Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: What limits nitrate reduction in situ?
Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: What limits nitrate reduction in situ?
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DOI：
10.1007/s004250050682
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发表时间：
2000-04-01
期刊：
影响因子：
4.3
通讯作者：
Glaab, J
中科院分区：
文献类型：
--
作者：
Kaiser, WM;Kandlbinder, A;Glaab, J
在存在 Mg2+ (NRact) 或 EDTA (NRmax) 的情况下，在底物饱和条件下，测量在蛋白磷酸酶抑制剂（50 μM 斑蝥素）存在下制备的菠菜叶提取物中的硝酸还原酶 (NR) 活性。将这些体外活性与硝酸盐充足的植物叶子中的硝酸盐还原率进行比较。每克鲜重含有高达 60 μmol 硝酸盐的菠菜叶在空气中用叶柄在水中照射。它们的硝酸盐含量随着雾凇的增加而减少，从而可以对硝酸盐的原位还原进行估计。硝酸盐消耗的初始速率（1-2 小时）通常低于 NRact，并且随着光照时间（4 小时）的延长，差异变得更大。当通过叶柄向叶片施用 30 mM 硝酸盐时，根据硝酸盐吸收和消耗计算的初始原位还原率仍然低于 NRact。然而，通过叶柄供给硝酸盐可以在较长时间内维持原位硝酸盐还原率。当叶子在 5% CO2 中照射时，原位硝酸盐还原的初始速率仅与 NRact 匹配。在不含CO2的空气中或在黑暗中，NRact和原位硝酸盐还原均下降，但NRact仍超过原位还原。更极端的是，在缺氧条件下或在黑暗中喂食5-氨基-4-咪唑甲酰胺核糖核苷后，NR被激活至高光水平；然而尽管如此，叶子中硝酸盐的减少量仍然很低。检查了 NRact 的标准测定是否会由于提取和稀释后非活性磷酸-NR-14-3-3 复合物的解离而高估原位速率，但没有发现任何证据。原位 NR 显然在底物饱和度以下运行，除了在高环境 CO2 的光照下。这表明在短期内（2小时），原位硝酸盐还原主要受到胞质NADH的限制，并且只有在液泡硝酸盐库部分清空后，胞质硝酸盐才变得有限。
Nitrate reductase (NR) activity in spinach leaf extracts prepared in the presence of a protein phosphatase inhibitor (50 mu M cantharidine) was measured in the presence of Mg2+ (NRact) or EDTA (NRmax), under substrate saturation. These in-vitro activities were compared with nitrate reduction rates in leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 mu mol nitrate per g fresh weight were illuminated in air with their petiole in water. Their nitrate content decreased with rime, permitting an estimation of nitrate reduction in situ. The initial rates (1-2 h) of nitrate consumption were usually lower than NRact, and with longer illumination time (4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitrate feeding through the petiole maintained the in situ-nitrate reduction rate for a longer time. Initial rates of nitrate reduction in situ only matched NRact when leaves were illuminated in 5% CO2. In CO2-free air or in the dark, both NRact and in-situ nitrate reduction decreased, but NRact still exceeded in-situ reduction. More extremely, under anoxia or after feeding 5-amino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated to the high light level; yet in spite of that, nitrate reduction in the leaf remained very low. It was examined whether the standard assay for NRact would overestimate the ia-situ rates due to a dissociation of the inactive phospho-NR-14-3-3 complex after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation, except in the light at high ambient CO2. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied.