Maize Mutant Research Articles

Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death.

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

Grasviq: an image analysis framework for automatically quantifying vein number and morphology in grass leaves.

Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image-based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high-throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species.

Quality protein maize (QPM): Importance, genetics, timeline of different events, breeding strategies and varietal adoption

AbstractLysine and tryptophan are two essential amino acids and these are deficit in maize grain thus posing the problem of nutritional deficiencies in the consumers. A wide range of deficiency symptoms like cognitive disorder, kwashiorkor disease, reduced appetite, impaired skeleton development, delayed growth and aberrant behaviour are associated with lysine and tryptophan deficiency. These amino acids are also important to cure the Pellagra disease. Researchers identified several mutants in maize especially opaque‐2 which are responsible for higher lysine and tryptophan contents. Few years later, it was observed that opaque‐2 mutant has several pleiotropic effects on maize grain and plant. Efforts of researchers are spanning over the period of four decades to develop quality protein maize (QPM). QPM is described as nutritionally superior maize with high lysine and tryptophan contents and desired kernel characteristics as compared to its normal maize counterparts. Biological value of QPM was almost equivalent to egg protein. Breeding of maize for quality protein is based on three genetic systems like opaque‐2 genetic system, endosperm modifier genetic system and associated gene systems. Keeping in view the importance of QPM, current review article is compiled to discuss the genetic basis, genetic systems and breeding strategies. Timeline for various events is also drafted like discovery of various mutants, several conventional and modern approaches for development and deployment of QPM varieties across the world. Despite its nutritional benefits, the rate of adoption of QPM is generally at low pace in the developing world and this review article discuss the challenges and potential opportunities for QPM adoption.

Genetic variability parameters of maize (Zea mays L.) mutant irradiated gamma-ray

Mutation induction often used to increase genetic variability in plant breeding for further proceed. Thirty inbred lines of maize mutants (M1) of were evaluated in a randomized complete block design experiment with three replications for morphology and agronomy of genetic variability parameters. Data were collected on days to anthesis and silking, anthesis-silking interval (ASI), plant height, leaves number, days to maturity, ear number, grain yield, grains number, weight of 100-grain. Mean values were used to determine characters’ phenotypic and genotypic variances, phenotypic, genotypic and environmental coefficients of variation. Moderate heritability estimates were observed for most traits. Otherwise based on genotypic coefficient of variance, for most traits had low genetic variability but generally high genetic variability was observed for most traits based on standard deviation of genotypic variance. Grain yield as important trait had moderate genetic variability based on heritability estimates, high based on genotypic coefficient of variance and standard deviation of genotypic variance. Hence provides better opportunities for selecting plant material regarding these traits.

Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize 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.

High spatial resolution imaging of the dynamics of cuticular lipid deposition during Arabidopsis flower development.

The extensive collection of glossy (gl) and eceriferum (cer) mutants of maize and Arabidopsis have proven invaluable in dissecting the branched metabolic pathways that support cuticular lipid deposition. This bifurcated pathway integrates a fatty acid elongation‐decarbonylative branch and a fatty acid elongation‐reductive branch, which collectively has the capacity to generate hundreds of cuticular lipid metabolites. In this study, a combined transgenic and biochemical strategy was implemented to explore and compare the physiological function of three homologous genes, Gl2, Gl2‐like, and CER2, in the context of this branched pathway. These biochemical characterizations integrated new extraction chromatographic procedures with high spatial resolution mass spectrometric imaging methods to profile the cuticular lipids on developing floral tissues transgenically expressing these transgenes in wild‐type or cer2 mutant lines of Arabidopsis. Collectively, these datasets establish that both the maize Gl2 and Gl2‐like genes are functional homologs of the Arabidopsis CER2 gene. In addition, the dynamic distribution of cuticular lipid deposition follows distinct floral organ localization patterns indicating that the fatty acid elongation‐decarbonylative branch of the pathway is differentially localized from the fatty acid elongation‐reductive branch of the pathway.

