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Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana.

Magnotta SM, Gogarten JP - BMC Plant Biol. (2002)

Bottom Line: Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated.Etiolation resulted in a slight increase in transcript levels.All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Hartford, West Hartford, CT, USA. smagnotta@mail.hartford.edu

ABSTRACT

Background: Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene.

Results: Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene.

Conclusions: Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

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Differential expression of subunit A transcripts in response to environmental stress conditions. Whole RNA was extracted from stress treated seedlings and control plants. Complementary DNA's were generated and transcripts amplified by PCR as described in materials and methods. All sixteen amplification products were fractionated on a single agarose gel, transferred to a solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. Multiple time exposures were taken and the best was used for each panel, thus this figure does not represent equivalent time exposures for all four transcripts. These results are of a single experiment which has not been replicated. Lanes correspond to the following: N-normal, untreated control plants, S-sodium chloride treated plants, C-cold, E-etiolation.
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Figure 7: Differential expression of subunit A transcripts in response to environmental stress conditions. Whole RNA was extracted from stress treated seedlings and control plants. Complementary DNA's were generated and transcripts amplified by PCR as described in materials and methods. All sixteen amplification products were fractionated on a single agarose gel, transferred to a solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. Multiple time exposures were taken and the best was used for each panel, thus this figure does not represent equivalent time exposures for all four transcripts. These results are of a single experiment which has not been replicated. Lanes correspond to the following: N-normal, untreated control plants, S-sodium chloride treated plants, C-cold, E-etiolation.

Mentions: The differential expression of subunit A transcripts was evaluated in response to salt, cold stress, and etiolation. Complementary DNA's were generated from seven-day-old seedlings subjected to individual stress conditions. Transcripts 1–4 were evaluated via RT-PCR using transcript specific primers. Amplification products were immobilized on solid support and hybridized with the transcript-4 amplification product. The results of this experiment indicate that all four transcripts behaved identically with respect to individual stress conditions (Fig. 7). All four transcripts showed approximately 2–4 fold increases in response to 100 mM sodium chloride stress compared to untreated controls. Similarly, treatment of plants at six degrees for four days resulted in a similar increase in signal for all four transcripts. Etiolation resulted in slightly higher message levels for transcripts -1, -2, and -3 compared to controls. The lesser degree of increase for transcript-3 might be due to near saturation of the signal. The latter might be due to the higher abundance of the transcript, or due to more efficient amplification of the transcript-3 primer set. Transcript-4 seems to show slightly less signal than control plants but the autoradiograph is somewhat under developed. These results are in agreement with the whole RNA stress blot analysis. Apparent quantitative differences in upregulation between transcripts shown in (Fig. 7) might be due to differential amplification efficiency of the four different primer sets and do not necessarily represent true differential upregulation of the four transcripts. The data can only be evaluated quantitatively between samples amplified with the same primer set. In summation, no significant differential regulation of the four subunit A transcripts was detected in seedlings in response to the three stress conditions evaluated.


Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana.

Magnotta SM, Gogarten JP - BMC Plant Biol. (2002)

Differential expression of subunit A transcripts in response to environmental stress conditions. Whole RNA was extracted from stress treated seedlings and control plants. Complementary DNA's were generated and transcripts amplified by PCR as described in materials and methods. All sixteen amplification products were fractionated on a single agarose gel, transferred to a solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. Multiple time exposures were taken and the best was used for each panel, thus this figure does not represent equivalent time exposures for all four transcripts. These results are of a single experiment which has not been replicated. Lanes correspond to the following: N-normal, untreated control plants, S-sodium chloride treated plants, C-cold, E-etiolation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103671&req=5

Figure 7: Differential expression of subunit A transcripts in response to environmental stress conditions. Whole RNA was extracted from stress treated seedlings and control plants. Complementary DNA's were generated and transcripts amplified by PCR as described in materials and methods. All sixteen amplification products were fractionated on a single agarose gel, transferred to a solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. Multiple time exposures were taken and the best was used for each panel, thus this figure does not represent equivalent time exposures for all four transcripts. These results are of a single experiment which has not been replicated. Lanes correspond to the following: N-normal, untreated control plants, S-sodium chloride treated plants, C-cold, E-etiolation.
Mentions: The differential expression of subunit A transcripts was evaluated in response to salt, cold stress, and etiolation. Complementary DNA's were generated from seven-day-old seedlings subjected to individual stress conditions. Transcripts 1–4 were evaluated via RT-PCR using transcript specific primers. Amplification products were immobilized on solid support and hybridized with the transcript-4 amplification product. The results of this experiment indicate that all four transcripts behaved identically with respect to individual stress conditions (Fig. 7). All four transcripts showed approximately 2–4 fold increases in response to 100 mM sodium chloride stress compared to untreated controls. Similarly, treatment of plants at six degrees for four days resulted in a similar increase in signal for all four transcripts. Etiolation resulted in slightly higher message levels for transcripts -1, -2, and -3 compared to controls. The lesser degree of increase for transcript-3 might be due to near saturation of the signal. The latter might be due to the higher abundance of the transcript, or due to more efficient amplification of the transcript-3 primer set. Transcript-4 seems to show slightly less signal than control plants but the autoradiograph is somewhat under developed. These results are in agreement with the whole RNA stress blot analysis. Apparent quantitative differences in upregulation between transcripts shown in (Fig. 7) might be due to differential amplification efficiency of the four different primer sets and do not necessarily represent true differential upregulation of the four transcripts. The data can only be evaluated quantitatively between samples amplified with the same primer set. In summation, no significant differential regulation of the four subunit A transcripts was detected in seedlings in response to the three stress conditions evaluated.

Bottom Line: Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated.Etiolation resulted in a slight increase in transcript levels.All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Hartford, West Hartford, CT, USA. smagnotta@mail.hartford.edu

ABSTRACT

Background: Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene.

Results: Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene.

Conclusions: Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

Show MeSH
Related in: MedlinePlus