A genome-wide association study uncovers a ZmRap2.7-ZCN9/ZCN10 module to regulate ABA signalling and seed vigour in maize.

Seed vigour, including rapid, uniform germination and robust seedling establishment under various field conditions, is becoming an increasingly essential agronomic trait for achieving high yield in crops. However, little is known about this important seed quality trait. In this study, we performed a genome-wide association study to identify a key transcription factor ZmRap2.7, which regulates seed vigour through transcriptionally repressing expressions of three ABA signalling genes ZmPYL3, ZmPP2C and ZmABI5 and two phosphatidylethanolamine-binding genes ZCN9 and ZCN10. In addition, ZCN9 and ZCN10 proteins could interact with ZmPYL3, ZmPP2C and ZmABI5 proteins, and loss-of-function of ZmRap2.7 and overexpression of ZCN9 and ZCN10 reduced ABA sensitivity and seed vigour, suggesting a complex regulatory network for regulation of ABA signalling mediated seed vigour. Finally, we showed that four SNPs in ZmRap2.7 coding region influenced its transcriptionally binding activity to the downstream gene promoters. Together with previously identified functional variants within and surrounding ZmRap2.7, we concluded that the distinct allelic variations of ZmRap2.7 were obtained independently during maize domestication and improvement, and responded separately for the diversities of seed vigour, flowering time and brace root development. These results provide novel genes, a new regulatory network and an evolutional mechanism for understanding the molecular mechanism of seed vigour.

ZmPTOX1, a plastid terminal oxidase, contributes to redox homeostasis during seed development and germination.

Maize plastid terminal oxidase1 (ZmPTOX1) plays a pivotal role in seed development by upholding redox balance within seed plastids. This study focuses on characterizing the white kernel mutant 3735 (wk3735) mutant, which yields pale-yellow seeds characterized by heightened protein but reduced carotenoid levels, along with delayed germination compared to wild-type (WT) seeds. We successfully cloned and identified the target gene ZmPTOX1, responsible for encoding maize PTOX-a versatile plastoquinol oxidase and redox sensor located in plastid membranes. While PTOX's established role involves regulating redox states and participating in carotenoid metabolism in Arabidopsis leaves and tomato fruits, our investigation marks the first exploration of its function in storage organs lacking a photosynthetic system. Through our research, we validated the existence of plastid-localized ZmPTOX1, existing as a homomultimer, and established its interaction with ferredoxin-NADP+ oxidoreductase 1 (ZmFNR1), a crucial component of the electron transport chain (ETC). This interaction contributes to the maintenance of redox equilibrium within plastids. Our findings indicate a propensity for excessive accumulation of reactive oxygen species (ROS) in wk3735 seeds. Beyond its known role in carotenoids' antioxidant properties, ZmPTOX1 also impacts ROS homeostasis owing to its oxidizing function. Altogether, our results underscore the critical involvement of ZmPTOX1 in governing seed development and germination by preserving redox balance within the seed plastids.

Transcriptome Analysis Revealed the Potential Molecular Mechanism of Anthocyanidins' Improved Salt Tolerance in Maize Seedlings.

