Phytyl Ester Synthases from Arabidopsis thaliana and Solanum lycopersicum
Phytyl Ester Synthases from Arabidopsis thaliana and Solanum lycopersicum
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DissertationPrüfungsdatum
15.03.2018Datum der Veröffentlichung
20.07.2018Erstgutachter
Dörmann, PeterZweitgutachter
Hochholdinger, FrankMetadaten
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Chlorophyll is the most abundant pigment in the biosphere, and its degradation leads to the release of large amounts of chlorophyll breakdown products, i.e. pheophorbide and phytol. Only a minor fraction of the released phytol is degraded. Phytol can be further processed by incorporation into fatty acid phytyl esters (FAPEs) or tocopherols. In A. thaliana, two phytyl ester synthases, PES1 and PES2, have been characterised which are expressed under senescence and which catalyse the esterification of chlorophyll-derived phytol and fatty acids. PES1 and PES2 belong to a family of six esterase/lipase/thioesterase (ELT) like proteins in A. thaliana. In this work, the analysis of PES2 as well as the characterisation of the remaining four ELT enzymes, ELT3, ELT4, ELT5, and ELT6, were presented. In addition, mutants of the closely related xanthophyll ester synthase PYP1 from Solanum lycopersicum were investigated.
Expression analysis of the ELT genes revealed that, similar to PES1 and PES2, the expression of ELT3 and ELT4 was increased under senescence. Furthermore, PES1, ELT4, ELT5, and ELT6 showed elevated expression levels in inflorescences. The FAPE analysis of elt single mutants and the newly generated double mutant elt4elt6 showed no differences compared to wild type under nitrogen deprivation induced senescence. Previous results revealed that the double mutant pes1pes2 contains decreased FAPE amounts under nitrogen deprivation compared to wild type. The introduction of a third mutation in the newly generated triple mutant pes1pes2elt4 did not lead to a further decrease in FAPE content. The analysis of the seed FAPE content of the multiple mutants revealed a decrease in FAPEs in elt4elt6 and pes1pes2elt4 demonstrating the involvement of ELT enzymes in seed FAPE production. FAPE accumulation under drought stress was investigated to study different stress conditions under which chlorophyll is degraded. Upon osmotically induced drought stress, A. thaliana accumulates FAPEs with the same fatty acid distribution as observed under nitrogen deprivation.
The heterologous expression of PES2 in E. coli cells and in leaves of N. benthamiana resulted in an increased accumulation of FAPEs compared to controls. The fatty acid distribution of FAPEs in PES2-expressing N. benthamiana leaves reflected the fatty acid preference of PES2 observed in A. thaliana. In addition to FAPEs, PES2 produced wax diesters (WDEs) when expressed in the chloroplasts of N. benthamiana leaves. These WDEs contained two fatty acids esterified to hexadecanediol as shown by Q-TOF MS/MS experiments. The backbone was identified by GC-MS and NMR as 1,6-hexadecanediol. This results represents the first report of 1,6-hexadecanediol in plants, and the first report of an alkanediol in chloroplasts. The fatty acid moieties in the WDEs were identified as mainly 12:0 and 14:0. The lipid analysis of isolated chloroplasts of infiltrated N. benthamiana leaves indicated that WDEs are first synthesised in the chloroplasts and then exported. Taken together these results unveil PES2 as the first plant enzyme that harbours wax diester synthase activity.
Screening of the FAPE composition of different plant species under senescence revealed the accumulation of unusual very long-chain FAPEs (VLC-FAPEs) which contain fatty acids with chain lengths longer than C20. These VLC-FAPEs were identified in different plant species from several families. The occurrence of VLC fatty acids is not typical for the chloroplast. Therefore, VLC-FAPE are presumably produced outside of chloroplasts, deduced from FAPE measurements of chloroplast isolated from senescent S. lycopersicum leaves.
