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C1-FDX (Complex I-ferredoxin) has been defined as a component of CI in a ferredoxin bridge in Arabidopsis mitochondria. However, its full function remains to be addressed. We created two c1-fdx mutants in Arabidopsis using the CRISPR-Cas9 methodology. The mutants show delayed seed germination. Over-expression of C1-FDX rescues the phenotype. Molecular analyses showed that loss of the C1-FDX function decreases the abundance and activity of both CI and subcomplexes of CV. In contrast, the over-expression of C1-FDX-GFP enhances the CI* (a sub-complex of CI) and CV assembly. Immunodetection reveals that the stoichiometric ratio of the α:β subunits in the F1 module of CV is altered in the c1-fdx mutant. In the complemented mutants, C1-FDX-GFP was found to be associated with the F' and α/β sub-complexes of CV. Protein interaction assays showed that C1-FDX could interact with the β, γ, δ, and ε subunits of the F1 module, indicating that C1-FDX, a structural component of CI, also functions as an assembly factor in the assembly of F' and α/β sub-complexes of CV. These results reveal a new role of C1-FDX in the CI and CV assembly and seed germination in Arabidopsis. Author summary: The mitochondrial oxidative phosphorylation (OXPHOS) system is composed of five large multiprotein complexes, complex I to V (CI to CV). However, their assembly is not fully understood in plants. C1-FDX, a ferredoxin protein, was found to form a ferredoxin bridge in CI. Here, we show that C1-FDX is also involved in the assembly of CI* and CV. CI* is an important intermedate of CI. Mutation of C1-FDX affects the assembly of these two complexes and delays the germination of Arabidopsis. C1-FDX interacts with several CV subunits, including the β, γ, δ, and ε subunits of the F1 sub-complex, and physically associates with the CV assembly intermediates, sub-complexes F' and α/β, but not with the mature CV, indicating that C1-FDX is dissociated when the CV is fully assembled. This study provides insights into the dual-function of CI-FDX in CI and CV assembly, facilitating the understanding of the CI and CV assembly mechanism in plants. [ABSTRACT FROM AUTHOR]

C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins). [ABSTRACT FROM AUTHOR]

The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3. [ABSTRACT FROM AUTHOR]

Plastid gene expression is essential to embryogenesis in higher plants, but the underlying mechanism is obscure. Through molecular characterization of an embryo defective 16 (emb16) locus, here we report that the requirement of plastid translation for embryogenesis is dependent on the genetic background in maize (Zea mays). The emb16 mutation arrests embryogenesis at transition stage and allows the endosperm to develop largely normally. Molecular cloning reveals that Emb16 encodes WHIRLY1 (WHY1), a DNA/RNA binding protein that is required for genome stability and ribosome formation in plastids. Interestingly, the previous why1 mutant alleles (why1-1 and why1-2) do not affect embryogenesis, only conditions albino seedlings. The emb16 allele of why1 mutation is in the W22 genetic background. Crosses between emb16 and why1-1 heterozygotes resulted in both defective embryos and albino seedlings in the F1 progeny. Introgression of the emb16 allele from W22 into A188, B73, Mo17, Oh51a and the why1-1 genetic backgrounds yielded both defective embryos and albino seedlings. Similar results were obtained with two other emb mutants (emb12 and emb14) that are impaired in plastid protein translation process. These results indicate that the requirement of plastid translation for embryogenesis is dependent on genetic backgrounds, implying a mechanism of embryo lethality suppression in maize. [ABSTRACT FROM AUTHOR]

The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3.