Modification Of Maize Research Articles

Bacillus amyloliquefaciens MZ895491: an endophytic bacterium as potential biocontrol agent in integrated pest management of fall armyworm in maize

ABSTRACT The fall armyworm, Spodoptera frugiperda is a major pest of maize crops and necessitates effective control measures. Excessive use of chemical pesticides often results in the development of resistant insect populations and environmental contamination, highlighting the need for environmentally friendly substitutes. This research aimed to estimate the potential of maize (COH6) apoplastic fluid bacterium Bacillus amyloliquefaciens (MZ895491), as a biocontrol agent against second instar S. frugiperda larvae under laboratory and pot culture conditions. The results suggest that the presence of B. amyloliquefaciens in the artificial diet could reduce the growth and development of S. frugiperda larvae. Metabolic profiling of extracellular components of B. amyloliquefaciens revealed the presence of numerous defensive and anti-feeding metabolites. A study conducted in pots demonstrated that foliar application of B. amyloliquefaciens to 5 d old maize resulted in a significant increase in antioxidant activity (83.3%), chlorophyll content (63.3%) and phenolic content (81.3%) in the leaves of 35 d old maize after 24 h of S. frugiperda infestation. Feeding bioassay further indicated significant reduction in the feeding capacity of S. frugiperda larvae on maize inoculated with B. amyloliquefaciens. The study observed that a metabolic modification in maize leaves likely led to reduced growth in larvae during the whole plant bioassay. This suggests that foliar application of B. amyloliquefaciens may effectively serve as one of the biocontrol agents against Spodoptera frugiperda. The application appears to alter the feeding behaviour of the larvae and modify maize leaf metabolism.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

Light Dependent Changes in Adenylate Methylation of the Promoter of the Mitochondrial Citrate Synthase Gene in Maize (Zea mays L.) Leaves.

Limited methyl-specific restriction of genomic DNA by endonuclease MAL1 revealed the changes in its methyl status caused by adenine modification in maize (Zea mays L.) leaves under different light conditions (dark, light, irradiation by red and far-red light). Incubation in the light and irradiation by red light exhibited an activating effect on DNA adenine methylase activity, which was reflected in an increase in the number of methylated adenines in GATC sites. Far-red light and darkness exhibited an opposite effect. The use of nitrite conversion of DNA followed by methyladenine-dependent restriction by MboI nuclease revealed a phytochrome B-dependent mechanism of regulation of the methyl status of adenine in the GATC sites in the promoter of the gene encoding the mitochondrial isoform of citrate synthase. Irradiation of plants with red light caused changes in the adenine methyl status of the analyzed amplicon, as evidenced by the presence of restriction products of 290, 254, and 121 nucleotides. Adenine methylation occurred at all three GATC sites in the analyzed DNA sequence. It is concluded that adenylate methylation is controlled by phytochrome B via the transcription factor PIF4 and represents an important mechanism for the tricarboxylic acid cycle regulation by light.

Physical Properties of Starches Modified by Phosphorylation and High-Voltage Electrical Discharge (HVED).

High-voltage electrical discharge (HVED) is considered as a novel, non-thermal process and is currently being researched regarding its effect on microorganisms (decontamination of food), waste water treatment, and modification of different compounds and food components. In this paper, four native starches (maize, wheat, potato, and tapioca) were treated with HVED, phosphorylated with Na2HPO4 and Na5P3O10, and modified by a combination of HVED with each phosphorylation reaction both prior and after chemical modification. Pasting properties, swelling power, solubility, gel texture, and particle size were analyzed. Although HVED induced lower contents of P in modified starches, it had an effect on analyzed properties. The results revealed that HVED treatment alone had a limited effect on pasting properties of starches, but it had an effect on properties of phosphorylated starches, both when it was conducted prior and after the chemical modification, reducing the influence of Na5P3O10 and Na2HPO4 on the decrease of pasting temperature. With minor exceptions, the gel strength of starches increased, and the rupture strength decreased by all modifications. HVED treatment resulted in a decrease of the particle size after the modification of maize and wheat starches, while potato and tapioca starches were not significantly influenced by the treatment.

