Edwards G E
Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706.
Plant Physiol. 1979 May;63(5):821-7. doi: 10.1104/pp.63.5.821.
A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C(4) plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O(2) evolution by chloroplasts isolated from protoplasts.Chloroplasts were isolated from protoplasts by several passages of the protoplasts through a 20-micrometer nylon mesh. Mesophyll chloroplasts were judged approximately 90 to 95% intact and bundle sheath chloroplasts 80 to 90% intact based on retention of chloroplast marker enzymes and the ferricyanide test for intactness. It was necessary to include 10 millimolar MgCl(2) in media for osmotically shocking the chloroplasts in order to obtain maximum and linear rates of ferricyanide-dependent O(2) evolution.Chloroplasts isolated from mesophyll protoplast preparations had low rates of light-dependent O(2) evolution in the presence of 10 millimolar NaHCO(3) (0.13 micromoles per milligram chlorophyll per minute) in comparison to bundle sheath chloroplasts (1 to 2.5 micromoles per milligram chlorophyll per minute). The mesophyll chloroplasts catalyze high rates of 3-phosphoglycerate-dependent O(2) evolution (2 to 4 micromoles per milligram chlorophyll per minute). Orthophosphate but not phosphoenolpyruvate inhibited the 3-phosphoglycerate-dependent O(2) evolution by the mesophyll chloroplasts. Rates of O(2) evolution were much higher with mesophyll than with bundle sheath chloroplasts in the presence of pyruvate plus oxaloacetate. The results are discussed in relation to the proposed function of these chloroplasts during C(4) photosynthesis.
本文描述了一种从C4植物黍稷中分离和纯化叶肉原生质体和维管束鞘原生质体的方法。对叶片组织进行酶解后，叶肉原生质体和维管束鞘原生质体被释放出来，并通过密度离心进行纯化。叶肉原生质体密度较低，使得两种原生质体类型能够快速分离。通过光学显微镜（基于叶绿体和原生质体的大小差异）、制剂中标记酶的水平（即叶肉中的丙酮酸、磷酸二激酶和磷酸烯醇式丙酮酸羧化酶以及维管束鞘中的核酮糖-1,5-二磷酸羧化酶）以及从原生质体中分离的叶绿体在底物依赖性氧气释放方面的差异，证明了叶肉原生质体和维管束鞘原生质体的分离（纯度高达95%）。通过使原生质体多次通过20微米的尼龙网从原生质体中分离叶绿体。基于叶绿体标记酶的保留情况和用于完整性检测的铁氰化物试验，判断叶肉叶绿体的完整性约为90%至95%，维管束鞘叶绿体的完整性为80%至90%。为了获得最大且呈线性的铁氰化物依赖性氧气释放速率，在用于渗透冲击叶绿体的培养基中必须加入10毫摩尔的氯化镁。与维管束鞘叶绿体（每分钟每毫克叶绿素1至2.5微摩尔）相比，从叶肉原生质体制剂中分离的叶绿体在10毫摩尔碳酸氢钠存在下光依赖性氧气释放速率较低（每分钟每毫克叶绿素0.13微摩尔）。叶肉叶绿体催化3-磷酸甘油酸依赖性氧气释放的速率较高（每分钟每毫克叶绿素2至4微摩尔）。正磷酸盐而非磷酸烯醇式丙酮酸抑制叶肉叶绿体的3-磷酸甘油酸依赖性氧气释放。在丙酮酸加草酰乙酸存在下，叶肉叶绿体的氧气释放速率比维管束鞘叶绿体高得多。结合这些叶绿体在C4光合作用中的拟议功能对结果进行了讨论。
