Genetic variation in ZmKW1 contributes to kernel weight and size in dent corn and popcorn.

Kernel weight is a critical factor that essentially affects maize (Zea mays) yield. In natural inbred lines, popcorn kernels exhibit overtly smaller sizes compared to dent corn kernels, and kernel weight, which is controlled by multiple genetic loci, varies widely. Here, we characterized a major quantitative trait locus on chromosome 1, responsible for controlling kernel weight (qKW1) and size. The qKW1 locus encodes a protein containing a seven in absentia domain with E3 ubiquitin ligase activity, expressed prominently from the top to the middle region of the endosperm. The presence and function of qKW1 were confirmed through ZmKW1 gene editing, where the mutations in ZmKW1 within dent corn significantly increased kernel weight, consistent with alterations in kernel size, while overexpression of ZmKW1 had the opposite effect. ZmKW1 acts as a negative regulator of kernel weight and size by reducing both the number and size of the endosperm cells and impacting endosperm filling. Notably, the popcorn allele qKW1N and the dent corn allele qKW1D encode identical proteins; however, the differences in promoter activity arise due to the insertion of an Indel-1346 sequence in the qKW1N promoter, resulting in higher expression levels compared to qKW1D, thus contributing to the variation in kernel weight and size between popcorn and dent corn kernels. Linkage disequilibrium analysis of the 2.8 kb promoter region of ZmKW1 in a dataset comprising 111 maize association panels identified two distinct haplotypes. Our results provide insight into the mechanisms underlying kernel development and yield regulation in dent corn and popcorn, with a specific focus on the role of the ubiquitination system.

ZmMPK6, a mitogen-activated protein kinase, regulates maize kernel weight.

Kernel weight is a critical agronomic trait in maize production. Many genes are related to kernel weight but only a few of them have been applied to maize breeding and cultivation. Here, we identify a novel function of maize mitogen-activated protein kinase 6 (ZmMPK6) in the regulation of maize kernel weight. Kernel weight was reduced in zmmpk6 mutants and increased in ZmMPK6-overexpressing lines. In addition, starch granules, starch content, protein content, and grain-filling characteristics were also affected by the ZmMPK6 expression level. ZmMPK6 is mainly localized in the nucleus and cytoplasm, widely distributed across various tissues, and is expressed during kernel development, which is consistent with its role in kernel weight. Thus, these results provide new insights into the role of ZmMPK6, a mitogen-activated protein kinase, in maize kernel weight, and could be applied to further molecular breeding for kernel quality and yield in maize.

An ARF24-ZmArf2 module influences kernel size in different maize haplotypes.

Members of the ADP-ribosylation factor family, which are GTP-binding proteins, are involved in metabolite transport, cell division, and expansion. Although there has been a significant amount of research on small GTP-binding proteins, their roles and functions in regulating maize kernel size remain elusive. Here, we identified ZmArf2 as a maize ADP-ribosylation factor-like family member that is highly conserved during evolution. Maize zmarf2 mutants showed a characteristic smaller kernel size. Conversely, ZmArf2 overexpression increased maize kernel size. Furthermore, heterologous expression of ZmArf2 dramatically elevated Arabidopsis and yeast growth by promoting cell division. Using expression quantitative trait loci (eQTL) analysis, we determined that ZmArf2 expression levels in various lines were mainly associated with variation at the gene locus. The promoters of ZmArf2 genes could be divided into two types, pS and pL, that were significantly associated with both ZmArf2 expression levels and kernel size. In yeast-one-hybrid screening, maize Auxin Response Factor 24 (ARF24) is directly bound to the ZmArf2 promoter region and negatively regulated ZmArf2 expression. Notably, the pS and pL promoter types each contained an ARF24 binding element: an auxin response element (AuxRE) in pS and an auxin response region (AuxRR) in pL, respectively. ARF24 binding affinity to AuxRR was much higher compared with AuxRE. Overall, our results establish that the small G-protein ZmArf2 positively regulates maize kernel size and reveals the mechanism of its expression regulation.

