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The plastid outer envelope protein OEP16 affects metabolic fluxes during ABA-controlled seed development and germination.

Pudelski B, Schock A, Hoth S, Radchuk R, Weber H, Hofmann J, Sonnewald U, Soll J, Philippar K - J. Exp. Bot. (2011)

Bottom Line: Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids.In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination.It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.

View Article: PubMed Central - PubMed

Affiliation: Department Biologie I, Botanik, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

ABSTRACT
Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids. Isolation of OEP16.2, a second OEP16 isoform from pea, in the current study allowed membrane localization and gene expression of OEP16 to be followed throughout seed development and germination of Arabidopsis thaliana and P. sativum. Thereby it can be shown on the transcript and protein level that the isoforms OEP16.1 and OEP16.2 in both plant species are alternating: whereas OEP16.1 is prominent in early embryo development and first leaves of the growing plantlet, OEP16.2 dominates in late seed development stages, which are associated with dormancy and desiccation, as well as early germination events. Further, OEP16.2 expression in seeds is under control of the phytohormone abscisic acid (ABA), leading to an ABA-hypersensitive phenotype of germinating oep16 knockout mutants. In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination. It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.

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Specific ABA induction of OEP16.2 expression in seeds. (A) Digital northern blot of At-OEP16.2 (black), At-OEP16.1 (grey), and At-OEP16.4 (white) expression (arbitrary units) in Arabidopsis endosperm and embryo tissue. Prior to dissection, seeds were germinated for 24 h without (0) and with 20 μM ABA (described in Penfield et al., 2004). Data used to create the expression profile were obtained from the analysis by Penfield et al. (2004) using NASCArrays database (http://affy.arabidopsis.info/narrays/experimentbrowse.pl), experiment NASCARRAYS 386. Signal intensities were averaged from three biological replicates (n=3, ±SD). (B) Western blot analysis of At-OEP16.2 in protein extracts (7.5 μg each) from Arabidopsis seeds, germinated for 48 h on medium containing 0, 2.5, and 20 μM ABA. (C) Transcript content of Ps-OEP16.2 (n=2, ±SD, arbitrary units) in developing pea seeds of the wild type (black) and the Vf-SnRK1-antisense line snf34 (white). The age of seeds is given in days after pollination (dap). According to the definition by Radchuk et al. (2006), delayed down-regulated genes are highly expressed in the pre-storage phase 13–15 dap (light grey area), and delayed up-regulated genes are continuously increased during seed maturation, starting in the transition phase at 19–22 dap (dark grey area). (D) Immunoblot analysis of Ps-OEP16.2 in protein extracts (4 μg each) from wild-type and VfSnRK1-antisense (snf34) pea seeds, isolated 20 dap. (B and D) Antiserum against the marker protein VDAC (outer membrane of mitochondria) was used as a loading control. Numbers indicate the molecular mass of proteins in kDa.
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fig5: Specific ABA induction of OEP16.2 expression in seeds. (A) Digital northern blot of At-OEP16.2 (black), At-OEP16.1 (grey), and At-OEP16.4 (white) expression (arbitrary units) in Arabidopsis endosperm and embryo tissue. Prior to dissection, seeds were germinated for 24 h without (0) and with 20 μM ABA (described in Penfield et al., 2004). Data used to create the expression profile were obtained from the analysis by Penfield et al. (2004) using NASCArrays database (http://affy.arabidopsis.info/narrays/experimentbrowse.pl), experiment NASCARRAYS 386. Signal intensities were averaged from three biological replicates (n=3, ±SD). (B) Western blot analysis of At-OEP16.2 in protein extracts (7.5 μg each) from Arabidopsis seeds, germinated for 48 h on medium containing 0, 2.5, and 20 μM ABA. (C) Transcript content of Ps-OEP16.2 (n=2, ±SD, arbitrary units) in developing pea seeds of the wild type (black) and the Vf-SnRK1-antisense line snf34 (white). The age of seeds is given in days after pollination (dap). According to the definition by Radchuk et al. (2006), delayed down-regulated genes are highly expressed in the pre-storage phase 13–15 dap (light grey area), and delayed up-regulated genes are continuously increased during seed maturation, starting in the transition phase at 19–22 dap (dark grey area). (D) Immunoblot analysis of Ps-OEP16.2 in protein extracts (4 μg each) from wild-type and VfSnRK1-antisense (snf34) pea seeds, isolated 20 dap. (B and D) Antiserum against the marker protein VDAC (outer membrane of mitochondria) was used as a loading control. Numbers indicate the molecular mass of proteins in kDa.

Mentions: Since OEP16.2 expression specifically correlates with seed desiccation and dormancy processes controlled by the phytohormone ABA, OEP16 transcript and protein content was monitored in germinating Arabidopsis seeds that were treated with ABA. In the analysis by Penfield et al. (2004), At-OEP16.2 transcripts were specifically and strongly increased upon germination for 24 h in the presence of ABA (Fig. 5A). In endosperm tissue, the induction was ∼5-fold, and in embryos even 9-fold. In contrast, At-OEP16.1 and At-OEP16.4 were not regulated in endosperm but responded slightly to ABA in embryos, showing ∼2-fold down-regulation (OEP16.1) and ∼2-fold up-regulation (OEP16.4). When testing for protein levels, it was found that after 48 h of ABA treatment during germination, OEP16.2 protein content also substantially increased (Fig. 5B).