PYP1 from S. lycopersicum, an ortholog of PES1 and PES2, has previously been identified as a xanthophyll ester synthase which is responsible for the synthesis of xanthophyll esters in petals of S. lycopersicum. Because PYP1 is closely related to phytyl ester synthases, the accumulation of FAPEs in different organs of S. lycopersicum was analysed. 14:0- and 16:0-phytol were strongly reduced in petals of pyp1 mutant plants indicating the involvement of PYP1 in FAPE synthesis in petals of S. lycopersicum. The fatty acid moieties of the decreased FAPE species reflects the fatty acid preference of PYP1 in xanthophyll ester synthesis as PYP1 mainly produces neoxanthin and violaxanthin esters with 14:0 and 16:0 fatty acids.
The further analysis of xanthophyll esters in petals of S. lycopersicum revealed the occurrence of a third group of xanthophyll esters besides mono- and diesters, e.g. neoxanthin triesters which have only been reported before for leaves of apple trees. The study of pyp1-1 petals showed that PYP1 is also responsible for the synthesis of neoxanthin triesters. In contrast to S. lycopersicum, the yellow petals of Camelina sativa and the senescent leaves of A. thaliana are devoid of xanthophyll esters.
In conclusion it could be shown that ELT enzymes are quite promiscuous regarding the acyl acceptors such as phytol, alcanediols and xanthophylls. On the other side, individual ELT enzymes are specific for certain fatty acid chain lengths.
Expression analysis of the ELT genes revealed that, similar to PES1 and PES2, the expression of ELT3 and ELT4 was increased under senescence. Furthermore, PES1, ELT4, ELT5, and ELT6 showed elevated expression levels in inflorescences. The FAPE analysis of elt single mutants and the newly generated double mutant elt4elt6 showed no differences compared to wild type under nitrogen deprivation induced senescence. Previous results revealed that the double mutant pes1pes2 contains decreased FAPE amounts under nitrogen deprivation compared to wild type. The introduction of a third mutation in the newly generated triple mutant pes1pes2elt4 did not lead to a further decrease in FAPE content. The analysis of the seed FAPE content of the multiple mutants revealed a decrease in FAPEs in elt4elt6 and pes1pes2elt4 demonstrating the involvement of ELT enzymes in seed FAPE production. FAPE accumulation under drought stress was investigated to study different stress conditions under which chlorophyll is degraded. Upon osmotically induced drought stress, A. thaliana accumulates FAPEs with the same fatty acid distribution as observed under nitrogen deprivation.
The heterologous expression of PES2 in E. coli cells and in leaves of N. benthamiana resulted in an increased accumulation of FAPEs compared to controls. The fatty acid distribution of FAPEs in PES2-expressing N. benthamiana leaves reflected the fatty acid preference of PES2 observed in A. thaliana. In addition to FAPEs, PES2 produced wax diesters (WDEs) when expressed in the chloroplasts of N. benthamiana leaves. These WDEs contained two fatty acids esterified to hexadecanediol as shown by Q-TOF MS/MS experiments. The backbone was identified by GC-MS and NMR as 1,6-hexadecanediol. This results represents the first report of 1,6-hexadecanediol in plants, and the first report of an alkanediol in chloroplasts. The fatty acid moieties in the WDEs were identified as mainly 12:0 and 14:0. The lipid analysis of isolated chloroplasts of infiltrated N. benthamiana leaves indicated that WDEs are first synthesised in the chloroplasts and then exported. Taken together these results unveil PES2 as the first plant enzyme that harbours wax diester synthase activity.
Screening of the FAPE composition of different plant species under senescence revealed the accumulation of unusual very long-chain FAPEs (VLC-FAPEs) which contain fatty acids with chain lengths longer than C20. These VLC-FAPEs were identified in different plant species from several families. The occurrence of VLC fatty acids is not typical for the chloroplast. Therefore, VLC-FAPE are presumably produced outside of chloroplasts, deduced from FAPE measurements of chloroplast isolated from senescent S. lycopersicum leaves.
