Poplar Genes Research Articles

MicroRNA828 negatively regulates lignin biosynthesis in stem of Populus tomentosa through MYB targets.

Lignin biosynthesis in the sclerenchyma cells is strictly controlled by a complex network of genetic and environmental signals. In the last decades, the transcriptional regulation of lignin synthesis in woody species has been established. However, the role of microRNA-mediated post-transcriptional modulation in secondary cell wall biosynthesis remains poorly understood. Here, we identified a microRNA, miR828, involved in the regulation specific to lignin biosynthesis during stem development in Populus tomentosa Carr. miR828 is preferentially expressed in the secondary vascular tissues during stem development. Two MYB genes (MYB171 and MYB011) were validated as direct targets of miR828 by degradome analysis and green fluorescent protein signal detection. Overexpression of miR828 in poplar downregulated genes for lignin biosynthesis, resulting in reduced lignin content in cell walls. Conversely, suppression of miR828 in plants by the short tandem target mimics elevated the expression of lignin biosynthetic genes and increased lignin deposition. We further revealed that poplar MYB171, as the most abundant miR828 target in the stem, is a positive regulator for lignin biosynthesis. Transient expression assays showed that both MYB171 and MYB011 activated PAL1 and CCR2 transcription, whereas the introduction of miR828 significantly suppressed their expression that was induced by MYB171 or MYB011. Collectively, our results demonstrate that the miR828-MYBs module precisely regulates lignin biosynthesis during the stem development in P. tomentosa through transcriptional and post-transcriptional manners.

Identification and Expression Analysis of the Populus trichocarpa GASA-Gene Family.

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays an important regulatory role in the growth and development of plants. In this study, we identified 19 GASA genes using bioinformatics-based methods in Populus trichocarpa, and these PtGASA genes could be divided into three categories based on their phylogenetic relationships. Based on an analysis of the structure and motifs of these genes, it was concluded that PtGASA class II members are more conserved than class I and class III members are, and the results of collinearity analysis showed that members of class II are collinearly related in poplar. Expression analysis of Populus trichocarpa roots, stems, and leaves showed that most of the PtGASA genes are expressed at higher levels in the stems or roots than in the leaves; a similar expression pattern was found in Vitis vinifera, indicating that the GASA-family members mainly play a role in the morphogenesis of poplar. Considering the phenomenon of gene amplification, we found that the higher the similarity of homologous genes was, the more similar the expression patterns. This study represents the first whole-genome identification and expression-profile analysis of the GASA-gene family in poplar, a model species, laying a foundation for functional studies of poplar GASA genes and serving as a reference for related research on other woody plant species.

Cloning and Functional Analysis of <i>PtATAF11</i> Transcription Factor Gene of <i>Populus trichocarpa</i>

The ATAF1 protein is a member of the NAC transcription factor family and plays an important regulatory role in plant development and stress response. In this study, using rapid amplification of cDNA ends, a full-length cDNA of  ATAF1  homolog was cloned from  Populus trichocarpa,  and the corresponding gene was named  PtATAF1-1 . The full-length cDNA sequence of  PtATAF1-1  is 1 242 bp, which consists of 3 exons and 2 introns, and an 873 bp coding region. The multiple amino acid sequences alignment showed that PtATAF1-1 protein contains the conserved NAM domain of NAC family. Transient expressing with poplar protoplast shows PtATAF1-1 protein localization in the nucleus. In addition, with the treatment of N-terminal conserved region of bacterial flagellin (Flg22) and abscisic acid (ABA) altered the expression level of  PtATAF1-1  gene. In this study, the homologous cloning and functional analysis of the  PtATAF1-1  gene were performed, which provided a reference for the further development of the function of  ATAF1  gene in  poplar .

Identification of <i>Populus tomentosa</i> OSCA Gene Family and Its Expression Analysis under Salt Stress

