Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays:NADH-linked and 14C-based assays of Rubisco activity

Sales, Cristina RG and Bernardes Da Silva, Anabela and Carmo-Silva, Elizabete (2020) Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays:NADH-linked and 14C-based assays of Rubisco activity. Journal of Experimental Botany. ISSN 0022-0957

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Abstract

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 and glycerolphosphate-dehydrogenase (GAPDH-GlyPDH); phosphoenolpyruvate-carboxylase and malate-dehydrogenase (PEPC-MDH); pyruvate-kinase and lactate-dehydrogenase (PK-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 (1) the 3-PGA concentration response curve of NADH oxidation, (2) the Michaelis-Menten constant for RuBP, (3) fully active and inhibited Rubisco activities, and (4) 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 where ADP/ATP might interfere with the assay. GAPDH-GlyPDH proved more laborious than the other methods. Thus, we recommend the PK-LDH as a reliable, cheaper and higher throughput method to phenotype Rubisco activity for crop improvement efforts.

Item Type:
Journal Article
Journal or Publication Title:
Journal of Experimental Botany
Uncontrolled Keywords:
/dk/atira/pure/subjectarea/asjc/1300/1314
Subjects:
ID Code:
145271
Deposited By:
Deposited On:
06 Jul 2020 12:30
Refereed?:
Yes
Published?:
Published
Last Modified:
25 Sep 2020 04:56