The Role of Acyl-ACP Thioesterases and Glycerophosphodiester Phosphodiesterases for Gametophyte Development in Arabidopsis thaliana
The Role of Acyl-ACP Thioesterases and Glycerophosphodiester Phosphodiesterases for Gametophyte Development in Arabidopsis thaliana
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DissertationPrüfungsdatum
21.08.2015Datum der Veröffentlichung
09.08.2016Erstgutachter
Dörmann, PeterZweitgutachter
Schreiber, LukasMetadaten
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Chloroplasts are the site of the de novo synthesis of the predominant proportion of fatty acids in plant cells. The growing fatty acid chain is bound to acyl carrier protein (ACP) during synthesis. Acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast releasing free fatty acids thereby regulating the chain lengths of fatty acids. The free fatty acids are activated by long chain fatty acid-CoA synthetases and exported from the plastid to serve as substrates for glycerolipid synthesis at the ER. In plants there are two types of acyl-ACP thioesterases that terminate the fatty acid synthesis, FatA and FatB. They differ in amino acid sequence and substrate specificity. FatA thioesterases are highly active towards oleoyl-ACPs (18:1-ACP), while FatB thioesterases show highest activity with saturated acyl-ACPs. Arabidopsis contains two genomic loci encoding FatA thioesterases, FatA1 and FatA2.
Arabidopsis FatA1 and FatA2 were heterologously expressed in the E. coli fadD mutant. The fadD mutant lacks the acyl-CoA synthetase leading to an accumulation of free fatty acids, since these are no longer re-esterified to CoA. After cultivation, the fatty acids were extracted from the cell pellet and analyzed via GC-FID, exhibiting an evident increase in 16:1Δ9 and 18:1Δ11 fatty acids, demonstrating acyl-ACP thioesterase A activities for both enzymes.
To characterize FatA1 and FatA2 in Arabidopsis T-DNA insertion lines were obtained and analyzed by using GC-MS and Q-TOF MS. The loss of function fata1-2 and fata2-2 mutants did not reveal any alterations either in fatty acid or in glycerolipid composition. After crossing the single mutants no double homozygous fata1-2-/-fata2-2-/- mutant could be obtained. Plants heterozygous for the FatA1 and homozygous for the FatA2 locus showed approximately 25 % of the seeds to be aborted in an early stage of the development. Microtome sections of these aborted seeds revealed that the embryo development is arrested at the morphological late heart to early torpedo stage, leading to the assumption that the loss of both FatA genes might be embryolethal. Fatty acid and lipid composition of heart stage mutant embryos showed, that the development comes to a halt when lipid export from the chloroplast becomes significant.
Glycerophosphodiester phosphodiesterases (GDPD) hydrolyze glycerophosphodiesters, phospholipid metabolites generated by phospholipase A activity, thereby releasing glycerol-3-phosphate (G3P) and the amino alcohol. Up to now, little is known about the physiological significance of GDPD in plants. Arabidopsis contains six GDPD genes most of which are highly expressed under low phosphate (Pi) conditions. Transient expression of Arabidopsis GDPD5 and GDPD6 in N. benthamiana revealed activities towards different glycerophosphodiesters with preference for glycerophosphocholine and glycerophosphoethanolamine. Different Arabidopsis T-DNA insertion lines for GDPD5 and GDPD6 (gdpd5-1, gdpd5-2, gdpd6-1, gdpd6-2) were subjected to Pi starvation experiments. Under Pi limiting conditions no significant differences in lipid composition were observed in the different gdpd5 and gdpd6 insertion lines, but a reduction in G3P levels was detected in gdpd5-2, gdpd6-1, as well as gdpd6-2 leaves. After crossing the gdpd5 and gpdp6 mutant lines in all combinations, double homozygous plants were obtained except for gdpd5-1gdpd6-1. Analysis by semi-quantitative RT-PCR showed residual expression of sequences upstream and downstream from the insertion sites except for gdpd6-1. The pollen formation was affected in the double heterozygous gdpd5-1+/-gdpd6-1+/- line, with differences in shape and size of the aberrant pollen, which were deformed and distorted. Lipid analysis of these pollen grains showed alterations in lipid composition. This phenotype was also confirmed by the characterization of two independent GDPD5 RNAi gdpd6-1 lines, providing independent evidence for the crucial role of GDPD5 and GDPD6 in male gametophyte development. These results suggest that GDPD5 and GDPD6 are essential for pollen development in Arabidopsis.