Detection of DNA methylation in DBF1 gene of maize inbred W64A and mutant vp14 exposed to drought stress

DNA methylation changes correlated with environmental stresses can confer a rapid adaptation to such challenges. Dehydration-responsive element-binding factor 1 (DBF1), a member of the AP2/EREBP transcriptional factor family, is induced by drought and salinity through an ABA-dependent pathway in maize seedlings. The present investigation was carried out to study the DNA methylation status of the DBF1 gene in two different maize genotypes, an inbred line W64A and a maize mutant vp14, by the methylation-sensitive restriction enzyme–polymerase chain reaction method. Stable DNA methylation upstream of the DBF1 gene in both W64A and vp14 was observed under non-stress and drought stress conditions; simultaneously, the methylation of particular cytosines in the proximal promoter or the transcribed regions was absent.

Identification of virulence associated milRNAs and their bidirectional targets in Rhizoctonia solani and maize during infection

BackgroundAnastomosis group 1 IA (AG1-IA) of Rhizoctonia solani is the major agent of banded leaf and sheath blight (BLSB) disease that causes severe yield loss in many worldwide crops. MicroRNAs (miRNAs) are ~ 22 nt non-coding RNAs that negatively regulate gene expression levels by mRNA degradation or translation inhibition. A better understanding of miRNA function during AG1-IA infection can expedite to elucidate the molecular mechanisms of fungi-host interactions.ResultsIn this study, we sequenced three small RNA libraries obtained from the mycelium of AG1-IA isolate, non-infected maize sheath and mixed maize sheath 3 days after inoculation. In total, 137 conserved and 34 novel microRNA-like small RNAs (milRNAs) were identified from the pathogen. Among these, one novel and 17 conserved milRNAs were identified as potential virulence-associated (VA) milRNAs. Subsequently, the prediction of target genes for these milRNAs was performed in both AG1-IA and maize, while functional annotation of these targets suggested a link to pathogenesis-related biological processes. Further, expression patterns of these virulence-associated milRNAs demonstrated that theyparticipate in the virulence of AG1-IA. Finally, regulation of one maize targeting gene, GRMZM2G412674 for Rhi-milRNA-9829-5p, was validated by dual-luciferase assay and identified to play a positive role in BLSB resistance in two maize mutants. These results suggest the global differentially expressed milRNAs of R. solani AG1-IA that participate in the regulation of target genes in both AG1-IA and maize to reinforce its pathogenicity.ConclusionsOur data have provided a comprehensive overview of the VA-milRNAs of R. solani and identified that they are probably the virulence factors by directly interfered in host targeting genes. These results offer new insights on the molecular mechanisms of R.solani-maize interactions during the process of infection.

A valine residue deletion in ZmSig2A, a sigma factor, accounts for a revertible leaf-color mutation in maize

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val480-Val481-Val482 to Val480-Val481, in the C-terminal domain σ4 of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A+ in WT plants, much higher expression of the mutant allele ZmSig2AΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A+ or ZmSig2AΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2AΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.

Indole-3-glycerolphosphate synthase, a branchpoint for the biosynthesis of tryptophan, indole, and benzoxazinoids in maize.

