BIBLIOS

Document type
Journal articles

Document subtype
Full paper

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

Participants in the publication
Cristina R G Sales (Author)
Anabela Bernardes da Silva (Author)
FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DE LISBOA
Dep. Biologia Vegetal
BioISI
Elizabete Carmo-Silva (Author)

Summary
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.


Institution
FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DE LISBOA

Where published
Journal of Experimental Botany

Publication Identifiers

Publisher
Oxford University Press (OUP)



Document Identifiers
DOI - https://doi.org/10.1093/jxb/eraa289
URL - http://dx.doi.org/10.1093/jxb/eraa289

Rankings
SCIMAGO Q1 (2020) - 6.992 - Physiology

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