Verification and fine mapping of qGW1.05, a major QTL for grain weight in maize (Zea mays L.).

Grain weight, one of the important factors to determine corn yield, is a typical quantitative inheritance trait. However, the molecular genetic basis of grain weight still remains limited. In our previous researches, a major QTL associated with grain weight, qGW1.05, has been identified between SSR markers umc1601 and umc1754 at bin locus 1.05-1.06 in maize. Here, its genetic and environmental stabiliteis were verified using a BC3F2 population to identify the effect of qGW1.05 on grain weight. Further, qGW1.05-NILs were obtained by MAS successfully. Via a large BC6F2 segregation population, together with polymorphic microsatellite markers developed between the parents to screen the genotype of the recombinant plants, qGW1.05 was positioned to a 1.11 Mb genome interval. Furthermore, the progenies of 15 recombinants were tested to confirm the effect of qGW1.05 on grain weight. Combining collinearity among cereal crops and genome annotation, the several candidate genes taking part in grain development were identified in the qGW1.05 region. In this study, qGW1.05 was limited to a 1.11 Mb region on chromosome 1, which established the foundation for understanding the molecular basis underlying kernel development and improving grain weight through MAS using the tightly flanking molecular markers in maize.

iTRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize.

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize.

A maize ADP-ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that regulate a wide variety of cell functions. Previously, we isolated a new ARF, ZmArf2, from maize (Zea mays). Sequence and expression characteristics indicated that ZmArf2 might play a critical role in the early stages of endosperm development. In this study, we investigated ZmArf2 function by analysis of its GTP-binding activity and subcellular localization. We also over-expressed ZmArf2 in Arabidopsis and measured organ and cell size and counted cell numbers. The expression levels of five organ size-associated genes were also determined in 35S::ZmArf2 transgenic and wild-type plants. Results showed that the recombinant ZmArf2 protein purified from Escherichia coli exhibited GTP-binding activity. Subcellular localization revealed that ZmArf2 was localized in the cytoplasm and plasma membrane. ZmArf2 over-expression in Arabidopsis showed that 35S::ZmArf2 transgenic plants were taller and had larger leaves and seeds compared to wild-type plants, which resulted from cell expansions, not an increase in cell numbers. In addition, three cell expansion-related genes, AtEXP3, AtEXP5 and AtEXP10, were upregulated in 35S::ZmArf2 transgenic lines, while the expression levels of AtGIF1 and AtGRF5, were unchanged. Collectively, our studies suggest that ZmArf2 has an active GTP-binding function, and plays a crucial role in growth and development in Arabidopsis through cell expansion mediated by cell expansion genes.

Structural characterization of helitrons and their stepwise capturing of gene fragments in the maize genome.

BACKGROUND: As a newly identified category of DNA transposon, helitrons have been found in a large number of eukaryotes genomes. Helitrons have contributed significantly to the intra-specific genome diversity in maize. Although many characteristics of helitrons in the maize genome have been well documented, the sequence of an intact autonomous helitrons has not been identified in maize. In addition, the process of gene fragment capturing during the transposition of helitrons has not been characterized. RESULTS: The whole genome sequences of maize inbred line B73 were analyzed, 1,649 helitron-like transposons including 1,515 helAs and 134 helBs were identified. ZmhelA1, ZmhelB1 and ZmhelB2 all encode an open reading frame (ORF) with intact replication initiator (Rep) motif and a DNA helicase (Hel) domain, which are similar to previously reported autonomous helitrons in other organisms. The putative autonomous ZmhelB1 and ZmhelB2 contain an extra replication factor-a protein1 (RPA1) transposase (RPA-TPase) including three single strand DNA-binding domains (DBD)-A/-B/-C in the ORF. Over ninety percent of maize helitrons identified have captured gene fragments. HelAs and helBs carry 4,645 and 249 gene fragments, which yield 2,507 and 187 different genes respectively. Many helitrons contain mutilple terminal sequences, but only one 3'-terminal sequence had an intact "CTAG" motif. There were no significant differences in the 5'-termini sequence between the veritas terminal sequence and the pseudo sequence. Helitrons not only can capture fragments, but were also shown to lose internal sequences during the course of transposing. CONCLUSIONS: Three putative autonomous elements were identified, which encoded an intact Rep motif and a DNA helicase domain, suggesting that autonomous helitrons may exist in modern maize. The results indicate that gene fragments captured during the transposition of many helitrons happen in a stepwise way, with multiple gene fragments within one helitron resulting from several sequential transpositions. In addition, we have proposed a potential mechanism regarding how helitrons with multiple termini are generated.

