Aoyagi K, Bassham J A
Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720.
Plant Physiol. 1986 Feb;80(2):322-33. doi: 10.1104/pp.80.2.322.
Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C(4) photosynthesis, for cell morphology, and for (14)C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later.Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C(3) leaves and from etiolated C(4) leaf tissues, but differing from the corresponding enzymes in the mature leaf.Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology.Analysis of the labeled products of (14)CO(2) fixation by nonchlorophyllous calli indicated beta-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in beta-carboxylation products was even greater. Analysis of enzyme levels in young leaf sections by protein blot and of (14)C-labeling patterns in the present study are in general agreement with enzyme activity determinations of previous studies, providing additional information about PPDK levels, and supporting the model proposed for developing young leaves.These results suggest that maize leaves begin to express C(4) enzymes during ontogeny through several stages from greening and cell differentiation as seen in the callus and then shoot formation, and finally acquire capacity for full C(4) photosynthesis during leaf development concomitant with the development of Kranz anatomy and accumulation of large amounts of enzymes involved in carbon metabolism.
对再生玉米A188组织培养物进行检测，以确定参与C4光合作用的酶的存在情况、细胞形态以及14C标记动力学，从而研究该途径在植物发育过程中的实施情况。作为对照，对玉米幼苗叶片切片进行了检测。使用针对叶片酶的抗体进行蛋白质印迹分析表明，在愈伤组织芽再生的早期阶段，这些酶的分布情况与在幼苗叶片中观察到的紧密协调的分布情况不同。在非绿色愈伤组织中发现了丙酮酸磷酸双激酶（PPDK）（EC 2.7.9.1）和磷酸烯醇式丙酮酸羧化酶（PEPC）（EC 4.1.1.31），而核酮糖1,5-二磷酸羧化酶（RuBPC，EC 4.1.1.39）和NADP特异性苹果酸酶（ME-NADP）（EC 1.3.1.37）直到后期才检测到。酶活性测定表明，非绿色愈伤组织中存在ME-NADP以及PEPC和PPDK。然而，ME-NADP和PEPC的活性特性与C3叶片和黄化C4叶片组织中的酶相似，但与成熟叶片中的相应酶不同。对从胚状体形成愈伤组织中提取的多聚腺苷酸RNA的体外翻译产物进行免疫沉淀，结果显示存在叶绿体PPDK的110千道尔顿前体和94千道尔顿多肽。因此，在叶片形态出现之前就已存在PPDK mRNA的叶绿体类型。对非绿色愈伤组织固定14CO2的标记产物进行分析表明，存在β羧化作用，生成三羧酸循环的酸，但没有掺入磷酸甘油酸中。随着愈伤组织变绿，出现了一些掺入磷酸甘油酸和糖磷酸的情况，并且随着芽的发育，这种情况在芽中增加，尽管对于较老的芽，β羧化产物的增加甚至更大。本研究中通过蛋白质印迹对幼叶切片中的酶水平进行分析以及对14C标记模式进行分析，总体上与先前研究的酶活性测定结果一致，提供了有关PPDK水平的额外信息，并支持了为发育中的幼叶提出的模型。这些结果表明，玉米叶片在个体发育过程中开始表达C4酶，经历从愈伤组织中观察到的变绿和细胞分化，然后形成芽，最后在叶片发育过程中伴随着花环结构的发育和参与碳代谢的大量酶的积累，获得完全进行C4光合作用的能力。
