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1.
Plant Physiol ; 181(4): 1404-1414, 2019 12.
Article in English | MEDLINE | ID: mdl-31636104

ABSTRACT

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.


Subject(s)
Gene Library , Genomics , Mutagenesis, Insertional/genetics , Mutation/genetics , Zea mays/genetics , Alleles , Base Sequence , Crosses, Genetic , DNA Transposable Elements/genetics , Genes, Plant
2.
J Exp Bot ; 70(15): 3795-3808, 2019 08 07.
Article in English | MEDLINE | ID: mdl-31020318

ABSTRACT

The splicing of organelle-encoded mRNA in plants requires proteins encoded in the nucleus. The mechanism of splicing and the factors involved are not well understood. Pentatricopeptide repeat (PPR) proteins are known to participate in such RNA-protein interactions. Maize defective kernel 41 (dek41) is a seedling-lethal mutant that causes developmental defects. In this study, the Dek41 gene was cloned by Mutator tag isolation and allelic confirmation, and was found to encode a P-type PPR protein that targets mitochondria. Analysis of the mitochondrial RNA transcript profile revealed that dek41 mutations cause reduced splicing efficiency of mitochondrial nad4 intron 3. Immature dek41 kernels exhibited severe reductions in complex I assembly and NADH dehydrogenase activity. Up-regulated expression of alternative oxidase genes and deformed inner cristae of mitochondria in dek41, as revealed by TEM, indicated that proper splicing of nad4 is essential for correct mitochondrial functioning and morphology. Consistent with this finding, differentially expressed genes in the dek41 endosperm included those related to mitochondrial function and activity. Our results indicate that DEK41 is a PPR protein that affects cis-splicing of mitochondrial nad4 intron 3 and is required for correct mitochondrial functioning and maize kernel development.


Subject(s)
Introns/genetics , Mitochondria/metabolism , Plant Proteins/metabolism , Zea mays/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Mitochondria/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Zea mays/genetics
3.
New Phytol ; 214(4): 1563-1578, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28277611

ABSTRACT

Mitochondria are semi-autonomous organelles that are the powerhouse of the cells. Plant mitochondrial RNA editing guided by pentatricopeptide repeat (PPR) proteins is essential for energy production. We identify a maize defective kernel mutant dek36, which produces small and collapsed kernels, leading to embryos and/or seedlings lethality. Seed filling in dek36 is drastically impaired, in line with the defects observed in the organization of endosperm transfer tissue. Positional cloning reveals that DEK36, encoding a mitochondria-targeted E+ subgroup PPR protein, is required for mitochondrial RNA editing at atp4-59, nad7-383 and ccmFN -302, thus resulting in decreased activities of mitochondrial complex I, complex III and complex IV in dek36. Loss-of-function of its Arabidopsis ortholog At DEK36 causes arrested embryo and endosperm development, leading to embryo lethality. At_dek36 also has RNA editing defects in atp4, nad7, ccmFN1 and ccmFN2 , but at the nonconserved sites. Importantly, efficiency of all editing sites in ccmFN1 , ccmFN2 and rps12 is severely decreased in At_dek36, probably caused by the impairment of their RNA stabilization. These results suggest that the DEK36 orthologue pair are essential for embryo and endosperm development in both maize and Arabidopsis, but through divergent function in regulating RNA metabolism of their mitochondrial targets.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Plant Proteins/metabolism , RNA Editing , Seeds/growth & development , Zea mays/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Cloning, Molecular , Genetic Complementation Test , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mutation , Plant Proteins/genetics , Plants, Genetically Modified , Seedlings/genetics , Seedlings/growth & development , Seeds/genetics , Zea mays/growth & development
4.
J Exp Bot ; 67(22): 6323-6335, 2016 12.
Article in English | MEDLINE | ID: mdl-27789589

ABSTRACT

Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB-ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs.


