Physiological function of a phosphatase dually targeted to the outer
membranes of chloroplasts and mitochondria
Boon Leong Lim
Introduction
In higher plants, purple acid phosphatases (PAPs) mainly
play roles in phosphorus metabolism. Our group has
discovered the first PAP (AtPAP2) that plays roles in carbon
metabolism [1]. We showed that AtPAP2 is targeted to the
outer membrane of both chloroplasts and mitochondria
by an additional transmembrane motif at its C-terminus
[2]. PAPs with a transmembrane motif at their C-termini
are conserved in green plants, including the smallest freeliving photosynthetic eukaryote, Ostreococcus tauri.
Transgenic Arabidopsis thaliana overexpressing AtPAP2
grew faster (Fig. 1), produced more seeds (37-57%) and
contained higher leaf sucrose content (up to 30%) and ATP
[1, 3 - 5]. Transgenic Camelina sativa overexpressing
AtPAP2 also grew faster and produced more seeds (Fig. 2)
[6] and transgenic potato overexpressing AtPAP2 also
grew faster and produced more tubers (Fig. 3) [7].
Overexpression of AtPAP2 in Arabidopsis thaliana
8 hrs light/day
16 hrs light/day
Fig. 1 Growth phenotypes of AtPAP2 overexpression lines (OE7 and OE21)
and T-DNA Arabidopsis lines (KO). (a) Six-wk-old plants grown under short
day (8 hours light/day) conditions. (b) 29-day-old plants grown under long
day (16 hours light/day) conditions [1].
Fig. 2 Growth phenotype of transgenic Camelina sativa at day 40.
All three OE lines produced more branches, grew faster, and
flowered earlier than the WT and null-lines. Western blotting
showed that the OE lines had higher expression of AtPAP2, while
there was a homologous protein in Camelina sativa [6].
Fig. 3 Growth phenotypes of AtPAP2 overexpression potato lines
(Experimental lines) and untransformed control potato (Control). (a)
Six-wk-old plants grown in greenhouse condition (b) Tubers were
collected after the decay of the shoots following a 4-month growth
period [7].
Objectives: To delineate the biological functions of AtPAP2 in chloroplasts and mitochondria
Experimental Results
5. AtPAP2 protein interacts with some mitochondrial proteins.
4. AtPAP2 protein interacts with many photosystem proteins.
1. AtPAP2 protein is targeted to both mitochondria and chloroplasts.
Fig. 4 Targeting of the GFP fusion proteins in Arabidopsis PSB-D protoplasts. Transient
expression in Arabidopsis PSB-D protoplasts showed that GFP fused with putative SP
region of AtPAP2 (SP-GFP) was directed to cytosol, whereas GFP constructs fused with
the TMD/CT region of AtPAP2, including SP-GFP-TMD/CT and GFP-TMD/CT, were colocalized with the mitochondrial (F1-RFP) and chloroplastic (plastid-mCherry) markers.
Bar = 50μm. The presence of AtPAP2 protein in both mitochondria and chloroplasts were
confirmed by Western blotting [1].
Fig. 7. AtPAP2 selectively interact with proteins at the acceptor side of PSI in Yeast-two
hybrid (A) and BIFC (B) assays. Interaction of AtPAP2 with pSSU is confirmed (C).
Fig. 8. AtPAP2 selectively interact with mitochondriaand chloroplast-targeted pMORF proteins in Yeast-two
hybrid (A) and BIFC (B) assays [8].
6. AtPAP2 plays a role in protein import into chloroplasts and mitochondria
2. Targeting of AtPAP2 protein to both mitochondria and
chloroplasts is essential for its plant growth promoting phenotypes
7. Overexpression of AtPAP2 modulates thylakoid and photosynthesis
A
B
Fig. 9. (A) The import rate of pSSU into pap2 chloroplasts is significantly lower than
that of the WT and OE7 chloroplasts (*P ≤ 0.05). (B) The import rate of pMORF3 into
pap2 mitochondria is significantly lower than that of the WT and OE7 mitochondria
(*P ≤ 0.05, **P ≤ 0.01 [8].
