Yunjun Liu

Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray

In order to identify genes induced during the water stress response in maize (Zea mays) seedlings, suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from maize seedlings treated with 20% PEG as testers and cDNAs from unstressed maize seedlings as drivers. A forward subtractive cDNA library was constructed, from which 960 recombinant colonies were picked and amplified. Through differential screening of the subtractive cDNA library, 533 clones were identified as water stress induced. After sequencing, 190 unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. The ESTs with significant protein homology were sorted into 13 functional categories. A cDNA marcoarray containing the 190 unique ESTs was used to analyze their expression profiles in maize seedling during both PEG treatment and natural drought. The results indicated that 67 ESTs in leaves and 113 ESTs in roots were significantly up-regulated by PEG-stress. 123 ESTs were found to be up-regulated for at least one time-course point in either maize leaves or roots. Correspondingly, 163 ESTs were significantly up-regulated by drought stress. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after PEG treatment and that there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts. A set of transcripts has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.

Genome-wide transcriptome analysis of two maize inbred lines under drought stress.

Drought stress greatly affects plant growth and crop yield. To understand the transcriptome dynamics during drought stress in maize seedlings, genome-wide gene expression profiling was compared between the drought-tolerant line Han21 and drought-sensitive line Ye478 using Affymetrix Maize Genome Array containing 17,555 probe sets. The results showed that in response to drought, the Han21 line had fewer probe sets with significant expression change than the Ye478 line and both lines had a common set of ~2,600 regulated probe sets under drought stress. The potential components of the abscisic acid signaling pathway were significantly identified from the common probe sets. A total of 827 probe sets with significantly differential expression between the two lines under drought stress were identified. The differential expression levels of cell wall-related and transporter genes may contribute to the different tolerances of the two lines. Additionally, we found that, compared to the sensitive line Ye478, the transcriptional levels of drought-responsive probe sets in the tolerant line Han21 recovered more quickly after re-watering, and more probe sets in the tolerant line Han21 were exclusively up-regulated at the re-watering stage. Our study provides a global gene expression dynamics of two maize inbred lines during drought stress and re-watering and will be valuable for further study of the molecular mechanisms of drought tolerance in maize.

Gene families of maize glutathione–ascorbate redox cycle respond differently to abiotic stresses

The glutathione-ascorbate (GSH-ASC) cycle in plants plays an important role in detoxifying reactive oxygen species. Little is known about how the enzymes and antioxidants in the maize GSH-ASC cycle respond to stress. We clarified the genome positions, exon-intron structures and predicted subcellular locations of the ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) families in maize. ABA treatment increased the transcript levels of most of the APX genes except ZmAPX3 and ZmAPX6, upregulated the transcription of ZmMDAR1 and downregulated the transcriptions of ZmMDAR3 and ZmMDAR4. However, it had little effect on the expressions of the ZmDHAR and ZmGR gene families. ABA treatment increased the activities of only 2 enzymes, ZmAPX and ZmDHAR. The PEG treatment led to similar expression patterns as that of ABA. ZmAPX1.1 and ZmAPX2 exhibited the same expression patterns under PEG treatment conditions. Enzyme activities were not affected by the PEG treatment with the exception of a significant decrease in MDAR activity that was observed after 6h. Compared to the ABA and PEG treatments, the NaCl treatment only slightly affected the transcription of the four gene families but significantly increased the activity of ZmGR. The ABA and PEG treatments elevate the ASC levels and decrease the GSSG level. Our results show that the gene families of the maize GSH-ASC redox cycle respond differently to abiotic stresses and suggest that APX and MDAR may play more important roles in stress tolerance in plants.

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

A putative maize zinc-finger protein gene, ZmAN13, participates in abiotic stress response

The maize ZnF-AN1 gene family has been phylogenetically studied. Here, we tried to characterize the possible function of ZmAN13, a putative member of the maize ZnF-AN1 gene family. The results showed that ZmAN13 had only one copy in the maize genome. The expression of ZmAN13 was higher in leaves than other organs and was induced by cold and abscisic acid (ABA), but down-regulated by NaCl. ZmAN13 protein fused to GFP was localized in cytoplast and also in the nucleus in both Arabidopsis protoplast and onion epidermic cells although no transcription factor activity was detected in the yeast system. Yeast two hybrid experiments demonstrated that the conserved A20 and AN1 domains of the ZmAN13 interacted with each other. Moreover, the conserved A20 domain of the ZmAN13 interacted with itself, but the AN1 domain could not. Overexpression of ZmAN13 in Arabidopsis conferred tolerance to cold, but increased their salt and drought sensitivity at seed germination and seedling stage compared to the wild-type.

