An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8‐bp insertion/deletion (InDel058) in the 5′ untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA‐sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR‐based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.     

Over‐expression of mutated ZmDA1 or ZmDAR1 gene improves maize kernel yield by enhancing starch synthesis

Grain weight and grain number are important crop yield determinants. DA1 and DAR1 are the ubiquitin receptors that function as the negative regulators of cell proliferation during development in Arabidopsis. An arginine to lysine mutant at amino acid site 358 could lead to the da1‐1 phenotype, which results in an increased organ size and larger seeds. In this study, the mutated ZmDA1 (Zmda1) and mutated ZmDAR1 (Zmdar1) driven by the maize ubiquitin promoter were separately introduced into maize elite inbred line DH4866. The grain yield of the transgenic plants was 15% greater than that of the wild‐type in 3 years of field trials due to improvements in the grain number, weight and starch content. Interestingly, the over‐expression of Zmda1 and Zmdar1 promoted kernel development, resulting in a more developed basal endosperm transfer cell layer (BETL) than WT and enhanced expression of starch synthase genes. This study suggests that the over‐expression of the mutated ZmDA1 or ZmDAR1 genes improves the sugar imports into the sink organ and starch synthesis in maize kernels.     

Robustness of central carbohydrate metabolism in developing maize kernels

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. ¹³C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-¹³C₆]glucose or [U-¹³C₁₂]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative ¹³C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.     

Improving the mineral reserves and protein quality of maize (Zea mays L.) kernels using unique genes

The effects of the maize genes, o ₂ and Mal, on the concentrations of mineral nutrient cations and amino acid levels in mature maize (Zea mays L) kernels of various inbred lines were studied. Previously, the o ₂ gene has been used to improve the protein quality and increase the mineral nutrient content of kernels from some inbred lines. Genotypes possessing the Mal (multiple aleurone layer) gene, contain more than one row of aleurone cells in their kernels and this gene enhances the effect of the o ₂gene on improving kernel protein quality. Incorporating these genes into the maize genome increased accumulation of several mineral nutrients (including Ca, Mg, Zn, Fe, Mn, Zn and Cu) in some of the experimental lines studied. The physiological basis for this increase of mineral nutrients in the kernels is discussed. The effect of the Mal gene on the kernel amino acid composition and protein quality was also examined. Possibly, these genes could be used in combination in breeding programs to improve kernel quality and nutritional value of maize.     

玉米籽粒淀粉含量遗传基础与调控机制*

玉米是世界上种植面积最大、总产量最高的粮食作物,其籽粒重量的70%来自于淀粉。淀粉不仅是人类及其他动物的主要能量来源,同时也是化工等行业的重要原料。利用拟南芥、水稻等模式植物,淀粉合成相关基因克隆与功能研究已取得较多进展。近年来,随着玉米淀粉含量相关遗传学研究的深入开展,通过数量性状位点(quantitative trait locus mapping,QTL)定位、全基因组关联分析(genome-wide association study, GWAS)及各种组学分析方法,发现了较多新的与淀粉含量相关的遗传位点及候选基因,但是尚缺乏归纳总结。综述了玉米籽粒淀粉合成与调控研究进展,对玉米籽粒淀粉含量相关的QTL和基因进行汇总和分析,通过构建一致性物理图谱,提炼玉米籽粒淀粉含量遗传定位热点区间,这为进一步解析玉米籽粒淀粉合成与代谢相关基因的功能提供参考,并为分子标记辅助育种提供遗传资源。     

Fusarium species complex and mycotoxins in grain maize from maize hybrid trials and from grower's fields

Aims: To quantify and to compare the occurrence of Fusarium species in maize kernels and stalk pieces, to analyse mycotoxins in kernels and maize crop residues, to evaluate two approaches to obtain kernel samples and to compare two methods for mycotoxin analyses. Methods and Results: The occurrence of Fusarium species in maize kernels and stalk pieces from a three‐year maize hybrid trial and 12 kernel samples from grower’s fields was assessed. Nine to 16 different Fusarium species were detected in maize kernels and stalks. In kernels, F. graminearum, F. verticillioides and F. proliferatum were the most prevalent species whereas in stalks, they were F. equiseti, F. proliferatum and F. verticillioides. In 2006, 68% of the kernel samples exceeded the recommended limit for pig feed for deoxynivalenol (DON) and 42% for zearalenone (ZON), respectively. Similarly, 75% of the samples from grower’s fields exceeded the limits for DON and 50% for ZON. In maize crop residues, toxin concentrations ranged from 2·6 to 15·3 mg kg^?1 for DON and from 0·7 to 7·4 mg kg^?1 for ZON. Both approaches to obtain maize kernel samples were valid, and a strong correlation between mycotoxin analysis using ELISA and LC‐MS/MS was found. Conclusions: The contamination of maize kernels, stalk pieces and remaining crop residues with various mycotoxins could pose a risk not only to animal health but also to the environment. With the hand‐picked sample, the entire Fusarium complex can be estimated, whereas combine harvested samples are more representative for the mycotoxin contents in harvested goods. Significance and Impact of the Study: This is the first multi‐year study investigating mycotoxin contamination in maize kernels as well as in crop residues. The results indicate a high need to identify cropping factors influencing the infection of maize by Fusarium species to establish recommendations for growers.     