Anthocyanin, a kind of flavonoid, plays a crucial role in plant resistance to abiotic stress. Salt stress is a kind of abiotic stress that can damage the growth and development of plant seedlings. However, limited research has been conducted on the involvement of maize seedlings in salt stress resistance via anthocyanin accumulation, and its potential molecular mechanism is still unclear. Therefore, it is of great significance for the normal growth and development of maize seedlings to explore the potential molecular mechanism of anthocyanin improving salt tolerance of seedlings via transcriptome analysis. In this study, we identified two W22 inbred lines (tolerant line pur-W22 and sensitive line bro-W22) exhibiting differential tolerance to salt stress during seedling growth and development but showing no significant differences in seedling characteristics under non-treatment conditions. In order to identify the specific genes involved in seedlings' salt stress response, we generated two recombinant inbred lines (RILpur-W22 and RILbro-W22) by crossing pur-W22 and bro-W22, and then performed transcriptome analysis on seedlings grown under both non-treatment and salt treatment conditions. A total of 6100 and 5710 differentially expressed genes (DEGs) were identified in RILpur-W22 and RILbro-W22 seedlings, respectively, under salt-stressed conditions when compared to the non-treated groups. Among these DEGs, 3160 were identified as being present in both RILpur-W22 and RILbro-W22, and these served as commonly stressed EDGs that were mainly enriched in the redox process, the monomer metabolic process, catalytic activity, the plasma membrane, and metabolic process regulation. Furthermore, we detected 1728 specific DEGs in the salt-tolerant RILpur-W22 line that were not detected in the salt-sensitive RILbro-W22 line, of which 887 were upregulated and 841 were downregulated. These DEGs are primarily associated with redox processes, biological regulation, and the plasma membrane. Notably, the anthocyanin synthesis related genes in RILpur-W22 were strongly induced under salt treatment conditions, which was consistented with the salt tolerance phenotype of its seedlings. In summary, the results of the transcriptome analysis not only expanded our understanding of the complex molecular mechanism of anthocyanin in improving the salt tolerance of maize seedlings, but also, the DEGs specifically expressed in the salt-tolerant line (RILpur-W22) provided candidate genes for further genetic analysis.

RNA polymerase common subunit ZmRPABC5b is transcriptionally activated by Opaque2 and essential for endosperm development in maize.

Maize (Zea mays) kernel size is an important factor determining grain yield; although numerous genes regulate kernel development, the roles of RNA polymerases in this process are largely unclear. Here, we characterized the defective kernel 701 (dek701) mutant that displays delayed endosperm development but normal vegetative growth and flowering transition, compared to its wild type. We cloned Dek701, which encoded ZmRPABC5b, a common subunit to RNA polymerases I, II and III. Loss-of-function mutation of Dek701 impaired the function of all three RNA polymerases and altered the transcription of genes related to RNA biosynthesis, phytohormone response and starch accumulation. Consistent with this observation, loss-of-function mutation of Dek701 affected cell proliferation and phytohormone homeostasis in maize endosperm. Dek701 was transcriptionally regulated in the endosperm by the transcription factor Opaque2 through binding to the GCN4 motif within the Dek701 promoter, which was subjected to strong artificial selection during maize domestication. Further investigation revealed that DEK701 interacts with the other common RNA polymerase subunit ZmRPABC2. The results of this study provide substantial insight into the Opaque2-ZmRPABC5b transcriptional regulatory network as a central hub for regulating endosperm development in maize.

Integrated Multi-Omics Reveals Significant Roles of Non-Additively Expressed Small RNAs in Heterosis for Maize Plant Height.

Heterosis is a complex biological phenomenon regulated by genetic variations and epigenetic changes. However, the roles of small RNAs (sRNAs), an important epigenetic regulatory element, on plant heterosis are still poorly understood. Here, an integrative analysis was performed with sequencing data from multi-omics layers of maize hybrids and their two homologous parental lines to explore the potential underlying mechanisms of sRNAs in plant height (PH) heterosis. sRNAome analysis revealed that 59 (18.61%) microRNAs (miRNAs) and 64,534 (54.00%) 24-nt small interfering RNAs (siRNAs) clusters were non-additively expressed in hybrids. Transcriptome profiles showed that these non-additively expressed miRNAs regulated PH heterosis through activating genes involved in vegetative growth-related pathways while suppressing those related to reproductive and stress response pathways. DNA methylome profiles showed that non-additive methylation events were more likely to be induced by non-additively expressed siRNA clusters. Genes associated with low-parental expression (LPE) siRNAs and trans-chromosomal demethylation (TCdM) events were enriched in developmental processes as well as nutrients and energy metabolism, whereas genes associated with high-parental expression (HPE) siRNAs and trans-chromosomal methylation (TCM) events were gathered in stress response and organelle organization pathways. Our results provide insights into the expression and regulation patterns of sRNAs in hybrids and help to elucidate their potential targeting pathways contributing to PH heterosis.

