Maize Ortholog Research Articles

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

Identification of the Teopod1, Teopod2, and Early Phase Change genes in maize.

Teopod1 (Tp1), Teopod2 (Tp2), and Early phase change (Epc) have profound effects on the timing of vegetative phase change in maize. Gain-of-function mutations in Tp1 and Tp2 delay all known phase-specific vegetative traits, whereas loss-of-function mutations in Epc accelerate vegetative phase change and cause shoot abortion in some genetic backgrounds. Here, we show that Tp1 and Tp2 likely represent cis-acting mutations that cause the overexpression of Zma-miR156j and Zma-miR156h, respectively. Epc is the maize ortholog of HASTY, an Arabidopsis gene that stabilizes miRNAs and promotes their intercellular movement. Consistent with its pleiotropic phenotype and epistatic interaction with Tp1 and Tp2, epc reduces the levels of miR156 and several other miRNAs.

A Systematic Investigation of Lipid Transfer Proteins Involved in Male Fertility and Other Biological Processes in Maize

Plant lipid transfer proteins (LTPs) play essential roles in various biological processes, including anther and pollen development, vegetative organ development, seed development and germination, and stress response, but the research progress varies greatly among Arabidopsis, rice and maize. Here, we presented a preliminary introduction and characterization of the whole 65 LTP genes in maize, and performed a phylogenetic tree and gene ontology analysis of the LTP family members in maize. We compared the research progresses of the reported LTP genes involved in male fertility and other biological processes in Arabidopsis and rice, and thus provided some implications for their maize orthologs, which will provide useful clues for the investigation of LTP transporters in maize. We predicted the functions of LTP genes based on bioinformatic analyses of their spatiotemporal expression patterns by using RNA-seq and qRT-PCR assays. Finally, we discussed the advances and challenges in substrate identification of plant LTPs, and presented the future research directions of LTPs in plants. This study provides a basic framework for functional research and the potential application of LTPs in multiple plants, especially for male sterility research and application in maize.

The evening complex promotes maize flowering and adaptation to temperate regions.

Maize (Zea mays) originated in southern Mexico and has spread over a wide latitudinal range. Maize expansion from tropical to temperate regions has necessitated a reduction of its photoperiod sensitivity. In this study, we cloned a quantitative trait locus (QTL) regulating flowering time in maize and show that the maize ortholog of Arabidopsis thaliana EARLY FLOWERING3, ZmELF3.1, is the causal locus. We demonstrate that ZmELF3.1 and ZmELF3.2 proteins can physically interact with ZmELF4.1/4.2 and ZmLUX1/2, to form evening complex(es; ECs) in the maize circadian clock. Loss-of-function mutants for ZmELF3.1/3.2 and ZmLUX1/2 exhibited delayed flowering under long-day and short-day conditions. We show that EC directly represses the expression of several flowering suppressor genes, such as the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) genes ZmCCT9 and ZmCCT10, ZmCONSTANS-LIKE 3, and the PSEUDORESPONSE REGULATOR (PRR) genes ZmPRR37a and ZmPRR73, thus alleviating their inhibition, allowing florigen gene expression and promoting flowering. Further, we identify two closely linked retrotransposons located in the ZmELF3.1 promoter that regulate the expression levels of ZmELF3.1 and may have been positively selected during postdomestication spread of maize from tropical to temperate regions during the pre-Columbian era. These findings provide insights into circadian clock-mediated regulation of photoperiodic flowering in maize and new targets of genetic improvement for breeding.

Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing.

U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5' end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

Genome-wide association identifies a missing hydrolase for tocopherol synthesis in plants

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

ZmEREB46, a maize ortholog of Arabidopsis WAX INDUCER1/SHINE1, is involved in the biosynthesis of leaf epicuticular very-long-chain waxes and drought tolerance

The aerial surfaces of plants are covered by a layer of cuticular wax that is composed of long-chain hydrocarbon compounds for protection against adverse environmental conditions. The current study identified a maize (Zea mays L.) APETALA2/ethylene-responsive element-binding protein (AP2/EREBP)-type transcription factor, ZmEREB46. Ectopic expression of ZmEREB46 in Arabidopsis increased the accumulation of epicuticular wax on the leaves and enhanced the drought tolerance of plants. The amounts of C24/C32 fatty acids, C32/C34 aldehydes, C32/C34 1-alcohols and C31 alkanes in zmereb46 (ZmEREB46 knockout mutant) leaves were reduced. The amount of leaf total epicuticular wax decreased approximately 50% in zmereb46. Compared to wild-type LH244 leaves, the cuticle permeability of zmereb46 leaves was increased, which resulted from decreased epicuticular wax load and a thinner cuticle layer. ZmEREB46 had transcriptional activation activity and directly bound to promoter regions of ZmCER2, ZmCER3.2 and ZmKCS1. The zmereb46 seedlings also exhibited reduced drought tolerance. These results, and the observations in ZmEREB46-overexpressing lines, suggest that ZmEREB46 is involved in cuticular metabolism by influencing the biosynthesis of very-long-chain waxes and participates in the cutin biosynthesis pathway. These results are helpful to further analyze the regulatory network of wax accumulation in maize.

