Non-transgenic Lines Research Articles

Comparative metabolic profiling of four transgenic maize lines and two non-transgenic maize lines using high-performance liquid chromatography mass spectrometry

We compared metabolic changes in transgenic maize lines (differing in genetic backgrounds and exogenous genes insertion) with non-transgenic counterparts. At the corn seedling stage, entirely expanding leaves were obtained from maize lines 1 (transgenic maize SK12-5; overexpressing cry1Ab/cry2Aj; parental maize ZD958), 2 (transgenic maize IE034; overexpressing cry1Ie; parental maize ZD958), 3 (transgenic maize Bt799; overexpressing cry1Ac-M; parental maize Z58), 4 (transgenic maize Bt799; overexpressing cry1Ac-M; parental maize ZD958), 5 (non-transgenic maize Z58), and 6 (non-transgenic maize ZD958). For each line, six biological replicates were prepared; each contained an entirely expanded leaf at the top of the plant. Metabolites were identified with high-performance liquid chromatography mass spectrometry in three comparison groups (4 transgenic maize lines vs. their corresponding controls, line 3 vs. 4, and transgenic vs. non-transgenic maize). Top 200 cationic and top 200 anionic metabolites with higher variable importance in projection value were chosen for principal component analysis, followed by pathway enrichment analysis. Annotation was possible for 227 of 400 metabolites. Twelve up- and 26 down-regulated overlapping metabolites were identified in line 1 vs. 6, line 2 vs. 6, and line 4 vs. 6 comparison groups. Altered metabolites significantly enriched in purine and glutathione metabolism pathways, but no pathways were enriched in line 3 vs. 4. Comparing transgenic and non-transgenic maize lines revealed 59 up- and 37 down-regulated metabolites, which were also significantly enriched in purine and glutathione metabolism pathways. Therefore, transgenic Bt maize differed metabolically from non-transgenic maize. Additionally, purine and glutathione metabolism pathways may be important in the transgenic maize metabolism.

Seed Metabolome Analysis of a Transgenic Rice Line Expressing Cholera Toxin B-subunit

Plant-based human vaccines have been actively developed in recent years, and rice (Oryza sativa L.) is one of the best candidate crops for their production and delivery. By expressing a modified cholera toxin B (CTB) subunit, we previously developed MucoRice-CTB, a rice-based vaccine against cholera, which is caused by infection of the intestine with the bacteria Vibrio cholerae. MucoRice-CTB lines have been extensively characterized by whole-genome sequencing and proteome analyses to evaluate the mutation profiles and proteome status, respectively. Here, we report non-targeted metabolomic profiling of the MucoRice-CTB transgenic rice line 51A (MR-CTB51A), MucoRice-RNAi (MR-RNAi), and their non-transgenic parent line by using gas chromatography–time-of-flight mass spectrometry. The levels of several amino acids, organic acids, carbohydrates, lipids, and secondary metabolites were significantly increased in MR-CTB51A compared with the non-transgenic parent line. These metabolomics results complement essential information obtained by genome sequencing and proteomics approaches, thereby contributing to comprehensive understanding of the properties of MucoRice-CTB as a plant-based vaccine.

Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley chi26 gene for fungal resistance

BackgroundThe main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker.ResultsMolecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events. Chitinase enzyme activity was detected using a standard enzymatic assay. The expression levels of chi26 gene in the different transgenic lines, compared to their respective controls, were determined using qRT-PCR. The transgene was silenced in some transgenic families across generations. Gene silencing in the present study seemed to be random and irreversible. The homozygous transgenic plants of T4, T5, T6, T8, and T9 generations were tested in the field for five growing seasons to evaluate their resistance against rusts and powdery mildew. The results indicated high chitinase activity at T0 and high transgene expression levels in few transgenic families. This resulted in high resistance against wheat rusts and powdery mildew under field conditions. It was indicated by proximate and chemical analyses that one of the transgenic families and the non-transgenic line were substantially equivalent.ConclusionTransgenic wheat with barley chi26 was found to be resistant even after five generations under artificial fungal infection conditions. One transgenic line was proved to be substantially equivalent as compared to the non-transgenic control.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Metagenomic analyses of bacterial endophytes associated with the phyllosphere of a Bt maize cultivar and its isogenic parental line from South Africa.

