Artificial chromosome formation in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15–30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Evgueni V. Ananiev, deceased Evgueni V. Ananiev and Chengcang Wu contributed equally to this work. Novel materials described in this publication may be available for noncommercial research purposes on acceptance and signing of a material transfer agreement. In some cases, such materials may contain or be derived from materials obtained from a third party. In such cases, the distribution of material will be subject to the requisite permission from any third-party owners, licensors, or controllers of all or parts of the material. Obtaining any permission will be the sole responsibility of the requestor.     

Isolation and Properties of Succinate Dehydrogenase Isozymes from Maize Scutellum (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Four electrophoretically homogeneous succinate dehydrogenase isoenzymes with specific activities of 0.041, 0.110, 0.030 and 0.037 U/mg protein were isolated and purified from maize scutellum (Zea mays L.). Their catalytic properties and pH optimums were studied.     

Genome-wide mutagenesis of <Emphasis Type="Italic">Zea mays</Emphasis> L. using <Emphasis Type="Italic">RescueMu</Emphasis> transposons

Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.     

Genetic dissection of the maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) MAMP response

Key message

Loci associated with variation in maize responses to two microbe-associated molecular patterns (MAMPs) were identified. MAMP responses were correlated. No relationship between MAMP responses and quantitative disease resistance was identified.

Abstract

Microbe-associated molecular patterns (MAMPs) are highly conserved molecules commonly found in microbes which can be recognized by plant pattern recognition receptors. Recognition triggers a suite of responses including production of reactive oxygen species (ROS) and nitric oxide (NO) and expression changes of defense-related genes. In this study, we used two well-studied MAMPs (flg22 and chitooctaose) to challenge different maize lines to determine whether there was variation in the level of responses to these MAMPs, to dissect the genetic basis underlying that variation and to understand the relationship between MAMP response and quantitative disease resistance (QDR). Naturally occurring quantitative variation in ROS, NO production, and defense genes expression levels triggered by MAMPs was observed. A major quantitative traits locus (QTL) associated with variation in the ROS production response to both flg22 and chitooctaose was identified on chromosome 2 in a recombinant inbred line (RIL) population derived from the maize inbred lines B73 and CML228. Minor QTL associated with variation in the flg22 ROS response was identified on chromosomes 1 and 4. Comparison of these results with data previously obtained for variation in QDR and the defense response in the same RIL population did not provide any evidence for a common genetic basis controlling variation in these traits.

     

Methane Yield and Feed Quality Parameters of Mixed Silages from Maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) and Common Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

European agricultural policy increasingly focuses on environmental friendly cropping systems. Intercropping of maize (Zea mays L.) and common beans (Phaseolus vulgaris L.) has been suggested as an alternative cropping system with environmental benefits. The aim of this study was to assess methane yield potential of mixed silages. Based on material from two field experiments at three sites in Germany, mixed silages were produced with proportions of individual components varying from 0 to 100 % of fresh matter in increments of 12.5 %. Chemical parameters (neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (XP), starch, sugar, and crude fat) were determined, and batch tests were performed to measure methane yield potential from silages. With increasing bean proportion, concentrations of XP increased while NDF, methane yield, and methane content decreased. While methane yield showed a negative relationship with XP content (R ² = 0.56***), a positive relation was found with NDF (R ² = 0.55***). The reduction of methane yield of circa 1 L of methane per each additional bean percentage in the silages could not be explained by the chemical parameters of the silages. It is hypothesized that other chemical compounds, such as lectins, which were not determined in the present study, may have influenced methane production.     

Overexpression of <Emphasis Type="Italic">Zm-HINT1</Emphasis> Confers Salt and Drought Tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Histidine triad nucleotide-binding protein 1 (HINT1) is highly conserved in many species and plays important roles in various biological processes. However, little is known about the responses of HINT1 to abiotic stress in plants. Salt and drought stress are major limiting factors for plant growth and development, and their negative effects on crop productivity may threaten the world’s food supply. Previously, we identified a maize gene, Zm-HINT1, which encodes a 138-amino-acid protein containing conserved domains including the HIT motif, helical regions, and β-strands. Here, we demonstrate that overexpression of Zm-HINT1 in Arabidopsis confers salt and drought tolerance to plants. Zm-HINT1 significantly regulated Na⁺ and K⁺ accumulation in plants under salt stress. The improve tolerance characteristics of Arabidopsis plants that were overexpressing Zm-HINT1 led to increased survival rates after salt and drought treatments. Compared with control plants, those plants that overexpressed Zm-HINT1 showed increased proline content and superoxide dismutase activity, as well as lower malondialdehyde and hydrogen peroxide accumulation under salt and drought treatments. The expression patterns of stress-responsive genes in Arabidopsis plants that overexpressed Zm-HINT1 significantly differed from those in control lines. Taken together, these results suggest that Zm-HINT1 has potential applications in breeding and genetic engineering strategies that are designed to produce new crop varieties with improved salt and drought tolerance.     

