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Physiological function of a phosphatase dually targeted to the outer membranes of chloroplasts and mitochondria Conclusions Experimental Results 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]. 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 co- localized 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]. 1. AtPAP2 protein is targeted to both mitochondria and chloroplasts. 2. Targeting of AtPAP2 protein to both mitochondria and chloroplasts is essential for its plant growth promoting phenotypes 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]. 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 mitochondria- and 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 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. Research Outputs 7. Overexpression of AtPAP2 modulates thylakoid and photosynthesis A B 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). 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]. 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]. WT Transgenic lines WT Transgenic lines Boon Leong Lim Introduction 8 hrs light/day 16 hrs light/day 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 free- living 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]. 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]. Overexpression of AtPAP2 in Arabidopsis thaliana 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]. 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]. Objectives: To delineate the biological functions of AtPAP2 in chloroplasts and mitochondria 4. AtPAP2 protein interacts with many photosystem proteins. 5. AtPAP2 protein interacts with some mitochondrial proteins.
