Limits...
A three-component gene expression system and its application for inducible flavonoid overproduction in transgenic Arabidopsis thaliana.

Feng Y, Cao CM, Vikram M, Park S, Kim HJ, Hong JC, Cisneros-Zevallos L, Koiwa H - PLoS ONE (2011)

Bottom Line: While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment.These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering.Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Vegetable and Fruit Improvement Center, Department of Horticultural Science, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.

Show MeSH

Related in: MedlinePlus

HPLC/PDA chromatograms showing the flavonoid profiles of PCcpl1 and B3cpl1 plants under normal growth condition (23°C) or after additional 3 weeks of cold treatment (4°C).“A” stands for cyanidin derivatives, and “F” stands for flavonoids, which were identified by LC-MS. The peaks were labeled according to Tohge et al (2005).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3050924&req=5

pone-0017603-g005: HPLC/PDA chromatograms showing the flavonoid profiles of PCcpl1 and B3cpl1 plants under normal growth condition (23°C) or after additional 3 weeks of cold treatment (4°C).“A” stands for cyanidin derivatives, and “F” stands for flavonoids, which were identified by LC-MS. The peaks were labeled according to Tohge et al (2005).

Mentions: In PCcpl1 plants, cold induction of anthocyanin biosynthesis pathway genes were accompanied with accumulation of anthocyanin pigments throughout the aerial part of plant bodies, indicating that the cold-induction system indeed increased biosynthetic capacity of anthocyanins in transgenic plants (Figure 3, bottom). In order to determine whether anthocyanin phytochemicals produced via three-component system is similar to those produced by constitutive overexpression of PAP1, profiles of anthocyanins and other flavonoids produced in transgenic plants were analyzed (Figure 5, Table 2). Phytochemicals were extracted from 3 weeks old PCcpl1 and B3cpl1 plants grown at room temperature (23°C) and plants after additional three weeks of cold treatment (4°C). Putative flavonoid compounds were identified by comparing their retention time and UV-visible absorption spectra in LC, and their molecular charge ratios and fragmentation patterns in MS/MS analyses, to the reported profiles [18]. Authentic standards were used to determine the amount of each compound using HPLC chromatogram. Five major anthocyanins (cyanidin derivatives) and six additional flavonoids (quercetin and kaempferol derivatives) were identified in cold-induced PCcpl1 plants, which were labeled according to Tohge et al (2005) (Figure 5).


A three-component gene expression system and its application for inducible flavonoid overproduction in transgenic Arabidopsis thaliana.

Feng Y, Cao CM, Vikram M, Park S, Kim HJ, Hong JC, Cisneros-Zevallos L, Koiwa H - PLoS ONE (2011)

HPLC/PDA chromatograms showing the flavonoid profiles of PCcpl1 and B3cpl1 plants under normal growth condition (23°C) or after additional 3 weeks of cold treatment (4°C).“A” stands for cyanidin derivatives, and “F” stands for flavonoids, which were identified by LC-MS. The peaks were labeled according to Tohge et al (2005).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3050924&req=5

pone-0017603-g005: HPLC/PDA chromatograms showing the flavonoid profiles of PCcpl1 and B3cpl1 plants under normal growth condition (23°C) or after additional 3 weeks of cold treatment (4°C).“A” stands for cyanidin derivatives, and “F” stands for flavonoids, which were identified by LC-MS. The peaks were labeled according to Tohge et al (2005).
Mentions: In PCcpl1 plants, cold induction of anthocyanin biosynthesis pathway genes were accompanied with accumulation of anthocyanin pigments throughout the aerial part of plant bodies, indicating that the cold-induction system indeed increased biosynthetic capacity of anthocyanins in transgenic plants (Figure 3, bottom). In order to determine whether anthocyanin phytochemicals produced via three-component system is similar to those produced by constitutive overexpression of PAP1, profiles of anthocyanins and other flavonoids produced in transgenic plants were analyzed (Figure 5, Table 2). Phytochemicals were extracted from 3 weeks old PCcpl1 and B3cpl1 plants grown at room temperature (23°C) and plants after additional three weeks of cold treatment (4°C). Putative flavonoid compounds were identified by comparing their retention time and UV-visible absorption spectra in LC, and their molecular charge ratios and fragmentation patterns in MS/MS analyses, to the reported profiles [18]. Authentic standards were used to determine the amount of each compound using HPLC chromatogram. Five major anthocyanins (cyanidin derivatives) and six additional flavonoids (quercetin and kaempferol derivatives) were identified in cold-induced PCcpl1 plants, which were labeled according to Tohge et al (2005) (Figure 5).

Bottom Line: While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment.These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering.Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Vegetable and Fruit Improvement Center, Department of Horticultural Science, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.

Show MeSH
Related in: MedlinePlus