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In JoVE (1)
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Articles by Leland J. Cseke in JoVE
JoVE General
En PCR-baseret genotypebestemmelse metode til at skelne mellem vildtype og Ornamental sorter af Imperata cylindrica
1Department of Biological Sciences, The University of Alabama, Huntsville, 2USDA-APHIS-PPQ, Center for Plant Health Science and Technology
Vi en omkostningseffektiv og hurtig molekylær genotypebestemmelse protokol, der anvender forskellige specifikke PCR-primere, som er rettet DNA-sekvensforskelle i chloroplasten trnL-F spacerregionen at skelne mellem varianter af
Other articles by Leland J. Cseke on PubMed
Characterization of PTM5 in Aspen Trees: a MADS-box Gene Expressed During Woody Vascular Development
The vascular component of trees possesses some of the most specialized processes active in the formation of roots, stems, and branches, and its wood component continues to be of primary importance to our daily lives. The molecular mechanisms of wood development, however, remain poorly understood with few well-characterized regulatory genes. We have identified a vascular tissue-specific MADS-box gene, Populus tremuloides MADS-box 5 (PTM5) that is expressed in differentiating primary and secondary xylem and phloem. Phylogenetic analysis has shown that PTM5 is a member of the SOC1/TM3 class of MADS-box genes. Temporal expression analysis of PTM5 in staged vascular cambium and other tissues indicated that PTM5 expression is seasonal and is limited to spring wood formation and rapidly expanding floral catkins. Spatial expression analysis using in situ hybridization revealed that PTM5 expression is localized within a few layers of differentiating vascular cambium and xylem tissues as well as the vascular bundles of expanding catkins. Since many MADS-box genes are known to act as transcription factors, these results suggest that the coordinated expression of PTM5 with other vascular developmental genes may be a hallmark of the complex events that lead to the formation of the woody plant body.
SEP-class Genes in Populus Tremuloides and Their Likely Role in Reproductive Survival of Poplar Trees
One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability.
Identification of PTM5 Protein Interaction Partners, a MADS-box Gene Involved in Aspen Tree Vegetative Development
In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees, Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonal expression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots. Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5 protein-protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like other SOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studies showed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cell growth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes with specific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.
High Efficiency Poplar Transformation
With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through beta-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3-4 months, rather than the 6-12 months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars.
Transcriptomic Comparison in the Leaves of Two Aspen Genotypes Having Similar Carbon Assimilation Rates but Different Partitioning Patterns Under Elevated [CO2]
This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO2] (e[CO2]) at the Aspen free-air CO2 enrichment site near Rhinelander, WI, USA. The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e[CO2], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO2], the transcriptional activity of leaf e[CO2]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons. An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO2-responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO2-unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening. This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO2]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO2 responsiveness.
Using Next Generation Transcriptome Sequencing to Predict an Ectomycorrhizal Metabolome
Mycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems. The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates. This union of plant and fungal metabolisms is the mycorrhizal metabolome. Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling.