Mutation of the nuclear pore complex component, aladin1, disrupts asymmetric cell division in Zea mays (maize).

The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here, we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.

Genetic architecture of embryo size and related traits in maize

The embryo in maize has a critical role in controlling kernel nutrition components and grain yield. We measured five embryo weight and size traits, six kernel weight and size traits, and five embryo-to-kernel ratio traits in a nested association mapping (NAM) population of 611 recombinant inbred lines (RILs) derived from four inbred lines including the high-oil, giant-embryo line BY815 as the common parent. Using three statistical methods, we identified 5–22 quantitative trait loci (QTL) for each trait, explaining 4.7%–46.7% of the phenotypic variation. The genetic architecture of maize embryo size and its related traits appeared to be dominated by multiple small-effect loci with little epistasis, and the genetic control underlying embryo size appeared to be distinct from that underlying kernel size. A trait–QTL association network included 205 nodes and 439 edges and revealed 28 key loci associated with at least three traits. Cloned maize genes including ZmUrb2, Emp12 and ZmBAM1d, maize orthologs of known rice genes that control seed size including BG1, XIAO and GS9, and 11 maize orthologs of Arabidopsis EMBRYO-DEFECTIVE (EMB) genes were identified as underlying these key loci. Further, the phenotypic and genetic relationships between embryo size and kernel size were evaluated, and genetic patterns for identified loci that control embryo size and its related traits were proposed. Our findings reveal distinct genetic architectures for embryo size, kernel size, and embryo-to-kernel ratio traits and establish a foundation for the improvement of embryo-size-mediated kernel nutrition and grain yield.

Identification and characterization of maize ACD6-like gene reveal ZmACD6 as the maize orthologue conferring resistance to Ustilago maydis

ABSTRACTEnhancing broad-spectrum resistance is a major goal of crop breeding. However, broad-spectrum resistance has not been thoroughly investigated, and its underlying molecular mechanisms remain elusive. In the model plant Arabidopsis (Arabidopsis thaliana), ACCELERATED CELL DEATH6 (ACD6) is a key component of broad-spectrum resistance that acts in a positive feedback loop with salicylic acid (SA) to regulate multiple pattern recognition receptors. However, the role of ACD6 in disease resistance in crop plants is unclear. Here, we show that the transcript of ANK23, one of the 15 ACD6-like genes in maize (Zea mays), is induced by SA and by infection with the pathogenic fungus Ustilago maydis. Heterologous expression of ANK23 restored disease resistance in the Arabidopsis mutant acd6-2. We show that ANK23 is a maize ortholog of ACD6 and therefore rename ANK23 as ZmACD6. Furthermore, using CRISPR/Cas9, we generated ZmACD6 knockout maize plants, which are more susceptible to U. maydis than wild-type plants. We also identified a maize line (SC-9) with relatively high ZmACD6 expression levels from a diverse natural maize population. SC-9 has increased disease resistance to U. maydis and defense activation, suggesting a practical approach to cultivate elite varieties with enhanced disease resistance.

SMK6 mediates the C-to-U editing at multiple sites in maize mitochondria

The recently identified PPR-E+/NVWA/DYW2 RNA editing complex provides insights into the mechanism of RNA editing in higher plant organelles. However, whether the complex works together with the previously identified editing factors RIPs/MORFs is unclear. In this paper, we identified a maize Smk6 gene, which encodes a mitochondrion-targeted PPR-E+protein with E1 and E2 domains at the C terminus. Loss of Smk6 function affects the C-to-U editing at nad1-740, nad4L-110, nad7-739, and mttB-138,139 sites, impairs mitochondrial activity and blocks embryogenesis and endosperm development. Genetic and molecular analysis indicated that SMK6 is the maize ortholog of the Arabidopsis SLO2, which is a component of the PPR-E+/NVWA/DYW2 editing complex. However, yeast two-hybrid analyses did not detect any interaction between SMK6 and any of the mitochondrion-targeted RIPs/MORFs, suggesting that RIPs/MORFs may not be a component of PPR-E+/NVWA/DYW2 RNA editing complex. Further analyses are required to provide evidence that RIP/MORFs and SMK6 do not physically interact in vivo.

