PMID- 32728715
OWN - NLM
STAT- MEDLINE
DCOM- 20210514
LR  - 20240329
IS  - 1460-2431 (Electronic)
IS  - 0022-0957 (Print)
IS  - 0022-0957 (Linking)
VI  - 71
IP  - 18
DP  - 2020 Sep 19
TI  - Measuring Rubisco activity: challenges and opportunities of NADH-linked 
      microtiter plate-based and 14C-based assays.
PG  - 5302-5312
LID - 10.1093/jxb/eraa289 [doi]
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 (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.
CI  - (c) The Author(s) 2020. Published by Oxford University Press on behalf of the 
      Society for Experimental Biology.
FAU - Sales, Cristina R G
AU  - Sales CRG
AD  - Lancaster Environment Centre, Lancaster University, Library Avenue, Lancaster, 
      UK.
FAU - da Silva, Anabela Bernardes
AU  - da Silva AB
AD  - BioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, 
      University of Lisbon, Lisbon, Portugal.
FAU - Carmo-Silva, Elizabete
AU  - Carmo-Silva E
AD  - Lancaster Environment Centre, Lancaster University, Library Avenue, Lancaster, 
      UK.
LA  - eng
GR  - BB/N020871/2/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Exp Bot
JT  - Journal of experimental botany
JID - 9882906
RN  - 0 (Carbon Radioisotopes)
RN  - 0U46U6E8UK (NAD)
RN  - 7V68J5677O (Carbon-14)
RN  - EC 4.1.1.39 (Ribulose-Bisphosphate Carboxylase)
SB  - IM
MH  - Carbon Radioisotopes
MH  - *NAD
MH  - Reproducibility of Results
MH  - *Ribulose-Bisphosphate Carboxylase
PMC - PMC7501812
OTO - NOTNLM
OT  - Crop improvement
OT  - GAPDH-GlyPDH
OT  - NADH
OT  - PEPC-MDH
OT  - PK-LDH
OT  - Rubisco
OT  - dPGM
OT  - enzyme activity assay
OT  - plant phenotyping
OT  - wheat
EDAT- 2020/07/31 06:00
MHDA- 2021/05/15 06:00
PMCR- 2020/06/30
CRDT- 2020/07/31 06:00
PHST- 2019/12/18 00:00 [received]
PHST- 2020/06/22 00:00 [accepted]
PHST- 2020/07/31 06:00 [pubmed]
PHST- 2021/05/15 06:00 [medline]
PHST- 2020/07/31 06:00 [entrez]
PHST- 2020/06/30 00:00 [pmc-release]
AID - 5864819 [pii]
AID - eraa289 [pii]
AID - 10.1093/jxb/eraa289 [doi]
PST - ppublish
SO  - J Exp Bot. 2020 Sep 19;71(18):5302-5312. doi: 10.1093/jxb/eraa289.