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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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Subunit A transcriptional response to environmental stress conditions in Arabidopsis seedlings. Each lane contained 20 μg whole RNA. The probe used was the same for organ level RNA blot analysis (1700 base pair subunit A cDNA labeled with digoxigenin). Lanes correspond to the following: N-normal, untreated control plants grown at 23°C with 16 hour photoperiod, S-sodium chloride treated plants subjected to 100 mM sodium chloride in the medium, E-etiolation, where plants were germinated in light for three days then transferred to total darkness for four days, C-cold, where plants were germinated under normal conditions for three days and then transferred to a cold room at 6°C for four days.
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Figure 4: Subunit A transcriptional response to environmental stress conditions in Arabidopsis seedlings. Each lane contained 20 μg whole RNA. The probe used was the same for organ level RNA blot analysis (1700 base pair subunit A cDNA labeled with digoxigenin). Lanes correspond to the following: N-normal, untreated control plants grown at 23°C with 16 hour photoperiod, S-sodium chloride treated plants subjected to 100 mM sodium chloride in the medium, E-etiolation, where plants were germinated in light for three days then transferred to total darkness for four days, C-cold, where plants were germinated under normal conditions for three days and then transferred to a cold room at 6°C for four days.

Mentions: Whole RNA blot analysis was used first to evaluate the overall response of subunit A to stress conditions. RNA was extracted from stress treated seedlings and non-treated control plants. Hybridization was performed as previously described. Results indicated a subunit A transcript response to stress. An approximate 2–4 fold increase in transcript level was detected in seedlings subjected to salt and chilling stress compared to control plants (Fig. 4). Etiolated seedlings showed essentially equivalent amounts of subunit A transcript compared to controls. Since etiolation results in rapid elongation of seedlings it was expected that there would be a concomitant increase in subunit A messenger RNA as seen with proteolipid subunit mRNA in A. thaliana[36] and subunit A message levels in rapidly expanding cotton (Gossypium hirsutum) fibers [73]. Equivalent loading of whole RNA was evaluated via spectrophotometry measuring absorption at 260 nm using a Perkin-Elmer Lambda-3 spectrophotometer and by visual confirmation using non denaturing ethidium bromide stained agarose gel electrophoresis (results not shown).


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)

Subunit A transcriptional response to environmental stress conditions in Arabidopsis seedlings. Each lane contained 20 μg whole RNA. The probe used was the same for organ level RNA blot analysis (1700 base pair subunit A cDNA labeled with digoxigenin). Lanes correspond to the following: N-normal, untreated control plants grown at 23°C with 16 hour photoperiod, S-sodium chloride treated plants subjected to 100 mM sodium chloride in the medium, E-etiolation, where plants were germinated in light for three days then transferred to total darkness for four days, C-cold, where plants were germinated under normal conditions for three days and then transferred to a cold room at 6°C for four days.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Subunit A transcriptional response to environmental stress conditions in Arabidopsis seedlings. Each lane contained 20 μg whole RNA. The probe used was the same for organ level RNA blot analysis (1700 base pair subunit A cDNA labeled with digoxigenin). Lanes correspond to the following: N-normal, untreated control plants grown at 23°C with 16 hour photoperiod, S-sodium chloride treated plants subjected to 100 mM sodium chloride in the medium, E-etiolation, where plants were germinated in light for three days then transferred to total darkness for four days, C-cold, where plants were germinated under normal conditions for three days and then transferred to a cold room at 6°C for four days.
Mentions: Whole RNA blot analysis was used first to evaluate the overall response of subunit A to stress conditions. RNA was extracted from stress treated seedlings and non-treated control plants. Hybridization was performed as previously described. Results indicated a subunit A transcript response to stress. An approximate 2–4 fold increase in transcript level was detected in seedlings subjected to salt and chilling stress compared to control plants (Fig. 4). Etiolated seedlings showed essentially equivalent amounts of subunit A transcript compared to controls. Since etiolation results in rapid elongation of seedlings it was expected that there would be a concomitant increase in subunit A messenger RNA as seen with proteolipid subunit mRNA in A. thaliana[36] and subunit A message levels in rapidly expanding cotton (Gossypium hirsutum) fibers [73]. Equivalent loading of whole RNA was evaluated via spectrophotometry measuring absorption at 260 nm using a Perkin-Elmer Lambda-3 spectrophotometer and by visual confirmation using non denaturing ethidium bromide stained agarose gel electrophoresis (results not shown).

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