Changhong Guo

Genome-wide survey and expression analysis of the MADS-box gene family in soybean

MADS-box genes encode important transcription factors in plants that are involved in many processes during plant growth and development. An investigation of the soybean genome revealed 106 putative MADS-box genes. These genes were classified into two classes, type I and type II, based on phylogenetic analysis. The soybean type II group has 72 members, which is higher than that of Arabidopsis, indicating that soybean type II genes have undergone a higher rate of duplication and/or a lower rate of gene loss after duplication. Soybean MADS-box genes are present on all chromosomes. Like Arabidopsis and rice MADS-box genes, soybean MADS-box genes expanded through tandem gene duplication and segmental duplication events. There are many duplicate genes distributed across the soybean genome, with two genomic regions, i.e., MADS-box gene hotspots, where MADS-box genes with high degrees of similarity are clustered. Analysis of high-throughput sequencing data from soybean at different developmental stages and in different tissues revealed that MADS-box genes are expressed in embryos of various stages and in floral buds. This expression pattern suggests that soybean MADS-box genes play an important role in soybean growth and floral development.

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.

Genotoxicity effect of nitrobenzene on soybean (Glycine max) root tip cells

Nitrobenzene is a synthetic compound widely used in industry which can lead to environmental pollution. While the toxicity and carcinogenicity of nitrobenzene on humans and animals have been studied, less is known about its genotoxicity to plants. In this study, the genotoxic effects of nitrobenzene were investigated with growing soybean seedlings in solution culture. Compared with the control, the growth of soybean seedlings (taproot length, longest lateral root length and lateral roots number) decreased and showed statistics difference at nitrobenzene test concentration of 50 and 100mg/L. Micronucleus, chromosomal bridge and others chromosomal aberrations were observed in soybean root tip cells exposed to nitrobenzene. Frequency of chromosomal aberrations increased linearly with nitrobenzene test concentration between 5 and 50mg/L and decreased at 100mg/L which showed significant difference between control and 25mg/L or higher test concentration. Results of the present study suggest that nitrobenzene has genotoxicity on soybean root tip cells. The mechanism of genotoxicity of NB needs further study. It is concluded that high environmental levels of nitrobenzene in rivers, lakes and dam waters are hazardous to aquatic species and to irrigated plants.

The effects of nitrobenzene on the genetic toxicity in tobacco seedling leaf cells by comet assay

Nitrobenzene (NB) is a synthetic hydrocarbon widely used in industries which is a highly toxic environmental pollutant. To investigate its genetic toxicity to plants, tobacco seedlings were exposed to varying concentration of NB solution for short-term (24 hours). The comet assay was used to examine the DNA damage in tobacco leaf cells under the stress of NB. The DNA migration increased with the dose of NB (from 10 to 100 mg/L). The values of tail intensity (TDNA), tail moment (TM), and olive tail moment (OTM) increased linearly with NB concentrations between 5 mg/L and 100 mg/L. The results suggest that NB has genotoxic effect on the molecular level in tobacco leaf cells. The comet parameters were significantly different from the control when NB concentrations were higher than 5 mg/L (P<0.05), suggesting that tobacco leaf cells have high sensitivity to NB stress. The phenomena demonstrated that the tobacco comet assay could be used efficiently monitor aquatic pollution caused by the toxic chemicals. In addition, a similar evaluation was performed using different NB and antioxidant ascorbic acid (Vc) mixed solutions. The DNA damage in the antioxidant group were significantly lower than those in the group without antioxidant, which illustrates that antioxidants could partially decrease the DNA damage induced by NB. These results suggest that oxidation reactions caused by NB might be one of the factors that lead to DNA damage in plants. However, to process the possible participation of mechanisms of DNA damage caused by NB, further investigation will need.

Biosafety assessment of GFP transplastomic tobacco to rhizosphere microbial community

Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry, and has many applications as a marker, especially in plant transformation system. Although a number of studies have been conducted to assess the toxify of this protein to specific organisms, little is known about GFP on rhizosphere microbial community, which is regarded as good indicator for environmental risk assessment. Chloroplast genetic engineering has shown superiority over traditional nuclear genetic engineering, and has been used in many aspects of plant genetic engineering. High levels of chloroplast-based protein accumulation make this technology as an ideal strategy to evaluate biosafety of transgenes. In the present study, the effects of field-released GFP transplastomic tobacco (Nicotiana tabacum) on rhizosphere microbes over a whole growth cycle were investigated by using both culture-dependent and culture-independent methods. Compared to wild-type control, transplastomic tobacco had no significant influence on the microbial population at the seedling, vegetative, flowering and senescing stages. However, developmental stages had more influence than ecotypes (GFP-transformed and wild-type). This was confirmed by colony forming unit, Biolog EcoTM and PCR-DGGE analysis. Thus, these results suggest chloroplast transformation with a GFP reporter gene has no significant influence on rhizosphere microbial community, and will be potential platform for plant biotechnology in future.