Combined population transcriptomic and genomic analysis reveals cis-regulatory differentiation of non-coding RNAs in maize.

KEY MESSAGE: Long intergenic non-coding RNA (lincRNA), cis-acting expression quantitative trait locus (cis-eQTL), maize, regulatory evolution. The law of genetic variation during domestication explains the evolutionary mechanism and provides a theoretical basis for improving existing varieties of maize. Previous studies focused on exploiting regulatory variations controlling the expression of protein-coding genes rather than of non-protein-coding genes. Here, we examined the genetic and evolutionary features of long non-coding RNAs from intergenic regions (long intergenic non-coding RNAs, lincRNAs) using population-scale transcriptome data and identified 1168 lincRNAs with cis-acting expression quantitative trait loci (cis-eQTLs). We found that lincRNAs are more likely to be regulated by cis-eQTLs, which exert stronger effects than the protein-coding genes. During maize domestication and improvement, upregulated alleles of lincRNAs, which originated from both standing variation and new mutation, accumulate more frequently and show larger effect sizes than the coding genes. A stronger signature of genetic differentiation was observed in their regulatory regions compared to those of randomly sampled lincRNAs. In addition, we found that cis-regulatory differentiation of lincRNAs is related to the sequence conservation of lincRNA transcripts. Non-conserved lincRNAs more tend to gain upregulated alleles and show a stronger relationship with selected traits than conserved lincRNAs between maize and its wild relatives. Our findings in maize improve the understanding of cis-regulatory variation in lincRNA genes during domestication and improvement and provide an effective approach for prioritizing candidates for further investigation.

The Anthocyanin Accumulation Related ZmBZ1, Facilitates Seedling Salinity Stress Tolerance via ROS Scavenging.

Anthocyanins are a class of antioxidants that scavenge free radicals in cells and play an important role in promoting human health and preventing many diseases. Here, we characterized a maize Bronze gene (BZ1) from the purple colored W22 introgression line, which encodes an anthocyanin 3-O-glucosyltransferase, a key enzyme in the anthocyanin synthesis pathway. Mutation of ZmBZ1 showed bronze-colored seeds and reduced anthocyanins in seeds aleurone layer, seedlings coleoptile, and stem of mature plants by comparison with purple colored W22 (WT). Furthermore, we proved that maize BZ1 is an aleurone layer-specific expressed protein and sub-located in cell nucleus. Real-time tracing of the anthocyanins in developing seeds demonstrated that the pigment was visible from 16 DAP (day after pollination) in field condition, and first deposited in the crown part then spread all over the seed. Additionally, it was transferred along with the embryo cell activity during seed germination, from aleurone layer to cotyledon and coleoptile, as confirmed by microscopy and real-time qRT-PCR. Finally, we demonstrated that the ZmBZ1 contributes to stress tolerance, especially salinity. Further study proved that ZmBZ1 participates in reactive oxygen scavenging (ROS) by accumulating anthocyanins, thereby enhancing the tolerance to abiotic stress.

PNGSeqR: An R Package for Rapid Candidate Gene Selection through Pooled Next-Generation Sequencing.

Although bulked segregant analysis (BSA) has been used extensively in genetic mapping, user-friendly tools which can integrate current algorithms for researchers with no background in bioinformatics are scarce. To address this issue, we developed an R package, PNGSeqR, which takes single-nucleotide polymorphism (SNP) markers from next-generation sequencing (NGS) data in variant call format (VCF) as the input file, provides four BSA algorithms to indicate the magnitude of genome-wide signals, and rapidly defines the candidate region through the permutation test and fractile quantile. Users can choose the analysis methods according to their data and experimental design. In addition, it also supports differential expression gene analysis (DEG) and gene ontology analysis (GO) to prioritize the target gene. Once the analysis is completed, the plots can conveniently be exported.

A model for genuineness detection in genetically and phenotypically similar maize variety seeds based on hyperspectral imaging and machine learning.