QTL analysis of popping fold and the consistency of QTLs under two environments in popcorn.

Popping fold (PF) is the most important quality trait in popcorn. In this study, a total of 259 F2:3 families, derived from the cross between a dent corn inbred Dan232 and a popcorn inbred N04, were evaluated for their popping folds in replicated experiments under two environments. Of 613 simple sequence repeat (SSR) primer pairs screened, 183 pairs were selected to construct a genetic linkage map with the genetic distance of 1,762.2 cM (centimorgan) and on average 9.63 cM every marker. Quantative trait loci (QTL) were identified, and their genetic effects were estimated using CIM (composite interval mapping) method. The interactions among QTLs detected were calculated using MIM (multiple interval mapping) method. In all, 22 QTLs were detected, and only 5 of them were common under two environments. Contribution to phenotypic variation of a single QTL varied from 3.07% to 12.84%, and total contributions of all QTLs under two environments were 66.46% and 51.90%, respectively. Three QTLs (qPF-6-1, qPF-8-1 and qPF-1-3) with more than 10% contributions were observed. The additive effects were larger than dominant effects for most QTLs. The amount of QTLs showing additive, partially dominant, dominant and over-dominant effects were 4, 5, 0, 2 in spring sowing and 2, 5, 2, 2 in summer sowing, respectively. There were only 2.60% pairs of QTLs or maker intervals expressing AA, DA or DD interactions.

[QTL analysis of starch content in maize kernels using the trisomic inheritance of the endosperm model].

Two hundred and fifty-nine F2:3 families, developed from the cross between a dent corn inbred line Dan232 and a popcorn inbred line N04, were evaluated for its starch content in the replicated experiments under two environments. One hundred and eighty three pairs of SSR markers were selected to construct a genetic linkage map, covering 1,762.2 cM (centiMorgan) and on the average distance of 9.63 cM between the markers. Quantative trait loci (QTL) were identified and its effects were analyzed, using the triploid endosperm genetic model and the interval mapping method. Ten QTL, detected on chromosome 1, 3, 4, 5 and 7 respectively, were mapped in spring and in summer. All detected QTL in spring were also identified in summer. The phenotypic variation of a single QTL varied from 4.74% to 11.26%, and the total contributions of all QTL under two environments were 36.84% and 72.65%, respectively. Two QTL of all identified QTL in spring showed over-dominant effects, but additive and partially dominant in summer respectively, the amounts of other QTL showing Additive, partially dominant and over-dominant effects were 2, 1, 5 respectively. The positive alleles of three QTL were contributed by the normal corn parent Dan232, and the other positive alleles were contributed by the popcorn parent N04.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

Verification of QTL for grain starch content and its genetic correlation with oil content using two connected RIL populations in high-oil maize.