Subject(s)
Endosperm/metabolism , Seed Storage Proteins/metabolism , Zea mays/metabolism , Endoplasmic Reticulum/metabolism , Endosperm/chemistry , Metabolic Networks and Pathways , Proteomics , Ribosomes/metabolism , Seed Storage Proteins/analysis , Seed Storage Proteins/isolation & purification , Zein/metabolism
5.
PLoS Genet ; 12(4): e1005991, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27077660

ABSTRACT

Zeins, the predominent storage proteins in maize endosperm, are encoded by multiple genes and gene families. However, only a few transcriptional factors for zein gene regulation have been functionally characterized. In this study, a MADS-box protein, namely ZmMADS47, was identified as an Opaque2 (O2) interacting protein via yeast two-hybrid screening. The N-terminal portion of ZmMADS47 contains a nuclear localization signal (NLS), and its C-terminal portion contains a transcriptional activation domain (AD). Interestingly, the transcriptional activation activity is blocked in its full length form, suggesting conformational regulation of the AD. Molecular and RNA-seq analyses of ZmMADS47 RNAi lines revealed down regulation of α-zein and 50-kD γ-zein genes. ZmMADS47 binds the CATGT motif in promoters of these zein genes, but ZmMADS47 alone is not able to transactivate the promoters. However, when both O2 and ZmMADS47 are present, the transactivation of these promoters was greatly enhanced. This enhancement was dependent on the AD function of ZmMADS47 and the interaction between ZmMADS47 and O2, but it was independent from the AD function of O2. Therefore, it appears interaction with O2 activates ZmMADS47 on zein gene promoters.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant/genetics , Nuclear Proteins/genetics , Plant Proteins/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , Zea mays/genetics , Zein/genetics , Electrophoretic Mobility Shift Assay , Molecular Sequence Data , Oryza/genetics , Phylogeny , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , RNA Interference , RNA, Small Interfering/genetics , Two-Hybrid System Techniques
7.
Plant Cell ; 27(3): 532-45, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25691733

ABSTRACT

Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we studied the transcriptome of o2 mutants using RNA sequencing (RNA-Seq) and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). The RNA-Seq analysis revealed 1605 differentially expressed genes (DEGs) and 383 differentially expressed long, noncoding RNAs. The DEGs cover a wide range of functions related to nutrient reservoir activity, nitrogen metabolism, stress resistance, etc. ChIP-Seq analysis detected 1686 O2 DNA binding sites distributed over 1143 genes. Overlay of the RNA-Seq and ChIP-Seq results revealed 35 O2-modulated target genes. We identified four O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16- and 18-kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. We built a hierarchical regulatory model for O2 that provides an understanding of its pleiotropic biological effects.


Subject(s)
DNA, Plant/genetics , Gene Expression Regulation, Plant , Genome, Plant , Plant Proteins/genetics , Promoter Regions, Genetic , Transcription, Genetic , Zea mays/genetics , Base Sequence , Binding Sites , Chromatin Immunoprecipitation , Down-Regulation/genetics , Gene Ontology , Genes, Plant , Molecular Sequence Data , Mutation , Nitrogen/metabolism , Nucleotide Motifs/genetics , Open Reading Frames/genetics , Plant Proteins/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Untranslated/genetics , Sequence Analysis, RNA , Stress, Physiological/genetics , Zein/genetics
8.
Plant Cell ; 26(6): 2582-2600, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24951479

ABSTRACT

Proline, an important amino acid, accumulates in many plant species. Besides its role in plant cell responses to environmental stresses, the potential biological functions of proline in growth and development are unclear. Here, we report cloning and functional characterization of the maize (Zea mays) classic mutant proline responding1 (pro1) gene. This gene encodes a Δ1-pyrroline-5- carboxylate synthetase that catalyzes the biosynthesis of proline from glutamic acid. Loss of function of Pro1 significantly inhibits proline biosynthesis and decreases its accumulation in the pro1 mutant. Proline deficiency results in an increased level of uncharged tRNApro AGG accumulation and triggers the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the pro1 mutant, leading to a general reduction in protein synthesis in this mutant. Proline deficiency also downregulates major cyclin genes at the transcriptional level, causing cell cycle arrest and suppression of cell proliferation. These processes are reversible when external proline is supplied to the mutant, suggesting that proline plays a regulatory role in the cell cycle transition. Together, the results demonstrate that proline plays an important role in the regulation of general protein synthesis and the cell cycle transition in plants.

