Transgenic poplar (Populus × euramericana 'Guariento') plants harboring five exogenous, stress-related genes exhibit increased tolerance to multiple stresses including drought, salt, waterlogging, and insect feeding, but the complex mechanisms underlying stress tolerance in these plants have not been elucidated. Here, we analyzed the differences in the transcriptomes of the transgenic poplar line D5-20 and the non-transgenic line D5-0 using high-throughput transcriptome sequencing techniques and elucidated the functions of the differentially expressed genes using various functional annotation methods.
Plants with heterosis/hybrid vigor perform better than their parents in many traits. However, the biological mechanisms underlying heterosis remain unclear. To investigate the significance of DNA methylation to heterosis, a comprehensive analysis of whole-genome DNA methylome profiles of Populus deltoides cl.'55/65' and '10/17' parental lines and their intraspecific F1 hybrids lines was performed using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing.
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 FST value (0.09189) for PnDREB69 than for PnDREB68 (0.07743). Nucleotide diversity analysis revealed a two-fold higher ?T 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.
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.
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.
Black poplar (Populus nigra L.) is an economically and ecologically important tree species and an ideal organism for studies of genetic variation. In the present work, we use a candidate gene approach to infer the patterns of DNA variation in natural populations of this species. A total of 312 single nucleotide polymorphisms (SNPs) are found among 8,056 bp sequenced from nine drought-adaptation and photosynthesis-related gene loci. The median SNP frequency is one site per 26 bp. The average nucleotide diversity is calculated to be theta(W) = 0.01074 and pi(T) = 0.00702, higher values than those observed in P. tremula, P. trichocarpa and most conifer species. Tests of neutrality for each gene reveal a general excess of low-frequency mutations, a greater number of haplotypes than expected and an excess of high-frequency derived variants in P. nigra, which is consistent with previous findings that genetic hitchhiking has occurred in this species. Linkage disequilibrium is low, decaying rapidly from 0.45 to 0.20 or less within a distance of 300 bp, although the declines of r(2) are variable among different loci. This is similar to the rate of decay reported in most other tree species. Our dataset is expected to enhance understanding of how evolutionary forces shape genetic variation, and it will contribute to molecular breeding in black poplar.
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.
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