Genetic modification of maize with Bacillus thuringiensis (Bt) cry proteins may predispose shifts in the bacterial endophytes' community associated with maize shoots. In this study, the diversity of bacterial endophytes associated with a Bt maize genotype (Mon810) and its isogenic non-transgenic parental line were investigated at pre-flowering (50days) and post-flowering (90days) developmental stages. PCR-DGGE and high throughput sequencing on the Illumina MiSeq sequencer were used to characterize bacterial 16S rRNA gene diversity in leaves, stems, seeds and tassels. PCR-DGGE profile revealed similarity as well as differences between bacterial communities of shoots in both cultivars and at both developmental stages. A total of 1771 operational taxonomic units (OTUs) were obtained from the MiSeq and assigned into 14 phyla, 27 classes, 58 orders, 116 families and 247 genera. Differences in alpha and beta diversity measures of OTUs between the phyllospheres of both genotypes were not significant (P > .05) at all developmental stages. In all cultivars, OTU diversity reduced with plant development. OTUs belonging to the phyla Proteobacteria were dominant in all maize phyllospheres. The class Gammaproteobacteria was dominant in Bt maize while, Alphaproteobacteria and Actinobacteria were dominant in non-Bt maize phyllospheres. Differences in the abundance of some genera, including Acidovorax, Burkerholderia, Brachybacterium, Enterobacter and Rhodococcus, whose species are known beneficial endophytes were observed between cultivars. Hierarchical cluster analysis further suggests that the bacterial endophyte communities of both maize genotypes associate differently (are dissimilar). Overall, the results suggest that bacterial endophytes community differed more across developmental stages than between maize genotypes.

A new transgenic rice line exhibiting enhanced ferric iron reduction and phytosiderophore production confers tolerance to low iron availability in calcareous soil.

Iron (Fe) deficiency is a critical agricultural problem, especially in calcareous soil, which is distributed worldwide. Rice plants take up Fe(II) from soil through a OsIRT1 transporter (Strategy I-related system) and also take up Fe(III) via a phytosiderophore-based system (Strategy II system). However, rice plants are susceptible to low-Fe conditions because they have low Fe(III) reduction activity and low-level phytosiderophore secretion. Previously, we produced transgenic rice plants expressing a mutationally reconstructed yeast ferric chelate reductase, refre1/372, under the control of the OsIRT1 promoter. This transgenic rice line exhibited higher Fe(III) chelate reductase activity and tolerance to Fe deficiency. In addition, we produced transgenic rice overexpressing the Fe deficiency-inducible transcription factor, OsIRO2, which regulates the expression of various genes involved in the strategy II Fe(III) uptake system, including OsNAS1, OsNAAT1, OsDMAS1, OsYSL15, and TOM1. This transgenic rice exhibited improved phytosiderophore secretion ability and tolerance to Fe deficiency. In the present research, transgenic rice plants that possess both the OsIRT1 promoter-refre1/372 and the 35S promoter-OsIRO2 (RI lines) were produced to enhance both Strategy I Fe(II) reductase ability and Strategy II phytosiderophore productivity. RI lines exhibited enhanced tolerance to Fe-deficient conditions at the early and middle-late stages of growth in calcareous soil, compared to both the non-transgenic line and lines harboring either OsIRT1 promoter-refre1/372 or 35S promoter-OsIRO2 alone. RI lines also exhibited a 9-fold higher yield than the non-transgenic line. Moreover, we successfully produced Fe-deficiency-tolerant Tachisugata rice, which is a high-biomass variety used as fodder. Collectively, our results demonstrate that combined enhancement of two Fe uptake systems in rice is highly effective in conferring tolerance to low Fe availability in calcareous soil.

A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures

In this work, transgenic lines of suspension cultured cells of Vitis vinifera cv. Monastrell containing the plasmid pMOG800-sts have been obtained. The cell growth of these transgenic cell lines decreased slightly as compared to non-transgenic suspension cultured cells, while cell viability was not affected. In addition, the elicitation with cyclodextrins and methyl jasmonate enhanced the production of trans-resveratrol, observing the highest levels of this compound in sts-expressing transgenic Vitis suspension cultured cells with the sts expression cassette in the forwards orientation. Moreover, the forwards 2 (F2) transgenic cell line produced the greater levels of trans-resveratrol in comparison with the non-transgenic cell line. In fact, when suspension cultured cells were treated with both elicitors, the accumulation of trans-resveratrol outside the cells in the F2 transgenic suspension cultured cells increased twice (1458 mg.L−1) as compared to non-transgenic cell lines (724 mg.L−1). In both cases, the levels of trans-resveratrol detected in the treatment with cyclodextrins and methyl jasmonate were greater than the sum of the individual treatments, and therefore we observed a synergistic effect in the presence of both elicitors. Moreover, the expression profile of sts gene in transgenic V. vinifera cell lines was similar to the expression profile detected for the endogenous sts gene in non-transgenic V. vinifera cell lines, being the expression levels greater in the treatment with methyl jasmonate and cyclodextrins, which was related to the high levels of trans-resveratrol found in the presence of both elicitors.

