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Genome-wide analysis of the Populus Hsp90 gene family reveals differential expression patterns, localization, and heat stress responses.

Zhang J, Li J, Liu B, Zhang L, Chen J, Lu M - BMC Genomics (2013)

Bottom Line: Furthermore, microarray and semi-quantitative real-time RT-PCR analyses show that a number of Populus Hsp90 genes are differentially expressed upon exposure to various stresses.Microarray and RT-PCR analyses show that most PtHsp90s were induced by various stresses, including heat stress.Collectively, these observations lay the foundation for future efforts to unravel the biological roles of PtHsp90 genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China.

ABSTRACT

Background: Members of the heat shock protein 90 (Hsp90) class of proteins are evolutionarily conserved molecular chaperones. They are involved in protein folding, assembly, stabilization, activation, and degradation in many normal cellular processes and under stress conditions. Unlike many other well-characterized molecular chaperones, Hsp90s play key roles in signal transduction, cell-cycle control, genomic silencing, and protein trafficking. However, no systematic analysis of genome organization, gene structure, and expression compendium has been performed in the Populus model tree genus to date.

Results: We performed a comprehensive analysis of the Populus Hsp90 gene family and identified 10 Populus Hsp90 genes, which were phylogenetically clustered into two major groups. Gene structure and motif composition are relatively conserved in each group. In Populus trichocarpa, we identified three paralogous pairs, among which the PtHsp90-5a/PtHsp90-5b paralogous pair might be created by duplication of a genome segment. Subcellular localization analysis shows that PtHsp90 members are localized in different subcellular compartments. PtHsp90-3 is localized both in the nucleus and in the cytoplasm, PtHsp90-5a and PtHsp90-5b are in chloroplasts, and PtHsp90-7 is in the endoplasmic reticulum (ER). Furthermore, microarray and semi-quantitative real-time RT-PCR analyses show that a number of Populus Hsp90 genes are differentially expressed upon exposure to various stresses.

Conclusions: The gene structure and motif composition of PtHsp90s are highly conserved among group members, suggesting that members of the same group may also have conserved functions. Microarray and RT-PCR analyses show that most PtHsp90s were induced by various stresses, including heat stress. Collectively, these observations lay the foundation for future efforts to unravel the biological roles of PtHsp90 genes.

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Subcellular localization of PtHsp90 proteins. A. Confocal image of an epidermal leaf cell expressing PtHsp90-3-YFP. B-C. Confocal images of epidermal leaf cells expressing PtHsp90-5a-YFP and PtHsp90-5b-YFP. The red channel shows autofluorescence of chlorophyll in photosynthetic tissues. D. Confocal images of epidermal leaf cells co-expressing YFP-PtHsp90-7 (red channel) and GFP-HDEL (green channel). Scale bar = 10 μm.
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Figure 3: Subcellular localization of PtHsp90 proteins. A. Confocal image of an epidermal leaf cell expressing PtHsp90-3-YFP. B-C. Confocal images of epidermal leaf cells expressing PtHsp90-5a-YFP and PtHsp90-5b-YFP. The red channel shows autofluorescence of chlorophyll in photosynthetic tissues. D. Confocal images of epidermal leaf cells co-expressing YFP-PtHsp90-7 (red channel) and GFP-HDEL (green channel). Scale bar = 10 μm.

