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Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum.

Yamaguchi H, Fukuoka H, Arao T, Ohyama A, Nunome T, Miyatake K, Negoro S - J. Exp. Bot. (2009)

Bottom Line: In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration.Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments.These transcriptional changes were also confirmed by real-time reverse transcription-PCR.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Physiology Research Team, National Institute of Vegetable and Tea Science, 360 Kusawa, Ano-cho, Tsu, Mie, Japan. hyamagu@affrc.go.jp

ABSTRACT
Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 muM CdCl(2) in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

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Classification of the TB unigenes based on a sequence similarity search against known plant sequence data sets. Figures indicate the number of TB unigenes that showed significant sequence similarity (E-value ≤10−5) by BLAST search. Figures in parentheses indicate the ratio of unigene number in each class to the total unigene number.
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fig4: Classification of the TB unigenes based on a sequence similarity search against known plant sequence data sets. Figures indicate the number of TB unigenes that showed significant sequence similarity (E-value ≤10−5) by BLAST search. Figures in parentheses indicate the ratio of unigene number in each class to the total unigene number.

Mentions: To attribute 24 bp tag sequences to expressed genes, cDNA sequences were collected from leaves and roots from TB plants that were treated or not treated with Cd. A Cd concentration of 0.89 μM was employed for cDNA library construction, instead of 0.1 μM used in tag expression analysis, in order to collect a diverse range of cDNAs related to Cd stress responses. In total, 18 816 cDNA clones were sequenced from both the 5’ and 3’ ends, and 28 379 high-quality sequences were obtained and registered in the DDBJ/EMBL/GenBank database (accession numbers FS098087–FS126465). These sequences were clustered into 6296 groups at the ‘locus’ level using the VISUALBIO-Clustering software, and consensus sequences were calculated for each cluster by Phrap. The resulting 6296 sequences were used for further analysis as a TB unigene set. The homology search against an Arabidopsis-predicted gene model revealed that 5230 unigenes (83.1%) had significant homology (E-value ≤10−5), while 1066 unigenes (16.9%) showed no significant correspondence. Similarly, 5302 unigenes (84.2%) corresponded to the plant genes in the RefSeq data set, and 5926 unigenes (94.1%) corresponded to potato and/or tomato unigene sets (Fig. 4). Finally, 330 unigenes were identified as being TB specific with no significant homology at the amino acid level to any of the data sets examined. In order to assign functional categories to the TB unigenes and to compare their distribution with that of tomato, a BLASTX search (E-value ≤10−5) against the data set of eukaryotic clusters of KOGs (Tatusov et al., 2003) was performed using both data sets as queries. In total, 3367 TB unigenes scored hits to KOG entries. As shown in Table 2, their functional categories showed a comparable distribution with that in tomato. This result suggested that the TB unigene set built in this study could represent the TB transcriptome without a significant bias. Because S. torvum was found to be most closely related to eggplant by ClustalW alignment analysis using 32 orthologue groups among Solanum species with available ESTs deposited in the database, the eggplant unigene set (16 245 independent unigenes) was also used for ascription of tag sequences to unigenes (Fukuoka et al., National Institute of Vegetable and Tea Science, unpublished results). The TB unigene set includes several genes highly related to Arabidopsis metal transporter genes. These include two P1B-ATPase family genes, HMA1 and HMA6; three ZIP metal transporter genes, ZIP4, ZIP5, and ZIP6; NRAMP1; MTPc4; and three MRP transporter family genes, MRP1, MRP2, and MRP4 (Supplementary Table S1 at JXB online). The TB unigene sequences (flat FASTA format) and a spreadsheet file containing annotation information based on BLAST results are available from the web-based database, VegMarks (http://vegmarks.nivot.affrc.go.jp/VegMarks/jsp/page.do?transition=link).


Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum.

Yamaguchi H, Fukuoka H, Arao T, Ohyama A, Nunome T, Miyatake K, Negoro S - J. Exp. Bot. (2009)

Classification of the TB unigenes based on a sequence similarity search against known plant sequence data sets. Figures indicate the number of TB unigenes that showed significant sequence similarity (E-value ≤10−5) by BLAST search. Figures in parentheses indicate the ratio of unigene number in each class to the total unigene number.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803209&req=5

fig4: Classification of the TB unigenes based on a sequence similarity search against known plant sequence data sets. Figures indicate the number of TB unigenes that showed significant sequence similarity (E-value ≤10−5) by BLAST search. Figures in parentheses indicate the ratio of unigene number in each class to the total unigene number.
Mentions: To attribute 24 bp tag sequences to expressed genes, cDNA sequences were collected from leaves and roots from TB plants that were treated or not treated with Cd. A Cd concentration of 0.89 μM was employed for cDNA library construction, instead of 0.1 μM used in tag expression analysis, in order to collect a diverse range of cDNAs related to Cd stress responses. In total, 18 816 cDNA clones were sequenced from both the 5’ and 3’ ends, and 28 379 high-quality sequences were obtained and registered in the DDBJ/EMBL/GenBank database (accession numbers FS098087–FS126465). These sequences were clustered into 6296 groups at the ‘locus’ level using the VISUALBIO-Clustering software, and consensus sequences were calculated for each cluster by Phrap. The resulting 6296 sequences were used for further analysis as a TB unigene set. The homology search against an Arabidopsis-predicted gene model revealed that 5230 unigenes (83.1%) had significant homology (E-value ≤10−5), while 1066 unigenes (16.9%) showed no significant correspondence. Similarly, 5302 unigenes (84.2%) corresponded to the plant genes in the RefSeq data set, and 5926 unigenes (94.1%) corresponded to potato and/or tomato unigene sets (Fig. 4). Finally, 330 unigenes were identified as being TB specific with no significant homology at the amino acid level to any of the data sets examined. In order to assign functional categories to the TB unigenes and to compare their distribution with that of tomato, a BLASTX search (E-value ≤10−5) against the data set of eukaryotic clusters of KOGs (Tatusov et al., 2003) was performed using both data sets as queries. In total, 3367 TB unigenes scored hits to KOG entries. As shown in Table 2, their functional categories showed a comparable distribution with that in tomato. This result suggested that the TB unigene set built in this study could represent the TB transcriptome without a significant bias. Because S. torvum was found to be most closely related to eggplant by ClustalW alignment analysis using 32 orthologue groups among Solanum species with available ESTs deposited in the database, the eggplant unigene set (16 245 independent unigenes) was also used for ascription of tag sequences to unigenes (Fukuoka et al., National Institute of Vegetable and Tea Science, unpublished results). The TB unigene set includes several genes highly related to Arabidopsis metal transporter genes. These include two P1B-ATPase family genes, HMA1 and HMA6; three ZIP metal transporter genes, ZIP4, ZIP5, and ZIP6; NRAMP1; MTPc4; and three MRP transporter family genes, MRP1, MRP2, and MRP4 (Supplementary Table S1 at JXB online). The TB unigene sequences (flat FASTA format) and a spreadsheet file containing annotation information based on BLAST results are available from the web-based database, VegMarks (http://vegmarks.nivot.affrc.go.jp/VegMarks/jsp/page.do?transition=link).

Bottom Line: In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration.Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments.These transcriptional changes were also confirmed by real-time reverse transcription-PCR.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Physiology Research Team, National Institute of Vegetable and Tea Science, 360 Kusawa, Ano-cho, Tsu, Mie, Japan. hyamagu@affrc.go.jp

ABSTRACT
Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 muM CdCl(2) in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

Show MeSH
Related in: MedlinePlus