M6A-Maize: Weakly supervised prediction of m6A-carrying transcripts and m6A-affecting mutations in maize (Zea mays)

With the rapid development of high-throughput sequencing techniques nowadays, extensive attention has been paid to epitranscriptomics, which covers more than 150 distinct chemical modifications to date. Among that, N6-methyladenosine (m6A) modification has the most abundant existence, and it is also significantly related to varieties of biological processes. Meanwhile, maize is the most important food crop and cultivated throughout the world. Therefore, the study of m6A modification in maize has both economic and academic value. In this research, we proposed a weakly supervised learning model to predict the situation of m6A modification in maize. The proposed model learns from low-resolution epitranscriptome datasets (e.g., MeRIP-seq), which predicts the m6A methylation status of given fragments or regions. By taking advantage of our prediction model, we further identified traits-associated SNPs that may affect (add or remove) m6A modifications in maize, which may provide potential regulatory mechanisms at epitranscriptome layer. Additionally, a centralized online-platform was developed for m6A study in maize, which contains 58,838 experimentally validated maize m6A-containing regions including training and testing datasets, and a database for 2,578 predicted traits-associated m6A-affecting maize mutations. Furthermore, the online web server based on proposed weakly supervised model is available for predicting putative m6A sites from user-uploaded maize sequences, as well as accessing the epitranscriptome impact of user-interested maize SNPs on m6A modification. In all, our work provided a useful resource for the study of m6A RNA methylation in maize species. It is freely accessible at www.xjtlu.edu.cn/biologicalsciences/maize.

Differential Gene Expression Responding to Low Phosphate Stress in Leaves and Roots of Maize by cDNA-SRAP.

Phosphate (Pi) deficiency in soil can have severe impacts on the growth, development, and production of maize worldwide. In this study, a cDNA-sequence-related amplified polymorphism (cDNA-SRAP) transcript profiling technique was used to evaluate the gene expression in leaves and roots of maize under Pi stress for seven days. A total of 2494 differentially expressed fragments (DEFs) were identified in response to Pi starvation with 1202 and 1292 DEFs in leaves and roots, respectively, using a total of 60 primer pairs in the cDNA-SRAP analysis. These DEFs were categorized into 13 differential gene expression patterns. Results of sequencing and functional analysis showed that 63 DEFs (33 in leaves and 30 in roots) were annotated to a total of 54 genes involved in diverse groups of biological pathways, including metabolism, photosynthesis, signal transduction, transcription, transport, cellular processes, genetic information, and organismal system. This study demonstrated that (1) the cDNA-SRAP transcriptomic profiling technique is a powerful method to analyze differential gene expression in maize showing advantageous features among several transcriptomic methods; (2) maize undergoes a complex adaptive process in response to low Pi stress; and (3) a total of seven differentially expressed genes were identified in response to low Pi stress in leaves or roots of maize and could be used in the genetic modification of maize.

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize 1507 for Cultivation, Import, Processing, Food and Feed Uses under Directive 2001/18/EC and Regulation (EC) No 1829/2003 (C/ES/01/01, C/NL/00/10, EFSA/GMO/NL/2004/02)

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize 1507 for Cultivation, Import, Processing, Food and Feed Uses under Directive 2001/18/EC and Regulation (EC) No 1829/2003 (C/ES/01/01, C/NL/00/10, EFSA/GMO/NL/2004/02)

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Natural Variation in RNA m6A Methylation and Its Relationship with Translational Status.

N 6 -methyladenosine (m6A) is the most abundant modification of eukaryotic mRNA. Although m6A has been demonstrated to affect almost all aspects of RNA metabolism, its global contribution to the post-transcriptional balancing of translational efficiency remains elusive in plants. In this study, we performed a parallel analysis of the transcriptome-wide mRNA m6A distribution and polysome profiling in two maize (Zea mays) inbred lines to assess the global correlation of m6A modification with translational status. m6A sites are widely distributed in thousands of protein-coding genes, confined to a consensus motif and primarily enriched in the 3' untranslated regions, and highly coordinated with alternative polyadenylation usage, suggesting a role of m6A modification in regulating alternative polyadenylation site choice. More importantly, we identified that the m6A modification shows multifaceted correlations with the translational status depending on its strength and genic location. Moreover, we observed a substantial intraspecies variation in m6A modification, and this natural variation was shown to be partly driven by gene-specific expression and alternative splicing. Together, these findings provide an invaluable resource for ascertaining transcripts that are subject to m6A modification in maize and pave the way to a better understanding of natural m6A variation in mediating gene expression regulation.