Mentions: In silico analyses using the protein subcellular localization prediction software WoLF PSORT (http://wolfpsort.org) enabled us to predict the likely protein localization of each of the different candidate Hsp90s in Populus. PtHsp90-3 is predicted to be localized in the nucleus or in the cytosol with high reliability, while PtHsp90-5a and PtHsp90-5b are predicted to be localized in chloroplasts, PtHsp90-6 is predicted to be localized in mitochondria, and PtHsp90-7 is predicted to be localized in the ER. For the other PtHsp90 proteins, the cytosol is predicted to be their most likely location (Table 1). To confirm their predicted localizations, some of these proteins were transiently expressed in tobacco leaf epidermal cells as fusions with the N-terminus of YFP. Four Hsp90 proteins were successfully expressed as fluorescent protein fusions (PtHsp90-3-YFP, PtHsp90-5a-YFP, PtHsp90-5b-YFP, and YFP-PtHsp90-7). Based on sequence analysis, PtHsp90-1a, PtHsp90-1b, PtHsp90-2, PtHsp90-3, PtHsp90-4a, and PtHsp90-4b contain the C-terminal pentapeptide MEEVD (Additional file 2), which is characteristic of cytoplasmic Hsp90 proteins both in plants and in animals. In Arabidopsis, it was confirmed that two cytoplasmic Hsp90s (AtHsp90-1 and AtHsp90-3) are localized both in the nucleus and in the cytoplasm [29]. As shown in Figure 3A, the fluorescent signal of PtHsp90-3-YFP is also detected both in the nucleus and in the cytoplasm. This is consistent with the subcellular localizations of cytoplasmic Arabidopsis Hsp90 proteins [4,15]. Using the autofluorescence of chlorophyll as a marker, we found that the fluorescent signals of both PtHsp90-5a-YFP and PtHsp90-5b-YFP are well co-localized with red chlorophyll autofluorescence (Figure 3B and 3C). A transit peptide for the import into mitochondria was identified in the N-terminal region of PtHsp90-6, but the intercellular localization of PtHsp90-6 remains to be confirmed experimentally. The PtHsp90-7 protein sequence contains a C-terminal KDEL ER-retention motif (Additional file 2). When YFP-PtHsp90-7 is co-expressed with the well-characterized luminal ER marker GFP-HDEL [30], it is co-localized with GFP-HDEL (Figure 3D), which confirms its ER localization. These results suggest that the localization of Hsp90s in the same subgroup is relatively conserved among different species. The conserved organelle localization of Hsp90 implies that they might play roles in organelle-specific development or stress response. It has been suggested that mutation of the chloroplast-localized AtHsp90-5 causes altered response to red light, chlorate resistance and constitutively delayed chloroplast development in the cr88 mutant [13,31,32]. In animals, a mitochondrial-localized Hsp90 appeared to have a critical role in cell cycle progression, cellular differentiation, and apoptosis [33,34]. In tobacco, mitochondrial-localized Hsp90 was involved in the N gene-dependent cell death by affecting downstream MAPK cascade function [35]. Mutation of the ER-localized AtHsp90-7 produced floral and shoot meristem phenotypes in the shepherd mutant that closely resemble that of the three clavata (clv) mutants in Arabidopsis[14,36,37]. The conserved subcellular localization of Hsp90s might provide clues for their specific cellular functions.


Genome-wide analysis of the Populus Hsp90 gene family reveals differential expression patterns, localization, and heat stress responses.

Zhang J, Li J, Liu B, Zhang L, Chen J, Lu M - BMC Genomics (2013)