The maize (Zea mays) genome encodes three indole-3-glycerolphosphate synthase enzymes (IGPS1, 2, and 3) catalyzing the conversion of 1-(2-carboxyphenylamino)-l-deoxyribulose-5-phosphate to indole-3-glycerolphosphate. Three further maize enzymes (BX1, benzoxazinoneless 1; TSA, tryptophan synthase alpha subunit; and IGL, indole glycerolphosphate lyase) convert indole-3-glycerolphosphate to indole, which is released as a volatile defense signaling compound and also serves as a precursor for the biosynthesis of tryptophan and defense-related benzoxazinoids. Phylogenetic analyses showed that IGPS2 is similar to enzymes found in both monocots and dicots, whereas maize IGPS1 and IGPS3 are in monocot-specific clades. Fusions of yellow fluorescent protein with maize IGPS enzymes and indole-3-glycerolphosphate lyases were all localized in chloroplasts. In bimolecular fluorescence complementation assays, IGPS1 interacted strongly with BX1 and IGL, IGPS2 interacted primarily with TSA, and IGPS3 interacted equally with all three indole-3-glycerolphosphate lyases. Whereas IGPS1 and IGPS3 expression was induced by insect feeding, IGPS2 expression was not. Transposon insertions in IGPS1 and IGPS3 reduced the abundance of both benzoxazinoids and free indole. Spodoptera exigua (beet armyworm) larvae show improved growth on igps1 mutant maize plants. Together, these results suggest that IGPS1 and IGPS3 function mainly in the biosynthesis of defensive metabolites, whereas IGPS2 may be involved in the biosynthesis of tryptophan. This metabolic channeling is similar to, though less exclusive than, that proposed for the three maize indole-3-glycerolphosphate lyases.

Transgenerational conditioned male fertility of HD-ZIP IV transcription factor mutant ocl4: impact on 21-nt phasiRNA accumulation in pre-meiotic maize anthers.

Maize Outer cell layer 4 (ocl4) encodes an HD-ZIP IV transcription factor required for robust male fertility and 21-nt phasiRNA biogenesis. ocl4 fertility is favored in warm conditions, and phasiRNAs are partially restored. Environment-sensitive male-sterile plants have been described before and can result from different molecular mechanisms and biological processes, but putative environment-conditioned, transgenerational rescue of their male fertility is a rather new mystery. Here, we report a derivative line of the male-sterile outer cell layer 4 (ocl4) mutant of maize, in which fertility was restored and perpetuated over several generations. Conditioned fertile ocl4 anthers exhibit the anatomical abnormality of a partially duplicated endothecial layer, just like their sterile counterparts. We profiled the dynamics of phased, small interfering RNAs (phasiRNAs) during pre-meiotic development in fully sterile and various grades of semi-fertile ocl4 anthers. The conditioned fertile anthers accumulated significantly higher 21-nt phasiRNAs compared to ocl4 sterile samples, suggesting a partial restoration of phasiRNAs in conditioned fertility. We found that the biogenesis of 21-nt phasiRNAs is largely dependent on Ocl4 at three key steps: (1) production of PHAS precursor transcripts, (2) expression of miR2118 that modulates precursor processing, and (3) accumulation of 21-nt phasiRNAs.

Nitrogen assimilation under osmotic stress in maize (Zea mays L.) seedlings

Nitrogen (N) is one of the most important nutrients affecting maize productivity. The effects of osmotic stress on nitrogen assimilation still remain unclear. The aim of this study is to characterize the physiological and biochemical responses to the N level and examine the expression of the genes involved in the N assimilation pathway under osmotic stress. Maize seedlings were supplied with three N levels; low N (LN, 0.5 mM), moderate N (MN, 10 mM) and high N (HN, 20 mM) with or without 10% of PEG6000. Results showed that osmotic stress reduced photosynthesis, and transpiration rate as well as stomatal conductance while increased N assimilation at HN. The activities of NR and GPT enzymes were negatively correlated with the N level. However, a positive correlation was observed between the activities of other N assimilation related enzymes and HN level under osmotic stress. The relative expression of genes involved directly in nitrate transport, for example, ZmNRT1.2, ZmNRT2.2, and ZmNRT2.3 were higher at LN level. At LN application, the genes involved in the N assimilation pathway, like ZmGln4, ZmGln5, ZmGs1.3, ZmGs1.4, and ZmSupp showed higher expression levels under osmotic stress. Overall, the expression level of osmotic-stress related genes was decreased at HN level. Taken together, we concluded that though the mRNA levels and enzymatic activities of N assimilation related enzymes were varied and not typically correlated at various nitrogen levels under osmotic stress. However, the expression level of major N assimilation related genes could be used as biomarkers for the identification of maize genotypes or mutants with high nitrogen assimilation under osmotic stress.

