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8. Cold acclimation in woody perennials
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Abstracts / Cryobiology 53 (2006) 367–446
or the homing ability after transplantation. At the same time, more intricate molecular mechanisms that lead to senescence and apoptosis, such as down regulation of telomerase expression and telomere shortening can be triggered as well. (See the poster by Kaprova and Abdulkadyrov at this meeting.) The SC content in CB determines whether samples should be cryopreserved. It is generally assumed that 50% of the cells will survive cryopreservation, but this may not always be the case. Even a 20% improvement in cryosurvival and cell functionality would add to the therapeutic value of the sample. Thus, understanding the mechanisms of cell cryodamage is important for clinical applications. (Conflict of interest: None declared. Source of funding: Burnham Stem Cell Center.) doi:10.1016/j.cryobiol.2006.10.007
Symposium 2: Molecular determinants of low temperature and dehydration tolerance 7. Acclimation to low temperatures in Arabidopsis thaliana. Arnd G. Heyer a, Dirk K. Hincha b, Matthew A. Hannah b, Ellen Zuther b, a Department of Botany, University of Stuttgart, 70569 Stuttgart, Germany; b Max Planck Institute of Molecular Plant Physiology, 14424 Potsdam, Germany Cold has large effects on crop production, restricting geographical distribution as well as quality and yield. Therefore, considerable effort has been directed towards understanding how plants respond to low temperature. In the model species Arabidopsis thaliana, we have used transgenic approaches, knockout mutants and natural genetic diversity to study the process of cold acclimation that substantially increases freezing tolerance of individuals exposed to low but non-freezing temperatures. At the gene expression level, we found that the number and magnitude of gene expression changes correlates with freezing tolerance, while at the metabolite level concentrations of specific compounds, but not gross changes, can be associated with the trait. For all the genotypes investigated, we found a strong correlation of soluble sugar levels with tolerance, however, no individual compound can be identified as the causative agent. Using in vitro models of cellular membranes, we tested possible mechanisms of protection during the freeze–thaw cycle and found that oligosaccharides would be good candidates as cryoprotectants. So far, little is known about subcellular distribution of individual sugars at different growth temperatures, and we have now started to explore the dynamics of compartment-specific sugar pools during the cold acclimation process. We are also using differentially cold adapted natural accessions of Arabidopsis to study the genetic basis of freezing tolerance. Because tolerance has proved to be a complex trait, we will use quantitative trait locus mapping to identify candidate genes involved in freezing tolerance of Arabidopsis. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.008
8. Cold acclimation in woody perennials. Vaughan Hurry, Catherine Benedict, Umea˚ Plant Science Centre, Umea˚ University, S907 81 Umea˚, Sweden
We investigated the changes in phenotype and transcript profile of transgenic Populus constitutively expressing CBF1 from Arabidopsis (AtCBF1). Ectopic expression of AtCBF1 was sufficient to significantly increase the freezing tolerance of non-acclimated leaves and stems, comparable to wild-type plants after one week of cold acclimation. cDNA microarray experiments identified genes upregulated by ectopic AtCBF1 expression in Populus and demonstrated the strong conservation of the CBF regulon between Populus and Arabidopsis. We also show that the four CBF paralogs identified from the Populus trichocarpa genome sequence (PtCBFs) are cold inducible but have different tissue specificities. Our results suggest that the central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus in the 50 million years since their divergence. However, the differential expression of the PtCBFs in annual (leaf) and perennial (stem) tissues, and the stem and leaf specific clusters of CBF-responsive genes, suggests that the perennial-driven evolution of winter dormancy may have given rise specific roles for these transcriptional activators in the different annual and perennial tissues of woody species. These results are discussed together with data from additional transcriptional activators induced in stems during autumn growth cessation. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.009
9. Involvement of plasma membrane microdomains in plant freezing tolerance. Anzu Minami a, Tomokazu Yamazaki a, Yukio Kawamura a, Akari Furuto b, Matsuo Uemura a,b, a The 21st Century Center of Excellence Program, Iwate University, 0208550 Morioka, Japan; b Cryobiosystem Research Center, Iwate University, 020-8550 Morioka, Japan Many temperate plants are known to increase their freezing tolerance when exposed to low temperatures, which is known as cold acclimation. The increase in freezing tolerance induced during cold acclimation is a result of many diverse alterations in association with cold-regulated gene expression. These alterations ultimately result in an increase in the cryostability of the plasma membrane, the primary site of freezing injury in plants. Complex aspects of compositional alterations (both lipids and proteins) in the plasma membrane have been reported with different plant species. To dissect the complexity of the plasma membrane changes in cold acclimation, we introduced the concept of existence of microdomains in the plasma membrane, which has been proposed to involve in signal transduction and membrane reorganization extensively in animal systems. Microdomains of the plasma membrane were isolated as a detergent-resistant membrane fraction (DRM) from purified plasma membrane from Arabidopsis thaliana leaves with Triton X-100 at 4 °C, according to protocols reported in the literature. Several proteins were specifically enriched in the DRM fraction, indicating the existence of microdomains with distinctive composition in the plasma membrane. In addition, immunofluorescence microscopy with an antibody to plasma membrane H+-ATPase, a plant DRM marker protein, revealed that localization of the protein was not uniform in the plasma membrane sheet prepared from a protoplast isolated from Arabidopsis leaves. These results strongly suggest the existence of microdomains with specific compositions in the plasma membrane in plants. During cold