9.
Plant Physiol ; 165(2): 582-594, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24706551

ABSTRACT

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19-kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ER-associated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 α-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

11.
Mol Biol Rep ; 41(1): 477-87, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24307252

ABSTRACT

A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.


Subject(s)
Arabidopsis/genetics , Chlorophyta/enzymology , Glutamate-Ammonia Ligase/genetics , Plant Proteins/genetics , Amino Acid Sequence , Arabidopsis/growth & development , Arabidopsis/metabolism , Cloning, Molecular , Glutamate-Ammonia Ligase/biosynthesis , Molecular Sequence Data , Nitrogen/metabolism , Phenotype , Phylogeny , Plant Proteins/biosynthesis , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid
12.
Gene ; 536(2): 407-15, 2014 Feb 25.
Article in English | MEDLINE | ID: mdl-24334123

ABSTRACT

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.


Subject(s)
Arabidopsis/genetics , Chlorophyta/genetics , Genes, Plant/genetics , Glutamate-Ammonia Ligase/genetics , Plants, Genetically Modified/genetics , Amino Acid Sequence , Arabidopsis/metabolism , Biomass , Chlorophyta/metabolism , Glutamate-Ammonia Ligase/metabolism , Mesophyll Cells/metabolism , Molecular Sequence Data , Nitrogen/metabolism , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plants, Genetically Modified/metabolism , RNA, Messenger/genetics , Sequence Alignment , Nicotiana/genetics , Nicotiana/metabolism
13.
J Exp Bot ; 65(4): 923-38, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24363426

ABSTRACT

The actin-based myosin system is essential for the organization and dynamics of the endomembrane system and transport network in plant cells. Plants harbour two unique myosin groups, class VIII and class XI, and the latter is structurally and functionally analogous to the animal and fungal class V myosin. Little is known about myosins in grass, even though grass includes several agronomically important cereal crops. Here, we identified 14 myosin genes from the genome of maize (Zea mays). The relatively larger sizes of maize myosin genes are due to their much longer introns, which are abundant in transposable elements. Phylogenetic analysis indicated that maize myosin genes could be classified into class VIII and class XI, with three and 11 members, respectively. Apart from subgroup XI-F, the remaining subgroups were duplicated at least in one analysed lineage, and the duplication events occurred more extensively in Arabidopsis than in maize. Only two pairs of maize myosins were generated from segmental duplication. Expression analysis revealed that most maize myosin genes were expressed universally, whereas a few members (XI-1, -6, and -11) showed an anther-specific pattern, and many underwent extensive alternative splicing. We also found a short transcript at the O1 locus, which conceptually encoded a headless myosin that most likely functions at the transcriptional level rather than via a dominant-negative mechanism at the translational level. Together, these data provide significant insights into the evolutionary and functional characterization of maize myosin genes that could transfer to the identification and application of homologous myosins of other grasses.


Subject(s)
Computational Biology , Gene Expression Regulation, Plant , Genome, Plant/genetics , Myosins/genetics , Zea mays/genetics , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Alternative Splicing , Gene Duplication , Genes, Reporter , Genetic Loci , Multigene Family , Myosins/metabolism , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Transport , RNA, Plant/genetics , Transcriptome , Zea mays/cytology , Zea mays/metabolism
14.
Genetica ; 140(7-9): 337-47, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23054225

ABSTRACT

Activator/Dissociation (Ac/Ds) transposable elements have been used in maize insertional mutagenesis as a complement to Mutator (Mu). In this study, to further improve the efficiency of the Ac/Ds mutagenesis system, we adopted apt1-m1 (Ac) on the long arm of chromosome 9 (9L) as a donor Ac to create an Ac insertion library. This system is based on the negative selection pressure against the donor Ac, and it was highly efficient for isolating new transposition events. We obtained 9,625 transposition events from 1083 F1 ears with an average transposition rate of 8.66 % (rates ranged from 1.11 to 29.73 %). We also adopted a modified PCR-based genome walking strategy to improve the efficiency of the new method for isolating transposon-flanking sequences. This method is more efficient than the Southern-based method that was used in previous studies. A validation step was developed to distinguish transposon tags derived from newly transposed Ac or Ds elements. Using this PCR-based method, we isolated 67 inheritable flanking sequences from the apt1-m1 (Ac) transposition library; of these, 51 were confirmed as tr-Ac-flanking sequences and 11 were tr-Ds-flanking sequences. Similar to other Ac donors from different loci, the apt1-m1 (Ac) system also exhibited a preference for short distance transposition. In this study, we have further improved the Ac mutagenesis system in maize for gene isolation and functional genomics studies.


