DEFECTIVE KERNEL 56 functions in mitochondrial RNA editing and maize seed development.

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.

A single silk- and multiple pollen-expressed PMEs at the Ga1 locus modulate maize unilateral cross-incompatibility.

The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross-incompatibility (UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene (PME) (ZmGa1F) and eight pollen-expressed PMEs (ZmGa1P and ZmGa1PL1-7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

ORF355 confers enhanced salinity stress adaptability to S-type cytoplasmic male sterility maize by modulating the mitochondrial metabolic homeostasis.

Moderate stimuli in mitochondria improve wide-ranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility (CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties.

A novel hybrid seed production technology based on a unilateral cross-incompatibility gene in maize.

Hybrid seed production technology (SPT) using genic recessive male sterility is of great importance in maize breeding. Here, we report a novel SPT based on a maize unilateral cross-incompatibility gene ZmGa1F with an extremely low transgene transmission rate (TTR). Proper pollen-specific ZmGa1F expression severely inhibits pollen tube growth leading to no fertilization. The maintainer line harbors a transgene cassette in an ipe1 male sterile background containing IPE1 to restore ipe1 male fertility, ZmGa1F to prevent transgenic pollen escape, the red fluorescence protein encoding gene DsRed2 for the separation of male sterile and fertile seeds, and the herbicide-resistant gene Bar for transgenic plant selection. When the maintainer line is selfed, gametes of ipe1/transgene and ipe1/- genotypes are produced, and pollen of the ipe1/transgene genotype is not able to fertilize female gametes due to pollen tube growth inhibition by ZmGa1F. Subsequently, seeds of ipe1/ipe1 and ipe1/transgene genotypes are produced at a 1:1 ratio and could be separated easily by fluorescence-based seed sorting. Not a single seed emitting fluorescence is detected in more than 200,000 seeds examined demonstrating that the pollen-tube-inhibition (PTI)-based TTR is lower than what has been reported for similar technologies to date. This PTI-based SPT shows promising potential for future maize hybrid seed production.

A pair of non-Mendelian genes at the Ga2 locus confer unilateral cross-incompatibility in maize.

Maize unilateral cross-incompatibility (UCI) that causes non-Mendelian segregation ratios has been documented for more than a century. Ga1, Ga2, and Tcb1 are three major UCI systems, described but not fully understood. Here, we report comprehensive genetic studies on the Ga2 locus and map-based cloning of the tightly linked male determinant ZmGa2P and female determinant ZmGa2F that govern pollen-silk compatibility among different maize genotypes. Both determinants encode putative pectin methylesterases (PME). A significantly higher degree of methyl esterification is detected in the apical region of pollen tubes growing in incompatible silks. No direct interaction between ZmGa2P and ZmGa2F is detected in the yeast two-hybrid system implying a distinct mechanism from that of self-incompatibility (SI). We also demonstrate the feasibility of Ga2 as a reproductive barrier in commercial breeding programs and stacking Ga2 with Ga1 could strengthen the UCI market potentials.

EAR APICAL DEGENERATION1 regulates maize ear development by maintaining malate supply for apical inflorescence.

Ear length (EL) is a key trait that contributes greatly to grain yield in maize (Zea mays). While numerous quantitative trait loci for EL have been identified, few causal genes have been studied in detail. Here we report the characterization of ear apical degeneration1 (ead1) exhibiting strikingly shorter ears and the map-based cloning of the casual gene EAD1. EAD1 is preferentially expressed in the xylem of immature ears and encodes an aluminum-activated malate transporter localizing to the plasma membrane. We show that EAD1 is a malate efflux transporter and loss of EAD1 leads to lower malate contents in the apical part of developing inflorescences. Exogenous injections of malate rescued the shortened ears of ead1. These results demonstrate that EAD1 plays essential roles in regulating maize ear development by delivering malate through xylem vessels to the apical part of the immature ear. Overexpression of EAD1 led to greater EL and kernel number per row and the EAD1 genotype showed a positive association with EL in two different genetic segregating populations. Our work elucidates the critical role of EAD1 in malate-mediated female inflorescence development and provides a promising genetic resource for enhancing maize grain yield.