Grain oil content is negatively correlated with starch content in maize in general. In this study, 282 and 263 recombinant inbred lines (RIL) developed from two crosses between one high-oil maize inbred and two normal dent maize inbreds were evaluated for grain starch content and its correlation with oil content under four environments. Single-trait QTL for starch content in single-population and joint-population analysis, and multiple-trait QTL for both starch and oil content were detected, and compared with the result obtained in the two related F(2∶3) populations. Totally, 20 single-population QTL for grain starch content were detected. No QTL was simultaneously detected across all ten cases. QTL at bins 5.03 and 9.03 were all detected in both populations and in 4 and 5 cases, respectively. Only 2 of the 16 joint-population QTL had significant effects in both populations. Three single-population QTL and 8 joint-population QTL at bins 1.03, 1.04-1.05, 3.05, 8.04-8.05, 9.03, and 9.05 could be considered as fine-mapped. Common QTL across F(2∶3) and RIL generations were observed at bins 5.04, 8.04 and 8.05 in population 1 (Pop.1), and at bin 5.03 in population 2 (Pop.2). QTL at bins 3.02-3.03, 3.05, 8.04-8.05 and 9.03 should be focused in high-starch maize breeding. In multiple-trait QTL analysis, 17 starch-oil QTL were detected, 10 in Pop.1 and 7 in Pop.2. And 22 single-trait QTL failed to show significance in multiple-trait analysis, 13 QTL for starch content and 9 QTL for oil content. However, QTL at bins 1.03, 6.03-6.04 and 8.03-8.04 might increase grain starch content and/or grain oil content without reduction in another trait. Further research should be conducted to validate the effect of these QTL in the simultaneous improvement of grain starch and oil content in maize.

Map-based cloning of zb7 encoding an IPP and DMAPP synthase in the MEP pathway of maize.

IspH is a key enzyme in the last step of the methyl-D-erythritol-4-phosphate (MEP) pathway. Loss of function of IspH can often result in complete yellow or albino phenotype in many plants. Here, we report the characterization of a recessive mutant of maize, zebra7 (zb7), showing transverse green/yellow striped leaves in young plants. The yellow bands of the mutant have decreased levels of chlorophylls and carotenoids with delayed chloroplast development. Low temperature suppressed mutant phenotype, while alternate light/dark cycle or high temperature enlarged the yellow section. Map-based cloning demonstrated that zb7 encodes the IspH protein with a mis-sense mutation in a conserved region. Transgenic silencing of Zb7 in maize resulted in complete albino plantlets that are aborted in a few weeks, confirming that Zb7 is important in the early stages of maize chloroplast development. Zb7 is constitutively expressed and its expression subject to a 16-h light/8-h dark cycle regulation. Our results suggest that the less effective or unstable IspH in zb7 mutant, together with its diurnal expression, are mechanistically accounted for the zebra phenotype. The increased IspH mRNA in the leaves of zb7 at the late development stage may explain the restoration of mutant phenotype in mature stages.

The genetic relationship among plant-height traits found using multiple-trait QTL mapping of a dent corn and popcorn cross.

Plant height (PH) is one of the most important traits in maize breeding programs. In popcorn, inferior plant traits can be improved with the dent/flint corn germplasm. In the current study, a total of 259 F2:3 families, developed from a cross between a dent corn inbred and a popcorn inbred, were evaluated for 4 PH traits. Quantitative trait loci (QTLs) for each trait were detected using composite interval mapping methods. In addition, genetic interrelationships were investigated using multiple-trait joint analysis for PH with ear height (EH), and for PH with top height (TH). In total, 6, 5, 2, and 6 QTLs were identified for PH, EH, TH, and TH/PH in single-trait analysis, respectively. Joint-analysis data suggest a strong and complex genetic relationship between PH and EH, and between PH and EH, with no QTLs controlling any single trait independently. In addition, 4 kinds of QTLs detected were classified as closely linked QTLs, pleiotropic QTLs, QTLs with opposite effects, and additional QTLs. It was, consequently, difficult to improve lodge resistance through selection on any individual PH trait. The current study demonstrates that multiple-trait joint analysis not only identified additional QTLs, but also revealed the genetic relationship among different highly correlated traits at the molecular level.