Phytol and Tocopherol Metabolism in Arabidopsis thaliana
Phytol and Tocopherol Metabolism in Arabidopsis thaliana
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DissertationDate of Exam
12.10.2015Date of Publication
04.12.2015Advisor
Dörmann, PeterCo-Referee
Schreiber, LukasMetadata
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Abstract
The ability of a plant to convert light energy into chemical energy relies on the integrity and functionality of the photosynthetic membranes of the chloroplasts. Chlorophyll is one of the key players in photosynthesis as it is involved in capturing of photons and forms part of the electron transport chain. Therefore, its synthesis and turnover have to be tightly regulated. Upon chlorotic stress, large amounts of chlorophyll are hydrolyzed in chloroplasts and the breakdown products, phytol and pheophorbide, are further metabolized. In this work, two pathways for the metabolism of phytol after chlorophyll dephytylation were investigated: phytol esterification and phytol phosphorylation, resulting in the synthesis of fatty acid phytyl esters and isoprenyl-phosphates (phytyl-P and phytyl-PP), respectively.
First, a set of analytical tools was established to obtain a comprehensive profile of phytol-containing metabolites in Arabidopsis. Highly sensitive methods for the detection of fatty acid phytyl esters and isoprenyl-phosphates were developed, including optimization of sample preparation and direct infusion or liquid chromatography Q-TOF MS/MS analysis. These methods were used to quantify fatty acid phytyl esters and isoprenyl-phosphates in minute sample amounts and tissues of low abundance, where previous methods were unsuccessful because of lack of sensitivity. This work includes the first profile of isoprenyl-phosphates in Arabidopsis tissues. Using this set of methods, changes of phytol metabolism in mutants or during chlorotic stress could be determined.
Previous work showed that PES1 and PES2 encode functional Phytyl Ester Synthases in Arabidopsis, and that the Arabidopsis pes1 pes2 mutant lacks the most abundant fatty acid phytyl esters. Therefore, the main route for fatty acid phytyl ester synthesis in Arabidopsis is via PES1 and PES2. In the present work, the biological function of fatty acid phytyl ester synthesis was investigated. When Arabidopsis was grown under nitrogen-deplete conditions, chlorophyll was degraded while fatty acid phytyl esters and tocopherol accumulated. When full nutrition was restored, fatty acid phytyl esters and tocopherol were degraded, possibly to provide phytol for synthesis of chlorophyll. Many photosynthetic organisms contain homologous sequences to PES1 and PES2, for example C. reinhardtii. The present work demonstrated that C. reinhardtii produces fatty acid phytyl esters under nitrogen starvation. Therefore, the fatty acid phytyl ester synthesis pathway seems to be preserved from green algae to plants.
The role of PES1 and PES2 in TAG biosynthesis in chlorotic Arabidopsis leaves was further investigated. The double mutant pes1 pes2 when grown under –N conditions accumulates 30% less TAG than WT plants under the same conditions. Therefore, a small proportion of TAG might be synthesized by PES1 and PES2. Other stress conditions (drought, osmotic stress) did not result in a changed TAG accumulation in pes1 pes2. These data indicate that plastidial TAG production by PES1 and PES2 contributes only little to the total TAG pool, which is mostly derived from extraplastidic enzymes. To study plastidial TAG biosynthesis by PES1/PES2 in detail, the enzymes could be purified from Arabidopsis or N. benthamiana after expression with a protein tag and used for an enzyme assay.
A candidate gene for a PHYTYL-P KINASE in Arabidopsis was characterized in this work to study its role in the phytol phosphorylation pathway. The Arabidopsis insertion mutants for the gene At1g78620 (vte6-1, vte6-2) are tocopherol-deficient. Therefore, this gene was named VITAMIN E DEFICIENT 6 (VTE6). Growth of vte6-1 and vte6-2 is strongly affected and homozygous seeds have a decreased longevity. Quantification of isoprenyl-phosphates using the method developed in this work revealed a strong accumulation of phytyl-P in vte6-1, while phytyl-PP was decreased. This indicates that At1g78620 encodes a phytyl-P kinase, because the level of phytyl-P increases as a result of a block in this pathway. Phytol and fatty acid phytyl ester levels were increased in vte6-1 in agreement with the scenario that the phytol phosphorylation pathway is blocked. Overexpression of VTE6 results in an increase in phytyl-PP and tocopherol in seeds of Arabidopsis, giving additional evidence to the identity of VTE6 as phytyl-P kinase. Introduction of an additional mutation in phytol kinase (VTE5) activity into vte6-1 by generating a vte5-2 vte6-1 double mutant strongly improved the growth defect of the vte6-1 plants. The double mutant plants are greener and larger than vte6-1 and can grow on soil. This double mutant does not accumulate phytyl-P, as phytol phosphorylation is blocked. The vte5-2 vte6-1 plants are tocopherol-deficient like vte6-1. Therefore, the tocopherol deficiency does not explain the severe phenotype of vte6-1 and vte6-2. Instead, the growth retardation of vte6-1 and vte6-2 might result from a toxic effect exerted by phytol or phytyl-P. In the future, a phytol and phytyl-P feeding experiment will be performed with Arabidopsis WT plants to determine whether high levels of phytol or phytyl-P affect plant growth.