Expression and functional analysis of <Emphasis Type="Italic">ZmDWF4</Emphasis>, an ortholog of <Emphasis Type="Italic">Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Development of versatile gene-based SNP assays in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

A large number of maize single nucleotide polymorphism (SNP) candidate sequences have been generated and deposited in public databases. However, very little work has been done to date to comprehensively characterize those SNPs and identify a set of markers, which potentially would have high impact in molecular genetics research and breeding programs. Here we describe a multi-step process to identify highly polymorphic gene-based SNPs among ~130,000 public markers. A set of 695 highly polymorphic SNPs (minor allele frequency value >0.3), identified within exons, 5′ and 3′ untranslated regions of genes, were converted into four of the most popular high-throughput genotyping assays that include Illumina’s GoldenGate and Infinium chemistries, Life Technologies’ TaqMan assay and KBioSciences’ KASPar assay. The term “versatile” was applied to 162 gene-based SNPs that were successfully converted into all four chemistries and had perfect genotypic clustering patterns. This subset of discovered versatile SNP markers represents a universal tool for application in various molecular genetics and breeding projects in maize, where genotyping is based on one of the four above-mentioned chemistries. This study demonstrated that despite the availability of millions of discovered SNPs in maize, only a very small portion of those polymorphisms could be utilized for the development of robust, versatile assays, and has real practical value in marker-assisted selection.     

A Novel Little Membrane Protein Confers Salt Tolerance in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

24-Epibrassinolide reduces stress in nematode-infected tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants cultured <Emphasis Type="Italic">in vitro</Emphasis>

24-Epibrassinolide (EBL) is considered the most probable brassinosteroid (BR) candidate that could be used for practical application in agriculture. EBL-induced stress-protective properties were evaluated in in vitro-grown tomato (Solanum lycopersicum L.) varieties Pusa Ruby (susceptible to nematodes) and PNR-7 (resistant to nematodes) during nematode pathogenesis. Sterilized tomato seeds treated with 10^?11, 10^?9, or 10^?7 M EBL and germinated in vitro were inoculated with second-stage juveniles of Meloidogyne incognita [(Kofoid and White) Chitwood]. Whole plant analyses of morphological and biochemical parameters 7 d after inoculation showed significant improvements in plant growth and development for both varieties and a highly significant reduction in the number of galls in the susceptible variety. Increased specific activities of antioxidative enzymes (catalase, ascorbate peroxidase, glutathione reductase, glutathione peroxidase, guaiacol peroxidase, and superoxide dismutase) were observed in EBL-treated seedlings of both varieties, but increases were higher in the resistant variety. A highly significant increase in antioxidants (ascorbic acid content, total flavonoid content, total glutathione content, and total phenolic content) was observed in EBL-treated Pusa Ruby seedlings, whereas in PNR-7, significant increases were found except for total flavonoid content, which increased non-significantly. Confocal microscopic images showed amelioration of stress in roots of EBL-treated seedlings as indicated by the decrease in level of green fluorescence in them as compared to untreated and nematode-inoculated roots.     

Genetic analysis and molecular mapping of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) stalk rot resistant gene<Emphasis Type="Italic"> Rfg</Emphasis>1

One single pathogen Fusarium graminearum Schw. was inoculated to maize inbred lines 1,145 (Resistant) and Y331 (Susceptive), and their progenies of F₁, F₂ and BC₁F₁ populations. Field statistical data revealed that all of the F₁ individuals were resistant to the disease and that the ratio of resistant plants to susceptive plants was 3:1 in the F₂ population, and 1:1 in the BC₁F₁ population. The results revealed that a single dominant gene controls the resistance to F. graminearum Schw.. The resistant gene to F. graminearum Schw. was denominated as Rfg1 according to the standard principle of the nomenclature of the plant disease resistant genes. RAPD (randomly amplified polymorphic DNA) combined with BSA (bulked segregant analysis) analysis was carried out in the developed F₂ and BC₁F₁ populations, respectively. Three RAPD products screened from the RAPD analysis with 820 Operon 10-mer primers showed the linkage relation with the resistant gene Rfg1. The three RAPD amplification products (OPD-20₁₀₀₀, OPA-04₁₁₀₀ and OPY-04₉₀₀) were cloned and their copy numbers were determined. The results indicated that only OPY-04₉₀₀ was a single-copy sequence. Then, OPY-04₉₀₀ was used as a probe to map the Rfg1 gene with a RIL F₇ mapping population provided by Henry Nguyen, which was developed from the cross S3×Mo17. Rfg1 was primarily mapped on chromosome 6 between the two linked markers OPY-04₉₀₀ and umc21 (Bin 6.04–6.05). In order to confirm the primary mapping result, 25 SSR (simple sequence repeat) markers and six RFLP (restriction fragment length polymorphism) markers in the Rfg1 gene-encompassing region were selected, and their linkage relation with Rfg1 was analyzed in our F₂ population. Results indicated that SSR marker mmc0241 and RFLP marker bnl3.03 are flanking the Rfg1 gene with a genetic distance of 3.0 cM and 2.0 cM, respectively. This is the first time to name and to map a single resistant gene of maize stalk rot through a single pathogen inoculation and molecular marker analysis.Communicated by H.F. Linskens     