OSCA  (Hyperosmdality-gate calcium-permeable channels) is a calcium-permeable cation channel protein. By participating in sensing changes in extracellular osmotic potential, it induces an increase in Ca 2+  concentration, regulates osmotic potential, and plays an important role in hypertonic stress induction. However, there are few studies on the  OSCA  genes of poplar. In this study, 16  OSCA  genes sequences were identified from  Populus tomentosa  genome, and renamed them according to their distribution on the chromosomes. Based on phylogenetic tree analysis, the  PtOSCAs  genes were divided into 4 groups, and the  OSCA  genes in each group had similar gene structures. Two repetitive events were identified between poplar OSCA family genes, one tandem repetitive event involving 3 genes and a fragment repetitive event involving 2 genes, and the Ka/Ks value of each pair of genes was below 1. Therefore, it can be seen that the OSCA family genes of poplar have a purification effect during evolution. By analyzing the expression of  PtOSCAs  gene under low and high concentration salt stress, it is found that there are the following three types: Type I, where the expression level increases under both low and high concentration salt stress; Type II, at low and high concentration salt stress, the expression level decreased under high concentration salt stress; Type Ⅲ, the expression level showed an upward trend under low concentration salt stress, but the expression level showed a downward trend under high concentration salt stress. This study provides a reference for the functional analysis of  PtOSCAs  gene and the genetic improvement of poplar.

Comprehensive analysis of the BES1 gene family and its expression under abiotic stress and hormone treatment in Populus trichocarpa

The BRI1 EMS SUPPRESSOR 1/BRASSINAZOLE RESISTANT 1 (BES1/BZR1) plays a vital role in plant growth and development and stress responses, but there are few studies on poplar BES1 genes. In this study, we identified 14 BES1 genes in the Populus trichocarpa genome and analyzed the expression under hormone treatment and abiotic stress. The PtrBES1 genes were classified into seven subgroups (I-VII) through phylogenetic analysis. All the paralogous gene pairs were shown to be subjected to expansion by segment duplication and purification selection during the PtrBES1 family evolution. Promoter cis-element analysis showed that the PtrBES1 promoter contains stress related cis-elements including ABRE-motif, MBS and TC-rich elements. Quantitative real time reverse transcription PCR (RT-qPCR) analysis showed that the PtrBES1 genes were upregulated upon NaCl, Polyethylene glycol 6000 (PEG6000) stress as well as the major stress hormone abscisic acid (ABA) treatment. Under the three treatments, PtrBES1-7 showed high expression levels in leaves and roots. Physiological experiments showed that the overexpression PtrBES1-7 line could enhance tolerance to drought stress in P. trichocarpa by improving the ability to scavenge ROS (reactive oxygen species). This is specifically reflected in the fact that the overexpression line contains less ROS (O2− and H2O2) and more antioxidant enzymes (1.42 times SOD and 1.5 times POD) than the control line. The preliminary results of this study provided a solid basis for the future functional studies of the BES1 gene family in P. trichocarpa.

A point mutation and large deletion at the candidate avirulence locus AvrMlp7 in the poplar rust fungus correlate with poplar RMlp7 resistance breakdown.

The deployment of plant varieties carrying resistance genes (R) exerts strong selection pressure on pathogen populations. Rapidly evolving avirulence genes (Avr) allow pathogens to escape R-mediated plant immunity through a variety of mechanisms, leading to virulence. The poplar rust fungus Melampsora larici-populina is a damaging pathogen of poplars in Europe. It underwent a major adaptive event in 1994, with the breakdown of the poplar RMlp7 resistance gene. Population genomics studies identified a locus in the genome of M. larici-populina that probably corresponds to the candidate avirulence gene AvrMlp7. Here, to further characterize this effector, we used a population genetics approach on a comprehensive set of 281 individuals recovered throughout a 28-year period encompassing the resistance breakdown event. Using two dedicated molecular tools, genotyping at the candidate locus highlighted two different alterations of a predominant allele found mainly before the resistance breakdown: a nonsynonymous mutation and a complete deletion of this locus. This results in six diploid genotypes: three genotypes related to the avirulent phenotype and three related to the virulent phenotype. The temporal survey of the candidate locus revealed that both alterations were found in association during the resistance breakdown event. They pre-existed before the breakdown in a heterozygous state with the predominant allele cited above. Altogether, these results suggest that the association of both alterations at the candidate locus AvrMlp7 drove the poplar rust adaptation to RMlp7-mediated immunity. This study demonstrates for the first time a case of adaptation from standing genetic variation in rust fungi during a qualitative resistance breakdown.

GEPSdb: The Gene Expression Database of Poplar under Stress.

