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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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Related in: MedlinePlus

Numbers of tag species differentially regulated during Cd treatment. The difference in the counts of individual tag species between the untreated library and Cd-treated libraries. Treatment with 0.1 μM CdCl2 for 3 h (Cd3h), 1 d (Cd1d), and 3 d (Cd3d) was evaluated by the SAGEbetaBin program with a 0.05 Bayes error rate (Vêncio et al., 2004). The numbers of significantly (A) up-regulated and (B) down-regulated tag species in three Cd-treated libraries are shown.
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fig6: Numbers of tag species differentially regulated during Cd treatment. The difference in the counts of individual tag species between the untreated library and Cd-treated libraries. Treatment with 0.1 μM CdCl2 for 3 h (Cd3h), 1 d (Cd1d), and 3 d (Cd3d) was evaluated by the SAGEbetaBin program with a 0.05 Bayes error rate (Vêncio et al., 2004). The numbers of significantly (A) up-regulated and (B) down-regulated tag species in three Cd-treated libraries are shown.

Mentions: Counts of individual tag species in the three Cd-treated libraries were compared with that in the untreated library by using the SAGEbetaBin program (Vêncio et al., 2004) with a Bayes error rate of 0.05. A total of 4011 tag species were differentially expressed during Cd treatment, and 2049 and 2022 tag species, respectively, increased and decreased in at least one Cd-treated library as compared with the untreated library. The numbers of differentially expressed tag species during Cd treatment are summarized in Fig. 6. After only 3 h of treatment, 580 up- and 652 down-regulated tags were found, indicating that transcriptional changes occur rapidly in the roots. Of these, the expression level of 322 up- and 271 down-regulated tags returned to an insignificant level after 1 d. These ‘rapid and transient’ responses may counteract cellular detrimental effects triggered by the sudden shock of Cd exposure. There were also some tags that reached significant levels again after 3 d of treatment. After 1 d of treatment, the number of differentially regulated tag species was the highest during Cd treatment, which included 2887 tags corresponding to 1423 up- and 1464 down-regulated tags. This may reflect the increased severity of Cd stress that accompanied the rapid accumulation of Cd in the roots (Fig. 3A). The number, however, decreased to 1376 tags comprising 655 up- and 721 down-regulated tags after 3 d of treatment. Thus, the expression levels of 1072 and 1028 differentially regulated tags at 1 d after treatment were no longer significantly different after 3 d of treatment. These ‘change and settle’ tags constituted more than half of the differentially regulated tags and are likely to play important roles in adaptation processes to Cd stress. The restitution of these genes indicates that TB plants might successfully acclimatize to Cd toxicity during 3 d of treatment. On the other hand, 110 and 173 tags had been up- and down-regulated, respectively, throughout the Cd treatment period. These ‘rapid and lasting’ tags may be involved in the cellular protective processes that respond to the intrinsic hazardous property of accumulated Cd in the cells, including systematic detoxification of Cd, antioxidative response, and related metabolic changes. It was not until after 3 d of treatment that the differential expression of 217 up- and 177 down-regulated tags became significant. Regulation of these ‘slow response’ tags might be due simply to the low severity of Cd stress, or they may be due to secondary effects caused by Cd. The diverse patterns of temporal changes described above could depend directly on the applied Cd concentration and might be partially explained by the Cd dose in the cells or tissues. The diverse patterns of gene expression, however, strongly suggest that the acclimation process consists of progressive phases characterized by regulation of sets of specific genes.


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)

Numbers of tag species differentially regulated during Cd treatment. The difference in the counts of individual tag species between the untreated library and Cd-treated libraries. Treatment with 0.1 μM CdCl2 for 3 h (Cd3h), 1 d (Cd1d), and 3 d (Cd3d) was evaluated by the SAGEbetaBin program with a 0.05 Bayes error rate (Vêncio et al., 2004). The numbers of significantly (A) up-regulated and (B) down-regulated tag species in three Cd-treated libraries are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Numbers of tag species differentially regulated during Cd treatment. The difference in the counts of individual tag species between the untreated library and Cd-treated libraries. Treatment with 0.1 μM CdCl2 for 3 h (Cd3h), 1 d (Cd1d), and 3 d (Cd3d) was evaluated by the SAGEbetaBin program with a 0.05 Bayes error rate (Vêncio et al., 2004). The numbers of significantly (A) up-regulated and (B) down-regulated tag species in three Cd-treated libraries are shown.
Mentions: Counts of individual tag species in the three Cd-treated libraries were compared with that in the untreated library by using the SAGEbetaBin program (Vêncio et al., 2004) with a Bayes error rate of 0.05. A total of 4011 tag species were differentially expressed during Cd treatment, and 2049 and 2022 tag species, respectively, increased and decreased in at least one Cd-treated library as compared with the untreated library. The numbers of differentially expressed tag species during Cd treatment are summarized in Fig. 6. After only 3 h of treatment, 580 up- and 652 down-regulated tags were found, indicating that transcriptional changes occur rapidly in the roots. Of these, the expression level of 322 up- and 271 down-regulated tags returned to an insignificant level after 1 d. These ‘rapid and transient’ responses may counteract cellular detrimental effects triggered by the sudden shock of Cd exposure. There were also some tags that reached significant levels again after 3 d of treatment. After 1 d of treatment, the number of differentially regulated tag species was the highest during Cd treatment, which included 2887 tags corresponding to 1423 up- and 1464 down-regulated tags. This may reflect the increased severity of Cd stress that accompanied the rapid accumulation of Cd in the roots (Fig. 3A). The number, however, decreased to 1376 tags comprising 655 up- and 721 down-regulated tags after 3 d of treatment. Thus, the expression levels of 1072 and 1028 differentially regulated tags at 1 d after treatment were no longer significantly different after 3 d of treatment. These ‘change and settle’ tags constituted more than half of the differentially regulated tags and are likely to play important roles in adaptation processes to Cd stress. The restitution of these genes indicates that TB plants might successfully acclimatize to Cd toxicity during 3 d of treatment. On the other hand, 110 and 173 tags had been up- and down-regulated, respectively, throughout the Cd treatment period. These ‘rapid and lasting’ tags may be involved in the cellular protective processes that respond to the intrinsic hazardous property of accumulated Cd in the cells, including systematic detoxification of Cd, antioxidative response, and related metabolic changes. It was not until after 3 d of treatment that the differential expression of 217 up- and 177 down-regulated tags became significant. Regulation of these ‘slow response’ tags might be due simply to the low severity of Cd stress, or they may be due to secondary effects caused by Cd. The diverse patterns of temporal changes described above could depend directly on the applied Cd concentration and might be partially explained by the Cd dose in the cells or tissues. The diverse patterns of gene expression, however, strongly suggest that the acclimation process consists of progressive phases characterized by regulation of sets of specific genes.

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