In conclusion, the highly sensitive methods developed in this work provided the means to establish a detailed overview of phytol metabolism under normal conditions, in stress and in Arabidopsis mutants. Moreover, it could be shown that phytol-P phosphorylation is essential for tocopherol biosynthesis in Arabidopsis. Phytol esterification is not essential for Arabidopsis metabolism but presumably represents a sink for small amounts of phytol during short-termed fluctuations in the chlorophyll content.
First, a set of analytical tools was established to obtain a comprehensive profile of phytol-containing metabolites in Arabidopsis. Highly sensitive methods for the detection of fatty acid phytyl esters and isoprenyl-phosphates were developed, including optimization of sample preparation and direct infusion or liquid chromatography Q-TOF MS/MS analysis. These methods were used to quantify fatty acid phytyl esters and isoprenyl-phosphates in minute sample amounts and tissues of low abundance, where previous methods were unsuccessful because of lack of sensitivity. This work includes the first profile of isoprenyl-phosphates in Arabidopsis tissues. Using this set of methods, changes of phytol metabolism in mutants or during chlorotic stress could be determined.
Previous work showed that PES1 and PES2 encode functional Phytyl Ester Synthases in Arabidopsis, and that the Arabidopsis pes1 pes2 mutant lacks the most abundant fatty acid phytyl esters. Therefore, the main route for fatty acid phytyl ester synthesis in Arabidopsis is via PES1 and PES2. In the present work, the biological function of fatty acid phytyl ester synthesis was investigated. When Arabidopsis was grown under nitrogen-deplete conditions, chlorophyll was degraded while fatty acid phytyl esters and tocopherol accumulated. When full nutrition was restored, fatty acid phytyl esters and tocopherol were degraded, possibly to provide phytol for synthesis of chlorophyll. Many photosynthetic organisms contain homologous sequences to PES1 and PES2, for example C. reinhardtii. The present work demonstrated that C. reinhardtii produces fatty acid phytyl esters under nitrogen starvation. Therefore, the fatty acid phytyl ester synthesis pathway seems to be preserved from green algae to plants.
The role of PES1 and PES2 in TAG biosynthesis in chlorotic Arabidopsis leaves was further investigated. The double mutant pes1 pes2 when grown under –N conditions accumulates 30% less TAG than WT plants under the same conditions. Therefore, a small proportion of TAG might be synthesized by PES1 and PES2. Other stress conditions (drought, osmotic stress) did not result in a changed TAG accumulation in pes1 pes2. These data indicate that plastidial TAG production by PES1 and PES2 contributes only little to the total TAG pool, which is mostly derived from extraplastidic enzymes. To study plastidial TAG biosynthesis by PES1/PES2 in detail, the enzymes could be purified from Arabidopsis or N. benthamiana after expression with a protein tag and used for an enzyme assay.