Identification of the Potential Genes Regulating Seed Germination Speed in Maize.

Seed germination is the crucial stage in plant life cycle. Rapid and uniform germination plays an essential role in plant development and grain yield improvement. However, the molecular mechanism underlying seed germination speed is largely unknown due to the complexity of the dynamic process and the difficulty in phenotyping. Here, we conducted a time-series comparative transcriptome study of two elite maize inbred lines, 72-3 and F9721, with striking difference in seed germination speed, and identified a major locus underlying maize germination speed through genome-wide association analysis (GWAS) of an F2 segregation population. Comparative transcriptome study identified 12 h after imbibition (HAI) as the critical stage responsible for the variation in germination speed. The differentially expressed genes (DEGs) between 72-3 and F9721 were mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, oxidoreductase activity pathways, hormone signal transduction, and amino acid transporter activity pathways. GWAS revealed that germination speed was controlled by a major locus on chromosome 1 with the leading SNP as AX-91332814, explaining 10.63% of phenotypic variation. A total of 87 proposed protein-coding genes surrounding the locus were integrated with DEGs. Combined with evidence from the gene expression database and gene synteny with other model species, we finally anchored three genes as the likely candidates regulating germination speed in maize. This study provides clues for the further exploration of genes controlling the maize seed germination speed, thus facilitating breeding of rapid germinated elite lines through marker assistant selection.

ZmPRD1 is essential for double-strand break formation, but is not required for bipolar spindle assembly during maize meiosis.

Homologs of PUTATIVE RECOMBINATION INITIATION DEFECT 1 (PRD1) are known to be essential for meiotic double-strand break (DSB) formation in mouse (Mus musculus), Arabidopsis, and rice (Oryza sativa). Recent research has shown that rice PRD1 also plays an unanticipated role in meiotic bipolar spindle assembly, revealing that PRD1 has multiple functions in plant meiosis. In this study, we characterize the meiotic function of PRD1 in maize (Zea mays; ZmPRD1). Our results show that Zmprd1 mutant plants display normal vegetative growth but have complete male and female sterility. Meiotic DSB formation is fully abolished in mutant meiocytes, leading to failure in homologous pairing, synapsis, and recombination. ZmPRD1 exhibits a different pattern of chromosome localization compared to its rice homologs. The ZmPRD1 protein interacts with several DSB-forming proteins, but does not directly interact with the kinetochore proteins REC8 and SGO1. Possibly as a result of this, there are no significant abnormalities of bipolar spindle assembly in Zmprd1 meiocytes. Overall, our results demonstrate that ZmPRD1 is essential for DSB formation and homologous recombination in maize meiosis. However, the recently-identified function of PRD1 in bipolar spindle assembly during rice meiosis is not conserved in maize.

ZmbHLH124 identified in maize recombinant inbred lines contributes to drought tolerance in crops.

Due to climate change, drought has become a severe abiotic stress that affects the global production of all crops. Elucidation of the complex physiological mechanisms underlying drought tolerance in crops will support the cultivation of new drought-tolerant crop varieties. Here, two drought-tolerant lines, RIL70 and RIL73, and two drought-sensitive lines, RIL44 and RIL93, from recombinant inbred lines (RIL) generated from maize drought-tolerant line PH4CV and drought-sensitive line F9721, were selected for a comparative RNA-seq study. Through transcriptome analyses, we found that gene expression differences existed between drought-tolerant and -sensitive lines, but also differences between the drought-tolerant lines, RIL70 and RIL73. ZmbHLH124 in RIL73, named as ZmbHLH124T-ORG which origins from PH4CV and encodes a bHLH type transcription factor, was specifically up-regulated during drought stress. In addition, we identified a substitution in ZmbHLH124 that produced an early stop codon in sensitive lines (ZmbHLH124S-ORG ). Overexpression of ZmbHLH124T-ORG , but not ZmbHLH124S-ORG , in maize and rice enhanced plant drought tolerance and up-regulated the expression of drought-responsive genes. Moreover, we found that ZmbHLH124T-ORG could directly bind the cis-acting elements in ZmDREB2A promoter to enhance its expression. Taken together, this work identified a valuable genetic locus and provided a new strategy for breeding drought-tolerant crops.