As a model tree species, poplar (Populus L.) has important economic and ecological value. Here, we constructed the GEPSdb (Gene Expression Database of Poplar under Stress; http://gepsdb.ahau-edu.cn/), which is an integrated database of poplar gene expression profiles derived from RNA-seq and microarray library data. This database provides a comprehensive collection of gene expression data from poplar exposed to 14 types of environmental stress from 11 high-quality RNA-seq experiments and 51 microarray libraries. The GEPSdb includes 56 genes from previous literature that have been examined in poplar and functionally verified. By incorporating data from numerous expression analyses, GEPSdb provides a user-friendly web interface for querying, browsing, and visualizing the expression profiles of related genes. Consequently, GEPSdb can be used to link transcription data with phenotypes and can enhance our understanding of important biological processes and mechanisms underlying complex agronomic traits in poplar.

Genome-Wide Analysis of Major Facilitator Superfamily and Its Expression in Response of Poplar to Fusarium oxysporum.

The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.

Harnessing an arbuscular mycorrhizal fungus to improve the adaptability of a facultative metallophytic poplar (Populus yunnanensis) to cadmium stress: Physiological and molecular responses

Populus yunnanensis Dode, a facultative metallophytic poplar, exhibits afforestation potential in barren mine tailing areas. However, the interactions and functional roles of arbuscular mycorrhizal fungus (AMF) in P. yunnanensis adaptability to heavy metal stress remain unclear. Physiological and molecular responses of P. yunnanensis plantlets to AMF (Funneliformis mosseae) under cadmium (Cd) stress (50 mg kg−1) were investigated. Results showed attenuation of Cd phytotoxicity effects on cell organelles upon AMF inoculation, which also reduced the Cd concentration in the poplar leaves, stems, and roots. Under Cd stress, AMF-blocking of metal transporter (e.g., Ca2+ channel) activity occurred, decreasing root cell Cd influx by reducing H+ efflux. Bioaugmentation of rhizosphere sediments by AMF to stabilize metals with a decreasing DTPA-extractable Cd also occurred. The AMF inoculation promoted Cd conversion into inactive, less phytotoxic forms, and helped to maintain ion homeostasis and relieve nutritional ion (e.g., Ca, Mg) disorders caused by excessive Cd. Leaf enzyme and non-enzyme antioxidant systems were triggered. Root and leaf physiological response patterns differed. The AMF regulated the poplar functional genes, and nine metal-responsive gene clusters were identified. We suggest that AMF is a functional component of P. yunnanensis phenotype extension, contributing to strong adaptability to unfavorable mine tailings conditions.

Genome-Wide Comparative Analysis of R2R3 MYB Gene Family in Populus and Salix and Identification of Male Flower Bud Development-Related Genes.

The MYB transcription factor (TF) family is one of the largest plant transcription factor gene family playing vital roles in plant growth and development, including defense, cell differentiation, secondary metabolism, and responses to biotic and abiotic stresses. As a model tree species of woody plants, in recent years, the identification and functional prediction of certain MYB family members in the poplar genome have been reported. However, to date, the characterization of the gene family in the genome of the poplar’s sister species willow has not been done, nor are the differences and similarities between the poplar and willow genomes understood. In this study, we conducted the first genome-wide investigation of the R2R3 MYB subfamily in the willow, identifying 216 R2R3 MYB gene members, and combined with the poplar R2R3 MYB genes, performed the first comparative analysis of R2R3 MYB genes between the poplar and willow. We identified 81 and 86 pairs of R2R3 MYB paralogs in the poplar and willow, respectively. There were 17 pairs of tandem repeat genes in the willow, indicating active duplication of willow R2R3 MYB genes. A further 166 pairs of poplar and willow orthologs were identified by collinear and synonymous analysis. The findings support the duplication of R2R3 MYB genes in the ancestral species, with most of the R2R3 MYB genes being retained during the evolutionary process. The phylogenetic trees of the R2R3 MYB genes of 10 different species were drawn. The functions of the poplar and willow R2R3 MYB genes were predicted using reported functional groupings and clustering by OrthoFinder. Identified 5 subgroups in general expanded in woody species, three subgroups were predicted to be related to lignin synthesis, and we further speculate that the other two subgroups also play a role in wood formation. We analyzed the expression patterns of the GAMYB gene of subgroup 18 (S18) related to pollen development in the male flower buds of poplar and willow at different developmental stages by qRT-PCR. The results showed that the GAMYB gene was specifically expressed in the male flower bud from pollen formation to maturity, and that the expression first increased and then decreased. Both the specificity of tissue expression specificity and conservation indicated that GAMYB played an important role in pollen development in both poplar and willow and was an ideal candidate gene for the analysis of male flower development-related functions of the two species.

Overexpression of antisense phosphatase 2C affords cold resistance in hybrid Populus davidiana × Populus bolleana.