Evaluation of genetic variability and interrelationships among M3 and M4 maize inbred lines in Kenya

The present study was conducted at Jomo Kenyatta University of Agriculture and Technology, Juja to assess the agronomic performance of mutant maize lines of 39 filial generation 3 (M3) and filial generation 4 (M4) maize lines and a check variety among agro-morphological traits with their association with grain yield. Data on various agro-morphological characters were recorded using morphological descriptors for maize and analyzed using Genstat Release 14.1. Data was also subjected to XLSTAT 2014 and DARwin 6.0.12 software for principal and cluster analyses. Results obtained differed significantly in herbicide tolerance days for both M3 and M4 (p≤0.01). However, plant height, maturity days, flag leaf length and width, grains ear-1, ear length, ear diameter and grain yield plant-1 differed significantly in M3 and M4 lines (p≤0.05). Grain yield plant-1 showed a strong significant positive correlation with anthesis days, plant height, grains ear-1 and ear diameter and length but negatively correlated with days to pollen shedding, tasseling, maturity and tolerance in M3 while flag leaf width, harvestable and total ears plant-1 showed positive and significant correlation but negatively correlated with tolerance days in M4. Principal component analysis showed variations among mutated maize lines in M3 and M4 with first seven principal components (PC) indicating that the first six PCs explained 78.69% and first six PCs contributing 71.28% respectively of the total variation. Cluster analysis showed three clusters and seven sub-clusters indicating differences in morphological diversity among the M3 inbred lines and two clusters with cluster one of hybrid 513 and three sub-clusters in cluster two of hybrid 520. Plant height, flowering days and ear length were crucial phenological traits determining grain yield among herbicide tolerant lines showing significant variability that could be considered in hybridization and development of herbicide tolerant hybrid genotypes in future maize breeding programmes.

Comparative transcriptome analysis of differentially expressed genes related to the physiological changes of yellow-green leaf mutant of maize.

Chlorophylls, green pigments in chloroplasts, are essential for photosynthesis. Reduction in chlorophyll content may result in retarded growth, dwarfism, and sterility. In this study, a yellow-green leaf mutant of maize, indicative of abnormity in chlorophyll content, was identified. The physiological parameters of this mutant were measured. Next, global gene expression of this mutant was determined using transcriptome analysis and compared to that of wild-type maize plants. The yellow-green leaf mutant of maize was found to contain lower contents of chlorophyll a, chlorophyll b and carotenoid compounds. It contained fewer active PSII centers and displayed lower values of original chlorophyll fluorescence parameters than the wild-type plants. The real-time fluorescence yield, the electron transport rate, and the net photosynthetic rate of the mutant plants showed reduction as well. In contrast, the maximum photochemical quantum yield of PSII of the mutant plants was similar to that of the wild-type plants. Comparative transcriptome analysis of the mutant plants and wild-type plants led to the identification of differentially expressed 1,122 genes, of which 536 genes were up-regulated and 586 genes down-regulated in the mutant. Five genes in the chlorophyll metabolism pathway, nine genes in the tricarboxylic acid cycle and seven genes related to the conversion of sucrose to starch displayed down-regulated expression. In contrast, genes encoding a photosystem II reaction center PsbP family protein and the PGR5-like protein 1A (PGRL1A) exhibited increased transcript abundance.

