35S-Methionine Incorporates Differentially into Polypeptides across Leaves of Spinach (Spinacia oleracea)

The distribution of proteins across leaves may have significant impact on optimal photosynthetic performance of leaves, however little is known about the distribution of proteins and protein synthesis across C3 leaves. We report here a detailed investigation of 35S-methionine incorporation into polypeptides and the steady-state polypeptide profiles at different leaf depths across spinach leaves. About 10 highly incorporating polypeptides (three with apparent molecular masses of 23 kDa, 21 kDa and 17 kDa were especially dominant) were detected in a few medial leaf sections. These highly incorporating polypeptides were soluble proteins, except for the 17 kDa polypeptide, which was associated with thylakoid membranes. All of the highly incorporating polypeptides were nuclearly encoded. Light significantly enhanced 35S-methionine incorporation into the highly incorporating polypeptides in “sun” grown leaves, but not in “shade” grown leaves. Microautoradiography showed that the highly incorporating polypeptides were associated mainly with the phloem tissue. A specific identity or function for the polypeptides is not known. The concentration of most polypeptides on an areal basis appeared to increase with leaf depth from the adaxial leaf surface, reaching a maximum around 25% of the leaf depth, and then declined gradually towards the abaxial surface. The periphery of cells exhibited high levels of 35S-methionine incorporation, and microautoradiography showed that the label was mainly located in the symplast. In general, polypeptides exhibited higher rates of 35S-methionine incorporation in the palisade mesophyll than in the spongy mesophy, probably due to cytoplasmic density and light. The data show that it may be possible to study vascular bundle proteins using paradermal leaf sections. In addition, we now can investigate how factors such as light or CO2 might control protein distribution across leaves, and further explore the complex interactions among photosynthesis, leaf anatomy, and light.