A candidate gene for a PHYTYL-P KINASE in Arabidopsis was characterized in this work to study its role in the phytol phosphorylation pathway. The Arabidopsis insertion mutants for the gene At1g78620 (vte6-1, vte6-2) are tocopherol-deficient. Therefore, this gene was named VITAMIN E DEFICIENT 6 (VTE6). Growth of vte6-1 and vte6-2 is strongly affected and homozygous seeds have a decreased longevity. Quantification of isoprenyl-phosphates using the method developed in this work revealed a strong accumulation of phytyl-P in vte6-1, while phytyl-PP was decreased. This indicates that At1g78620 encodes a phytyl-P kinase, because the level of phytyl-P increases as a result of a block in this pathway. Phytol and fatty acid phytyl ester levels were increased in vte6-1 in agreement with the scenario that the phytol phosphorylation pathway is blocked. Overexpression of VTE6 results in an increase in phytyl-PP and tocopherol in seeds of Arabidopsis, giving additional evidence to the identity of VTE6 as phytyl-P kinase. Introduction of an additional mutation in phytol kinase (VTE5) activity into vte6-1 by generating a vte5-2 vte6-1 double mutant strongly improved the growth defect of the vte6-1 plants. The double mutant plants are greener and larger than vte6-1 and can grow on soil. This double mutant does not accumulate phytyl-P, as phytol phosphorylation is blocked. The vte5-2 vte6-1 plants are tocopherol-deficient like vte6-1. Therefore, the tocopherol deficiency does not explain the severe phenotype of vte6-1 and vte6-2. Instead, the growth retardation of vte6-1 and vte6-2 might result from a toxic effect exerted by phytol or phytyl-P. In the future, a phytol and phytyl-P feeding experiment will be performed with Arabidopsis WT plants to determine whether high levels of phytol or phytyl-P affect plant growth.
In conclusion, the highly sensitive methods developed in this work provided the means to establish a detailed overview of phytol metabolism under normal conditions, in stress and in Arabidopsis mutants. Moreover, it could be shown that phytol-P phosphorylation is essential for tocopherol biosynthesis in Arabidopsis. Phytol esterification is not essential for Arabidopsis metabolism but presumably represents a sink for small amounts of phytol during short-termed fluctuations in the chlorophyll content.
Classification (DDC)
580 Pflanzen (Botanik)vom Dorp, Katharina: Phytol and Tocopherol Metabolism in Arabidopsis thaliana. - Bonn, 2015. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-42126
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-42126
@phdthesis{handle:20.500.11811/6577,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-42126,
author = {{Katharina vom Dorp}},
title = {Phytol and Tocopherol Metabolism in Arabidopsis thaliana},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2015,
month = dec,
note = {The ability of a plant to convert light energy into chemical energy relies on the integrity and functionality of the photosynthetic membranes of the chloroplasts. Chlorophyll is one of the key players in photosynthesis as it is involved in capturing of photons and forms part of the electron transport chain. Therefore, its synthesis and turnover have to be tightly regulated. Upon chlorotic stress, large amounts of chlorophyll are hydrolyzed in chloroplasts and the breakdown products, phytol and pheophorbide, are further metabolized. In this work, two pathways for the metabolism of phytol after chlorophyll dephytylation were investigated: phytol esterification and phytol phosphorylation, resulting in the synthesis of fatty acid phytyl esters and isoprenyl-phosphates (phytyl-P and phytyl-PP), respectively.
First, a set of analytical tools was established to obtain a comprehensive profile of phytol-containing metabolites in Arabidopsis. Highly sensitive methods for the detection of fatty acid phytyl esters and isoprenyl-phosphates were developed, including optimization of sample preparation and direct infusion or liquid chromatography Q-TOF MS/MS analysis. These methods were used to quantify fatty acid phytyl esters and isoprenyl-phosphates in minute sample amounts and tissues of low abundance, where previous methods were unsuccessful because of lack of sensitivity. This work includes the first profile of isoprenyl-phosphates in Arabidopsis tissues. Using this set of methods, changes of phytol metabolism in mutants or during chlorotic stress could be determined.
Previous work showed that PES1 and PES2 encode functional Phytyl Ester Synthases in Arabidopsis, and that the Arabidopsis pes1 pes2 mutant lacks the most abundant fatty acid phytyl esters. Therefore, the main route for fatty acid phytyl ester synthesis in Arabidopsis is via PES1 and PES2. In the present work, the biological function of fatty acid phytyl ester synthesis was investigated. When Arabidopsis was grown under nitrogen-deplete conditions, chlorophyll was degraded while fatty acid phytyl esters and tocopherol accumulated. When full nutrition was restored, fatty acid phytyl esters and tocopherol were degraded, possibly to provide phytol for synthesis of chlorophyll. Many photosynthetic organisms contain homologous sequences to PES1 and PES2, for example C. reinhardtii. The present work demonstrated that C. reinhardtii produces fatty acid phytyl esters under nitrogen starvation. Therefore, the fatty acid phytyl ester synthesis pathway seems to be preserved from green algae to plants.