Conclusions
Fig. 5 (A) Phenotype of forty-day-old plants (n=12) of WT, P2NS, OE7, P2NC and
AtPAP2 T-DNA plants. (B) AtPAP2 protein band identification in WT, P2NS, OE7,
P2NC and AtPAP2 T-DNA plants by Western blot analysis [1].
3. AtPAP2 protein is located on the outer membranes of both
mitochondria and chloroplasts
Fig. 6. AtPAP2 protein import experiments. (A) Mitochondrial import experiments
showed that only AOX was imported into mitochondria but AtPAP2 was not imported.
(B) Chloroplast import experiments showed that only SSU was imported into
mitochondria but AtPAP2 was not imported [2].
Many nuclear-encoded proteins are imported into organelles and their
transit peptides/presequences are phosphorylated by STY kinases before
import. AtPAP2 selectively interacts with some proteins and plays a role in
their import into organelles. By selective import of certain proteins (e.g.
proteins at electron acceptor side of PSI, MORF proteins, etc), AtPAP2
modulates the activities of chloroplasts and mitochondria. Overexpression
of AtPAP2 in both organelles results in fast growth, high ATP, sugars,
biomass and seed yield, but when AtPAP2 is only overexpressed in
mitochondria, the transgenic plants exhibited early senescence, high ATP,
low sugars and low seed yield [1, 3-5, 8].
Fig. 10. (A) OE lines have thinner thylakoid stacks than WT. Values (n>30) marked by
different letters are significantly different (P < 0.05). (B) The OE line exhibited higher
ETR, NPQ and PSII(f). Asterisks indicate significant difference between WT and OE (P
< 0.05).
Research Outputs
1.
F. Sun, P. K. Suen, Y. Zhang, C. Liang, C Carrie, J Whelan, J. Ward, N.D. Hawkins, L Jiang and B. L.
Lim (2012) A dual-targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon
metabolism and its overexpression leads to faster plant growth and higher seed yield. New Phytologist
194: 206–219.
2.
F. Sun., C. Carrie, S. Law, M. Murcha, R. Zhang, Y. Law, P.K. Suen, J. Whelan, and B. L. Lim (2012)
AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria. Plant
Signaling & Behavior 7:927-932.
3.
F. Sun, C. Liang, J. Whelan, J. Yang, P. Zhang and B. L. Lim (2013) Global transcriptome analysis of
AtPAP2 - overexpressing Arabidopsis thaliana with elevated ATP. BMC Genomics 14:752-763.
4.
C. Liang, X. Liu, Y. Sun, S. Yiu, B. L. Lim (2014) Global small RNA analysis in fast-growing
Arabidopsis thaliana with elevated concentrations of ATP and sugars. BMC Genomics 15:116-128.
5.
C. Liang, Y. Zhang, S. Cheng, S. Osorio, Y. Sun, A. Fernie, C.Y.M. Cheung, B. L. Lim (2015) Impacts of
high ATP supply from chloroplasts and mitochondria on the leaf metabolism of Arabidopsis thaliana.
Frontiers in Plant Science 6:922.
6.
Y. Zhang., L.Yu, K. Yung, Y. Leung, F. Sun and B. L. Lim (2012). Over-expression of AtPAP2 in
Camelina sativa leads to faster plant growth and higher seed yield. Biotechnology for Biofuels 5:19-28.
7.
Y. Zhang, F. Sun, J. Fettke, M. A. Schöttler, L. Ramsden, A. R. Fernie, B. L. Lim (2014) Heterologous
expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development. FEBS
Letters 588(20):3726-3731.
8.
Y. Law, R. Zhang, X. Guan, S. Cheng, F. Sun, O. Duncan, M. W. Murcha, J. Whelan, B. L. Lim (2015)
Phosphorylation and dephosphorylation of the presequence of pMORF3 during import into
mitochondria from Arabidopsis thaliana. Plant Physiology 169:1344-55.
WT
Transgenic lines
WT
Transgenic lines