Abstracts / Cryobiology 53 (2006) 367–446
or the homing ability after transplantation. At the same time, more intricate molecular mechanisms that lead to senescence and apoptosis, such as down regulation of telomerase expression and telomere shortening can be triggered as well. (See the poster by Kaprova and Abdulkadyrov at this meeting.) The SC content in CB determines whether samples should be cryopreserved. It is generally assumed that 50% of the cells will survive cryopreservation, but this may not always be the case. Even a 20% improvement in cryosurvival and cell functionality would add to the therapeutic value of the sample. Thus, understanding the mechanisms of cell cryodamage is important for clinical applications. (Conflict of interest: None declared. Source of funding: Burnham Stem Cell Center.) doi:10.1016/j.cryobiol.2006.10.007
Symposium 2: Molecular determinants of low temperature and dehydration tolerance 7. Acclimation to low temperatures in Arabidopsis thaliana. Arnd G. Heyer a, Dirk K. Hincha b, Matthew A. Hannah b, Ellen Zuther b, a Department of Botany, University of Stuttgart, 70569 Stuttgart, Germany; b Max Planck Institute of Molecular Plant Physiology, 14424 Potsdam, Germany Cold has large effects on crop production, restricting geographical distribution as well as quality and yield. Therefore, considerable effort has been directed towards understanding how plants respond to low temperature. In the model species Arabidopsis thaliana, we have used transgenic approaches, knockout mutants and natural genetic diversity to study the process of cold acclimation that substantially increases freezing tolerance of individuals exposed to low but non-freezing temperatures. At the gene expression level, we found that the number and magnitude of gene expression changes correlates with freezing tolerance, while at the metabolite level concentrations of specific compounds, but not gross changes, can be associated with the trait. For all the genotypes investigated, we found a strong correlation of soluble sugar levels with tolerance, however, no individual compound can be identified as the causative agent. Using in vitro models of cellular membranes, we tested possible mechanisms of protection during the freeze–thaw cycle and found that oligosaccharides would be good candidates as cryoprotectants. So far, little is known about subcellular distribution of individual sugars at different growth temperatures, and we have now started to explore the dynamics of compartment-specific sugar pools during the cold acclimation process. We are also using differentially cold adapted natural accessions of Arabidopsis to study the genetic basis of freezing tolerance. Because tolerance has proved to be a complex trait, we will use quantitative trait locus mapping to identify candidate genes involved in freezing tolerance of Arabidopsis. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.008
8. Cold acclimation in woody perennials. Vaughan Hurry, Catherine Benedict, Umea˚ Plant Science Centre, Umea˚ University, S907 81 Umea˚, Sweden
We investigated the changes in phenotype and transcript profile of transgenic Populus constitutively expressing CBF1 from Arabidopsis (AtCBF1). Ectopic expression of AtCBF1 was sufficient to significantly increase the freezing tolerance of non-acclimated leaves and stems, comparable to wild-type plants after one week of cold acclimation. cDNA microarray experiments identified genes upregulated by ectopic AtCBF1 expression in Populus and demonstrated the strong conservation of the CBF regulon between Populus and Arabidopsis. We also show that the four CBF paralogs identified from the Populus trichocarpa genome sequence (PtCBFs) are cold inducible but have different tissue specificities. Our results suggest that the central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus in the 50 million years since their divergence. However, the differential expression of the PtCBFs in annual (leaf) and perennial (stem) tissues, and the stem and leaf specific clusters of CBF-responsive genes, suggests that the perennial-driven evolution of winter dormancy may have given rise specific roles for these transcriptional activators in the different annual and perennial tissues of woody species. These results are discussed together with data from additional transcriptional activators induced in stems during autumn growth cessation. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.009
9. Involvement of plasma membrane microdomains in plant freezing tolerance. Anzu Minami a, Tomokazu Yamazaki a, Yukio Kawamura a, Akari Furuto b, Matsuo Uemura a,b, a The 21st Century Center of Excellence Program, Iwate University, 0208550 Morioka, Japan; b Cryobiosystem Research Center, Iwate University, 020-8550 Morioka, Japan Many temperate plants are known to increase their freezing tolerance when exposed to low temperatures, which is known as cold acclimation. The increase in freezing tolerance induced during cold acclimation is a result of many diverse alterations in association with cold-regulated gene expression. These alterations ultimately result in an increase in the cryostability of the plasma membrane, the primary site of freezing injury in plants. Complex aspects of compositional alterations (both lipids and proteins) in the plasma membrane have been reported with different plant species. To dissect the complexity of the plasma membrane changes in cold acclimation, we introduced the concept of existence of microdomains in the plasma membrane, which has been proposed to involve in signal transduction and membrane reorganization extensively in animal systems. Microdomains of the plasma membrane were isolated as a detergent-resistant membrane fraction (DRM) from purified plasma membrane from Arabidopsis thaliana leaves with Triton X-100 at 4 °C, according to protocols reported in the literature. Several proteins were specifically enriched in the DRM fraction, indicating the existence of microdomains with distinctive composition in the plasma membrane. In addition, immunofluorescence microscopy with an antibody to plasma membrane H+-ATPase, a plant DRM marker protein, revealed that localization of the protein was not uniform in the plasma membrane sheet prepared from a protoplast isolated from Arabidopsis leaves. These results strongly suggest the existence of microdomains with specific compositions in the plasma membrane in plants. During cold