The Arabidopsis pentatricopeptide repeat protein LPE1 and its maize ortholog are required for translation of the chloroplast psbJ RNA.

The expression of chloroplast genes relies on a host of nucleus-encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the pentatricopeptide repeat protein LPE1, reported to stimulate translation of the chloroplast psbA mRNA in Arabidopsis. Mutation of the maize LPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplast psbJ open reading frame (ORF) but has no effect on psbA expression. To reflect this function, we named the maize LPE1 ortholog Translation of psbJ 1 (TPJ1). Arabidopsis lpe1 mutants likewise exhibit a loss of psbJ translation, and have, in addition, a decrease in psbN translation. We detected a small decrease in ribosome occupancy on the psbA mRNA in Arabidopsis lpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 and hcf173) in conjunction with invitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of the psbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 in psbN translation contributes to the PSII deficiency in lpe1 mutants.

Nitrate affects transcriptional regulation of UPBEAT1 and ROS localisation in roots of Zea mays L.

Nitrogen (N) is an indispensable nutrient for crops but its availability in agricultural soils is subject to considerable fluctuation. Plants have developed plastic responses to external N fluctuations in order to optimise their development. The coordinated action of nitric oxide and auxin seems to allow the cells of the root apex transition zone (TZ) of N-deprived maize to rapidly sense nitrate (NO3 - ). Preliminary results support the hypothesis that reactive oxygen species (ROS) signalling might also have a role in this pathway, probably through a putative maize orthologue of UPBEAT1 (UPB1). To expand on this hypothesis and better understand the different roles played by different root portions, we investigated the dynamics of ROS production, and the molecular and biochemical regulation of the main components of ROS production and scavenging in tissues of the meristem, transition zone, elongation zone and maturation zone of maize roots. The results suggest that the inverse regulation of ZmUPB1 and ZmPRX112 transcription observed in cells of the TZ in response to nitrogen depletion or NO3 - supply affects the balance between superoxide (O2 •- ) and hydrogen peroxide (H2 O2 ) in the root apex and consequently triggers differential root growth. This explanation is supported by additional results on the overall metabolic and transcriptional regulation of ROS homeostasis.

Arabidopsis MATE45 antagonizes local abscisic acid signaling to mediate development and abiotic stress responses.

Anthocyanins provide ideal visual markers for the identification of mutations that disrupt molecular responses to abiotic stress. We screened Arabidopsis mutants of ABC (ATP‐Binding Cassette) and MATE (Multidrug And Toxic compound Extrusion) transporter genes under nutritional stress and identified four genes (ABCG25,ABCG9,ABCG5, and MATE45) required for normal anthocyanin pigmentation. ABCG25 was previously demonstrated to encode a vascular‐localized cellular exporter of abscisic acid (ABA). Our results show that MATE45 encodes an aerial meristem‐ and a vascular‐localized transporter associated with the trans‐Golgi, and that it plays an important role in controlling the levels and distribution of ABA in growing aerial meristems and non‐meristematic tissues. MATE45 promoter‐GUS reporter fusions revealed the activity localized to the leaf and influorescence meristems and the vasculature. Loss‐of‐function mate45 mutants exhibited accelerated rates of aerial organ initiation suggesting at least partial functional conservation with the maize ortholog bige1. The aba2‐1 mutant, which is deficient in ABA biosynthesis, exhibited a number of phenotypes that were rescued in the mate45‐1 aba2‐1 double mutant. mate45 exhibited enhanced the seed dormancy, and germination was hypersensitive to ABA. Enhanced frequency of leaf primordia growth in mate45 seedlings grown in nutrient imbalance stress was ABA‐dependent. The ABA signaling reporter construct pRD29B::GUS revealed elevated levels of ABA signaling in the true leaf primordia of mate45 seedlings grown under nutritional stress, and gradually reduced signaling in surrounding cotyledon and hypocotyl tissues concomitant with reduced expressions of ABCG25. Our results suggest a role of MATE45 in reducing meristematic ABA and in maintaining ABA distribution in adjacent non‐meristematic tissues.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

The CLAVATA receptor FASCIATED EAR2 responds to distinct CLE peptides by signaling through two downstream effectors.

