Plant Molecular Biology 3:431 444 (1984). © Martinus N!ihqlf/Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands'. Molecular biology of C 4 photosynthesis in Zea mays: differential localization of proteins and mRNAs in the two leaf cell types Richard Broglie, Gloria Coruzzi, Brian Keith, & Nam-Hai Chua Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, U.S.A. Summary We have investigated the molecular basis of differential localization of enzyme activities in mesophyll (M) and bundle-sheath (B) cells of maize leaves. M protoplasts and B strands were prepared by enzymatic digestions and mechanical treatment of secondary leaves. Soluble and thylakoid membrane proteins from the two cell types were compared by one- and two-dimensional gel electrophoresis and quantitative rocket immunoelectrophoresis. In addition, several thylakoid polypeptides were identified by crossed immunoelec- trophoresis using monospecific antibodies. M and B thylakoids show quantitative and qualitative differences in their polypeptide compositions. While the M thylakoids contain the normal complement of polypeptides, the B thylakoids are deficient in ferredoxin-NADP+ reductase, photosystem II reaction center polypeptides, and the light-harvesting chlorophyll a/b-protein complex. Comparison of the soluble proteins by two-di- mensional gel electrophoresis revealed marked differences between M and B cells. The major proteins of one cell type are clearly absent from the other. These differences are paralleled by differences in the in vitro translation products of poly A + RNA isolated from the two cell types. Immunoprecipitation experiments showed that mRNA encoding the small subunit of ribulose-l,5-bisphosphate carboxylase (rbcS) is localized exclusively in B cells, whereas mRNA encoding phosphoenolpyruvate carboxylase is detected only in M cells. cDNA clones encoding the carboxylase rbcS and the chlorophyll a/b binding protein were used as probes in Northern blot analysis. M ceils contain no detectable RNA encoding rbcS but have a higher steady state level of RNA encoding the chlorophyll a/b-binding polypeptide compared to B cells. Taken together, our results demonstrate that differential gene expression in the two leaf cell types is regulated at the level of translatable mRNA, and, for at least two proteins, at the level of steady-state RNA. Introduction Higher plants may be divided into two groups, C3 and C4, depending on their photosynthetic carbon assimilation pathway. The primary carbon fixation reaction in C3 plants is catalyzed by ribulose 1,5- bisposphate carboxylase/oxygenase (RUBISCO), while that in Ca plants is catalyzed by phospho- enolpyruvate carboxylase (PEPC). C4 plants in- clude such economically important crops such as maize, sorghum and sugarcane. A characteristic feature of Ca plants is the differ- entiation of their photosynthetic cells into two dis- tinct types, deemed mesophyll (M) and bundle sheath (B). These cells contain different comple- ments of photosynthetically important enzymatic activities and display a precise spatial organization within the leaf (for a review see Ref. 21). In this 'Krantz' type anatomy, B cells form a layer sur- rounding the vascular bundle and M cells form a concentric layer surrounding the B cells. This un- usual anatomy serves to compartmentalize the bio- chemical reactions and metabolites characteristic of C4 photosynthesis. Primary carbon fixation, mediated by PEPC, occurs in the cytosol of M cells. In maize, the fixed CO 2 is transported as malate to B cell chloroplasts, where it is decarboxylated and the released CO2 refixed via the photosynthetic carbon reduction cycle. This scheme results in ele- vated CO2 concentrations at the site of net carbon