Bingyu Zhang

Expression of Multiple Resistance Genes Enhances Tolerance to Environmental Stressors in Transgenic Poplar (Populus × euramericana ‘Guariento’)

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana ‘Guariento’) harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.

Expression of jasmonic ethylene responsive factor gene in transgenic poplar tree leads to increased salt tolerance

The stress resistance of plants can be enhanced by regulating the expression of multiple downstream genes associated with stress resistance. We used the Agrobacterium method to transfer the tomato jasmonic ethylene responsive factors (JERFs) gene that encodes the ethylene response factor (ERF) like transcription factor to the genome of a hybrid poplar (Populus alba x Populus berolinensis). Eighteen resistant plants were obtained, of which 13 were identified by polymerase chain reaction (PCR), reverse transcriptase PCR and Southern blot analyses as having incorporated the JERFs gene and able to express it at the transcriptional level. Salinity tests were conducted in a greenhouse with 0, 100, 200 and 300 mM NaCl. In the absence of NaCl, the transgenic plants were significantly taller than the control plants, but no statistically significant differences in the concentrations of proline and chlorophyll were observed. With increasing salinity, the extent of damage was significantly less in transgenic plants than that in control plants, and the reductions in height, basal diameter and biomass were less in transgenic plants than those in control plants. At 200 and 300 mM NaCl concentrations, transgenic plants were 128.9% and 98.8% taller, respectively, and had 199.8% and 113.0% more dry biomass, respectively, than control plants. The saline-induced reduction in leaf water content and increase in root/crown ratio were less in transgenic plants than in control plants. Foliar proline concentration increased more in response to salt treatment in transgenic plants than in control plants. Foliar Na(+) concentration was higher in transgenic plants than in control plants. In the coastal area in Panjin of Liaoning where the total soil salt concentration is 0.3%, a salt tolerance trial of transgenic plants indicated that 3-year-old transgenic plants were 14.5% and 33.6% taller than the control plants at two field sites. The transgenic plants at the two field sites were growing vigorously, had dark green leaves and showed no symptoms of salt damage, implying that the JERFs gene enhanced their salt tolerance.

Expression and molecular evolution of two DREB1 genes in black poplar (Populus nigra).

Environmental stresses such as low temperature, drought, and high salinity significantly affect plant growth and yield. As selective forces, these adverse factors play essential roles in shaping phenotypic variation in plant populations. Black poplar (Populus nigra) is an economically and ecologically important forest tree species with widely distributed populations and is thus suitable for experiments detecting evolutionary footprints left by stress. Here, we performed expression and evolutionary analysis of two duplicated DREB A1-subgroup (DREB1) genes, PnDREB68 and PnDREB69, encoding transcription factors that are involved in stress responses. The two genes showed partially overlapping but distinct expression patterns in response to stresses. These genes were strongly and rapidly induced by cold stress in leaves, stems, and roots. In leaf tissue, dehydration stress induced the expression of PnDREB68 but not PnDREB69. PnDREB69 displayed more rapid responses and longer expression durations than PnDREB68 under salt and ABA stress, respectively. Based on single nucleotide polymorphism (SNP) analysis, we found significant population genetic differentiation, with a greater F ST value (0.09189) for PnDREB69 than for PnDREB68 (0.07743). Nucleotide diversity analysis revealed a two-fold higher πT for PnDREB68 than for PnDREB69 (0.00563 vs. 0.00243), reflecting strong purifying selection acting on the former. The results suggest that positive selection acted on PnDREB69, as evidenced by neutral testing using Tajima’s D statistic. The distinct selective forces to which each of the genes was subjected may be associated with expression divergence. Linkage disequilibrium (LD) was low for the sequenced region, with a higher level for PnDREB68 than for PnDREB69. Additionally, analysis of the relationship among carbon isotope ratios, SNP classes and gene expression, together with motif and domain analysis, suggested that 14 polymorphisms within the two genes may be candidates for an association study of important traits such as water use efficiency/drought tolerance in black poplar.

Molecular cloning and functional analysis of the Populus deltoides remorin gene PdREM

Remorins play vital roles in signal transduction, energy transformation, ion flow and transport in plants. Upregulation of remorins correlates with dehiscence and cell maturation; however, no studies have been performed to elucidate the function of remorins in tree species. In this study, a Populus deltoides (Marsh.) plasma membrane-binding protein remorin gene (PdREM) was cloned and characterized by investigating its expression pattern and creating transgenic hybrid poplar (P. davidiana Dode × P. bolleana Lauche) lines expressing sense or antisense PdREM. PdREM was specifically expressed in leaf buds, and immature and mature phloem in P. deltoides. Downregulation of PdREM increased plant height, stem diameter, number of leaves, size of the xylem and phloem zones and induced expression of cell wall biosynthesis- and microfibril angle (MFA)-related genes. Overexpression of PdREM retarded vegetative growth. PdREM may negatively regulate vascular growth by inhibiting secondary cell wall expansion in poplar. In addition, antisense PdREM transgenic poplar had a lower MFA, suggesting that PdREM might contribute to sheet strength and wood properties in poplar. This study sheds light on the molecular mechanism of PdREM in P. deltoides growth and development, and lays the foundation for future functional genomics research into wood formation and the genetic engineering of forest trees with improved wood quality traits.