Evaluation of phytotoxicity and genotoxicity of nitrobenzene with A battery of Vicia Faba assay system

Nitrobenzene (NB) is an important organic compound intermediate that is used widely in industry. In the present study, to evaluate the phytotoxicity and genotoxicity of NB on plants, Vicia faba was exposed to increasing concentrations of NB (5 mg L(-1) , 10 mg L(-1) , 25 mg L(-1) , 50 mg L(-1) , and 100 mg L(-1) ). The data revealed that germination rate and radicle length of V. faba seedlings were promoted by low NB concentrations and short exposure periods, whereas these parameters were inhibited at greater NB concentrations and longer exposures. When assessed by mitotic index, micronucleus, and chromosomal aberration assays, NB showed dose-dependent genotoxicity at 0 mg L(-1) to 50 mg L(-1).

The effect of nitrobenzene on antioxidative enzyme activity and DNA damage in tobacco seedling leaf cells.

Nitrobenzene, although widely used in industry, is a highly toxic environmental pollutant. To evaluate the toxicity of nitrobenzene to tobacco seedlings, seedlings were exposed to varying concentrations of nitrobenzene (0-100 mg/L) for 24 h. The contents of reactive oxygen species (hydrogen peroxide [H(2)O(2)] and superoxide anion [O2(-)]) and the activities of antioxidative enzymes (superoxide dismutase [SOD], guaiacol peroxidase [POD], and catalase [CAT]) were measured in leaf cells. Damage to DNA was assessed by single-cell gel electrophoresis (comet assay). Compared with the control, the contents of H(2) O(2) increased significantly with nitrobenzene concentrations ranging from 5 to 100 mg/L. Activity of SOD was induced by 50 to 100 mg/L of nitrobenzene but not by 10 to 25 mg/L. Activity of POD was stimulated by nitrobenzene at 10 to 50 mg/L but inhibited at 100 mg/L. Activity of CAT was increased significantly only by 100 mg/L. Lipid peroxidation increased with 50 to 100 mg/L, which indicated that nitrobenzene induced oxidative stress in tobacco leaf cells. Comet assay of the leaf cells showed a significant enhancement of the head DNA (H-DNA), tail DNA (T-DNA), and olive tail moment (OTM) with increasing doses of nitrobenzene. The values of H-DNA, T-DNA, and OTM exhibited significant differences from the control when stress concentrations were higher than 10 mg/L. The results indicated that nitrobenzene caused oxidative stress, which may be one of the mechanisms through which nitrobenzene induces DNA damage.

Uncovering key small RNAs associated with gametocidal action in wheat

Gametocidal (Gc) chromosomes can kill gametes that lack them by causing chromosomal breakage to ensure their preferential transmission, and they have been exploited in genetic breeding. The present study investigated the possible roles of small RNAs (sRNAs) in Gc action. By sequencing two small RNA libraries from the anthers of Triticum aestivum cv. Chinese Spring (CS) and the Chinese Spring-Gc 3C chromosome monosomic addition line (CS-3C), we identified 239 conserved and 72 putative novel miRNAs, including 135 differentially expressed miRNAs. These miRNAs were predicted to target multiple genes with various molecular functions relevant to the features of Gc action, including sterility and genome instability. The transgenic overexpression of miRNA, which was up-regulated in CS-3C, reduced rice fertility. The CS-3C line exhibited a genome-wide reduction in 24 nt siRNAs compared with that of the CS line, particularly in transposable element (TE) and repetitive DNA sequences. Corresponding to this reduction, the bisulfite sequencing analysis of four retro-TE sequences showed a decrease in CHH methylation, typical of RNA-directed DNA methylation (RdDM). These results demonstrate that both miRNA-directed regulation of gene expression and siRNA-directed DNA methylation of target TE loci could play a role in Gc action.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

Genome-Wide Analysis of Oligopeptide Transporters Gene Family in Medicago truncatula

Oligopeptide transporters (OPTs) are a group of membrane localized proteins that have a broad range of substrate transport capabilities and contribute to numerous biological processes. However, limited information has been reported on OPTs in higher plants. In this study, a comprehensive analysis of the OPT gene family in Medicago truncatula was performed. A total of 26 OPT genes (MtOPT01-MtOPT26) have been identified in the Medicago truncatula genome. Phylogenetic analyses indicated that MtOPTs consisted of two distinct subgroups, 12 MtOPTs belonged to the peptide transport subgroup (PT-OPT) based on their predicted amino acid sequences containing the two highly conserved motifs (NPG and KIPPR) and 14 MtOPTs belonged to yellow stripe subgroup (YS-OPT). The MtOPTs distributed on each of 8 chromosomes in Medicago truncatula. Sequence analysis verified that MtOPTs significant similar to those in other plants. The copy number of MtOPTs was low and the multiply of MtOPTs was simple relatively. Gene structure analysis showed that most of the MtOPTs have various numbers of introns. The multiple of MtOPTs might play different biological roles which were supported by the fact that MtOPTs have a distinct tissue-specific expression pattern. The data obtained in this study will help to better understand the complexity of the MtOPTs gene family and provide new evidence for the function and evolution of the OPT gene family in higher plants.