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A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides).

ABSTRACT: BACKGROUND:The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches. RESULTS:We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter. CONCLUSIONS:The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.

SUBMITTER: Takata N

PROVIDER: S-EPMC3476444 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides).

Takata Naoki N Eriksson Maria E ME

Plant methods 20120807 1

<h4>Background</h4>The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is ...[more]

PMID: 22871142

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Project description:Plants have evolved heavy metal tolerance mechanisms to adapt and cope with nickel (Ni) toxicity. Decrypting whole gene expression of Trembling Aspen (Pinus tremuloides) under nickel stress could elucidate the nickel resistance/tolerance mechanisms. The main objectives of the present research were to 1) characterize the P. tremuloides transcriptome, and 2) compare gene expression dynamics between nickel-resistant and nickel-susceptible P. tremuloides genotypes with Whole Transcriptome (WT) sequencing. Illumina Sequencing generated 27-45 million 2X150 paired-end reads of raw data per sample. The alignment performed with StringTie Software added two groups of transcripts to the draft genome annotation. One group contained 32,677 new isoforms that match to 17,254 genes. The second group contained 17,349 novel transcripts that represent 16,157 novel genes. Overall, 52,987 genes were identified from which 36,770 genes were selected as differently expressed. With the high stringency (two-fold change, FDR value ≤ 0.05 and logFC value ≥1 (upregulated) or ≤ -1 (downregulated), after GSEA analysis and filtering for gene set size, 575 gene sets were upregulated and 146 were downregulated in nickel resistant phenotypes compared to susceptible genotypes. For biological process, genes associated with translation were significantly upregulated while signal transduction and cellular protein process genes were downregulated in resistant compared to susceptible genotypes. For molecular function, there was a significant downregulation of genes associated with DNA binding in resistant compared to susceptible lines. Significant upregulation was observed in genes located in ribosome while downregulation of genes in chloroplast and mitochondrion were preponderant in resistant genotypes compared to susceptible. Hence, from a whole transcriptome level, an upregulation in ribosomal and translation activities was identified as the main response to Ni toxicity in the resistant plants. More importantly, this study revealed that a metal transport protein (Potrs038704g29436 -ATOX1-related copper transport) was among the top upregulated genes in resistant genotypes when compared to susceptible plants. Other identified upregulated genes associated with abiotic stress include genes coding for Dirigent Protein 10, GATA transcription factor, Zinc finger protein, Auxin response factor, Bidirectional sugar transporter, and thiamine thiazole synthase.

Project description:Quaking aspen (Populus tremuloides Michx.), a widespread and keystone tree species in North America, experienced heat and drought stress in the years 2002 and 2003 in the southwestern United States. This led to widespread aspen mortality that has altered the composition of forests, and is expected to occur again if climate change continues. Understanding interactions between aspen and its environments is essential to understanding future mortality risk in forests. Polyploidy, which is common in aspen, can modify plant structure and function and therefore plant-environment interactions, but the influence of polyploidy on aspen physiology is still not well understood. Furthermore, the ploidy types of aspen have different biogeographies, with triploids being most frequent at lower latitudes in generally warmer and drier climates, while the northerly populations are virtually 100% diploid. This suggests that ploidy-environment interactions differ, and could mean that the ploidy types have different vulnerabilities to environmental stress. In this study, to understand aspen ploidy-environment interactions, we measured 38 different traits important to carbon uptake, water loss and water-use efficiency in diploid and triploid aspen in Colorado. We found that triploid aspen had lower stand density, and greater leaf area, leaf mass, leaf mass per area, percent nitrogen content, chlorophyll content and stomatal size. These differences corresponded to greater potential net carbon assimilation (A, measured using A/Ci curves, and chlorophyll fluorescence) and stomatal conductance (gs) in triploids than diploids. While triploid aspen had higher intrinsic water-use efficiency (iWUE, calculated from measurements of δ13C in leaf tissue), they also had greater potential water loss from higher measured gs and lower stomatal sensitivity to increasing vapor pressure deficit. Therefore, despite greater iWUE, triploids may have lower resilience to climate-induced stress. We conclude that ploidy type strongly influences physiological traits and function, and mediates drought stress responses in quaking aspen.

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A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides).

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A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides).

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