Genome-wide analysis of day/night DNA methylation differences in Populus nigra

DNA methylation is an important mechanism of epigenetic modification. Methylation changes during stress responses and developmental processes have been well studied; however, their role in plant adaptation to the day/night cycle is poorly understood. In this study, we detected global methylation patterns in leaves of the black poplar Populus nigra ‘N46’ at 8:00 and 24:00 by methylated DNA immunoprecipitation sequencing (MeDIP-seq). We found 10,027 and 10,242 genes to be methylated in the 8:00 and 24:00 samples, respectively. The methylated genes appeared to be involved in multiple biological processes, molecular functions, and cellular components, suggesting important roles for DNA methylation in poplar cells. Comparing the 8:00 and 24:00 samples, only 440 differentially methylated regions (DMRs) overlapped with genic regions, including 193 hyper- and 247 hypo-methylated DMRs, and may influence the expression of 137 downstream genes. Most hyper-methylated genes were associated with transferase activity, kinase activity, and phosphotransferase activity, whereas most hypo-methylated genes were associated with protein binding, ATP binding, and adenyl ribonucleotide binding, suggesting that different biological processes were activated during the day and night. Our results indicated that methylated genes were prevalent in the poplar genome, but that only a few of these participated in diurnal gene expression regulation.

PoplarGene: poplar gene network and resource for mining functional information for genes from woody plants

Poplar is not only an important resource for the production of paper, timber and other wood-based products, but it has also emerged as an ideal model system for studying woody plants. To better understand the biological processes underlying various traits in poplar, e.g., wood development, a comprehensive functional gene interaction network is highly needed. Here, we constructed a genome-wide functional gene network for poplar (covering ~70% of the 41,335 poplar genes) and created the network web service PoplarGene, offering comprehensive functional interactions and extensive poplar gene functional annotations. PoplarGene incorporates two network-based gene prioritization algorithms, neighborhood-based prioritization and context-based prioritization, which can be used to perform gene prioritization in a complementary manner. Furthermore, the co-functional information in PoplarGene can be applied to other woody plant proteomes with high efficiency via orthology transfer. In addition to poplar gene sequences, the webserver also accepts Arabidopsis reference gene as input to guide the search for novel candidate functional genes in PoplarGene. We believe that PoplarGene (http://bioinformatics.caf.ac.cn/PoplarGene and http://124.127.201.25/PoplarGene) will greatly benefit the research community, facilitating studies of poplar and other woody plants.

Overexpression of the novel Zygophyllum xanthoxylum C2H2-type zinc finger gene ZxZF improves drought tolerance in transgenic Arabidopsis and poplar

Abstract Zygophyllum xanthoxylum (Bunge) is a perennial woody succulent xerophyte that is one of the most drought-tolerant plant species identified to date. In this study, the gene encoding the novel C2H2-type zinc finger protein (ZFP) ZxZF was cloned from Z. xanthoxylum and expressed in both Arabidopsis thaliana and poplar (Populus × euramericana cl. Bofeng 1) under the control of the drought-inducible promoter rd29A. Overexpression in Arabidopsis resulted in a higher survival rate and enhanced root growth compared with wild type (WT) plants under osmotic stress conditions induced by mannitol. Overexpression in poplar under stress conditions induced by PEG6000 improved photosynthetic function as evidenced by a higher maximum photochemical activity of photosystem II (PSII) (Fv/Fm) and elevated chlorophyll content. Furthermore, leaf tissue of transgenic poplar accumulated less malondialdehyde (MDA), and both superoxide dismutase (SOD) and peroxidase (POD) activities were elevated in transgenic plants. These results suggest that ZxZF overexpression played an essential role in drought tolerance in both Arabidopsis and poplar. Incorporating overexpression of ZxZF and other A1 subgroup C2H2 zinc finger proteins in plant breeding programs may result in enhanced drought tolerance.

Small GTP-binding protein PdRanBP regulates vascular tissue development in poplar

BackgroundPrevious research has demonstrated that ectopic expression of Ran-binding protein (RanBP) in Arabidopsis results in more axillary buds and reduced apical dominance compared to WT plants. However, the function of RanBP in poplar, which has very typical secondary growth, remains unclear. Here, the Populus deltoides (Marsh.) RanBP gene (PdRanBP) was isolated and functionally characterized by ectopic expression in a hybrid poplar (P. davidiana Dode × P. bolleana Lauche).ResultsPdRanBP was predominantly expressed in leaf buds and tissues undergoing secondary wall expansion, including immature xylem and immature phloem in the stem. Overexpression of PdRanBP in poplar increased the number of sylleptic branches and the proportion of cells in the G2 phase of the cell cycle, retarded plant growth, consistently decreased the size of the secondary xylem and secondary phloem zones, and reduced the expression levels of cell wall biosynthesis genes. The downregulation of PdRanBP facilitated secondary wall expansion and increased stem height, the sizes of the xylem and phloem zones, and the expression levels of cell wall biosynthesis genes.ConclusionsThese results suggest that PdRanBP influences the apical and radial growth of poplar trees and that PdRanBP may regulate cell division during cell cycle progression. Taken together, our results demonstrated that PdRanBP is a nuclear, vascular tissue development-associated protein in P. deltoides.