IRREGULAR POLLEN EXINE2 Encodes a GDSL Lipase Essential for Male Fertility in Maize.

Anther cuticle and pollen exine are two physical barriers protecting plant reproductive cells against environmental stresses; defects in either often cause male sterility. Here, we report the characterization of a male-sterile mutant irregular pollen exine2 (ipe2) of maize (Zea mays), which displays shrunken anthers and no starch accumulation in mature pollen grains. We cloned the causal gene IPE2 and confirmed its role in male fertility in maize with a set of complementary experiments. IPE2 is specifically expressed in maize developing anthers during stages 8 to 9 and encodes an endoplasmic-reticulum-localized GDSL lipase. Dysfunction of IPE2 resulted in delayed degeneration of tapetum and middle layer, leading to defective formation of anther cuticle and pollen exine, and complete male sterility. Aliphatic metabolism was greatly altered, with the contents of lipid constituents, especially C16/C18 fatty acids and their derivatives, significantly reduced in ipe2 developing anthers. Our study elucidates GDSL function in anther and pollen development and provides a promising genetic resource for breeding hybrid maize.

Activation of Mitochondrial orf355 Gene Expression by a Nuclear-Encoded DREB Transcription Factor Causes Cytoplasmic Male Sterility in Maize.

Coordination between mitochondria and the nucleus is crucial for fertility determination in plants with cytoplasmic male sterility (CMS). Using yeast one-hybrid screening, we identified a transcription factor, ZmDREB1.7, that is highly expressed in sterile microspores at the large vacuole stage and activates the expression of mitochondria-encoded CMS gene orf355. Δpro, a weak allele of ZmDREB1.7 with the loss of a key unfolded protein response (UPR) motif in the promoter, partially restores male fertility of CMS-S maize. ZmDREB1.7 expression increases rapidly in response to antimycin A treatment, but this response is attenuated in the Δpro allele. Furthermore, we found that expression of orf355 in mitochondria activates mitochondrial retrograde signaling, which in turn induces ZmDREB1.7 expression. Taken together, these findings demonstrate that positive-feedback transcriptional regulation between a nuclear regulator and a mitochondrial CMS gene determines male sterility in maize, providing new insights into nucleus-mitochondria communication in plants.

Maize YSL2 is required for iron distribution and development in kernels.

Iron (Fe) is an essential micronutrient and plays an irreplaceable role in plant growth and development. Although its uptake and translocation are important biological processes, little is known about the molecular mechanism of Fe translocation within seed. Here, we characterized a novel small kernel mutant yellow stripe like 2 (ysl2) in maize (Zea mays). ZmYSL2 was predominantly expressed in developing endosperm and was found to encode a plasma membrane-localized metal-nicotianamine (NA) transporter ZmYSL2. Analysis of transporter activity revealed ZmYSL2-mediated Fe transport from endosperm to embryo during kernel development. Dysfunction of ZmYSL2 resulted in the imbalance of Fe homeostasis and abnormality of protein accumulation and starch deposition in the kernel. Significant changes of nitric oxide accumulation, mitochondrial Fe-S cluster content, and mitochondrial morphology indicated that the proper function of mitochondria was also affected in ysl2. Collectively, our study demonstrated that ZmYSL2 had a pivotal role in mediating Fe distribution within the kernel and kernel development in maize.

A PECTIN METHYLESTERASE gene at the maize Ga1 locus confers male function in unilateral cross-incompatibility.

Unilateral cross-incompatibility (UCI) is a unidirectional inter/intra-population reproductive barrier when both parents are self-compatible. Maize Gametophyte factor1 (Ga1) is an intraspecific UCI system and has been utilized in breeding. However, the mechanism underlying maize UCI specificity has remained mysterious for decades. Here, we report the cloning of ZmGa1P, a pollen-expressed PECTIN METHYLESTERASE (PME) gene at the Ga1 locus that can confer the male function in the maize UCI system. Homozygous transgenic plants expressing ZmGa1P in a ga1 background can fertilize Ga1-S plants and can be fertilized by pollen of ga1 plants. ZmGa1P protein is predominantly localized to the apex of growing pollen tubes and may interact with another pollen-specific PME protein, ZmPME10-1, to maintain the state of pectin methylesterification required for pollen tube growth in Ga1-S silks. Our study discloses a PME-mediated UCI mechanism and provides a tool to manipulate hybrid breeding.

Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize.

In maize (Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination (G_GG, G_GB, B_BB, B_BG). A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S.

MALE STERILE6021 (MS6021) is required for the development of anther cuticle and pollen exine in maize.

The anther cuticle and pollen wall function as physical barriers that protect genetic material from various environmental stresses. The anther cuticle is composed of wax and cutin, the pollen wall includes exine and intine, and the components of the outer exine are collectively called sporopollenin. Other than cuticle wax, cutin and sporopollenin are biopolymers compounds. The precise constituents and developmental mechanism of these biopolymeric are poorly understood. Here, we reported a complete male sterile mutant, male sterile6021, in maize. The mutant displayed a smooth anther surface and irregular pollen wall formation before anthesis, and its tapetum was degraded immaturely. Gas chromatography-mass spectrometry analysis revealed a severe reduction of lipid derivatives in the mutant anther. We cloned the gene by map based cloning. It encoded a fatty acyl carrier protein reductase that was localized in plastids. Expression analysis indicated that MS6021 was mainly expressed in the tapetum and microspore after the microspore was released from the tetrad. Functional complementation of the orthologous Arabidopsis mutant demonstrated that MS6021 is conserved between monocots and dicots and potentially even in flowering plants. MS6021 plays a conserved, essential role in the successful development of anther cuticle and pollen exine in maize.

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation.

Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize.

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize.

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum-specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate-shaped compared with the three-dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild-type. The wild-type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2-Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co-localized with the endoplasmic reticulum (ER) signal. RNA-Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.

Identification of Drought Tolerant Mechanisms in Maize Seedlings Based on Transcriptome Analysis of Recombination Inbred Lines.

Zea mays is an important crop that is sensitive to drought stress, but survival rates and growth status remain strong in some drought-tolerant lines under stress conditions. Under drought conditions, many biological processes, such as photosynthesis, carbohydrate metabolism and energy metabolism, are suppressed, while little is known about how the transcripts of genes respond to drought stress in the genome-wide rang in the seedling stage. In our study, the transcriptome profiles of two maize recombination inbred lines (drought-tolerant RIL70 and drought-sensitive RIL93) were analyzed at different drought stages to elucidate the dynamic mechanisms underlying drought tolerance in maize seedlings during drought conditions. Different numbers of differentially expressed genes presented in the different stages of drought stress in the two RILs, for the numbers of RIL93 vs. RIL70 were: 9 vs. 358, 477 vs. 103, and 5207 vs. 152 respectively in DT1, DT2, and DT5. Gene Ontology enrichment analysis revealed that in the initial drought-stressed stage, the primary differentially expressed genes involved in cell wall biosynthesis and transmembrane transport biological processes were overrepresented in RIL70 compared to RIL93. On the contrary, differentially expressed genes profiles presented at 2 and 5 day-treatments, the primary differentially expressed genes involved in response to stress, protein folding, oxidation-reduction, photosynthesis and carbohydrate metabolism, were overrepresented in RIL93 compared to RIL70. In addition, the transcription of genes encoding key members of the cell cycle and cell division processes were blocked, but ABA- and programmed cell death-related processes responded positively in RIL93. In contrast, the expression of cell cycle genes, ABA- and programmed cell death-related genes was relatively stable in RIL70. The results we obtained supported the working hypothesis that signaling events associated with turgor homeostasis, as established by cell wall biosynthesis regulation- and aquaporin-related genes, responded early in RIL70, which led to more efficient detoxification signaling (response to stress, protein folding, oxidation-reduction) during drought stress. This energy saving response at the early stages of drought should facilitate more cell activity under stress conditions and result in drought tolerance in RIL70.