BACKGROUND: Variety genuineness and purity are essential indices of maize seed quality that affect yield. However, detection methods for variety genuineness are time-consuming, expensive, require extensive training, or destroy the seeds in the process. Here, we present an accurate, high-throughput, cost-effective, and non-destructive method for screening variety genuineness that uses seed phenotype data with machine learning to distinguish between genetically and phenotypically similar seed varieties. Specifically, we obtained image data of seed morphology and hyperspectral reflectance for Jingke 968 and nine other closely-related varieties (non-Jingke 968). We then compared the robustness of three common machine learning algorithms in distinguishing these varieties based on the phenotypic imaging data. RESULTS: Our results showed that hyperspectral imaging (HSI) combined with a multilayer perceptron (MLP) or support vector machine (SVM) model could distinguish Jingke 968 from varieties that differed by as few as two loci, with a 99% or higher accuracy, while machine vision imaging provided ~ 90% accuracy. Through model validation and updating with varieties not included in the training data, we developed a genuineness detection model for Jingke 968 that effectively discriminated between genetically similar and distant varieties. CONCLUSIONS: This strategy has potential for wide adoption in large-scale variety genuineness detection operations for internal quality control or governmental regulatory agencies, or for accelerating the breeding of new varieties. Besides, it could easily be extended to other target varieties and other crops.

Epigenetic Mutation in a Tubulin-Folding Cofactor B (ZmTFCB) Gene Arrests Kernel Development in Maize.

Epialleles, the heritable epigenetic variants that are not caused by changes in DNA sequences, can broaden genetic and phenotypic diversity and benefit to crop breeding, but very few epialleles related to agricultural traits have been identified in maize. Here, we cloned a small kernel mutant, smk-wl10, from maize, which encoded a tubulin-folding cofactor B (ZmTFCB) protein. Expression of the ZmTFCB gene decreased in the smk-wl10 mutant, which arrested embryo, endosperm and basal endosperm transfer layer developments. Overexpression of ZmTFCB could complement the defective phenotype of smk-wl10. No nucleotide sequence variation in ZmTFCB could be found between smk-wl10 and wild type (WT). Instead, we detected hypermethylation of nucleotide CHG (where H is A, C or T nucleotide) sequence contexts and increased level of histone H3K9me2 methylation in the upstream sequence of ZmTFCB in smk-wl10 compared with WT, which might respond to the attenuating transcription of ZmTFCB. In addition, yeast two-hybrid and bimolecular fluorescence complementation assays identified a strong interaction between ZmTFCB and its homolog ZmTFCE. Thus, our work identifies a novel epiallele of the maize ZmTFCB gene, which might represent a common phenomenon in the epigenetic regulation of important traits such as kernel development in maize.

Dissection of the Genetic Basis of Yield Traits in Line per se and Testcross Populations and Identification of Candidate Genes for Hybrid Performance in Maize.

Dissecting the genetic basis of yield traits in hybrid populations and identifying the candidate genes are important for molecular crop breeding. In this study, a BC1F3:4 population, the line per se (LPS) population, was constructed by using elite inbred lines Zheng58 and PH4CV as the parental lines. The population was genotyped with 55,000 SNPs and testcrossed to Chang7-2 and PH6WC (two testers) to construct two testcross (TC) populations. The three populations were evaluated for hundred kernel weight (HKW) and yield per plant (YPP) in multiple environments. Marker-trait association analysis (MTA) identified 24 to 151 significant SNPs in the three populations. Comparison of the significant SNPs identified common and specific quantitative trait locus/loci (QTL) in the LPS and TC populations. Genetic feature analysis of these significant SNPs proved that these SNPs were associated with the tested traits and could be used to predict trait performance of both LPS and TC populations. RNA-seq analysis was performed using maize hybrid varieties and their parental lines, and differentially expressed genes (DEGs) between hybrid varieties and parental lines were identified. Comparison of the chromosome positions of DEGs with those of significant SNPs detected in the TC population identified potential candidate genes that might be related to hybrid performance. Combining RNA-seq analysis and MTA results identified candidate genes for hybrid performance, providing information that could be useful for maize hybrid breeding.

