Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation Chisato Masumotoa,1, Shin-Ichi Miyazawaa,1, Hiroshi Ohkawaa,2, Takuya Fukudaa, Yojiro Taniguchia,3, Seiji Murayamaa,4, Miyako Kusanob, Kazuki Saitob, Hiroshi Fukayamaa,5, and Mitsue Miyaoa,6 aPhotobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, Japan; and bRIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan Edited by Elisabeth Gantt, University of Maryland, College Park, MD, and approved February 4, 2010 (received for review November 12, 2009) Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary encoded by a small gene family (5). Treatments that lead to ele- metabolism in bacteria, algae, and vascular plants, and is believed to vation of the PEPC activity, such as enhanced nitrogen assimilation Oryza sativa be cytosolic. Here we show that rice ( L.) has a plant-type and Pi starvation in C3 plant leaves (6, 7), induce expression of PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was ex- PEPC kinase gene(s) and phosphorylation of PEPC (8, 9). These pressed in all organs tested and showed high expression in the leaves. mechanisms strictly regulate PEPC activity, which is essential for Its expression in the leaves was confined to mesophyll cells, and plants because of the irreversible nature of the reaction. Osppc4 accounted for approximately one-third of total PEPC protein In addition to the plant-type PEPC, vascular plants have in theleaf blade.RecombinantOsppc4 wasactivein thePEPCreaction, another isozyme, a bacterial-type PEPC that lacks the conserved V showing max comparable to cytosolic isozymes. Knockdown of phosphorylatable Ser residue (10). Whereas most PEPCs exist as Osppc4 expression by the RNAi technique resulted in stunting at homotetramers (class 1 PEPC) (1), bacterial-type PEPCs associate the vegetative stage, which was much more marked when rice plants with plant-type isozymes to form a heteromeric complex (class 2 were grown with ammonium than with nitrate as the nitrogen PEPC) in unicellular green algae (11) and castor oilseeds (12). It source. Comparison ofleaf metabolomesofammonium-grown plants has been recently shown that Class-2 PEPC is composed of four PLANT BIOLOGY suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic each of the bacterial- and plant-type isozymes (13). acids, which are carbon skeleton donors for these processes. We also Although PEPC genes and proteins have been extensively identified the chloroplastic PEPC gene in other Oryza species, all of studied in a variety of organisms, the enzymes reported so far are which are adapted to waterlogged soil where the major nitrogen all cytosolic (2). Here we report a previously undescribed plant- source is ammonium. This suggests that, in addition to glycolysis, type PEPC targeted to the chloroplast of rice. It is also unique in its the genus Oryza has a unique route to provide organic acids for primary structure, not belonging to any of known groups of the ammonium assimilation that involves a chloroplastic PEPC, and that plant-type PEPCs. This chloroplastic isozyme is one of the major this route is crucial for growth with ammonium. This work provides PEPCs in leaf mesophyll cells and plays a crucial role in ammo- evidence for diversity of primary ammonium assimilation in the nium assimilation in rice plants. leaves of vascular plants. Results amino acid synthesis | glycolysis | nitrogen assimilation | organic acid Rice Gene Encoding PEPC Targeted to Chloroplast. Searches of rice synthesis | Oryza genome databases identified six putative PEPC (Osppc) genes (Fig. S1). Five (Osppc1, 2a, 2b, 3, and 4) encode the plant-type hosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) cata- PEPC that has the conserved phosphorylation domain and the C- Plyzes irreversible β-carboxylation of phosphoenolpyruvate terminal tetrapeptide QNTG (1, 3, 14), and the other (Osppc-b) − 2+ (PEP) in the presence of HCO3 and Mg to yield oxaloacetate encodes a bacterial-type enzyme with the C-terminal tetrapeptide (OAA) and inorganic phosphate (Pi). PEPC is widely distributed RNTG (15). As reported for Arabidopsis (10), all of the plant-type in all photosynthetic organisms including vascular plants, algae, Osppc genes have similar exon-intron structures that basically cyanobacteria, and photosynthetic bacteria, and also in non- contain 10 exons, whereas the bacterial type has a distinct structure photosynthetic bacteria and protozoa (1). In vascular plants, the (Fig. 1A), an indication of a different evolutionary lineage. The six reaction catalyzed by PEPC is the primary fixation step of photo- synthetic CO2 assimilation in C4 photosynthesis and crassulacean acid metabolism (CAM). In most nonphotosynthetic tissues and in Author contributions: C.M., S.-I.M., H.O., H.F., and M.M. designed research; C.M., S.