Meristems contain groups of indeterminate stem cells, which are maintained by a feedback loop between CLAVATA (CLV) and WUSCHEL (WUS) signaling. CLV signaling involves the secretion of the CLV3 peptide and its perception by a number of Leucine-Rich-Repeat (LRR) receptors, including the receptor-like kinase CLV1 and the receptor-like protein CLV2 coupled with the CORYNE (CRN) pseudokinase. CLV2, and its maize ortholog FASCIATED EAR2 (FEA2) appear to function in signaling by CLV3 and several related CLV3/EMBRYO-SURROUNDING REGION (CLE) peptide ligands. Nevertheless, how signaling specificity is achieved remains unknown. Here we show that FEA2 transmits signaling from two distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) through two different candidate downstream effectors, the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN. Our data provide a novel framework to understand how diverse signaling peptides can activate different downstream pathways through common receptor proteins.

Characterization of the ZmbHLH122 transcription factor and its potential collaborators in maize male reproduction

OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.

A plastidial pantoate transporter with a potential role in pantothenate synthesis.

The pantothenate (vitamin B5) synthesis pathway in plants is not fully defined because the subcellular site of its ketopantoate → pantoate reduction step is unclear. However, the pathway is known to be split between cytosol, mitochondria, and potentially plastids, and inferred to involve mitochondrial or plastidial transport of ketopantoate or pantoate. No proteins that mediate these transport steps have been identified. Comparative genomic and transcriptomic analyses identified Arabidopsis thaliana BASS1 (At1g78560) and its maize (Zea mays) ortholog as candidates for such a transport role. BASS1 proteins belong to the bile acid : sodium symporter family and share similarity with the Salmonella enterica PanS pantoate/ketopantoate transporter and with predicted bacterial transporters whose genes cluster on the chromosome with pantothenate synthesis genes. Furthermore, Arabidopsis BASS1 is co-expressed with genes related to metabolism of coenzyme A, the cofactor derived from pantothenate. Expression of Arabidopsis or maize BASS1 promoted the growth of a S. enterica panB panS mutant strain when pantoate, but not ketopantoate, was supplied, and increased the rate of [3H]pantoate uptake. Subcellular localization of green fluorescent protein fusions in Nicotiana tabacum BY-2 cells demonstrated that Arabidopsis BASS1 is targeted solely to the plastid inner envelope. Two independent Arabidopsis BASS1 knockout mutants accumulated pantoate ∼10-fold in leaves and had smaller seeds. Taken together, these data indicate that BASS1 is a physiologically significant plastidial pantoate transporter and that the pantoate reduction step in pantothenate biosynthesis could be at least partly localized in plastids.

Candidate Loci for Yield-Related Traits in Maize Revealed by a Combination of MetaQTL Analysis and Regional Association Mapping.

Maize grain yield and related traits are complex and are controlled by a large number of genes of small effect or quantitative trait loci (QTL). Over the years, a large number of yield-related QTLs have been identified in maize and deposited in public databases. However, integrating and re-analyzing these data and mining candidate loci for yield-related traits has become a major issue in maize. In this study, we collected information on QTLs conferring maize yield-related traits from 33 published studies. Then, 999 of these QTLs were iteratively projected and subjected to meta-analysis to obtain metaQTLs (MQTLs). A total of 76 MQTLs were found across the maize genome. Based on a comparative genomics strategy, several maize orthologs of rice yield-related genes were identified in these MQTL regions. Furthermore, three potential candidate genes (Gene ID: GRMZM2G359974, GRMZM2G301884, and GRMZM2G083894) associated with kernel size and weight within three MQTL regions were identified using regional association mapping, based on the results of the meta-analysis. This strategy, combining MQTL analysis and regional association mapping, is helpful for functional marker development and rapid identification of candidate genes or loci.

Cloning, molecular evolution and functional characterization of ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142).

ABSTRACTThe transcription factor ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), was isolated in the present study. Tissue expression analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs. Subcellular location analysis of ZmbHLH16 via rice protoplast indicated that it is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 single nucleotide polymorphisms and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. Yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. Genome-wide coexpression analysis showed that 395 genes were coexpressed with ZmbHLH16. Among these genes, the transcription factor ZmbHLH51 had similar expression pattern and identical subcellular localization to those of ZmbHLH16. Subsequently, the interaction between ZmbHLH51 and ZmbHLH16 was verified by yeast two-hybrid experiment. Through yeast two-hybrid analysis of series truncated ZmbHLH16 fragments, we found not only the typical bHLH domain [175-221 amino acids (a.a.)], but also that the 81-160 a.a. and 241-365 a.a. of ZmbHLH16 could interact with ZmbHLH51. All these results lay the foundation for further understanding the functions of ZmbHLH16.