Subcellular localization of PtHsp90 proteins. A. Confocal image of an epidermal leaf cell expressing PtHsp90-3-YFP. B-C. Confocal images of epidermal leaf cells expressing PtHsp90-5a-YFP and PtHsp90-5b-YFP. The red channel shows autofluorescence of chlorophyll in photosynthetic tissues. D. Confocal images of epidermal leaf cells co-expressing YFP-PtHsp90-7 (red channel) and GFP-HDEL (green channel). Scale bar = 10 μm.
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Figure 3: Subcellular localization of PtHsp90 proteins. A. Confocal image of an epidermal leaf cell expressing PtHsp90-3-YFP. B-C. Confocal images of epidermal leaf cells expressing PtHsp90-5a-YFP and PtHsp90-5b-YFP. The red channel shows autofluorescence of chlorophyll in photosynthetic tissues. D. Confocal images of epidermal leaf cells co-expressing YFP-PtHsp90-7 (red channel) and GFP-HDEL (green channel). Scale bar = 10 μm.
Mentions: In silico analyses using the protein subcellular localization prediction software WoLF PSORT (http://wolfpsort.org) enabled us to predict the likely protein localization of each of the different candidate Hsp90s in Populus. PtHsp90-3 is predicted to be localized in the nucleus or in the cytosol with high reliability, while PtHsp90-5a and PtHsp90-5b are predicted to be localized in chloroplasts, PtHsp90-6 is predicted to be localized in mitochondria, and PtHsp90-7 is predicted to be localized in the ER. For the other PtHsp90 proteins, the cytosol is predicted to be their most likely location (Table 1). To confirm their predicted localizations, some of these proteins were transiently expressed in tobacco leaf epidermal cells as fusions with the N-terminus of YFP. Four Hsp90 proteins were successfully expressed as fluorescent protein fusions (PtHsp90-3-YFP, PtHsp90-5a-YFP, PtHsp90-5b-YFP, and YFP-PtHsp90-7). Based on sequence analysis, PtHsp90-1a, PtHsp90-1b, PtHsp90-2, PtHsp90-3, PtHsp90-4a, and PtHsp90-4b contain the C-terminal pentapeptide MEEVD (Additional file 2), which is characteristic of cytoplasmic Hsp90 proteins both in plants and in animals. In Arabidopsis, it was confirmed that two cytoplasmic Hsp90s (AtHsp90-1 and AtHsp90-3) are localized both in the nucleus and in the cytoplasm [29]. As shown in Figure 3A, the fluorescent signal of PtHsp90-3-YFP is also detected both in the nucleus and in the cytoplasm. This is consistent with the subcellular localizations of cytoplasmic Arabidopsis Hsp90 proteins [4,15]. Using the autofluorescence of chlorophyll as a marker, we found that the fluorescent signals of both PtHsp90-5a-YFP and PtHsp90-5b-YFP are well co-localized with red chlorophyll autofluorescence (Figure 3B and 3C). A transit peptide for the import into mitochondria was identified in the N-terminal region of PtHsp90-6, but the intercellular localization of PtHsp90-6 remains to be confirmed experimentally. The PtHsp90-7 protein sequence contains a C-terminal KDEL ER-retention motif (Additional file 2). When YFP-PtHsp90-7 is co-expressed with the well-characterized luminal ER marker GFP-HDEL [30], it is co-localized with GFP-HDEL (Figure 3D), which confirms its ER localization. These results suggest that the localization of Hsp90s in the same subgroup is relatively conserved among different species. The conserved organelle localization of Hsp90 implies that they might play roles in organelle-specific development or stress response. It has been suggested that mutation of the chloroplast-localized AtHsp90-5 causes altered response to red light, chlorate resistance and constitutively delayed chloroplast development in the cr88 mutant [13,31,32]. In animals, a mitochondrial-localized Hsp90 appeared to have a critical role in cell cycle progression, cellular differentiation, and apoptosis [33,34]. In tobacco, mitochondrial-localized Hsp90 was involved in the N gene-dependent cell death by affecting downstream MAPK cascade function [35]. Mutation of the ER-localized AtHsp90-7 produced floral and shoot meristem phenotypes in the shepherd mutant that closely resemble that of the three clavata (clv) mutants in Arabidopsis[14,36,37]. The conserved subcellular localization of Hsp90s might provide clues for their specific cellular functions.

Bottom Line: Furthermore, microarray and semi-quantitative real-time RT-PCR analyses show that a number of Populus Hsp90 genes are differentially expressed upon exposure to various stresses.Microarray and RT-PCR analyses show that most PtHsp90s were induced by various stresses, including heat stress.Collectively, these observations lay the foundation for future efforts to unravel the biological roles of PtHsp90 genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China.

ABSTRACT

Background: Members of the heat shock protein 90 (Hsp90) class of proteins are evolutionarily conserved molecular chaperones. They are involved in protein folding, assembly, stabilization, activation, and degradation in many normal cellular processes and under stress conditions. Unlike many other well-characterized molecular chaperones, Hsp90s play key roles in signal transduction, cell-cycle control, genomic silencing, and protein trafficking. However, no systematic analysis of genome organization, gene structure, and expression compendium has been performed in the Populus model tree genus to date.

Results: We performed a comprehensive analysis of the Populus Hsp90 gene family and identified 10 Populus Hsp90 genes, which were phylogenetically clustered into two major groups. Gene structure and motif composition are relatively conserved in each group. In Populus trichocarpa, we identified three paralogous pairs, among which the PtHsp90-5a/PtHsp90-5b paralogous pair might be created by duplication of a genome segment. Subcellular localization analysis shows that PtHsp90 members are localized in different subcellular compartments. PtHsp90-3 is localized both in the nucleus and in the cytoplasm, PtHsp90-5a and PtHsp90-5b are in chloroplasts, and PtHsp90-7 is in the endoplasmic reticulum (ER). Furthermore, microarray and semi-quantitative real-time RT-PCR analyses show that a number of Populus Hsp90 genes are differentially expressed upon exposure to various stresses.

Conclusions: The gene structure and motif composition of PtHsp90s are highly conserved among group members, suggesting that members of the same group may also have conserved functions. Microarray and RT-PCR analyses show that most PtHsp90s were induced by various stresses, including heat stress. Collectively, these observations lay the foundation for future efforts to unravel the biological roles of PtHsp90 genes.

Show MeSH
Related in: MedlinePlus