TY - JOUR

T1 - Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays

T2 - NADH-linked and 14C-based assays of Rubisco activity

AU - Sales, Cristina RG

AU - Bernardes Da Silva, Anabela

AU - Carmo-Silva, Elizabete

PY - 2020/9/19

Y1 - 2020/9/19

N2 - 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.

AB - 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.

KW - crop improvement

KW - plant phenotyping

KW - wheat

KW - Rubisco

KW - dPGM

KW - NADH

KW - GAPDH-GlyPDH

KW - PEPC-MDH

KW - PK-LDH

KW - enzyme activity assay

U2 - 10.1093/jxb/eraa289

DO - 10.1093/jxb/eraa289

M3 - Journal article

VL - 71

SP - 5302

EP - 5312

JO - Journal of Experimental Botany

JF - Journal of Experimental Botany

SN - 0022-0957

IS - 18

ER -