Overexpression of the abiotic and biotic stress-resistance genes of the plant signaling pathway is well known for its significant role in the regulation of plant growth and enhancement of the productivity of agricultural land under changing climatic conditions. This research aimed to clone Populus davidiana × Populus bolleana PP2C (PdPP2C) gene and analyze its structure and function, and downregulate PdPP2C by overexpression of its antisense PdPP2C (AS-PdPP2C) gene for enhancing cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. PdPP2C was cloned and transformed to identify its function, and its antisense was overexpressed via downregulation to increase the cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. Antisense inhibition of protein phosphatase 2C accelerates the cold acclimation of Poplar (P. davidiana × P. bolleana) gene in terms of antifreeze. PdPP2C was expressed in the roots, stems, and leaves of P. davidiana × P. bolleana, and the expression was higher in the leaves. The expression of PdPP2C was also significantly downregulated at low-temperature (0°C and 4°C) stress. The relative conductivity and malondialdehyde content of non-transgenic lines were higher than those of AS-PdPP2C lines after 2days of cold treatment at - 1°C. The leaves of the transgenic lines were not wilted and showed no chlorosis compared with those of the non-transgenic lines. The AS-PdPP2C transgenic lines also showed higher freezing resistance than the non-transgenic lines. AS-PdPP2C participated in the regulation of freezing resistance.

Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties.

Woody cells generate lignocellulosic biomass, which is a promising sustainable bioresource for wide industrial applications. Woody cell differentiation in vascular plants, including the model plant poplar (Populus trichocarpa), is regulated by a set of NAC family transcription factors, the VASCULAR-RELATED NAC-DOMAIN (VND), NAC SECONDARY CELL WALL THICKENING PROMOTING FACTOR (NST)/SND, and SOMBRERO (SMB) (VNS)-related proteins, but the precise contributions of each VNS protein to wood quality are unknown. Here, we performed a detailed functional analysis of the poplar SMB-type VNS proteins PtVNS13-PtVNS16. PtVNS13-PtVNS16 were preferentially expressed in the roots of young poplar plantlets, similar to the Arabidopsis thalianaSMB gene. PtVNS13 and PtVNS14, as well as the NST-type PtVNS11, suppressed the abnormal root cap phenotype of the Arabidopsis sombrero-3 mutant, whereas the VND-type PtVNS07 gene did not, suggesting a functional gap between SMB- or NST-type VNS proteins and VND-type VNS proteins. Overexpressing PtVNS13-PtVNS16 in Arabidopsis seedlings and poplar leaves induced ectopic xylem-vessel-like cells with secondary wall deposition, and a transient expression assay showed that PtVNS13-16 transactivated woody-cell-related genes. Interestingly, although any VNS protein rescued the pendant stem phenotype of the Arabidopsis nst1-1 nst3-1 mutant, the resulting inflorescence stems exhibited distinct cell wall properties: poplar VNS genes generated woody cell walls with higher enzymatic saccharification efficiencies compared with Arabidopsis VNS genes. Together, our data reveal clear functional diversity among VNS proteins in woody cell differentiation and demonstrate a novel VNS-based strategy for modifying woody cell wall properties toward enhanced utilization of woody biomass.

Molecular Cloning, Transcriptional Profiling, Subcellular Localization, and miRNA-Binding Site Analysis of Six SCL9 Genes in Poplar.

The SCL9 subfamily is a key member of the GRAS family that regulates plant development and stress responses. Nevertheless, the functional role of these genes in the growth and development of poplar still unclear. Here, we reported the six SCL9 genes, which were found to be differentially expressed during poplar adventitious root formation. The full-length sequences of PeSCL9 genes of ‘Nanlin895’ poplar (Populus deltoids × Populus euramericana) were cloned by the RACE technique All PeSCL9 genes lacked introns. RT-qPCR revealed that PeSCL9 genes displayed a dynamic expression pattern in the adventitious root of poplar, according to RT-qPCR data. A series of comprehensive genes characteristics analysis were carried out for six genes by bioinformation. Meanwhile, transient expression analysis of the Populus protoplasts showed that all the PeSCL9 proteins were localized in the nucleus. In addition, the degradome and sRNA of ‘Nanlin895’ poplar in combination were used to predict miRNAs that regulate PeSCL9. It was found that miR396a and miR396c may affect PeSCL9 expression via cleavage, which was further verified by a transient expression experiment in Populus protoplasts. Overall, the development of poplar adventitious root and other tissues was closely related to these six SCL9 genes, and they serve as a starting point for further research into the mechanisms regulating poplar growth and development.

