Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, 10021-6399, New York, NY, U.S.A..
Plant Mol Biol. 1984 Nov;3(6):431-44. doi: 10.1007/BF00033391.
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 immunoelectrophoresis 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-dimensional 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-1,5-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 cells 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.
我们研究了酶活性在玉米叶片中叶肉（M）和束鞘（B）细胞中差异定位的分子基础。通过酶消化和次生叶片的机械处理制备 M 原生质体和 B 束。比较了来自两种细胞类型的可溶性和类囊体膜蛋白的一维和二维凝胶电泳和定量火箭免疫电泳。此外，使用单特异性抗体通过交叉免疫电泳鉴定了几种类囊体多肽。M 和 B 类囊体在它们的多肽组成上表现出定量和定性的差异。虽然 M 类囊体含有正常的多肽组成，但 B 类囊体缺乏铁氧还蛋白-NADP(+)还原酶、光系统 II 反应中心多肽和光捕获叶绿素 a/b-蛋白复合物。通过二维凝胶电泳比较可溶性蛋白，发现 M 和 B 细胞之间存在明显差异。一种细胞类型的主要蛋白质明显不存在于另一种细胞类型中。这些差异与从两种细胞类型分离的 poly A(+) RNA 的体外翻译产物的差异相平行。免疫沉淀实验表明，编码核酮糖-1,5-二磷酸羧化酶（rbcS）小亚基的 mRNA 仅在 B 细胞中定位，而编码磷酸烯醇丙酮酸羧化酶的 mRNA 仅在 M 细胞中检测到。用于 Northern blot 分析的编码羧化酶 rbcS 和叶绿素 a/b 结合蛋白的 cDNA 克隆被用作探针。M 细胞中未检测到编码 rbcS 的 RNA，但与 B 细胞相比，编码叶绿素 a/b 结合多肽的 RNA 的稳态水平更高。总之，我们的结果表明，两种叶片细胞类型的差异基因表达是在可翻译的 mRNA 水平上调节的，并且对于至少两种蛋白质，是在稳态 RNA 水平上调节的。