PYP1 from S. lycopersicum, an ortholog of PES1 and PES2, has previously been identified as a xanthophyll ester synthase which is responsible for the synthesis of xanthophyll esters in petals of S. lycopersicum. Because PYP1 is closely related to phytyl ester synthases, the accumulation of FAPEs in different organs of S. lycopersicum was analysed. 14:0- and 16:0-phytol were strongly reduced in petals of pyp1 mutant plants indicating the involvement of PYP1 in FAPE synthesis in petals of S. lycopersicum. The fatty acid moieties of the decreased FAPE species reflects the fatty acid preference of PYP1 in xanthophyll ester synthesis as PYP1 mainly produces neoxanthin and violaxanthin esters with 14:0 and 16:0 fatty acids.
The further analysis of xanthophyll esters in petals of S. lycopersicum revealed the occurrence of a third group of xanthophyll esters besides mono- and diesters, e.g. neoxanthin triesters which have only been reported before for leaves of apple trees. The study of pyp1-1 petals showed that PYP1 is also responsible for the synthesis of neoxanthin triesters. In contrast to S. lycopersicum, the yellow petals of Camelina sativa and the senescent leaves of A. thaliana are devoid of xanthophyll esters.
In conclusion it could be shown that ELT enzymes are quite promiscuous regarding the acyl acceptors such as phytol, alcanediols and xanthophylls. On the other side, individual ELT enzymes are specific for certain fatty acid chain lengths.
Schlagwörter
Phytylester, Phytylestersynthasen, Wachsdiester, Pflanzenlipide, Arabidopsis thaliana, Nicotiana benthamiana, Solanum lycopersicum, phytyl ester, phytyl ester synthases, wax diester, plant lipids
Klassifikation (DDC)
570 Biowissenschaften, Biologie 580 Pflanzen (Botanik)Wehler, Regina Daniela: Phytyl Ester Synthases from Arabidopsis thaliana and Solanum lycopersicum. - Bonn, 2018. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-51342
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-51342
@phdthesis{handle:20.500.11811/7598,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-51342,
author = {{Regina Daniela Wehler}},
title = {Phytyl Ester Synthases from Arabidopsis thaliana and Solanum lycopersicum},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2018,
month = jul,
note = {Chlorophyll is the most abundant pigment in the biosphere, and its degradation leads to the release of large amounts of chlorophyll breakdown products, i.e. pheophorbide and phytol. Only a minor fraction of the released phytol is degraded. Phytol can be further processed by incorporation into fatty acid phytyl esters (FAPEs) or tocopherols. In A. thaliana, two phytyl ester synthases, PES1 and PES2, have been characterised which are expressed under senescence and which catalyse the esterification of chlorophyll-derived phytol and fatty acids. PES1 and PES2 belong to a family of six esterase/lipase/thioesterase (ELT) like proteins in A. thaliana. In this work, the analysis of PES2 as well as the characterisation of the remaining four ELT enzymes, ELT3, ELT4, ELT5, and ELT6, were presented. In addition, mutants of the closely related xanthophyll ester synthase PYP1 from Solanum lycopersicum were investigated.
Expression analysis of the ELT genes revealed that, similar to PES1 and PES2, the expression of ELT3 and ELT4 was increased under senescence. Furthermore, PES1, ELT4, ELT5, and ELT6 showed elevated expression levels in inflorescences. The FAPE analysis of elt single mutants and the newly generated double mutant elt4elt6 showed no differences compared to wild type under nitrogen deprivation induced senescence. Previous results revealed that the double mutant pes1pes2 contains decreased FAPE amounts under nitrogen deprivation compared to wild type. The introduction of a third mutation in the newly generated triple mutant pes1pes2elt4 did not lead to a further decrease in FAPE content. The analysis of the seed FAPE content of the multiple mutants revealed a decrease in FAPEs in elt4elt6 and pes1pes2elt4 demonstrating the involvement of ELT enzymes in seed FAPE production. FAPE accumulation under drought stress was investigated to study different stress conditions under which chlorophyll is degraded. Upon osmotically induced drought stress, A. thaliana accumulates FAPEs with the same fatty acid distribution as observed under nitrogen deprivation.
The heterologous expression of PES2 in E. coli cells and in leaves of N. benthamiana resulted in an increased accumulation of FAPEs compared to controls. The fatty acid distribution of FAPEs in PES2-expressing N. benthamiana leaves reflected the fatty acid preference of PES2 observed in A. thaliana. In addition to FAPEs, PES2 produced wax diesters (WDEs) when expressed in the chloroplasts of N. benthamiana leaves. These WDEs contained two fatty acids esterified to hexadecanediol as shown by Q-TOF MS/MS experiments. The backbone was identified by GC-MS and NMR as 1,6-hexadecanediol. This results represents the first report of 1,6-hexadecanediol in plants, and the first report of an alkanediol in chloroplasts. The fatty acid moieties in the WDEs were identified as mainly 12:0 and 14:0. The lipid analysis of isolated chloroplasts of infiltrated N. benthamiana leaves indicated that WDEs are first synthesised in the chloroplasts and then exported. Taken together these results unveil PES2 as the first plant enzyme that harbours wax diester synthase activity.
Screening of the FAPE composition of different plant species under senescence revealed the accumulation of unusual very long-chain FAPEs (VLC-FAPEs) which contain fatty acids with chain lengths longer than C20. These VLC-FAPEs were identified in different plant species from several families. The occurrence of VLC fatty acids is not typical for the chloroplast. Therefore, VLC-FAPE are presumably produced outside of chloroplasts, deduced from FAPE measurements of chloroplast isolated from senescent S. lycopersicum leaves.
PYP1 from S. lycopersicum, an ortholog of PES1 and PES2, has previously been identified as a xanthophyll ester synthase which is responsible for the synthesis of xanthophyll esters in petals of S. lycopersicum. Because PYP1 is closely related to phytyl ester synthases, the accumulation of FAPEs in different organs of S. lycopersicum was analysed. 14:0- and 16:0-phytol were strongly reduced in petals of pyp1 mutant plants indicating the involvement of PYP1 in FAPE synthesis in petals of S. lycopersicum. The fatty acid moieties of the decreased FAPE species reflects the fatty acid preference of PYP1 in xanthophyll ester synthesis as PYP1 mainly produces neoxanthin and violaxanthin esters with 14:0 and 16:0 fatty acids.
The further analysis of xanthophyll esters in petals of S. lycopersicum revealed the occurrence of a third group of xanthophyll esters besides mono- and diesters, e.g. neoxanthin triesters which have only been reported before for leaves of apple trees. The study of pyp1-1 petals showed that PYP1 is also responsible for the synthesis of neoxanthin triesters. In contrast to S. lycopersicum, the yellow petals of Camelina sativa and the senescent leaves of A. thaliana are devoid of xanthophyll esters.
In conclusion it could be shown that ELT enzymes are quite promiscuous regarding the acyl acceptors such as phytol, alcanediols and xanthophylls. On the other side, individual ELT enzymes are specific for certain fatty acid chain lengths.},
url = {https://hdl.handle.net/20.500.11811/7598}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-51342,
author = {{Regina Daniela Wehler}},
title = {Phytyl Ester Synthases from Arabidopsis thaliana and Solanum lycopersicum},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2018,
month = jul,
note = {Chlorophyll is the most abundant pigment in the biosphere, and its degradation leads to the release of large amounts of chlorophyll breakdown products, i.e. pheophorbide and phytol. Only a minor fraction of the released phytol is degraded. Phytol can be further processed by incorporation into fatty acid phytyl esters (FAPEs) or tocopherols. In A. thaliana, two phytyl ester synthases, PES1 and PES2, have been characterised which are expressed under senescence and which catalyse the esterification of chlorophyll-derived phytol and fatty acids. PES1 and PES2 belong to a family of six esterase/lipase/thioesterase (ELT) like proteins in A. thaliana. In this work, the analysis of PES2 as well as the characterisation of the remaining four ELT enzymes, ELT3, ELT4, ELT5, and ELT6, were presented. In addition, mutants of the closely related xanthophyll ester synthase PYP1 from Solanum lycopersicum were investigated.
