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The 4-Coumarate:coenzyme A ligases from Nicotiana tabacum and Arabidopsis thaliana : characterization of cDNA clones, gene families, recombinant proteins, and antisense transgenic-plants Lee, Diana
Abstract
The cDNAs encoding 4-coumarate:coenzyme A ligase (4CL), an enzyme in the general phenylpropanoid pathway, were cloned from Nicotiana tabacum and Arabidopsis thaliana. In tobacco, 4CL was encoded by a gene family and northern blot analysis demonstrated that the steady-state RNA levels were highest in stems, ovaries, and non-pigmented portions of the corolla. Two 4CL cDNAs, which were 80% identical to each other at the nucleotide level, were expressed in E. coli. The relative abilities of the recombinant-4CL proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were comparable to that of the 4CL activity found in tobacco-stem extracts. Both recombinant-4CL proteins utilized cinnamate as a substrate, an activity not observed in tobacco extracts. This activity towards cinnamate was inhibited by a modifying-component found in tobacco extracts and the evidence suggests that the substrate specificity of 4CL is, in part, determined by post-translational modification such as phosphorylation. In Arabidopsis, 4CL was shown to be encoded by a single gene. Northern blot analysis indicated that, like tobacco, 4CL steady-state RNA levels were highest in the bolting stem. The Arabidopsis-4CL cDNA was inserted in antisense orientation behind the CaMV 35S or parsley 4CL1 promoters and introduced into Arabidopsis. Transgenic plants were analyzed by western blot analysis and plants with severely suppressed-4CL protein levels were further analyzed. One transgenic-Arabidopsis line had greater than 90% decrease in 4CL enzyme activity and accumulated significantly less (50%) lignin in the bolting stem as compared to wild-type untransformed plants. Despite the decrease in 4CL mRNA, 4CL protein, and 4CL enzyme activity, anthocyanin accumulation was unaffected in the antisense-4CL Arabidopsis-lines. Mature, fully-expanded Arabidopsis-leaves were wounded and this resulted in a coordinated increase in RNA transcripts from genes encoding enzymes in the oxidative pentose phosphate pathway, the shikimic acid pathway, and the general phenylpropanoid pathway. The coordinated activation of gene expression was observed in the wild-type and antisense-4CL transgenic lines suggesting that wound-induced gene expression is not dependent on the carbon flow through the 4CLcatalyzed step.
Item Metadata
Title |
The 4-Coumarate:coenzyme A ligases from Nicotiana tabacum and Arabidopsis thaliana : characterization of cDNA clones, gene families, recombinant proteins, and antisense transgenic-plants
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1996
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Description |
The cDNAs encoding 4-coumarate:coenzyme A ligase (4CL), an enzyme in the
general phenylpropanoid pathway, were cloned from Nicotiana tabacum and
Arabidopsis thaliana. In tobacco, 4CL was encoded by a gene family and northern
blot analysis demonstrated that the steady-state RNA levels were highest in stems,
ovaries, and non-pigmented portions of the corolla. Two 4CL cDNAs, which were 80%
identical to each other at the nucleotide level, were expressed in E. coli. The relative
abilities of the recombinant-4CL proteins to utilize 4-coumarate, ferulate, and caffeate
as substrates were comparable to that of the 4CL activity found in tobacco-stem
extracts. Both recombinant-4CL proteins utilized cinnamate as a substrate, an activity
not observed in tobacco extracts. This activity towards cinnamate was inhibited by a
modifying-component found in tobacco extracts and the evidence suggests that the
substrate specificity of 4CL is, in part, determined by post-translational modification
such as phosphorylation.
In Arabidopsis, 4CL was shown to be encoded by a single gene. Northern blot
analysis indicated that, like tobacco, 4CL steady-state RNA levels were highest in the
bolting stem. The Arabidopsis-4CL cDNA was inserted in antisense orientation
behind the CaMV 35S or parsley 4CL1 promoters and introduced into Arabidopsis.
Transgenic plants were analyzed by western blot analysis and plants with severely
suppressed-4CL protein levels were further analyzed. One transgenic-Arabidopsis
line had greater than 90% decrease in 4CL enzyme activity and accumulated
significantly less (50%) lignin in the bolting stem as compared to wild-type
untransformed plants. Despite the decrease in 4CL mRNA, 4CL protein, and 4CL
enzyme activity, anthocyanin accumulation was unaffected in the antisense-4CL
Arabidopsis-lines. Mature, fully-expanded Arabidopsis-leaves were wounded and this
resulted in a coordinated increase in RNA transcripts from genes encoding enzymes in
the oxidative pentose phosphate pathway, the shikimic acid pathway, and the general
phenylpropanoid pathway. The coordinated activation of gene expression was
observed in the wild-type and antisense-4CL transgenic lines suggesting that wound-induced
gene expression is not dependent on the carbon flow through the 4CLcatalyzed
step.
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Extent |
14398642 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-03-30
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0088129
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1996-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.