The role of PES1 and PES2 in TAG biosynthesis in chlorotic Arabidopsis leaves was further investigated. The double mutant pes1 pes2 when grown under –N conditions accumulates 30% less TAG than WT plants under the same conditions. Therefore, a small proportion of TAG might be synthesized by PES1 and PES2. Other stress conditions (drought, osmotic stress) did not result in a changed TAG accumulation in pes1 pes2. These data indicate that plastidial TAG production by PES1 and PES2 contributes only little to the total TAG pool, which is mostly derived from extraplastidic enzymes. To study plastidial TAG biosynthesis by PES1/PES2 in detail, the enzymes could be purified from Arabidopsis or N. benthamiana after expression with a protein tag and used for an enzyme assay.
A candidate gene for a PHYTYL-P KINASE in Arabidopsis was characterized in this work to study its role in the phytol phosphorylation pathway. The Arabidopsis insertion mutants for the gene At1g78620 (vte6-1, vte6-2) are tocopherol-deficient. Therefore, this gene was named VITAMIN E DEFICIENT 6 (VTE6). Growth of vte6-1 and vte6-2 is strongly affected and homozygous seeds have a decreased longevity. Quantification of isoprenyl-phosphates using the method developed in this work revealed a strong accumulation of phytyl-P in vte6-1, while phytyl-PP was decreased. This indicates that At1g78620 encodes a phytyl-P kinase, because the level of phytyl-P increases as a result of a block in this pathway. Phytol and fatty acid phytyl ester levels were increased in vte6-1 in agreement with the scenario that the phytol phosphorylation pathway is blocked. Overexpression of VTE6 results in an increase in phytyl-PP and tocopherol in seeds of Arabidopsis, giving additional evidence to the identity of VTE6 as phytyl-P kinase. Introduction of an additional mutation in phytol kinase (VTE5) activity into vte6-1 by generating a vte5-2 vte6-1 double mutant strongly improved the growth defect of the vte6-1 plants. The double mutant plants are greener and larger than vte6-1 and can grow on soil. This double mutant does not accumulate phytyl-P, as phytol phosphorylation is blocked. The vte5-2 vte6-1 plants are tocopherol-deficient like vte6-1. Therefore, the tocopherol deficiency does not explain the severe phenotype of vte6-1 and vte6-2. Instead, the growth retardation of vte6-1 and vte6-2 might result from a toxic effect exerted by phytol or phytyl-P. In the future, a phytol and phytyl-P feeding experiment will be performed with Arabidopsis WT plants to determine whether high levels of phytol or phytyl-P affect plant growth.
In conclusion, the highly sensitive methods developed in this work provided the means to establish a detailed overview of phytol metabolism under normal conditions, in stress and in Arabidopsis mutants. Moreover, it could be shown that phytol-P phosphorylation is essential for tocopherol biosynthesis in Arabidopsis. Phytol esterification is not essential for Arabidopsis metabolism but presumably represents a sink for small amounts of phytol during short-termed fluctuations in the chlorophyll content.},
url = {https://hdl.handle.net/20.500.11811/6577}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-42126,
author = {{Katharina vom Dorp}},
title = {Phytol and Tocopherol Metabolism in Arabidopsis thaliana},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2015,
month = dec,
note = {The ability of a plant to convert light energy into chemical energy relies on the integrity and functionality of the photosynthetic membranes of the chloroplasts. Chlorophyll is one of the key players in photosynthesis as it is involved in capturing of photons and forms part of the electron transport chain. Therefore, its synthesis and turnover have to be tightly regulated. Upon chlorotic stress, large amounts of chlorophyll are hydrolyzed in chloroplasts and the breakdown products, phytol and pheophorbide, are further metabolized. In this work, two pathways for the metabolism of phytol after chlorophyll dephytylation were investigated: phytol esterification and phytol phosphorylation, resulting in the synthesis of fatty acid phytyl esters and isoprenyl-phosphates (phytyl-P and phytyl-PP), respectively.
First, a set of analytical tools was established to obtain a comprehensive profile of phytol-containing metabolites in Arabidopsis. Highly sensitive methods for the detection of fatty acid phytyl esters and isoprenyl-phosphates were developed, including optimization of sample preparation and direct infusion or liquid chromatography Q-TOF MS/MS analysis. These methods were used to quantify fatty acid phytyl esters and isoprenyl-phosphates in minute sample amounts and tissues of low abundance, where previous methods were unsuccessful because of lack of sensitivity. This work includes the first profile of isoprenyl-phosphates in Arabidopsis tissues. Using this set of methods, changes of phytol metabolism in mutants or during chlorotic stress could be determined.