Characterization of a drought tolerance-related gene of Chinese cabbage in a transgenic tobacco plant

Drought is a major abiotic stress that affects plant growth and productivity in many regions of the world. As climate change has increased the incidence of drought, research of the genes related to drought stress and development of drought-tolerant plants is necessary now more than ever. In this study, genes related to drought tolerance were screened from Brassica rapa 135k microarray data and a gene with full-length sequence and unknown function was selected and named BrDSR28 (B. rapa drought stress resistance 28). The expression of BrDSR28 was over 4-fold higher in drought-tolerant Chinese cabbage than in wild-type controls. This gene contains a 483 bp open reading frame encoding a 160 amino acid polypeptide and it contains a senescence regulator domain. To characterize the function of BrDSR28, Nicotiana tabacum was transformed with over-expression and down-regulation vectors of the gene. Transgenic tobacco plants were confirmed by PCR, Southern hybridization, and RT-PCR analyses. The expression levels and phenotypes of the transgenic tobacco plants were analyzed under drought stress. The BrDSR28 over-expression lines showed higher expression of BrDSR28 in all stages of drought treatment and showed significant tolerance to drought when compared to the non-transgenic lines and BrDSR28 down-regulated tobacco plants.

Characterization and Functional Analysis of Five MADS-Box B Class Genes Related to Floral Organ Identification in Tagetes erecta.

According to the floral organ development ABC model, B class genes specify petal and stamen identification. In order to study the function of B class genes in flower development of Tagetes erecta, five MADS-box B class genes were identified and their expression and putative functions were studied. Sequence comparisons and phylogenetic analyses indicated that there were one PI-like gene—TePI, two euAP3-like genes—TeAP3-1 and TeAP3-2, and two TM6-like genes—TeTM6-1 and TeTM6-2 in T. erecta. Strong expression levels of these genes were detected in stamens of the disk florets, but little or no expression was detected in bracts, receptacles or vegetative organs. Yeast hybrid experiments of the B class proteins showed that TePI protein could form a homodimer and heterodimers with all the other four B class proteins TeAP3-1, TeAP3-2, TeTM6-1 and TeTM6-2. No homodimer or interaction was observed between the euAP3 and TM6 clade members. Over-expression of five B class genes of T. erecta in Nicotiana rotundifolia showed that only the transgenic plants of 35S::TePI showed altered floral morphology compared with the non-transgenic line. This study could contribute to the understanding of the function of B class genes in flower development of T. erecta, and provide a theoretical basis for further research to change floral organ structures and create new materials for plant breeding.

Transgenic sorghum with improved digestibility of storage proteins obtained by Agrobacterium-mediated transformation

Development of transgenic plants with modified seed storage protein composition and increased nutritive value is one of the most promising areas of genetic engineering. This task is especially important for sorghum—a unique drought tolerant cereal crop that is characterized, however, by a relatively poor nutritive value in comparison with other cereals. It is considered that one of the reasons of the low nutritive value of the sorghum grain is the resistance of one of its seed storage proteins, γ-kafirin, located in the outer layer of endosperm protein bodies, to protease digestion. Using Agrobacterium-mediated genetic transformation, we obtained transgenic sorghum plants (Sorghum bicolor (L.) Moench) harboring a genetic construct for RNAi silencing of the γ-kafirin gene. In the T1 generation, the plants with almost floury or modified endosperm texture of kernels were found. In these kernels, the vitreous endosperm layer has been reduced and/or covered by a thin layer of floury endosperm. In vitro protein digestibility (IVPD) analysis showed that the amount of undigested protein in transgenic plants from the T3 generation was reduced by 2.9–3.2 times, in comparison with the original non-transgenic line, and the digestibility index reached 85–88% (in comparison with 59% in the original line). In T2 families, the plants combining high IVPD with vitreous endosperm type were found. In the electrophoretic spectra of endosperm proteins of transgenic plants with increased digestibility, the proportion of 20 kD protein that is encoded by the γ-kafirin gene, was significantly reduced, in comparison with the original non-transgenic line. HPLC analysis showed total amino acid content in two out of the three studied transgenic plants from the T2 generation was reduced in comparison with the original non-transgenic line, while the lysine proportion increased by 1.6–1.7 times. The mechanisms conditioning improved digestibility of storage proteins in transgenic plants are discussed. The results of experiments demonstrate that it is feasible to develop sorghum lines combining high protein digestibility and vitreous endosperm that has a high breeding value.

Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation.

Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34-22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals.

Time Course Field Analysis of COMT-Downregulated Switchgrass: Lignification, Recalcitrance, and Rust Susceptibility

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38 % more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2 years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11–16 % less lignin, 33–40 % lower S/G (syringyl-to-guaiacyl) ratios, and 15–42 % higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. These results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.

Functional analysis of diacylglycerol acyltransferase1 genes from Camelina sativa and effects of CsDGAT1B overexpression on seed mass and storage oil content in C. sativa

Camelina (Camelina sativa), which belongs to the Brassicaeae family, is an emerging oilseed crop with the potential to expand biodiesel production to arid land. During storage oil synthesis, diacylglycerol acyltrasferase1 (DGAT1) catalyzes the conversion of diacylglycerol (DAG) and free fatty acids to triacylglycerol (TAG). In this study, three DGAT1 genes (CsDGAT1A, CsDGAT1B, and CsDGAT1C) were isolated from developing C. sativa seeds. The deduced amino acid sequences of the three CsDGAT1 genes shared more than 84 % identity with those of DGAT1 genes from Arabidopsis thaliana and Brassica napus. CsDGAT1A, B, and C transcripts were detected in various C. sativa organs, including developing seeds. Fluorescent protein-fused CsDGAT1A, B, and C were localized in the endoplasmic reticulum (ER) of tobacco epidermal cells. When the CsDGAT1A, B, and C genes under the control of the BnNapin promoter were expressed in an Arabidopsis AS11 mutant, which is defective in DGAT1, the amounts and composition of total fatty acids in dry seeds were restored to those of the wild type, indicating the three CsDGAT1 genes to be functionally active. In transgenic C. sativa plants overexpressing CsDGAT1B, the levels of total seed oils were increased by ~24 % compared with non-transgenic lines. Transgenic C. sativa embryos with enhanced seed oil contents harbored larger embryonic cells and a greater number of cells compared with the wild type. Transgenic Camelina plants with increased oil contents can be used as renewable resources for the production of biodiesel and non-petroleum-based biomaterials.

Generation of transgenic watermelon resistance to Cucumber mosaic virus facilitated by an effective Agrobacterium-mediated transformation method

Watermelon is an annual vegetable crop that suffers from various diseases during growth. Genetic engineering is an effective tool for improving plant disease resistance. This paper presents an effective transformation protocol designed for a female parent watermelon line mediated by Agrobacterium. In this protocol, 2 times MS Fe-EDTA were supplemented into Murashige and Skoog (MS) medium to suppress the chlorosis during watermelon explants culturing, and the optimal preculture time (5days) and coculture time (5days) were screened out to improve the frequency of shoots regeneration. Using our protocol, 17 putative transgenic plants expressing artificial microRNAs that target Cucumber mosaic virus (CMV) 2a/2b genes were recovered, and seven of them were succeeded in obtaining T1 generation seeds because of the technique difficulties of self-pollination of the transgenic watermelon lines. PCR and Southern blot analyses confirmed that the transferred fragment was successfully imported and integrated into the genome of three of these seven transgenic lines. DAS-ELISA assay indicated that the CMV can be detected in the nontransgenic line but not in the transgenic lines, and the transgenic lines displayed resistance to CMV infection.

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.

Soybean Salt Tolerance 1 (GmST1) Reduces ROS Production, Enhances ABA Sensitivity, and Abiotic Stress Tolerance in Arabidopsis thaliana.

Abiotic stresses, including high soil salinity, significantly reduce crop production worldwide. Salt tolerance in plants is a complex trait and is regulated by multiple mechanisms. Understanding the mechanisms and dissecting the components on their regulatory pathways will provide new insights, leading to novel strategies for the improvement of salt tolerance in agricultural and economic crops of importance. Here we report that soybean salt tolerance 1, named GmST1, exhibited strong tolerance to salt stress in the Arabidopsis transgenic lines. The GmST1-overexpressed Arabidopsis also increased sensitivity to ABA and decreased production of reactive oxygen species under salt stress. In addition, GmST1 significantly improved drought tolerance in Arabidopsis transgenic lines. GmST1 belongs to a 3-prime part of Glyma.03g171600 gene in the current version of soybean genome sequence annotation. However, comparative reverse transcription-polymerase chain reaction analysis around Glyma.03g171600 genomic region confirmed that GmST1 might serve as an intact gene in soybean leaf tissues. Unlike Glyma.03g171600 which was not expressed in leaves, GmST1 was strongly induced by salt treatment in the leaf tissues. By promoter analysis, a TATA box was detected to be positioned close to GmST1 start codon and a putative ABRE and a DRE cis-acting elements were identified at about 1 kb upstream of GmST1 gene. The data also indicated that GmST1-transgenic lines survived under drought stress and showed a significantly lower water loss than non-transgenic lines. In summary, our results suggest that overexpression of GmST1 significantly improves Arabidopsis tolerance to both salt and drought stresses and the gene may be a potential candidate for genetic engineering of salt- and drought-tolerant crops.