Subject(s)
Chromosomes, Plant/genetics , DNA Transposable Elements , Zea mays/genetics , Base Sequence , DNA, Plant/chemistry , Genes, Plant , Genome, Plant , Molecular Sequence Data , Mutagenesis, Insertional , Polymerase Chain Reaction
15.
Plant Cell ; 24(8): 3447-62, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22892319

ABSTRACT

Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.


Subject(s)
Cell Movement , Endoplasmic Reticulum/metabolism , Endosperm/metabolism , Myosins/metabolism , Plant Proteins/metabolism , Zea mays/metabolism , Alleles , Cloning, Molecular , Endoplasmic Reticulum/genetics , Endosperm/genetics , Genes, Plant , HSP70 Heat-Shock Proteins/metabolism , Intracellular Membranes/metabolism , Mutation , Myosins/genetics , Phenotype , Phylogeny , Plant Cells/metabolism , Plant Proteins/genetics , Protein Interaction Mapping , RNA, Plant/genetics , RNA, Plant/metabolism , Nicotiana/genetics , Nicotiana/metabolism , Two-Hybrid System Techniques , Zea mays/genetics , Zein/genetics , Zein/metabolism
16.
Genetica ; 139(9): 1109-18, 2011 Sep.
Article in English | MEDLINE | ID: mdl-22057628

ABSTRACT

Homologous recombination in meiosis provides the evolutionary driving force in eukaryotic organisms by generating genetic variability. Meiotic recombination does not always occur evenly across the chromosome, and therefore genetic and physical distances are not consistently in proportion. We discovered a 278 kb interval on the long arm of chromosome 10 (10 L) through analyzed 13,933 descendants of backcross population. The recombinant events distributed unevenly in the interval. The ratio of genetic to physical distance in the interval fluctuated about 47-fold. With the assistance of molecular markers, the interval was divided into several subintervals for further characterization. In agreement with previous observations, high gene-density regions such as subinterval A and B were also genetic recombination hot subintervals, and repetitive sequence-riched region such as subinterval C was also found to be recombination inert at the detection level of the study. However, we found an unusual subinterval D, in which the 72-kb region contained 6 genes. The gene-density of subinterval D was 5.8 times that of the genome-wide average. The ratio of genetic to physical distance in subinterval D was 0.58 cM/Mb, only about 3/4 of the genome average. We carried out an analysis of sequence polymorphisms and methylation status in subinterval D, and the potential causes of recombination suppression were discussed. This study was another case of a detailed genetic analysis of an unusual recombination region in the maize genome.


Subject(s)
Chromosomes, Plant , Genomics , Recombination, Genetic , Zea mays/genetics , DNA Methylation , Gene Order , Genes, Plant , Polymorphism, Genetic
17.
Genetics ; 189(4): 1281-95, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21954158

ABSTRACT

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.


Subject(s)
Plant Proteins/genetics , Zea mays/metabolism , Alleles , Genes, Plant , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Mutation , Plant Proteins/biosynthesis , Plants, Genetically Modified , RNA Interference , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Zea mays/genetics
18.
Microbiol Res ; 166(1): 27-35, 2011 Jan 20.
Article in English | MEDLINE | ID: mdl-20116227