DNA methylation changes stimulated by drought stress in ABA-deficient maize mutant vp10

Plants are constantly challenged with several biotic and abiotic stresses, and the adaptation to these stresses requires molecular and morphological changes. Epigenetic regulation provides effective control that enables plants to tolerate stress, which results in improved survivability. The distinct role of abscisic acid (ABA) in controlling numerous stress-responsive genes and enhancing respiration metabolism is well known; however, whether DNA methylation is associated with the regulation of ABA-dependent gene expression remains unclear. This study was conducted to identify the changes in DNA methylation induced by drought stress in ABA-deficient maize mutant vp10 using the amplified methylation polymorphism–polymerase chain reaction (AMP–PCR) technique. Differentially methylated DNA fragments were mapped to intragenic regions of zinc finger, amino acid catabolic enzymes, and other genes implicated in DNA repair and plant survival, in addition to several demethylated transposable elements.

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays)

Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize (Zea mays) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 (anther ear 2), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.

Understanding Ustilago maydis Infection of Multiple Maize Organs.

Ustilago maydis is a smut fungus that infects all aerial maize organs, namely, seedling leaves, tassels, and ears. In all organs, tumors are formed by inducing hypertrophy and hyperplasia in actively dividing cells; however, the vast differences in cell types and developmental stages for different parts of the plant requires that U. maydis have both general and organ-specific strategies for infecting maize. In this review, we summarize how the maize–U. maydis interaction can be studied using mutant U. maydis strains to better understand how individual effectors contribute to this interaction, either through general or specific expression in a cell type, tissue, or organ. We also examine how male sterile maize mutants that do not support tumor formation can be used to explore key features of the maize anthers that are required for successful infection. Finally, we discuss key unanswered questions about the maize–U. maydis interaction and how new technologies can potentially be used to answer them.

Empty Pericarp24 and Empty Pericarp25 Are Required for the Splicing of Mitochondrial Introns, Complex I Assembly, and Seed Development in Maize

RNA splicing is an essential post-transcriptional regulation in plant mitochondria and chloroplasts. As the mechanism of RNA splicing remains obscure, identification and functional elucidation of new splicing factors are necessary. Through a characterization of two maize mutants, we cloned Empty pericarp 24 (Emp24) and Empty pericarp 25 (Emp25). Both Emp24 and Emp25 encode mitochondrion-targeted P-type PPR proteins. EMP24 is required for the splicing of nad4 introns 1 and 3, which was reported (Ren Z. et al., 2019), and EMP25 functions in the splicing of nad5 introns 1, 2, and 3. Absence of either Nad4 or Nad5 proteins blocks the assembly of mitochondrial complex I, resulting in the formation of a sub-sized complex I of similar size in both mutants. Mass spectrometry identification revealed that the subcomplexes in both mutants lack an identical set of proteins of complex I. These results indicate that EMP24 and EMP25 function in the splicing of nad4 and nad5 introns, respectively, and are essential to maize kernel development. The identification of the subcomplexes provides genetic and molecular insights into the modular complex I assembly pathway in maize.

Identification of a locus associated with genic male sterility in maize via EMS mutagenesis and bulked-segregant RNA-seq

Genic male sterility (GMS) is one of the most important resources for exploiting heterosis in crop breeding, so that identifying genomic loci regulating GMS is desirable. However, many regulatory genes controlling GMS have not yet been characterized in maize, owing partly to a lack of genetic materials. We generated a recessive male-sterile maize mutant in the Jing 724 genetic background via ethyl methanesulfonate treatment, and found the male sterility to be due to a single gene mutation. Bulk-segregant RNA sequencing of three replicates indicated that one genomic region located at the end of chromosome 4 was associated with the observed mutant phenotype. Among genes with nonsynonymous mutations, Zm00001d053895 (bHLH51) showed abolished expression in the sterile bulks and was annotated as a bHLH transcription factor orthologous to Arabidopsis AMS, suggesting an association with the male sterility of the mutant. Kompetitive Allele-Specific PCR assays further validated the exclusive correlation of male sterility with the single C-to-T mutation in the fifth exon. The new maize mutant and the potential SNP locus provide novel genetic material for investigating the molecular mechanism underlying tapetal development and may facilitate the improvement of hybrid production systems.