The plastid outer envelope protein OEP16 affects metabolic fluxes during ABA-controlled seed development and germination.

Pudelski B, Schock A, Hoth S, Radchuk R, Weber H, Hofmann J, Sonnewald U, Soll J, Philippar K - J. Exp. Bot. (2011)

Specific ABA induction of OEP16.2 expression in seeds. (A) Digital northern blot of At-OEP16.2 (black), At-OEP16.1 (grey), and At-OEP16.4 (white) expression (arbitrary units) in Arabidopsis endosperm and embryo tissue. Prior to dissection, seeds were germinated for 24 h without (0) and with 20 μM ABA (described in Penfield et al., 2004). Data used to create the expression profile were obtained from the analysis by Penfield et al. (2004) using NASCArrays database (http://affy.arabidopsis.info/narrays/experimentbrowse.pl), experiment NASCARRAYS 386. Signal intensities were averaged from three biological replicates (n=3, ±SD). (B) Western blot analysis of At-OEP16.2 in protein extracts (7.5 μg each) from Arabidopsis seeds, germinated for 48 h on medium containing 0, 2.5, and 20 μM ABA. (C) Transcript content of Ps-OEP16.2 (n=2, ±SD, arbitrary units) in developing pea seeds of the wild type (black) and the Vf-SnRK1-antisense line snf34 (white). The age of seeds is given in days after pollination (dap). According to the definition by Radchuk et al. (2006), delayed down-regulated genes are highly expressed in the pre-storage phase 13–15 dap (light grey area), and delayed up-regulated genes are continuously increased during seed maturation, starting in the transition phase at 19–22 dap (dark grey area). (D) Immunoblot analysis of Ps-OEP16.2 in protein extracts (4 μg each) from wild-type and VfSnRK1-antisense (snf34) pea seeds, isolated 20 dap. (B and D) Antiserum against the marker protein VDAC (outer membrane of mitochondria) was used as a loading control. Numbers indicate the molecular mass of proteins in kDa.
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Related In: Results  -  Collection

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fig5: Specific ABA induction of OEP16.2 expression in seeds. (A) Digital northern blot of At-OEP16.2 (black), At-OEP16.1 (grey), and At-OEP16.4 (white) expression (arbitrary units) in Arabidopsis endosperm and embryo tissue. Prior to dissection, seeds were germinated for 24 h without (0) and with 20 μM ABA (described in Penfield et al., 2004). Data used to create the expression profile were obtained from the analysis by Penfield et al. (2004) using NASCArrays database (http://affy.arabidopsis.info/narrays/experimentbrowse.pl), experiment NASCARRAYS 386. Signal intensities were averaged from three biological replicates (n=3, ±SD). (B) Western blot analysis of At-OEP16.2 in protein extracts (7.5 μg each) from Arabidopsis seeds, germinated for 48 h on medium containing 0, 2.5, and 20 μM ABA. (C) Transcript content of Ps-OEP16.2 (n=2, ±SD, arbitrary units) in developing pea seeds of the wild type (black) and the Vf-SnRK1-antisense line snf34 (white). The age of seeds is given in days after pollination (dap). According to the definition by Radchuk et al. (2006), delayed down-regulated genes are highly expressed in the pre-storage phase 13–15 dap (light grey area), and delayed up-regulated genes are continuously increased during seed maturation, starting in the transition phase at 19–22 dap (dark grey area). (D) Immunoblot analysis of Ps-OEP16.2 in protein extracts (4 μg each) from wild-type and VfSnRK1-antisense (snf34) pea seeds, isolated 20 dap. (B and D) Antiserum against the marker protein VDAC (outer membrane of mitochondria) was used as a loading control. Numbers indicate the molecular mass of proteins in kDa.
Mentions: Since OEP16.2 expression specifically correlates with seed desiccation and dormancy processes controlled by the phytohormone ABA, OEP16 transcript and protein content was monitored in germinating Arabidopsis seeds that were treated with ABA. In the analysis by Penfield et al. (2004), At-OEP16.2 transcripts were specifically and strongly increased upon germination for 24 h in the presence of ABA (Fig. 5A). In endosperm tissue, the induction was ∼5-fold, and in embryos even 9-fold. In contrast, At-OEP16.1 and At-OEP16.4 were not regulated in endosperm but responded slightly to ABA in embryos, showing ∼2-fold down-regulation (OEP16.1) and ∼2-fold up-regulation (OEP16.4). When testing for protein levels, it was found that after 48 h of ABA treatment during germination, OEP16.2 protein content also substantially increased (Fig. 5B).

Bottom Line: Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids.In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination.It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.

View Article: PubMed Central - PubMed

Affiliation: Department Biologie I, Botanik, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

ABSTRACT
Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids. Isolation of OEP16.2, a second OEP16 isoform from pea, in the current study allowed membrane localization and gene expression of OEP16 to be followed throughout seed development and germination of Arabidopsis thaliana and P. sativum. Thereby it can be shown on the transcript and protein level that the isoforms OEP16.1 and OEP16.2 in both plant species are alternating: whereas OEP16.1 is prominent in early embryo development and first leaves of the growing plantlet, OEP16.2 dominates in late seed development stages, which are associated with dormancy and desiccation, as well as early germination events. Further, OEP16.2 expression in seeds is under control of the phytohormone abscisic acid (ABA), leading to an ABA-hypersensitive phenotype of germinating oep16 knockout mutants. In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination. It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.

Show MeSH
Related in: MedlinePlus