ABSTRACT

Methanogenesis from the biomass in the anoxic biogas reactors is catalyzed by syntrophic cooperation between anaerobic bacteria, syntrophic acetogenic bacteria and methanogenic archaea. Understanding of microbial community composition within the biogas reactors may improve the methane production from biomass fermentation. In this study, methanogenic archaea diverity of a biogas reactor supplied with swine feces as mono-substrate under mesophilic conditions was investigated. Community composition was determined by analysis of methyl coenzyme reductase subunit A gene (mcrA) clone library consisting of 123 clones. Statistical analysis of mcrA library indicated that all major groups of methanogens from our biogas reactor were detected. In the library, 57.7% clones were affiliated to Methanobacteriales, 34.2% to Methanomicrobiales, 2.4% to Methanosarcinales and about 5.7% clones belonged to unclassified euryarchaeota. Over 90% of the methanogenic archaea from our biogas reactor were postulated to be hydrogenotrophic methanogens. Comparing with other previous studies reporting that hydrogenotrophic methanogens are dominant species in the biogas plants, this study firstly reported that Methanobacteriales instead of Methanomicrobiales are the most predominant methanogenic archaea in the biogas reactor fed with swine feces as sole substrate.


Subject(s)
Biodiversity , Euryarchaeota/genetics , Euryarchaeota/metabolism , Methanobacteriales/genetics , Oxidoreductases/genetics , Animals , Biofuels , Biomass , Bioreactors , DNA, Archaeal/analysis , Feces , Methane/biosynthesis , Methanobacteriales/metabolism , Methanomicrobiales/genetics , Methanomicrobiales/metabolism , Methanosarcinales/genetics , Methanosarcinales/metabolism , Molecular Sequence Data , Phylogeny , Swine
19.
Plant Cell Rep ; 29(6): 545-59, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20336461

ABSTRACT

Maize seeds are an important source of food, animal feed, and industrial raw materials. To understand global gene expression and regulation during maize seed development, a normalized cDNA library, covering most of the developmental stages of maize seeds, was constructed. Sequencing analysis of 10,848 randomly selected clones identified 6,630 unique ESTs. Among them, 57 putative transcription factors (TFs) were identified. The TFs belong to seven different super-families, specifically 17 Zinc-finger, 13 bZIP, 8 bHLH, 6 MADS, 7 MYB, 3 Homedomain, and 3 AP2/EREBP. The spatial and temporal expression of the TFs was analyzed by semi-quantitative RT-PCR with representative tissue types and seeds at different developmental stages, revealing their diverse expression patterns and expression levels. One-third (19) of the maize TFs was found their putative orthologs in Arabidopsis. Similar expression patterns were observed in both maize and Arabidopsis for the majority of orthologous pairs (15 out of 19), suggesting their conserved functions during seed development. In conclusion, the systematic analysis of maize seed TFs has provided valuable insight into transcriptional regulation during maize seed development.


Subject(s)
Expressed Sequence Tags , Plant Proteins/genetics , Seeds/genetics , Transcription Factors/genetics , Zea mays/genetics , Chromosomes, Plant/genetics , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Gene Library , Oligonucleotide Array Sequence Analysis , Phylogeny , RNA, Plant/genetics , Seeds/growth & development
20.
Plant Mol Biol ; 69(6): 649-59, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19112555

ABSTRACT

The alpha-zein super gene family encodes the most predominant storage protein in maize (Zea mays) endosperm. In maize inbred line B73, it consists of four gene families with 41 member genes. In this study, we combined quantitative real-time PCR and random clone sequencing to successfully profile the expression of alpha-zein super gene family during endosperm development. We found that only 18 of the 41 member genes were expressed, and their expression levels diverge greatly. At the gene family level, all families had characteristic "up-and-down" oscillating expressional patterns that diverged into two major groups. At the individual gene level, member genes showed dramatic divergence of expression patterns, indicating fast differentiation of their expression regulation. A comparison study among different inbred lines revealed significantly different expressed gene sets, indicating the existence of highly diverged haplotypes. Large gene families containing long gene clusters, e.g. z1A or z1C, mainly contributed the highly divergent haplotypes. In addition, allelic genes also showed significant divergence in their expressional levels. These results indicated a highly dynamic and fast evolving nature to the maize alpha-zein super gene family, which might be a common feature for other large gene families.


Subject(s)
Gene Expression Profiling , Seeds/genetics , Zea mays/genetics , Zein/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genetic Variation , Multigene Family , Reverse Transcriptase Polymerase Chain Reaction , Seeds/embryology , Species Specificity , Zea mays/classification , Zea mays/embryology
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