Transcriptome Analysis of Near-Isogenic Lines Provides Novel Insights into Genes Associated with Seed Low-Temperature Germination Ability in Maize (Zea mays L.).

Maize originated from tropical regions and is extremely sensitive to low temperature during germination. Our previous work identified a major QTL, qp1ER1-1, for low temperature germination ability (LTGA) of maize. Here, we introgressed qp1ER1-1 from the tolerant line L220 into the sensitive line PH4CV to generate two near isogenic lines NIL220-3 and NIL220-25. When germinated under cold temperature for 25 days (Cold-25), the NILs showed similar seedling root length and shoot length to L220, but significantly higher than PH4CV. However, when germinated under cold temperature for 15 days (Cold-15) or under normal temperature (25 °C) for 3 days (CK-3), all lines showed similar seedling performance, indicating that introgression of qp1ER1-1 from L220 into PH4CV could improve LTGA of NIL220-3 and NIL220-25. The whole seedlings, including root and shoot, of Cold-15 and CK-3 were harvested for transcriptome analysis, when both stayed at a similar developmental stage. Dry seed embryo was sequenced as a non-germination control (CK-0). Compared with PH4CV, the tolerant line (L220, NIL220-3, and NIL220-25) specifically expressed genes (different expressed genes, DEGs) were identified for CK-0, Cold-15, and CK-3. Then, DEGs identified from Cold-15, but not from CK-0 or CK-3, were defined as tolerant line specifically expressed LTGA genes. Finally, 1786, 174, and 133 DEGs were identified as L220, NIL220-3, and NIL220-25 specifically expressed LTGA genes, respectively. Of them, 27 were common LTGA genes that could be identified from all three tolerant lines, with two (Zm00001d031209 and Zm00001d031292) locating in the confidence interval of qp1ER1-1. In addition, GO analysis revealed that L220 specifically expressed LTGA genes were majorly enriched in the cell division process and plasma membrane related categories. Taken together, these results provided new insight into the molecular mechanism of maize seed LTGA and facilitated the cloning of the qp1ER1-1 gene.

The ideal harvest time for seed production in maize (Zea mays L.) varieties of different maturity groups.

BACKGROUND: The correct time for harvesting is a key factor contributing to the production of high-quality maize seeds. We conducted field experiments to harvest seeds at 11 developmental stages for 3 years, to investigate seed vigor traits in three early maturity maize varieties and two late maturity varieties in one location. RESULTS: Significant correlations (r = 0.72 ~ 0.89) were found among six seed-related traits: standard germination (SG), accelerated aging germination (AAG), cold test germination (CTG), hundred-seed weight (HSW), seed moisture content (SMC), and ≥ 10 °C accumulated temperature from pollination to harvest (AT10). Analysis of variance showed that harvest stage, year, and variety had significant effects on all traits, and harvest stage displayed the greatest effect. The responses of SG, AAG, CTG, HSW and SMC to harvest stage fitted quadratic models, and AT10 fitted a linear model. From the quadratic models, an ideal harvest time (IHT, the final date to reach maximum SG, AAG, and CTG) could be calculated for each variety. The three early maturity varieties reached their IHT at 54.94-58.44 days after pollination (DAP); the two later maturity varieties reached IHT several days later (at 59.87-59.90 DAP). The early maturity varieties consistently required less AT10 to reach the IHT than the later maturity varieties. However, all of the varieties reached the IHT at similar SMC levels of about 35%. CONCLUSION: The later maturity varieties reached the IHT at later DAPs when they acquired more AT10 than the early maturity varieties but both reached it at similar SMC levels. © 2022 Society of Chemical Industry.

Dek504 Encodes a Mitochondrion-Targeted E+-Type Pentatricopeptide Repeat Protein Essential for RNA Editing and Seed Development in Maize.

In flowering plants, RNA editing is a post-transcriptional process that selectively deaminates cytidines (C) to uridines (U) in organellar transcripts. Pentatricopeptide repeat (PPR) proteins have been identified as site-specific recognition factors for RNA editing. Here, we report the map-based cloning and molecular characterization of the defective kernel mutant dek504 in maize. Loss of Dek504 function leads to delayed embryogenesis and endosperm development, which produce small and collapsed kernels. Dek504 encodes an E+-type PPR protein targeted to the mitochondria, which is required for RNA editing of mitochondrial NADH dehydrogenase 3 at the nad3-317 and nad3-44 sites. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the mitochondrial NADH dehydrogenase complex I activity, indicating that the alteration of the amino acid sequence at nad3-44 and nad3-317 through RNA editing is essential for NAD3 function. Moreover, the amino acids are highly conserved in monocots and eudicots, whereas the events of C-to-U editing are not conserved in flowering plants. Thus, our results indicate that Dek504 is essential for RNA editing of nad3, which is critical for NAD3 function, mitochondrial complex I stability, and seed development in maize.

Transcriptome Analysis Identifies Novel Genes Associated with Low-Temperature Seed Germination in Sweet Corn.

Typically, sweet corn, particularly sh2 sweet corn, has low seed vigor owing to its high sugar and low starch content, which is a major problem in sweet corn production, particularly at low temperatures. There is considerable variation in the germination rates among sweet corn varieties under low-temperature conditions, and the underlying mechanisms behind this phenomenon remain unclear. In this study, we screened two inbred sweet corn lines (tolerant line L282 and sensitive line L693) differing in their low-temperature germination rates; while no difference was observed in their germination rates at normal temperatures. To identify the specifically induced genes influencing the germination capacity of sweet corn at low temperatures, a transcriptome analysis of the two lines was conducted at both normal and low temperatures. Compared to the lines at a normal temperature, 3926 and 1404 differently expressed genes (DEGs) were identified from L282 and L693, respectively, under low-temperature conditions. Of them, 830 DEGs were common DEGs (cDEGs) that were identified from both L282 and L693, which were majorly enriched in terms of microtubule-based processes, histone H3-K9 modification, single-organism cellular processes, and carbohydrate metabolic processes. In addition, 3096 special DEGs (sDEGs), with 2199 upregulated and 897 downregulated, were detected in the tolerant line L282, but not in the sensitive line L693. These sDEGs were primarily related to plasma membranes and oxygen-containing compounds. Furthermore, electric conductivity measurements demonstrated that the membrane of L282 experienced less damage, which is consistent with its strong tolerance at low temperatures. These results expand our understanding of the complex mechanisms involved in the cold germination of sweet corn and provide a set of candidate genes for further genetic analysis.

Genomic Prediction across Structured Hybrid Populations and Environments in Maize.

Genomic prediction (GP) across different populations and environments should be enhanced to increase the efficiency of crop breeding. In this study, four populations were constructed and genotyped with DNA chips containing 55,000 SNPs. These populations were testcrossed to a common tester, generating four hybrid populations. Yields of the four hybrid populations were evaluated in three environments. We demonstrated by using real data that the prediction accuracies of GP across structured hybrid populations were lower than those of within-population GP. Including relatives of the validation population in the training population could increase the prediction accuracies of GP across structured hybrid populations drastically. G × E models (including main and genotype-by-environment effect) had better performance than single environment (within environment) and across environment (including only main effect) GP models in the structured hybrid population, especially in the environment where yields had higher heritability. GP by implementing G × E models in two cross-validation schemes indicated that, to increase the prediction accuracy of a new hybrid line, it would be better to field-test the hybrid line in at least one environment. Our results would be helpful for designing training population and planning field testing in hybrid breeding.

Small kernel 501 (smk501) encodes the RUBylation activating enzyme E1 subunit ECR1 (E1 C-TERMINAL RELATED 1) and is essential for multiple aspects of cellular events during kernel development in maize.

