Rubisco is central to carbon assimilation, and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of ¹⁴CO₂ into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerolphosphate dehydrogenase (GlyPDH); phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH); and pyruvate kinase (PK) and lactate dehydrogenase (LDH). To date there has been no thorough evaluation of their reliability by comparison with the ¹⁴C-based method. The three NADH-linked assays were used in parallel to estimate (i) the 3-PGA concentration–response curve of NADH oxidation, (ii) the Michaelis–Menten constant for ribulose-1,5-bisphosphate, (iii) fully active and inhibited Rubisco activities, and (iv) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the ¹⁴C-based method, and the PK–LDH method showed a strong correlation and was the cheapest method. PEPC–MDH would be a suitable option for situations in which ADP/ATP might interfere with the assay. GAPDH–GlyPDH proved more laborious than the other methods. Thus, we recommend the PK–LDH method as a reliable, cheaper, and higher throughput method to phenotype Rubisco activity for crop improvement efforts.

中文翻译：

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测量Rubisco活性：基于NADH的微量滴定板和基于14C的测定法的挑战和机遇。

Rubisco在碳同化中起着至关重要的作用，提高作物生产效率和可持续性的努力激发了人们对Rubisco活性表型的兴趣。我们测试了以下假设，即基于微量滴定板的方法可提供与辐射测定法（可测量¹⁴ CO ₂掺入3-磷酸甘油酸酯（3-PGA）的方法）获得的结果相当的结果。测试了使用替代偶联酶的三种与NADH连锁的测定法：3-磷酸甘油醛脱氢酶（GAPDH）和三磷酸甘油磷酸脱氢酶（GlyPDH）；磷酸烯醇丙酮酸羧化酶（PEPC）和苹果酸脱氢酶（MDH）；丙酮酸激酶（PK）和乳酸脱氢酶（LDH）。迄今为止，与^14个相比，还没有对其可靠性进行全面评估基于C的方法。并行使用了三种与NADH联用的测定方法，以估计（i）NADH氧化的3-PGA浓度-响应曲线，（ii）核糖-1,5-双磷酸酯的Michaelis-Menten常数，（iii）完全活性和抑制了Rubisco的活性，以及​​（iv）在充分光照和阴影的叶片中Rubisco的初始和总活性。这三种方法都与基于¹⁴ C的方法密切相关，而PK–LDH方法显示出很强的相关性，并且是最便宜的方法。对于ADP / ATP可能干扰测定的情况，PEPC-MDH将是合适的选择。与其他方法相比，GAPDH–GlyPDH被证明更加费力。因此，我们建议使用PK-LDH方法作为可靠的，便宜的，高通量的表型Rubisco活性表型，以促进作物改良。