Arabidopsis FatA1 and FatA2 were heterologously expressed in the E. coli fadD mutant. The fadD mutant lacks the acyl-CoA synthetase leading to an accumulation of free fatty acids, since these are no longer re-esterified to CoA. After cultivation, the fatty acids were extracted from the cell pellet and analyzed via GC-FID, exhibiting an evident increase in 16:1Δ9 and 18:1Δ11 fatty acids, demonstrating acyl-ACP thioesterase A activities for both enzymes.
To characterize FatA1 and FatA2 in Arabidopsis T-DNA insertion lines were obtained and analyzed by using GC-MS and Q-TOF MS. The loss of function fata1-2 and fata2-2 mutants did not reveal any alterations either in fatty acid or in glycerolipid composition. After crossing the single mutants no double homozygous fata1-2-/-fata2-2-/- mutant could be obtained. Plants heterozygous for the FatA1 and homozygous for the FatA2 locus showed approximately 25 % of the seeds to be aborted in an early stage of the development. Microtome sections of these aborted seeds revealed that the embryo development is arrested at the morphological late heart to early torpedo stage, leading to the assumption that the loss of both FatA genes might be embryolethal. Fatty acid and lipid composition of heart stage mutant embryos showed, that the development comes to a halt when lipid export from the chloroplast becomes significant.
Glycerophosphodiester phosphodiesterases (GDPD) hydrolyze glycerophosphodiesters, phospholipid metabolites generated by phospholipase A activity, thereby releasing glycerol-3-phosphate (G3P) and the amino alcohol. Up to now, little is known about the physiological significance of GDPD in plants. Arabidopsis contains six GDPD genes most of which are highly expressed under low phosphate (Pi) conditions. Transient expression of Arabidopsis GDPD5 and GDPD6 in N. benthamiana revealed activities towards different glycerophosphodiesters with preference for glycerophosphocholine and glycerophosphoethanolamine. Different Arabidopsis T-DNA insertion lines for GDPD5 and GDPD6 (gdpd5-1, gdpd5-2, gdpd6-1, gdpd6-2) were subjected to Pi starvation experiments. Under Pi limiting conditions no significant differences in lipid composition were observed in the different gdpd5 and gdpd6 insertion lines, but a reduction in G3P levels was detected in gdpd5-2, gdpd6-1, as well as gdpd6-2 leaves. After crossing the gdpd5 and gpdp6 mutant lines in all combinations, double homozygous plants were obtained except for gdpd5-1gdpd6-1. Analysis by semi-quantitative RT-PCR showed residual expression of sequences upstream and downstream from the insertion sites except for gdpd6-1. The pollen formation was affected in the double heterozygous gdpd5-1+/-gdpd6-1+/- line, with differences in shape and size of the aberrant pollen, which were deformed and distorted. Lipid analysis of these pollen grains showed alterations in lipid composition. This phenotype was also confirmed by the characterization of two independent GDPD5 RNAi gdpd6-1 lines, providing independent evidence for the crucial role of GDPD5 and GDPD6 in male gametophyte development. These results suggest that GDPD5 and GDPD6 are essential for pollen development in Arabidopsis.
Schlagwörter
glycerophosphodiester, phosphodiesterase, acyl-ACP thioesterase, Arabidopsis, gametophyte development, fatty acid modification, lipid, G3P
Klassifikation (DDC)
570 Biowissenschaften, BiologieSiebers, Meike: The Role of Acyl-ACP Thioesterases and Glycerophosphodiester Phosphodiesterases for Gametophyte Development in Arabidopsis thaliana. - Bonn, 2016. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44384
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44384
@phdthesis{handle:20.500.11811/6858,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44384,
author = {{Meike Siebers}},
title = {The Role of Acyl-ACP Thioesterases and Glycerophosphodiester Phosphodiesterases for Gametophyte Development in Arabidopsis thaliana},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = aug,
note = {Chloroplasts are the site of the de novo synthesis of the predominant proportion of fatty acids in plant cells. The growing fatty acid chain is bound to acyl carrier protein (ACP) during synthesis. Acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast releasing free fatty acids thereby regulating the chain lengths of fatty acids. The free fatty acids are activated by long chain fatty acid-CoA synthetases and exported from the plastid to serve as substrates for glycerolipid synthesis at the ER. In plants there are two types of acyl-ACP thioesterases that terminate the fatty acid synthesis, FatA and FatB. They differ in amino acid sequence and substrate specificity. FatA thioesterases are highly active towards oleoyl-ACPs (18:1-ACP), while FatB thioesterases show highest activity with saturated acyl-ACPs. Arabidopsis contains two genomic loci encoding FatA thioesterases, FatA1 and FatA2.
Arabidopsis FatA1 and FatA2 were heterologously expressed in the E. coli fadD mutant. The fadD mutant lacks the acyl-CoA synthetase leading to an accumulation of free fatty acids, since these are no longer re-esterified to CoA. After cultivation, the fatty acids were extracted from the cell pellet and analyzed via GC-FID, exhibiting an evident increase in 16:1Δ9 and 18:1Δ11 fatty acids, demonstrating acyl-ACP thioesterase A activities for both enzymes.
To characterize FatA1 and FatA2 in Arabidopsis T-DNA insertion lines were obtained and analyzed by using GC-MS and Q-TOF MS. The loss of function fata1-2 and fata2-2 mutants did not reveal any alterations either in fatty acid or in glycerolipid composition. After crossing the single mutants no double homozygous fata1-2-/-fata2-2-/- mutant could be obtained. Plants heterozygous for the FatA1 and homozygous for the FatA2 locus showed approximately 25 % of the seeds to be aborted in an early stage of the development. Microtome sections of these aborted seeds revealed that the embryo development is arrested at the morphological late heart to early torpedo stage, leading to the assumption that the loss of both FatA genes might be embryolethal. Fatty acid and lipid composition of heart stage mutant embryos showed, that the development comes to a halt when lipid export from the chloroplast becomes significant.
Glycerophosphodiester phosphodiesterases (GDPD) hydrolyze glycerophosphodiesters, phospholipid metabolites generated by phospholipase A activity, thereby releasing glycerol-3-phosphate (G3P) and the amino alcohol. Up to now, little is known about the physiological significance of GDPD in plants. Arabidopsis contains six GDPD genes most of which are highly expressed under low phosphate (Pi) conditions. Transient expression of Arabidopsis GDPD5 and GDPD6 in N. benthamiana revealed activities towards different glycerophosphodiesters with preference for glycerophosphocholine and glycerophosphoethanolamine. Different Arabidopsis T-DNA insertion lines for GDPD5 and GDPD6 (gdpd5-1, gdpd5-2, gdpd6-1, gdpd6-2) were subjected to Pi starvation experiments. Under Pi limiting conditions no significant differences in lipid composition were observed in the different gdpd5 and gdpd6 insertion lines, but a reduction in G3P levels was detected in gdpd5-2, gdpd6-1, as well as gdpd6-2 leaves. After crossing the gdpd5 and gpdp6 mutant lines in all combinations, double homozygous plants were obtained except for gdpd5-1gdpd6-1. Analysis by semi-quantitative RT-PCR showed residual expression of sequences upstream and downstream from the insertion sites except for gdpd6-1. The pollen formation was affected in the double heterozygous gdpd5-1+/-gdpd6-1+/- line, with differences in shape and size of the aberrant pollen, which were deformed and distorted. Lipid analysis of these pollen grains showed alterations in lipid composition. This phenotype was also confirmed by the characterization of two independent GDPD5 RNAi gdpd6-1 lines, providing independent evidence for the crucial role of GDPD5 and GDPD6 in male gametophyte development. These results suggest that GDPD5 and GDPD6 are essential for pollen development in Arabidopsis.},
url = {https://hdl.handle.net/20.500.11811/6858}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44384,
author = {{Meike Siebers}},
title = {The Role of Acyl-ACP Thioesterases and Glycerophosphodiester Phosphodiesterases for Gametophyte Development in Arabidopsis thaliana},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = aug,
note = {Chloroplasts are the site of the de novo synthesis of the predominant proportion of fatty acids in plant cells. The growing fatty acid chain is bound to acyl carrier protein (ACP) during synthesis. Acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast releasing free fatty acids thereby regulating the chain lengths of fatty acids. The free fatty acids are activated by long chain fatty acid-CoA synthetases and exported from the plastid to serve as substrates for glycerolipid synthesis at the ER. In plants there are two types of acyl-ACP thioesterases that terminate the fatty acid synthesis, FatA and FatB. They differ in amino acid sequence and substrate specificity. FatA thioesterases are highly active towards oleoyl-ACPs (18:1-ACP), while FatB thioesterases show highest activity with saturated acyl-ACPs. Arabidopsis contains two genomic loci encoding FatA thioesterases, FatA1 and FatA2.
Arabidopsis FatA1 and FatA2 were heterologously expressed in the E. coli fadD mutant. The fadD mutant lacks the acyl-CoA synthetase leading to an accumulation of free fatty acids, since these are no longer re-esterified to CoA. After cultivation, the fatty acids were extracted from the cell pellet and analyzed via GC-FID, exhibiting an evident increase in 16:1Δ9 and 18:1Δ11 fatty acids, demonstrating acyl-ACP thioesterase A activities for both enzymes.
To characterize FatA1 and FatA2 in Arabidopsis T-DNA insertion lines were obtained and analyzed by using GC-MS and Q-TOF MS. The loss of function fata1-2 and fata2-2 mutants did not reveal any alterations either in fatty acid or in glycerolipid composition. After crossing the single mutants no double homozygous fata1-2-/-fata2-2-/- mutant could be obtained. Plants heterozygous for the FatA1 and homozygous for the FatA2 locus showed approximately 25 % of the seeds to be aborted in an early stage of the development. Microtome sections of these aborted seeds revealed that the embryo development is arrested at the morphological late heart to early torpedo stage, leading to the assumption that the loss of both FatA genes might be embryolethal. Fatty acid and lipid composition of heart stage mutant embryos showed, that the development comes to a halt when lipid export from the chloroplast becomes significant.
Glycerophosphodiester phosphodiesterases (GDPD) hydrolyze glycerophosphodiesters, phospholipid metabolites generated by phospholipase A activity, thereby releasing glycerol-3-phosphate (G3P) and the amino alcohol. Up to now, little is known about the physiological significance of GDPD in plants. Arabidopsis contains six GDPD genes most of which are highly expressed under low phosphate (Pi) conditions. Transient expression of Arabidopsis GDPD5 and GDPD6 in N. benthamiana revealed activities towards different glycerophosphodiesters with preference for glycerophosphocholine and glycerophosphoethanolamine. Different Arabidopsis T-DNA insertion lines for GDPD5 and GDPD6 (gdpd5-1, gdpd5-2, gdpd6-1, gdpd6-2) were subjected to Pi starvation experiments. Under Pi limiting conditions no significant differences in lipid composition were observed in the different gdpd5 and gdpd6 insertion lines, but a reduction in G3P levels was detected in gdpd5-2, gdpd6-1, as well as gdpd6-2 leaves. After crossing the gdpd5 and gpdp6 mutant lines in all combinations, double homozygous plants were obtained except for gdpd5-1gdpd6-1. Analysis by semi-quantitative RT-PCR showed residual expression of sequences upstream and downstream from the insertion sites except for gdpd6-1. The pollen formation was affected in the double heterozygous gdpd5-1+/-gdpd6-1+/- line, with differences in shape and size of the aberrant pollen, which were deformed and distorted. Lipid analysis of these pollen grains showed alterations in lipid composition. This phenotype was also confirmed by the characterization of two independent GDPD5 RNAi gdpd6-1 lines, providing independent evidence for the crucial role of GDPD5 and GDPD6 in male gametophyte development. These results suggest that GDPD5 and GDPD6 are essential for pollen development in Arabidopsis.},
url = {https://hdl.handle.net/20.500.11811/6858}
}
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