Expression analysis of the ELT genes revealed that, similar to PES1 and PES2, the expression of ELT3 and ELT4 was increased under senescence. Furthermore, PES1, ELT4, ELT5, and ELT6 showed elevated expression levels in inflorescences. The FAPE analysis of elt single mutants and the newly generated double mutant elt4elt6 showed no differences compared to wild type under nitrogen deprivation induced senescence. Previous results revealed that the double mutant pes1pes2 contains decreased FAPE amounts under nitrogen deprivation compared to wild type. The introduction of a third mutation in the newly generated triple mutant pes1pes2elt4 did not lead to a further decrease in FAPE content. The analysis of the seed FAPE content of the multiple mutants revealed a decrease in FAPEs in elt4elt6 and pes1pes2elt4 demonstrating the involvement of ELT enzymes in seed FAPE production. FAPE accumulation under drought stress was investigated to study different stress conditions under which chlorophyll is degraded. Upon osmotically induced drought stress, A. thaliana accumulates FAPEs with the same fatty acid distribution as observed under nitrogen deprivation.
The heterologous expression of PES2 in E. coli cells and in leaves of N. benthamiana resulted in an increased accumulation of FAPEs compared to controls. The fatty acid distribution of FAPEs in PES2-expressing N. benthamiana leaves reflected the fatty acid preference of PES2 observed in A. thaliana. In addition to FAPEs, PES2 produced wax diesters (WDEs) when expressed in the chloroplasts of N. benthamiana leaves. These WDEs contained two fatty acids esterified to hexadecanediol as shown by Q-TOF MS/MS experiments. The backbone was identified by GC-MS and NMR as 1,6-hexadecanediol. This results represents the first report of 1,6-hexadecanediol in plants, and the first report of an alkanediol in chloroplasts. The fatty acid moieties in the WDEs were identified as mainly 12:0 and 14:0. The lipid analysis of isolated chloroplasts of infiltrated N. benthamiana leaves indicated that WDEs are first synthesised in the chloroplasts and then exported. Taken together these results unveil PES2 as the first plant enzyme that harbours wax diester synthase activity.
Screening of the FAPE composition of different plant species under senescence revealed the accumulation of unusual very long-chain FAPEs (VLC-FAPEs) which contain fatty acids with chain lengths longer than C20. These VLC-FAPEs were identified in different plant species from several families. The occurrence of VLC fatty acids is not typical for the chloroplast. Therefore, VLC-FAPE are presumably produced outside of chloroplasts, deduced from FAPE measurements of chloroplast isolated from senescent S. lycopersicum leaves.
PYP1 from S. lycopersicum, an ortholog of PES1 and PES2, has previously been identified as a xanthophyll ester synthase which is responsible for the synthesis of xanthophyll esters in petals of S. lycopersicum. Because PYP1 is closely related to phytyl ester synthases, the accumulation of FAPEs in different organs of S. lycopersicum was analysed. 14:0- and 16:0-phytol were strongly reduced in petals of pyp1 mutant plants indicating the involvement of PYP1 in FAPE synthesis in petals of S. lycopersicum. The fatty acid moieties of the decreased FAPE species reflects the fatty acid preference of PYP1 in xanthophyll ester synthesis as PYP1 mainly produces neoxanthin and violaxanthin esters with 14:0 and 16:0 fatty acids.
The further analysis of xanthophyll esters in petals of S. lycopersicum revealed the occurrence of a third group of xanthophyll esters besides mono- and diesters, e.g. neoxanthin triesters which have only been reported before for leaves of apple trees. The study of pyp1-1 petals showed that PYP1 is also responsible for the synthesis of neoxanthin triesters. In contrast to S. lycopersicum, the yellow petals of Camelina sativa and the senescent leaves of A. thaliana are devoid of xanthophyll esters.
In conclusion it could be shown that ELT enzymes are quite promiscuous regarding the acyl acceptors such as phytol, alcanediols and xanthophylls. On the other side, individual ELT enzymes are specific for certain fatty acid chain lengths.},
url = {https://hdl.handle.net/20.500.11811/7598}
}
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