Poplar aquaporin PIP1;1 promotes Arabidopsis growth and development

BackgroundRoot hydraulic conductance is primarily determined by the conductance of living tissues to radial water flow. Plasma membrane intrinsic proteins (PIPs) in root cortical cells are important for plants to take up water and are believed to be directly involved in cell growth.ResultsIn this study, we found that constitutive overexpression of the poplar root-specific gene PtoPIP1;1 in Arabidopsis accelerated bolting and flowering. At the early stage of the developmental process, PtoPIP1;1 OE Arabidopsis exhibited faster cell growth in both leaves and roots. The turgor pressure of plants was correspondingly increased in PtoPIP1;1 OE Arabidopsis, and the water status was changed. At the same time, the expression levels of flowering-related genes (CRY1, CRY2 and FCA) and hub genes in the regulatory networks underlying floral timing (FT and SOC1) were significantly upregulated in OE plants, while the floral repressor FLC gene was significantly downregulated.ConclusionsTaken together, the results of our study indicate that constitutive overexpression of PtoPIP1;1 in Arabidopsis accelerates bolting and flowering through faster cell growth in both the leaf and root at an early stage of the developmental process. The autonomous pathway of flowering regulation may be executed by monitoring developmental age. The increase in turgor and changes in water status with PtoPIP1;1 overexpression play a role in promoting cell growth.

Genome-Wide and Comprehensive Analysis of the Multiple Stress-Related CAF1 (CCR4-Associated Factor 1) Family and Its Expression in Poplar.

Poplar is one of the most widely used tree in afforestation projects. However, it is susceptible to abiotic and biotic stress. CCR4-associated factor 1 (CAF1) is a major member of CCR4-NOT, and it is mainly involved in transcriptional regulation and mRNA degradation in eukaryotes. However, there are no studies on the molecular phylogeny and expression of the CAF1 gene in poplar. In this study, a total of 19 PtCAF1 genes were identified in the Populus trichocarpa genome. Phylogenetic analysis of the PtCAF1 gene family was performed with two closely related species (Arabidopsis thaliana and Oryza sativa) to investigate the evolution of the PtCAF1 gene. The tissue expression of the PtCAF1 gene showed that 19 PtCAF1 genes were present in different tissues of poplar. Additionally, the analysis of the expression of the PtCAF1 gene showed that the CAF1 family was up-regulated to various degrees under biotic and abiotic stresses and participated in the poplar stress response. The results of our study provide a deeper understanding of the structure and function of the PtCAF1 gene and may contribute to our understanding of the molecular basis of stress tolerance in poplar.

Genome-Wide Characterization of Dirigent Proteins in Populus: Gene Expression Variation and Expression Pattern in Response to Marssonina brunnea and Phytohormones

Marssonina brunnea causes a major disease that limits poplar growth. Lignin and lignan play essential roles in protecting plants from various biological stresses. Dirigent (DIR) proteins are thought to control the stereoselective coupling of coniferyl alcohol in the formation of lignan and lignin. DIR family members have been well studied in several plant species, but no previous detailed genome-wide analysis has been carried out in forest trees, such as poplar. We identified 40 PtDIR genes in Populus trichocarpa and classified them into three subgroups (DIR-a, DIR-b/d, and DIR-e) based on phylogenetic analyses. These genes are distributed on 11 poplar chromosomes, and 80% of PtDIRs (32/40) are intronless. The cis-element analysis inferred that PtDIRs possess many types of biological and abiotic stress-response cis-elements. We also analyzed intra- and inter-specific collinearity, which provided deep insights into the evolutionary characteristics of the poplar DIR genes. Analyses of the protein tertiary structure and critical amino acid residues showed that PtDIR7–10 and PtDIR13–16, which belong to the DIR-a subfamily, might be involved in the regio- and stereo-selectivity of bimolecular phenoxy radical coupling in poplars. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed different expression patterns for the PtDIR genes of P. trichocarpa and the PeDIR genes of ‘Nanlin 895’ in various tissues. Additionally, we analyzed responses of PeDIRs to M. brunnea and different phytohormone treatments (abscisic acid, salicylic acid, methyl jasmonate, and ethylene) in ‘Nanlin 895’. The results showed that at least 18 genes responded strongly to M. brunnea, and these PeDIRs also showed significant responses to phytohormones. These results suggest that DIR genes are involved in the poplar defense response against M. brunnea, and this study will provide fundamental insights for future research on poplar DIR genes.