RUBylation plays essential roles in plant growth and development through regulating Cullin-RING ubiquitin E3 ligase (CRL) activities and the CRL-mediated protein degradations. However, the function of RUBylation in regulating kernel development remains unclear. Through genetic and molecular analyses of a small kernel 501 (smk501) mutant in maize (Zea mays), we cloned the smk501 gene, revealed its molecular function, and defined its roles in RUBylation pathway and seed development. Smk501 encodes a RUBylation activating enzyme E1 subunit ZmECR1 (E1 C-TERMINAL RELATED 1) protein. Destruction in RUBylation by smk501 mutation resulted in less embryo and endosperm cell number and smaller kernel size. The transcriptome and proteome profiling, hormone evaluation and cell proliferation observation revealed that disturbing ZmECR1 expression mainly affects pathways on hormone signal transduction, cell cycle progression and starch accumulation during kernel development. In addition, mutant in zmaxr1 (Auxin resistant 1), another RUB E1 subunit, also showed similar defects in kernel development. Double mutation of zmecr1 and zmaxr1 lead to empty pericarp kernel phenotype. RUBylation is a novel regulatory pathway affecting maize kernel development, majorly through its functions in modifying multiple cellular progresses.

Nuclear-Encoded Maturase Protein 3 Is Required for the Splicing of Various Group II Introns in Mitochondria during Maize (Zea mays L.) Seed Development.

Splicing of plant organellar group II introns from precursor-RNA transcripts requires the assistance of nuclear-encoded splicing factors. Maturase (nMAT) is one such factor, as its three homologs (nMAT1, 2 and 4) have been identified as being required for the splicing of various mitochondrial introns in Arabidopsis. However, the function of nMAT in maize (Zea mays L.) is unknown. In this study, we identified a seed development mutant, empty pericarp 2441 (emp2441) from maize, which showed severely arrested embryogenesis and endosperm development. Positional cloning and transgenic complementation assays revealed that Emp2441 encodes a maturase-related protein, ZmnMAT3. ZmnMAT3 is highly expressed during seed development and its protein locates to the mitochondria. The loss of function of ZmnMAT3 resulted in the reduced splicing efficiency of various mitochondrial group II introns, particularly of the trans-splicing of nad1 introns 1, 3 and 4, which consequently abolished the transcript of nad1 and severely impaired the assembly and activity of mitochondrial complex I. Moreover, the Zmnmat3 mutant showed defective mitochondrial structure and exhibited expression and activity of alternative oxidases. These results indicate that ZmnMAT3 is essential for mitochondrial complex I assembly during kernel development in maize.

Maize Defective Kernel605 Encodes a Canonical DYW-Type PPR Protein that Edits a Conserved Site of nad1 and Is Essential for Seed Nutritional Quality.

Pentatricopeptide repeat (PPR) proteins involved in mitochondrial RNA cytidine (C)-to-uridine (U) editing mostly result in stagnant embryo and endosperm development upon loss of function. However, less is known about PPRs that are involved in farinaceous endosperm formation and maize quality. Here, we cloned a maize DYW-type PPR Defective Kernel605 (Dek605). Mutation of Dek605 delayed seed and seedling development. Mitochondrial transcript analysis of dek605 revealed that loss of DEK605 impaired C-to-U editing at the nad1-608 site and fails to alter Ser203 to Phe203 in NAD1 (dehydrogenase complex I), disrupting complex I assembly and reducing NADH dehydrogenase activity. Meanwhile, complexes III and IV in the cytochrome pathway, as well as AOX2 in the alternative respiratory pathway, are dramatically increased. Interestingly, the dek605 mutation resulted in opaque endosperm and increased levels of the free amino acids alanine, aspartic acid and phenylalanine. The down- and upregulated genes mainly involved in stress response-related and seed dormancy-related pathways, respectively, were observed after transcriptome analysis of dek605 at 12 d after pollination. Collectively, these results indicate that Dek605 specifically affects the single nad1-608 site and is required for normal seed development and resulted in nutritional quality relevant amino acid accumulation.