Genome-wide identification,systematic analysis and characterization of <Emphasis Type="Italic">SRO</Emphasis> family genes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

SIMILAR TO RCD ONE (SRO) is a small plant-specific gene family, which play essential roles in plant growth and development as well as in abiotic stresses. However, the function of SROs in maize is still unknown. In our study, six putative SRO genes were isolated from the maize genome. A systematic analysis was performed to characterize the ZmSRO gene family. The ZmSRO gene family was divided into two groups according to the motif and intron/exon analysis. Phylogenetic analysis of them with other plants showed that the clades of SROs along with the divergence of monocot and dicot and ZmSROs were more closely with OsSROs. Many abiotic stress response and hormone-induced cis-regulatory elements were identified from the promoter region of ZmSROs. Furthermore, RNA-seq analysis indicated that SRO genes were widely expressed in different tissues and development stages in maize, and the expression divergence was also obviously observed. Analyses of expression in response to PEG6000 and NaCl treatment, in addition to exogenous application of ABA and GA hormones showed that the majority of the members display stress-induced expression patterns. Taken together, our results provide valuable reference for further functional analysis of the SRO gene family in maize, especially in abiotic stress responses.     

Leaf area prediction for corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) cultivars with multiregression analysis

Leaf area is one of the most important parameter for plant growth. Reliable equations were offered to predict leaf area for Zea mays L. cultivars. All equations produced for leaf area were derived as affected by leaf length and leaf width. As a result of ANOVA and multiregression analysis, it was found that there was a close relationship between actual and predicted growth parameters. The produced leaf-area prediction model in the present study is LA = a + b L + c W + d LZ where LA is leaf area, L is leaf length, W is maximum leaf width, LZ is leaf zone and a, b, c, d are coefficients. R ² values were between 0.88–0.97 and standard errors were found to be significant at the p<0.001 significance level.     

Characterization of phenylpropanoid pathway genes within European maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbreds

Background  

Forage quality of maize is influenced by both the content and structure of lignins in the cell wall. Biosynthesis of monolignols, constituting the complex structure of lignins, is catalyzed by enzymes in the phenylpropanoid pathway.     

A Phytocystatin Gene from <Emphasis Type="Italic">Malus prunifolia</Emphasis> (Willd.) Borkh., <Emphasis Type="Italic">MpCYS5</Emphasis>, Confers Salt Stress Tolerance and Functions in Endoplasmic Reticulum Stress Response in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cystatins, or phytocystatins (PhyCys), comprise a family of plant-specific inhibitors of cysteine proteinases. They are thought to help regulate endogenous processes and protect plants against biotic or abiotic stresses, such as heat, salinity, cold, water deficit, chilling, and abscisic acid (ABA) treatment. We isolated and identified a novel cystatin gene from Malus prunifolia, MpCYS5. Its expression was typically induced by salt stress treatment; ectopic expression in Arabidopsis enhanced salt tolerance. Physiological parameters confirmed this phenotype, with the transgenics having remarkably lower electrolyte leakage (EL) values, higher chlorophyll concentrations, and lower levels of malondialdehyde (MDA) upon salt treatment. In addition, the accumulation of reactive oxygen species was markedly regulated by MpCYS5 under stress conditions, as shown by fluctuations in the concentrations of hydrogen peroxide and superoxide radicals and the activities of antioxidant enzymes. We also noted that this gene modulated tunicamycin (TM)-induced endoplasmic reticulum (ER) stress tolerance and functioned in the unfolded protein response (UPR)-signaling pathway in Arabidopsis. This was confirmed by the expression of eight ER stress-responsive genes. All marker genes examined were strongly induced in the wild type, while most of them maintained relatively stable over time in the transgenics. These results demonstrated that ectopic expression of a cystatin gene is associated with salt-tolerant and TM-tolerant phenotypes. Therefore, the discovery of MpCYS5 from M. prunifolia might establish a molecular link between the ER stress response and salt tolerance in plants.