Pyruvate orthophosphate dikinase (PPDK) was found in varis immature seeds of C3 planRts (wheat, pea, green bean, plm, ad castor bean) in some C3 eaves (tobacco, spinach, sunflower, an wheat and in C (maize) kernels. The Protein Blot. PPDK was isolated from maize and purified as previously described (29). Antiserum to PPDK was prepared by injecting New eland white rabbits with 150 pg of the purified enzyme and Freund's complete adjuvant, followed after 26 d by a booster containing the same amount of enzyme and Freund's incomplete adjuvant and a second such booster at 40 d. The rabbits were bled at 41 d. The crude antiserum was used for the protein blot. Nonimmunized serum showed no cross-reaction with PPDK, and PPDK antiserum cross-reacted only with PPDK (1). This antiserum was used as a probe for the presence ofPPDK in.the various tissues studied.Each plant tissue studied (3 g) was ground in 6 ml of 0.1 M Tris buffer, pH 7.4, containing 10 mM MgC2, 18% w/v sucrose, and 1% P-mercaptoethanol. The homogenate was filtered and then centrifuged at 15,000g and OC for 20 min. Total soluble protein was determined by the method of Bradford (5). Crossreacting protein subunits similar in size to that of maize PPDK were detected by the protein blot method (6, 21).SDS-PAGE of each plant extract was carried out in a gradient gel (6.4 to 12.8%). Protein was transferred to cyanogen bromide paper prepared by the method of Clarke et al. (6). The tansfer paper was probed with anti-PPDK serum and then with J125 protein A. An autoradiograph was prepared using Kodak AR5 x-ray film with an intensifying screen at -70°C overnight.Densitometry. Relative levels of PPDK in preparations from different plant samples were estimated by densitometry of the xray film by comparison of the peak areas.

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.

Mesophyll protoplasts and bundle sheath strands were isolated from maize leaves. Light microscopic observation showed the preparations were pure and without cross contamination. Protein blot analysis of mesophyll and bundle sheath cell soluble protein showed that the concentration of pyruvate orthophosphate dikinase (EC 2.7.9 The C4 pathway is characterized by the initial assimilation of CO2 and C4 acids in mesophyll cells and subsequent decarboxylation and reduction of the carbon by the RPP2 pathway in the bundle sheath cells (4, 7). Previous studies have provided evidence for the differential localization ofcertain enzymes involved in the C4 pathway (7). For example, PEPC, PPDK, and NADPdependent MDH are predominantly present in mesophyll cells and RuBPC and NADP-dependent ME are predominantly present in bundle sheath cells.

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