L, Brennicke A, Schuster W. The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera. Nucleic Acids Res. 1992;20(21):5641–5646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Wischmann C, Schuster W. Transfer of rps10 from the mitochondrion to the nucleus in Arabidopsis thaliana: evidence for RNA mediated transfer and exon shuffling at the integration site. FEBS Lett. 1995;374(2):152–156. [DOI] [PubMed] [Google Scholar]
  • [43].Kadowaki KI, Kubo N, Ozawa K, et al. Targeting presequence acquisition after mitochondrial gene transfer to the nucleus occurs by duplication of existing targeting signals. Embo J. 1996;15(23):6652–6661. [PMC free article] [PubMed] [Google Scholar]
  • [44].McCarty DR, Settles AM, Suzuki M, et al. Steady-state transposon mutagenesis in inbred maize. Plant J. 2005;44(1):52–61. . [DOI] [PubMed] [Google Scholar]
  • [45].Yang YZ, Ding S, Wang HC, et al. The pentatricopeptide repeat protein EMP9 is required for mitochondrial ccmB and rps4 transcript editing, mitochondrial complex biogenesis and seed development in maize. New Phytol. 2017;214(2):782–795. . [DOI] [PubMed] [Google Scholar]
  • [46].Gutierrez-Marcos JF, Dal Pra M, Giulini A, et al. empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize. Plant Cell. 2007;19(1):196–210. . [DOI] [PMC free article] [PubMed] [Google Scholar]

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