Ground beetle (Coleoptera: Carabidae) inventories: a comparison of light and pitfall trapping

Carabid inventories gained via pitfall trapping were compared to manual samples from light towers. A comparison of the two methods indicated that pitfall traps recorded a significantly higher diversity of carabids and were efficient in indicating changes of habitat conditions. Nevertheless, this method failed to give near-complete inventories of all carabid species present. Manual sampling at light towers resulted in far greater sample sizes, and this method was particularly efficient in monitoring potential pest species, but again failed to record all species present. Both methods hence showed different strengths, and only a combination, potentially also including further sampling techniques, will enable the generation of complete species inventories.

Cloning and characterization of the stress-induced bZIP gene ZmbZIP60 from maize

AbstractsZmbZIP60 is a member of the bZIP transcription factor family in maize. Expression of ZmbZIP60 is strongly induced by a wide spectrum of stresses, including dehydration, high salinity, abscisic acid and tunicamycin treatments. A truncated form of ZmbZIP60, without a transmembrane domain (ZmbZIP60ΔC) and fused with GFP, is localized in the nucleus, suggesting the translocation of the native protein to the nucleus by release from the membrane. Yeast one-hybrid analysis showed that both ZmbZIP60 and ZmbZIP60ΔC had transcriptional activity. The expression of ZmbZIP60 in Arabidopsis bzip60 mutant partially restored the induction of BiP3 transcription under TM treatment, which indicated that ZmbZIP60 may play a role in the signal transduction of endoplasmic reticulum stress. Overexpression of ZmbZIP60 in wild-type Arabidopsis displayed enhanced bolting trends when subjected to dithiothreitol stress. Real-time PCR analysis revealed that some key genes in floral transition, including CO, FT, and AP1, were up- or down-regulated in ZmbZIP60-overexpressing plants, which may reveal the functional difference of ZmbZIP60 from AtbZIP60.

Metabolic effects of glyphosate on transgenic maize expressing a G2-EPSPS gene from Pseudomonas fluorescens

Transgenic glyphosate-tolerant maize expressing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene has been commercialized since 1996. However, it is not very clear how glyphosate treatment affects metabolite pathway in transgenic glyphosate-tolerant maize. Here, we obtained numerous of glyphosate-tolerant transgenic maize expressing a Pseudomonas fluorescens G2-EPSPS gene. The expression and integration site of G2-EPSPS in transgenic maize event Aro203, which can tolerate 3 folds of field usage of glyphosate, were investigated. Metabolite analysis was performed with Aro203 leaf samples using GC/MS method. The results showed that total 58 metabolites were identified. Over-expression of G2-EPSPS led to the increase of glutamate, malate, hydroxylamine and trehalose contents, but the decrease of glyoxylate, ribose and sucrose, compared to wild type plants. Twenty-two and 13 metabolites were up-regulated and down-regulated in non-transgenic maize by glyphosate treatments, respectively, whereas fewer metabolites (10 up-regulated and 4 down-regulated) were affected in transgenic maize. Glyphosate treatment significantly stimulated the accumulation of most amino acids but decreased lots of sugars in non-transgenic plants. The PCA analysis results showed that wild type plant cluster treated with glyphosate was clearly separated with other three clusters. The results in this study provide evidence to understand how genetic modification or glyphosate treatment affects the metabolite pathway in maize.

Field trials to evaluate the effects of transgenic cry1Ie maize on the community characteristics of arthropod natural enemies

Possible non-target effect of transgenic cry1Ie maize exerts on natural enemy community biodiversity in the field is unresolved. In the present study, a 2-yr comparison of transgenic cry1Ie maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) on natural enemy community biodiversity were compared with whole plant inspections, pitfall traps and suction sampler. Natural enemy diversity indices (Shannon-Wiener’, Simpson’s and Pielou’s index) and abundance suggested there were no significant differences between the two types of maize. The only exceptions were the Pielou’s index for whole plant inspections in 2013 and abundance for pitfall traps in 2012, which were significantly higher in Bt maize than those of non-Bt maize. The main species of natural enemies were identical in Bt and non-Bt maize plots for each method and the three methods combined. For whole plant inspections, Bt maize had no time-dependent effect on the entire arthropod natural enemy community, and also no effect on community dissimilarities between Bt and non-Bt maize plots. These results suggested that despite the presence of a relatively minor difference in natural enemy communities between Bt and non-Bt maize, transgenic cry1Ie maize had little, if any, effect on natural enemy community biodiversity.

Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene

Abstract5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.