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

Time-course expressions of transgene and flavonoid biosynthetic pathway genes in B3cpl1 and in PCcpl1 lines during cold treatment.(a) Total RNAs were extracted from leaves of 3-week-old plants grown at room temperature (23°C) and plants treated with cold (4°C) for additional 2 days, 1 week, 2 weeks and 3 weeks. Expression levels of each gene were shown relative to the levels of B3cpl1 grown at 23°C. Bars indicate standard errors. Experiments were conducted two times with similar results. Results from one experiment were shown. PAL1; phenylalanine ammonia lyase 1, CHS; chalcone synthase, CHI; chalcone isomerase, F3′H; flavonoid 3′-hydroxylase, DFR; dihydroflavonol reductase, ANS; anthocyanidin synthase, FLS1; flavonol synthase 1, FLS3; flavonol synthase 3, UGT73B2; UDP-glucosyltransferase 73B2 (flavonol 3-O-glucosyltransferase activity), UGT78D2; UDP-glucosyltransferase 78D2 (anthocyanidin 3-O-glucosyltransferase). (b) Structure of phenylpropanoid pathway. Transcript levels of the enzymes in the marked steps were analyzed in (a).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3050924&req=5

pone-0017603-g004: Time-course expressions of transgene and flavonoid biosynthetic pathway genes in B3cpl1 and in PCcpl1 lines during cold treatment.(a) Total RNAs were extracted from leaves of 3-week-old plants grown at room temperature (23°C) and plants treated with cold (4°C) for additional 2 days, 1 week, 2 weeks and 3 weeks. Expression levels of each gene were shown relative to the levels of B3cpl1 grown at 23°C. Bars indicate standard errors. Experiments were conducted two times with similar results. Results from one experiment were shown. PAL1; phenylalanine ammonia lyase 1, CHS; chalcone synthase, CHI; chalcone isomerase, F3′H; flavonoid 3′-hydroxylase, DFR; dihydroflavonol reductase, ANS; anthocyanidin synthase, FLS1; flavonol synthase 1, FLS3; flavonol synthase 3, UGT73B2; UDP-glucosyltransferase 73B2 (flavonol 3-O-glucosyltransferase activity), UGT78D2; UDP-glucosyltransferase 78D2 (anthocyanidin 3-O-glucosyltransferase). (b) Structure of phenylpropanoid pathway. Transcript levels of the enzymes in the marked steps were analyzed in (a).

Mentions: In order to understand the efficacy of the cold-inducible three-component system, a time course of gene expression was determined for transgenes and genes encoding the flavonoid biosynthesis pathway during a long-term cold induction (Figure 4). Three-week old PCcpl1 and B3cpl1 plants were exposed to 4°C for up to additional 3 weeks. Cold treatments longer than 3 weeks induced senescence of plants and therefore were not included in the analysis. In PCcpl1, expression of CBF3 and PAP1 reached their highest levels (1,600 fold and 37 fold, respectively) after 2 days and slowly declined after 1 week. After 3 weeks of cold treatment, the PAP1 level was similar to that of vector control plants. Expression of PAL1 (phenylalanine ammonia lyase 1) was induced both in cold-treated PCcpl1 and B3cpl1 plants, albeit PCcpl1 plants showed slightly faster response and higher expression level. Genes that lead to anthocyanin biosynthesis, such as CHS (chalcone synthase), CHI (chalcone isomerase), F3′H (flavonoid 3′-hydroxylase), DFR (dihydroflavonol reductase), and ANS (anthocyanidin synthase) were all expressed at higher levels in cold-treated PCcpl1 plants. In contrast, cold treatment induced expression of FLS (flavonol synthase) and UGT73B2 (flavonol glucosyltransferases) both in PCcpl1 and B3cpl1 plants to the similar levels. These results indicated that the three-component system effectively activated the anthocyanin biosynthesis pathway, whereas cold treatment itself induced flavonol biosynthesis pathway genes independent of the three-component system. In addition, the induction of the anthocyanin biosynthesis pathway persisted until plants started to senesce after three weeks of cold treatment.