Community composition and functions of endophytic bacteria of Bt maize

We investigated the potential effects of genetic modification of Bt maize on the community composition and functions of bacterial endophytes associated with transgenic maize (Bt MON 810) in comparison with its isogenic parental line at two developmental stages. Bacterial isolates were obtained from transgenic (Bt) and non-transgenic (non-Bt) maize at 50- and 90-day-old developmental stages. Isolated bacterial endophytes were screened for their capabilities in phosphate solubilisation, nitrogen fixation, production of antifungal metabolites and production of indole acetic acid. After molecular identification, 60 isolates were obtained and clustered into 19 and 18 operational taxonomic units from 50- and 90-day-old maize, respectively. The isolates belonged to the genera Bacillus, Pantoea, Serratia, Yersinia, Enterobacter, Pseudomonas, Acinetobacter and Stenotrophomonas. Functional attributes and diversity of the isolated endophytes at both developmental stages were not significantly different for both maize varieties. However, functional attributes were significantly affected by plant growth stage. Isolates from younger plants were more efficient producers of indole acetic acid, but exhibited little or no capabilities for nitrogen fixation, phosphate solubilisation and antifungal activity in both maize genotypes. Based on these outcomes, Bt modification in maize does not seem to affect the community composition or functional attributes of bacterial endophytes.Significance:• Bt modification in maize does not affect the ecological guild or functional attributes of cultivable bacterial endophytes.

Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.

Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

A Computational Workflow to Identify Allele-specific Expression and Epigenetic Modification in Maize

Allele-specific expression refers to the preferential expression of one of the two alleles in a diploid genome, which has been thought largely attributable to the associated cis-element variation and allele-specific epigenetic modification patterns. Allele-specific expression may contribute to the heterosis (or hybrid vigor) effect in hybrid plants that are produced from crosses of closely-related species, subspecies and/or inbred lines. In this study, using Illumina high-throughput sequencing of maize transcriptomics, chromatic H3K27me3 histone modification and DNA methylation data, we developed a new computational framework to identify allele-specifically expressed genes by simultaneously tracking allele-specific gene expression patterns and the epigenetic modification landscape in the seedling tissues of hybrid maize. This approach relies on detecting nucleotide polymorphisms and any genomic structural variation between two parental genomes in order to distinguish paternally or maternally derived sequencing reads. This computational pipeline also incorporates a modified Chi-square test to statistically identify allele-specific gene expression and epigenetic modification based on the Poisson distribution.

Enzymatic esterification of starch using recovered coconut oil

Modification of maize and cassava starches was done using recovered coconut oil and microbial lipase. Microwave esterification was advantageous as it gave a DS 1.55 and 1.1 for maize starch and cassava starch, respectively. Solution state esterification of cassava starch for 36 h at 60 °C gave a DS of 0.08 and semi-solid state esterification gave a DS of 0.43. TGA and DSC studies showed that the higher DS attributed to the thermostability, since onset of decomposition is at a higher temperature (492 °C) than the unmodified (330 °C) and was stable above 600 °C. α-Amylase digestibility and viscosity reduced for modified starch.

Investigations on Genetically Modified Maize (Bt-Maize) in Pig Nutrition: Chemical Composition and Nutritional Evaluation

The objective of the present study was to determine the composition and the nutritional value of parental and transgenic maize seeds fed to pigs. The parental maize line was genetically modified to incorporate a gene from Bacillus thuringiensis (Bt) expressing a toxin against the European corn borer (Ostrinia nubilalis). Both (parental and transgenic) maize lines were analyzed for crude nutrients, starch, sugar, non-starch polysaccharides (NSP), amino acids, fatty acids, as well as for selected minerals. Furthermore, four complete diets were mixed and were analyzed for the same nutrients and some selected ingredients. The diets contained 70% maize to attain a high effect level. To evaluate the feeding value of one variety of genetically modified maize (transgenic) compared to the feeding value of the unmodified maize (parental) line, a balance study with twelve pigs was designed. Three collecting periods were used for each maize line each with six animals. The collected faeces were analyzed for crude nutrients. All measured parameters were virtually the same ( e.g. crude protein 11.59% vs. 11.06% in DM), especially the digestibility of crude protein (85.8 ± 2.3% vs. 86.1 ± 1.8%), the amount of nitrogen-free-extract (92.8 ± 0.6% vs. 93.2 ± 0.6%) and the metabolizable energy (15.7 ± 0.2% vs. 15.8 ± 0.2% MJ/kg DM) for both maize lines. Compared to the parental line, the chemical composition and digestibility of crude nutrients and energy content were not significantly affected by the genetic modification of maize. Therefore, from the view of a nutritional assessment, the genetically modified maize can be regarded as substantially equivalent to the parental maize line.