BpMADS12 gene role in lignin biosynthesis of Betula platyphylla Suk by transcriptome analysis

MADS-box transcription factors show highly diverse regulatory functions in a wide variety of organisms. In this study, we characterized a MADS-box gene (BpMADS12) from the white birch (Betula platyphylla Suk). This gene is a member of the suppressor of overexpression of CO 1/tomato MADS 3 class of MADS-box genes. We generated lines overexpressing BpMADS12 and found that these had higher levels of lignin compared to that observed in nontransgenic lines. Transcriptome analysis revealed numerous changes in gene expression patterns. In total, 8794 differentially expressed genes were identified, including 5006 upregulated unigenes and 3788 downregulated unigenes in BpMADS-overexpression lines. Differentially expressed genes involved in the pathways for lignin and brassinosteroid biosynthesis were significantly enriched and may have contributed to phenotypic changes. The results from a quantitative RT-PCR analysis were consistent those obtained with the transcriptome analysis. Our transcriptome analysis, in combination with measurement of lignin level, indicated that BpMADS12 promotes lignin synthesis through regulation of key enzymes in response to brassinosteroid signaling. These results suggest that this MADS-box protein is crucial to all subsequent structural events and provide a good foundation for studies aiming to elucidate the developmental mechanisms underlying formation of wood.

Effective Identification of Low-Gliadin Wheat Lines by Near Infrared Spectroscopy (NIRS): Implications for the Development and Analysis of Foodstuffs Suitable for Celiac Patients.

ScopeThe aim of this work was to assess the ability of Near Infrared Spectroscopy (NIRS) to distinguish wheat lines with low gliadin content, obtained by RNA interference (RNAi), from non-transgenic wheat lines. The discriminant analysis was performed using both whole grain and flour. The transgenic sample set included 409 samples for whole grain sorting and 414 samples for flour experiments, while the non-transgenic set consisted of 126 and 156 samples for whole grain and flour, respectively.Methods and ResultsSamples were scanned using a Foss-NIR Systems 6500 System II instrument. Discrimination models were developed using the entire spectral range (400–2500 nm) and ranges of 400–780 nm, 800–1098 nm and 1100–2500 nm, followed by analysis of means of partial least square (PLS). Two external validations were made, using samples from the years 2013 and 2014 and a minimum of 99% of the flour samples and 96% of the whole grain samples were classified correctly.ConclusionsThe results demonstrate the ability of NIRS to successfully discriminate between wheat samples with low-gliadin content and wild types. These findings are important for the development and analysis of foodstuff for celiac disease (CD) patients to achieve better dietary composition and a reduction in disease incidence.

Proteomic analysis of mature barley grains from C-hordein antisense lines

Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.

Cry1Ac Transgenic Sugarcane Does Not Affect the Diversity of Microbial Communities and Has No Significant Effect on Enzyme Activities in Rhizosphere Soil within One Crop Season.

Cry1Ac transgenic sugarcane provides a promising way to control stem-borer pests. Biosafety assessment of soil ecosystem for cry1Ac transgenic sugarcane is urgently needed because of the important role of soil microorganisms in nutrient transformations and element cycling, however little is known. This study aimed to explore the potential impact of cry1Ac transgenic sugarcane on rhizosphere soil enzyme activities and microbial community diversity, and also to investigate whether the gene flow occurs through horizontal gene transfer. We found no horizontal gene flow from cry1Ac sugarcane to soil. No significant difference in the population of culturable microorganisms between the non-GM and cry1Ac transgenic sugarcane was observed, and there were no significant interactions between the sugarcane lines and the growth stages. A relatively consistent trend at community-level, represented by the functional diversity index, was found between the cry1Ac sugarcane and the non-transgenic lines. Most soil samples showed no significant difference in the activities of four soil enzymes: urease, protease, sucrose, and acid phosphate monoester between the non-transgenic and cry1Ac sugarcane lines. We conclude, based on one crop season, that the cry1Ac sugarcane lines may not affect the microbial community structure and functional diversity of the rhizosphere soil and have few negative effects on soil enzymes.