-I.M., the leaves of C3 plants, the primary function of PEPC is anapler- H.O., T.F., Y.T., S.M., M.K., K.S., H.F., and M.M. performed research; C.M., S.-I.M., M.K., otic, replenishing the tricarboxylic acid (TCA) cycle with inter- H.F., and M.M. analyzed data; and C.M., S.-I.M., M.K., H.F., and M.M. wrote the paper. mediates that are withdrawn for a variety of biosynthetic pathways The authors declare no conflict of interest. and nitrogen assimilation (1, 2). PEPC also plays roles in the This article is a PNAS Direct Submission. provision of malate in guard cells and legume root nodules (3). Freely available online through the PNAS open access option. Consistent with this wide range of roles, PEPC is encoded by a 1C.M. and S-I.M. contributed equally to this work. small gene family and expressed in various tissues of plants (4). 2Present address: Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036- Most PEPCs are allosteric enzymes the activity of which is regu- 8561, Japan. lated by a variety of metabolic effectors (1). The activity of vascular 3Present address: National Institute of Crop Science, Tsukuba 305-8517, Japan. plant PEPC is further regulated through reversible phosphorylation 4Present address: National Agricultural Research Center for Hokkaido Region, Sapporo of a conserved Ser residue near the N terminus (3). Upon phos- 062-8555, Japan. phorylation, PEPC becomes less sensitive to its feedback inhibitor 5Present address: Graduate School of Agriculture, Kobe University, Kobe 657-8501, Japan. malate and more sensitive to the activator glucose 6-phosphate 6To whom correspondence should be addressed. E-mail: [email protected]. (Glu6P), thereby attaining higher activity (3). Phosphorylation of This article contains supporting information online at www.pnas.org/cgi/content/full/ PEPC is catalyzed by a dedicated PEPC kinase, which is also 0913127107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0913127107 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 A Osppc1 Osppc2a 3.7 kb Osppc2b 1 kb Osppc3 Osppc4 Osppc-b B LB LS Rt Sp C Ro to type Foxtail Osppc4 Osppc1 25 millet Maize Osppc1 Osppc2a 25 root 100 1.0 M zia e C3 Osppc2b Whe ta 2255 74 100 77 Sugarcane Osppc2b 96 100 100 C Osppc3 30 100 100 4 Osppc2a 100 95 Sorghum Osppc4 25 C4 Sugarcane Maize C4 Osppc-b C 30 3 Osppc3 C4 type C type OsRAc1 3 Foxt ia l Rhodes 25 mill te C4 grass Fig. 1. Comparison of rice PEPC (Osppc) genes. (A) Exon–intron structures. The coding and noncoding regions are represented by white/colored and black boxes, respectively. Thin lines show splitting/conjunction of exons among plant-type genes. (B) Expression in rice organs. Total RNA was subjected to RT-PCR. Expression of the rice actin gene (OsRAc1) was examined as a control. The numbers of PCR cycles are indicated on the right. LB, leaf blade; LS, leaf sheath; Rt, root; Sp, spikelet. (C) Phylogenetic relationships of plant-type PEPCs from Gramineae. Presence or absence of the N-terminal extension of Osppc4 does not affect the result. Bootstrap values are shown at nodes. Details in SI Text. putative PEPC genes are all expressed in rice plants, but with Osppc4, a Major PEPC in Leaf Mesophyll Cells. RT-PCR analyses (Fig. different organ specificities (Fig. 1B). 1B) showed that Osppc4 was expressed in all organs tested, with the The deduced amino acid sequences of the plant-type genes are highest expression in the leaf blade. The cellular specificity of highly homologous to one another (Fig. S1), sharing amino acid Osppc4 expression was examined by histochemical staining of identities above 73%. One apparent difference among them is a β-glucuronidase (GUS) activity in transgenic rice introduced with deletion of 42 amino acid residues in the N-terminal region of the Osppc4::GUS chimeric gene (Fig. 2C). In the leaf blade and the Osppc3. Another notable feature is an extension of about 40 leaf sheath, Osppc4 expression was confined to green parenchymal amino acid residues at the N terminus of Osppc4. The plant-type cells (mesophyll cells), and no expression was detected in epidermis, fi PEPCs studied so far are classi ed into three groups, the C3,C4, vascular bundles, or guard cells. We also detected expression in and root types (2). Phylogenetic analysis of plant-type PEPCs from green cross cells of the ovary, although the expression was low. No Gramineae (Fig. 1C) indicates that Osppc2a, 2b, and 3 belong to the C3 type and Osppc1 to the root type, whereas Osppc4 does not belong to any of known types. To examine whether the N-terminal extension of Osppc4 acts as A B Chloroplasts a transit peptide, we expressed a protein fusion between the N- kDa TMS fl 115 terminal portion of Osppc4 and green uorescent protein (GFP) 111 in dayflower epidermal cells.