Quantitative trait loci affecting amylose,amylopectin and starch content in maize recombinant inbred lines

The loci explaining the variability of quantitative traits related to starch content and composition (amylose, amylopectin and water soluble fraction) were searched for in maize kernels. Multifactorial genetic methods were used to detect and locate QTLs (quantitative trait loci) on a genetic map consisting mainly of RFLP markers for genes with known function. The genetic material was recombinant inbred lines originating from parents differing in starch structure (dent vs. flint). Kernels were harvested from field grown plants for two successive years and under two pollination systems. Main effect and epistasis QTLs were detected using two methods, composite interval mapping (MQTL) and ANOVA. Despite large year-to-year differences, physiologically meaningful co-locations were observed between trait QTLs. Moreover, the number of expressed sequences on our map allowed the search for co-locations between QTLs and genes involved in carbohydrate metabolism. The main co-location was between an amylose QTL and Shrunken 2 (SH2) locus, on chromosome 3 (SH2 encoding for the large subunit of ADPglucose pyrophosphorylase). The importance of this locus as a candidate gene for a starch QTL is in agreement with previous studies based either on QTL co-locations or on revertant analysis. Other co-locations were observed between amylose and amylopectin QTLs and the two loci of IVR1 invertase genes on chromosomes 2 and 10. Further comparison with previously detected QTLs for carbohydrate metabolism in maize leaves showed consistent co-location in map regions devoid of candidate genes, such as near chromosome 1S telomere. The possible contribution of regulatory genes in this region is discussed.     

Combined linkage mapping and association analysis reveals genetic control of maize kernel moisture content

The free moisture in crop kernels after being naturally dried is referred to as kernel moisture content (KMC). Maize KMC reflects grain quality and influences transportation and storage of seeds. We used an IBM Syn10 DH maize population consisting of 249 lines and an association panel comprising 310 maize inbred lines to identify the genetic loci affecting maize KMC in three environments. Using the IBM population detected 13 QTL on seven chromosomes, which were clustered into nine common QTL. Genome-wide association analysis (GWAS) identified 16 significant SNPs across the 3 environments, which were linked to 158 genes across the three environments. Combined QTL mapping and GWAS found two SNPs that were located in two of the mapped QTL, respectively. Twenty-three genes were linked with the loci co-localized in both populations. Of these 181 genes, five have previously been reported to be associated with KMC or to regulate seed development. These associations were verified by candidate gene association analysis. Two superior alleles and one favorable haplotype for Zm00001d007774 and Zm00001d047868 were found to influence KMC. These findings provide insights into molecular mechanisms underlying maize KMC and contribute to the use of marker-assisted selection for breeding low-KMC maize.     

A transposable element insertion disturbed starch synthase gene SSIIb in maize

Transposable elements, as the most active genetic factors, have driven genome evolution in maize and reshaped certain key loci responsible for maize domestication, exemplified by an inserted transposon in teosinte branched1 (tb1), which controls plant architecture. In this study, we detected an insertion of a transposable element in the second exon of the coding sequence of the maize starch synthase gene SSIIb, leading to a splicing modification and gene frameshift. This insertion provided a means of determining the function of SSIIb for starch synthesis during maize domestication. Association and quantitative trait locus (QTL) mappings showed that SSIIb was not associated with starch eating quality and total starch content of kernel, and two maize near-isogenic-line-like lines with and without the insertion of the transposable element further exhibited the same starch content of kernel and leaf; in addition, nucleotide diversity analysis revealed that maize SSIIb was not under selection during domestication. All these results demonstrated that maize SSIIb might serve as a very minor genetic factor or a functional redundancy gene in starch synthesis. Global BLAST showed that the maize genome harbored 1,387 copies of this transposable element, of which 135 copies were located in genic regions. At least three genes beside maize SSIIb were disturbed by this transposable element. Five patterns of transposition, according to the insertion sites close to or within genes such as maize SSIIb in this study, are under discussion and a large quantity of present/absent variations due to the insertion of varieties of transposable elements, discovered by revolutionary next-generation sequencing, would rapidly accelerate QTL and association mappings for maize domestication through candidate gene tactics in the near future.     

Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

A genome-wide survey of maize lipid-related genes: candidate genes mining,digital gene expression profiling and co-location with QTL for maize kernel oil

Lipids play an important role in plants due to their abundance and their extensive participation in many metabolic processes. Genes involved in lipid metabolism have been extensively studied in Arabidopsis and other plant species. In this study, a total of 1003 maize lipid-related genes were cloned and annotated, including 42 genes with experimental validation, 732 genes with full-length cDNA and protein sequences in public databases and 229 newly cloned genes. Ninety-seven maize lipid-related genes with tissue-preferential expression were discovered by in silico gene expression profiling based on 1984483 maize Expressed Sequence Tags collected from 182 cDNA libraries. Meanwhile, 70 QTL clusters for maize kernel oil were identified, covering 34.5% of the maize genome. Fifty-nine (84%) QTL clusters co-located with at least one lipid-related gene, and the total number of these genes amounted to 147. Interestingly, thirteen genes with kernel-preferential expression profiles fell within QTL clusters for maize kernel oil content. All the maize lipid-related genes identified here may provide good targets for maize kernel oil QTL cloning and thus help us to better understand the molecular mechanism of maize kernel oil accumulation.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Accumulation of stem sugar and its remobilisation in response to drought stress in a sweet sorghum genotype and its near‐isogenic lines carrying different stay‐green loci

• Near isogenic lines (NILs) of sweet sorghum genotype S35 into which individual stay green loci were introgressed, were used to understand the contribution of Stay green loci to stem sugar accumulation and its remobilization under drought stress exposure.
• Sugar and starch content, activities of sugar metabolism enzymes and levels of their expression were studied in the 3rd (source) leaf from panicle and the 5th (sugar storing) internode of the three lines, in irrigated plants and in plants exposed to a brief drought exposure at the panicle emergence stage. Annotation of genes in the respective Stay green loci introgressed in the NILs was carried out using bioinformatics tools.
• The leaves of NILs accumulated more photoassimilates and the internodes accumulated more sugar, as compared to the parent S35 line. Drought stress exposure led to a decrease in the starch and sugar levels in leaves of all three lines, while an increase in sugar levels was observed in internodes of the NILs. Sugar fluxes were accompanied by alterations in the activities of sugar metabolizing enzymes as well as the expression of genes related to sugar metabolism and transport.
• Remobilization of sugars from the stem internodes was apparent in the NILs when subjected to drought stress, since the peduncle, which supports the panicle, showed an increase in the sugar content, even when photoassimation in source leaves was reduced. Several genes related to carbohydrate metabolism were located in the Stay green loci, which probably contributed to variation in the parameters studied.

     

Bioinformatics Analysis of Microarray Data to Reveal Novel Genes Related to Cold-Resistance of Maize

Low temperature has become a major abiotic stress factor that can reduce maize yield and cause a number of economic loss. This study was designed to identify key genes and pathways associated with coldresistance of maize. The gene expression profile GSE46704, including 4 control temperature treated plants and 4 low temperature treated plants, was downloaded from the Gene Expression Omnibus database. Differentially-expressed genes (DEGs) were identified by limma package. Then, protein-protein interaction (PPI) network and module selection were constructed using Cytoscape. Moreover, the DEGs were re-matched based on the Zea mays L. gene ID and symbol data from PlantRegMap. Finally, the re-matched DEGs were performed functional and pathway enrichment analyses by the DAVID online tool. A total of 750 DEGs were screened (including 387 up-regulated and 363 down-regulated genes) In the PPI network, GRMZM2G070837_P01 and GRMZM2G114578_P01 had higher degrees. Besides, carbohydrate metabolic process, starch and sucrose metabolism and biosynthesis of secondary metabolites were significantly enriched in functional and pathway enrichment analysis. GRMZM2G070837_P01 and GRMZM2G114578_P01 might play a critical role in cold-resistance of maize. Meanwhile, carbohydrate metabolic process, starch and sucrose metabolism and biosynthesis of secondary metabolites might function in cold-resistance of maize.     

Dissection of maize kernel composition and starch production by candidate gene association

Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified.