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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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RNA blot analysis of subunit A in A. thaliana. The probe utilized was approximately 1700 base pairs in length corresponding to the vacuolar type H+-ATPase subunit A cDNA from Arabidopsis thaliana. Probe DNA was labeled with digoxigenin via the Polymerase Chain Reaction. Each lane contained 20 μg of whole RNA. Lanes correspond to the following organs: S-seedlings, R-roots, L-leaves, B-bolts, Si-siliques, and F-flowers. EF-Tu is a nuclear encoded chloroplast gene transcribed in all organs of the plant. Hybridization to a digoxigenin labeled probe corresponding to EF-Tu was performed to evaluate the RNA isolated from each organ to determine its suitability for RNA blot analysis.
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Figure 2: RNA blot analysis of subunit A in A. thaliana. The probe utilized was approximately 1700 base pairs in length corresponding to the vacuolar type H+-ATPase subunit A cDNA from Arabidopsis thaliana. Probe DNA was labeled with digoxigenin via the Polymerase Chain Reaction. Each lane contained 20 μg of whole RNA. Lanes correspond to the following organs: S-seedlings, R-roots, L-leaves, B-bolts, Si-siliques, and F-flowers. EF-Tu is a nuclear encoded chloroplast gene transcribed in all organs of the plant. Hybridization to a digoxigenin labeled probe corresponding to EF-Tu was performed to evaluate the RNA isolated from each organ to determine its suitability for RNA blot analysis.

Mentions: RNA blot analysis was performed to determine the organ level expression pattern of the subunit A gene. Vacuolar type ATPases are housekeeping enzymes critical for the maintenance of homeostasis. Therefore, it is expected that all living plant cells require V-ATPase function. Since A. thaliana seemed to possess only a single gene for subunit A of the V-ATPase it was expected that this gene should be expressed in all organs tested. Whole RNA was harvested from leaves, bolts, siliques, flowers, axenically grown seedlings, and axenically grown roots. RNA was denatured with glyoxal, fractionated via agarose gel electrophoresis, and immobilized on a positively charged nylon membrane. Hybridization was conducted at high stringency with a 1700 base pair homologous cDNA probe labeled with digoxigenin. In agreement with our expectations, results indicated high levels of expression in all organs tested (Fig. 2) with slightly higher levels of subunit A mRNA present in seedlings. This is most likely due to the fact that seedlings are the most rapidly growing and metabolizing stage of the plant tested. Seedlings are undergoing dramatic and rapid vacuole biogenesis to allow for rapid growth at this stage of development and therefore would be expected to require a higher level of subunit A expression than other more terminally differentiated organs. Roots, leaves, bolts, and flowers showed approximately equivalent amounts of signal with somewhat lower levels in siliques.


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)

RNA blot analysis of subunit A in A. thaliana. The probe utilized was approximately 1700 base pairs in length corresponding to the vacuolar type H+-ATPase subunit A cDNA from Arabidopsis thaliana. Probe DNA was labeled with digoxigenin via the Polymerase Chain Reaction. Each lane contained 20 μg of whole RNA. Lanes correspond to the following organs: S-seedlings, R-roots, L-leaves, B-bolts, Si-siliques, and F-flowers. EF-Tu is a nuclear encoded chloroplast gene transcribed in all organs of the plant. Hybridization to a digoxigenin labeled probe corresponding to EF-Tu was performed to evaluate the RNA isolated from each organ to determine its suitability for RNA blot analysis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC103671&req=5

Figure 2: RNA blot analysis of subunit A in A. thaliana. The probe utilized was approximately 1700 base pairs in length corresponding to the vacuolar type H+-ATPase subunit A cDNA from Arabidopsis thaliana. Probe DNA was labeled with digoxigenin via the Polymerase Chain Reaction. Each lane contained 20 μg of whole RNA. Lanes correspond to the following organs: S-seedlings, R-roots, L-leaves, B-bolts, Si-siliques, and F-flowers. EF-Tu is a nuclear encoded chloroplast gene transcribed in all organs of the plant. Hybridization to a digoxigenin labeled probe corresponding to EF-Tu was performed to evaluate the RNA isolated from each organ to determine its suitability for RNA blot analysis.
Mentions: RNA blot analysis was performed to determine the organ level expression pattern of the subunit A gene. Vacuolar type ATPases are housekeeping enzymes critical for the maintenance of homeostasis. Therefore, it is expected that all living plant cells require V-ATPase function. Since A. thaliana seemed to possess only a single gene for subunit A of the V-ATPase it was expected that this gene should be expressed in all organs tested. Whole RNA was harvested from leaves, bolts, siliques, flowers, axenically grown seedlings, and axenically grown roots. RNA was denatured with glyoxal, fractionated via agarose gel electrophoresis, and immobilized on a positively charged nylon membrane. Hybridization was conducted at high stringency with a 1700 base pair homologous cDNA probe labeled with digoxigenin. In agreement with our expectations, results indicated high levels of expression in all organs tested (Fig. 2) with slightly higher levels of subunit A mRNA present in seedlings. This is most likely due to the fact that seedlings are the most rapidly growing and metabolizing stage of the plant tested. Seedlings are undergoing dramatic and rapid vacuole biogenesis to allow for rapid growth at this stage of development and therefore would be expected to require a higher level of subunit A expression than other more terminally differentiated organs. Roots, leaves, bolts, and flowers showed approximately equivalent amounts of signal with somewhat lower levels in siliques.

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