Previous work showed that PES1 and PES2 encode functional Phytyl Ester Synthases in Arabidopsis, and that the Arabidopsis pes1 pes2 mutant lacks the most abundant fatty acid phytyl esters. Therefore, the main route for fatty acid phytyl ester synthesis in Arabidopsis is via PES1 and PES2. In the present work, the biological function of fatty acid phytyl ester synthesis was investigated. When Arabidopsis was grown under nitrogen-deplete conditions, chlorophyll was degraded while fatty acid phytyl esters and tocopherol accumulated. When full nutrition was restored, fatty acid phytyl esters and tocopherol were degraded, possibly to provide phytol for synthesis of chlorophyll. Many photosynthetic organisms contain homologous sequences to PES1 and PES2, for example C. reinhardtii. The present work demonstrated that C. reinhardtii produces fatty acid phytyl esters under nitrogen starvation. Therefore, the fatty acid phytyl ester synthesis pathway seems to be preserved from green algae to plants.
The role of PES1 and PES2 in TAG biosynthesis in chlorotic Arabidopsis leaves was further investigated. The double mutant pes1 pes2 when grown under –N conditions accumulates 30% less TAG than WT plants under the same conditions. Therefore, a small proportion of TAG might be synthesized by PES1 and PES2. Other stress conditions (drought, osmotic stress) did not result in a changed TAG accumulation in pes1 pes2. These data indicate that plastidial TAG production by PES1 and PES2 contributes only little to the total TAG pool, which is mostly derived from extraplastidic enzymes. To study plastidial TAG biosynthesis by PES1/PES2 in detail, the enzymes could be purified from Arabidopsis or N. benthamiana after expression with a protein tag and used for an enzyme assay.
A candidate gene for a PHYTYL-P KINASE in Arabidopsis was characterized in this work to study its role in the phytol phosphorylation pathway. The Arabidopsis insertion mutants for the gene At1g78620 (vte6-1, vte6-2) are tocopherol-deficient. Therefore, this gene was named VITAMIN E DEFICIENT 6 (VTE6). Growth of vte6-1 and vte6-2 is strongly affected and homozygous seeds have a decreased longevity. Quantification of isoprenyl-phosphates using the method developed in this work revealed a strong accumulation of phytyl-P in vte6-1, while phytyl-PP was decreased. This indicates that At1g78620 encodes a phytyl-P kinase, because the level of phytyl-P increases as a result of a block in this pathway. Phytol and fatty acid phytyl ester levels were increased in vte6-1 in agreement with the scenario that the phytol phosphorylation pathway is blocked. Overexpression of VTE6 results in an increase in phytyl-PP and tocopherol in seeds of Arabidopsis, giving additional evidence to the identity of VTE6 as phytyl-P kinase. Introduction of an additional mutation in phytol kinase (VTE5) activity into vte6-1 by generating a vte5-2 vte6-1 double mutant strongly improved the growth defect of the vte6-1 plants. The double mutant plants are greener and larger than vte6-1 and can grow on soil. This double mutant does not accumulate phytyl-P, as phytol phosphorylation is blocked. The vte5-2 vte6-1 plants are tocopherol-deficient like vte6-1. Therefore, the tocopherol deficiency does not explain the severe phenotype of vte6-1 and vte6-2. Instead, the growth retardation of vte6-1 and vte6-2 might result from a toxic effect exerted by phytol or phytyl-P. In the future, a phytol and phytyl-P feeding experiment will be performed with Arabidopsis WT plants to determine whether high levels of phytol or phytyl-P affect plant growth.
In conclusion, the highly sensitive methods developed in this work provided the means to establish a detailed overview of phytol metabolism under normal conditions, in stress and in Arabidopsis mutants. Moreover, it could be shown that phytol-P phosphorylation is essential for tocopherol biosynthesis in Arabidopsis. Phytol esterification is not essential for Arabidopsis metabolism but presumably represents a sink for small amounts of phytol during short-termed fluctuations in the chlorophyll content.},
url = {https://hdl.handle.net/20.500.11811/6577}
}
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