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)

Time-course expressions of transgene and flavonoid biosynthetic pathway genes in B3cpl1 and in PCcpl1 lines during cold treatment.(a) Total RNAs were extracted from leaves of 3-week-old plants grown at room temperature (23°C) and plants treated with cold (4°C) for additional 2 days, 1 week, 2 weeks and 3 weeks. Expression levels of each gene were shown relative to the levels of B3cpl1 grown at 23°C. Bars indicate standard errors. Experiments were conducted two times with similar results. Results from one experiment were shown. PAL1; phenylalanine ammonia lyase 1, CHS; chalcone synthase, CHI; chalcone isomerase, F3′H; flavonoid 3′-hydroxylase, DFR; dihydroflavonol reductase, ANS; anthocyanidin synthase, FLS1; flavonol synthase 1, FLS3; flavonol synthase 3, UGT73B2; UDP-glucosyltransferase 73B2 (flavonol 3-O-glucosyltransferase activity), UGT78D2; UDP-glucosyltransferase 78D2 (anthocyanidin 3-O-glucosyltransferase). (b) Structure of phenylpropanoid pathway. Transcript levels of the enzymes in the marked steps were analyzed in (a).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017603-g004: Time-course expressions of transgene and flavonoid biosynthetic pathway genes in B3cpl1 and in PCcpl1 lines during cold treatment.(a) Total RNAs were extracted from leaves of 3-week-old plants grown at room temperature (23°C) and plants treated with cold (4°C) for additional 2 days, 1 week, 2 weeks and 3 weeks. Expression levels of each gene were shown relative to the levels of B3cpl1 grown at 23°C. Bars indicate standard errors. Experiments were conducted two times with similar results. Results from one experiment were shown. PAL1; phenylalanine ammonia lyase 1, CHS; chalcone synthase, CHI; chalcone isomerase, F3′H; flavonoid 3′-hydroxylase, DFR; dihydroflavonol reductase, ANS; anthocyanidin synthase, FLS1; flavonol synthase 1, FLS3; flavonol synthase 3, UGT73B2; UDP-glucosyltransferase 73B2 (flavonol 3-O-glucosyltransferase activity), UGT78D2; UDP-glucosyltransferase 78D2 (anthocyanidin 3-O-glucosyltransferase). (b) Structure of phenylpropanoid pathway. Transcript levels of the enzymes in the marked steps were analyzed in (a).
Mentions: In order to understand the efficacy of the cold-inducible three-component system, a time course of gene expression was determined for transgenes and genes encoding the flavonoid biosynthesis pathway during a long-term cold induction (Figure 4). Three-week old PCcpl1 and B3cpl1 plants were exposed to 4°C for up to additional 3 weeks. Cold treatments longer than 3 weeks induced senescence of plants and therefore were not included in the analysis. In PCcpl1, expression of CBF3 and PAP1 reached their highest levels (1,600 fold and 37 fold, respectively) after 2 days and slowly declined after 1 week. After 3 weeks of cold treatment, the PAP1 level was similar to that of vector control plants. Expression of PAL1 (phenylalanine ammonia lyase 1) was induced both in cold-treated PCcpl1 and B3cpl1 plants, albeit PCcpl1 plants showed slightly faster response and higher expression level. Genes that lead to anthocyanin biosynthesis, such as CHS (chalcone synthase), CHI (chalcone isomerase), F3′H (flavonoid 3′-hydroxylase), DFR (dihydroflavonol reductase), and ANS (anthocyanidin synthase) were all expressed at higher levels in cold-treated PCcpl1 plants. In contrast, cold treatment induced expression of FLS (flavonol synthase) and UGT73B2 (flavonol glucosyltransferases) both in PCcpl1 and B3cpl1 plants to the similar levels. These results indicated that the three-component system effectively activated the anthocyanin biosynthesis pathway, whereas cold treatment itself induced flavonol biosynthesis pathway genes independent of the three-component system. In addition, the induction of the anthocyanin biosynthesis pathway persisted until plants started to senesce after three weeks of cold treatment.

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