Genome-Wide Analysis of Poplar SQUAMOSA-Promoter-Binding Protein (SBP) Family under Salt Stress

SQUAMOSA promoter binding protein (SBP) is a kind of plant-specific transcription factor, which plays a crucial role in stress responses and plant growth and development by activating and inhibiting the transcription of multiple target genes. In this study, a total of 30 SBP genes were identified from Populus trichocarpa genome and randomly distributed on 16 chromosomes in poplar. According to phylogenetic analysis, the PtSBPs can be divided into six categories, and 14 out of the genes belong to VI. Furthermore, the SBP genes in VI were proved to have a targeting relationship with miR156. The homeopathic element analysis showed that the promoters of poplar SBP genes mainly contain the elements involved in growth and development, abiotic stress and hormone response. In addition, there existed 10 gene segment duplication events in the SBP gene duplication analysis. Furthermore, there were four poplar and Arabidopsis orthologous gene pairs among the poplar SBP members. What is more, poplar SBP gene family has diverse gene expression pattern under salt stress. As many as nine SBP members were responding to high salt stress and six members possibly participated in growth development and abiotic stress. Yeast two-hybrid experiments indicated that PtSBPs can form heterodimers to interact in the transcriptional regulatory networks. The genome-wide analysis of poplar SBP family will contribute to function characterization of SBP genes in woody plants.

Galactinol synthase confers salt-stress tolerance by regulating the synthesis of galactinol and raffinose family oligosaccharides in poplar

Plants respond to abiotic stress through a series of transcriptional profile and metabolic process modifications through signaling events. Raffinose family oligosaccharides (RFOs) play a role in the regulatory mechanisms of abiotic stress tolerance and are catalyzed by the key enzyme, galactinol synthase (GolS), in plants. The amino acids sequences of GolS of Populus trichocarpa contained the common features of GolS proteins in other plant species, namely, a hydrophobic pentapeptide (APSAA). The PtrGolS3 promoter region contains several cis-elements for stress responses, including abscisic acid responsive element (ABRE), the dehydration and cold responsive elements (DRE/CRT), the low-temperature responsive element (LTRE), and the transcription factor MYB binding sites. Overexpression of AtGolS2 and PtrGolS3 enhanced abiotic stress tolerance along with the expression of stress-related genes in transgenic poplar. In addition, overexpression of AtGolS2 and PtrGolS3 resulted in higher contents of soluble sugars and other stress-associated metabolites (proline, salicylic acid, phenylalanine, and so forth) in two GolS overexpressing poplar lines compared with wild-type poplar under salt stress. Based on these results, a promising PtrGolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in poplar was identified and it may increase the understanding of RFO metabolism in woody plants under short-term salt treatment.

Construction of a Full-Length cDNA Over-Expressing Library to Identify Valuable Genes from Populus tomentosa.

Poplar wood is the main source of renewable biomass energy worldwide, and is also considered to be a model system for studying woody plants. The Full-length cDNA Over-eXpressing (FOX) gene hunting system is an effective method for generating gain-of-function mutants. Large numbers of novel genes have successfully been identified from many herbaceous plants according to the phenotype of gain-of-function mutants under normal or abiotic stress conditions using this system. However, the system has not been used for functional gene identification with high-throughput mutant screening in woody plants. In this study, we constructed a FOX library from the Chinese white poplar, Populus tomentosa. The poplar cDNA library was constructed into the plant expression vector pEarleyGate101 and further transformed into Arabidopsis thaliana (thale cress). We collected 1749 T1 transgenic plants identified by PCR. Of these, 593 single PCR bands from different transgenic lines were randomly selected for sequencing, and 402 diverse sequences of poplar genes were isolated. Most of these genes were involved in photosynthesis, environmental adaptation, and ribosome biogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. We characterized in detail two mutant lines carrying PtoCPCa or PtoWRKY13 cDNA insertions. Phenotypic characterization showed that overexpression of these genes in A. thaliana affected trichome development or secondary cell wall (SCW) deposition, respectively. Together, the Populus-FOX-Arabidopsis library generated in our experiments will be helpful for efficient discovery of novel genes in poplar.

An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

BackgroundForest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar.MethodsA total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin.ResultsWe greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation.ConclusionsThe application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.