Carbonic anhydrase activity in seaweeds: overview and recommendations for measuring activity with an electrometric method, using Macrocystis pyrifera as a model species
Carbonic anhydrase (CA) plays an important physiological role in all biological systems by accelerating the interconversion of CO2 and HCO3−. In algae, CA is essential for photosynthesis: external CA (CAext) dehydrates HCO3−, enhancing the supply of CO2 to the cell surface, and internal CA (CAint) interconverts HCO3− and CO2 to maintain the inorganic carbon (Ci) pool and supply CO2 to RuBisCO. We first conducted a literature review comparing the conditions in which CA extraction and measurement have been carried out, using the commonly used Wilbur–Anderson method. We found that the assay has been widely modified since its introduction in 1948, mostly without being optimized for the species tested. Based on the review, an optimized protocol for measuring CA in Macrocystis pyrifera was developed, which showed that the assay conditions can strongly affect CA activity. Tris–HCl buffer gave the highest levels of CA activity, but phosphate buffer reduced activity significantly. Buffers containing polyvinylpyrrolidone (PVP) and dithiothreitol (DTT) stabilized CA. Using the optimized assay, CAext and CAint activities were readily measured in Macrocystis with higher precision compared to the non-optimized method. The CAint activity was 2 × higher than CAext, which is attributed to the Ci uptake mechanisms of Macrocystis. This study suggests that the CA assay needs to be optimized for each species prior to experimental work to obtain both accurate and precise results.
We acknowledge the support of a PhD scholarship from the Chilean government to Pamela A. Fernández (BECAS CHILE–CONICYT), a grant from The Royal Society of New Zealand Mardsen fund (UOO0914) to Catriona L. Hurd, and a German Research Foundation grant (RA 2030/1,1) to Ralf Rautenberger. The authors are grateful to Pablo Leal for his help with seaweed collection.
This study was funded by the Chilean program for BECAS CHILE-CONICYT, for a Royal Society of New Zealand Mardsen grant (UOO0914), and a German Research Foundation grant (RA 2030/1,1).
Compliance with ethical standard
Conflict of interest
Pamela A. Fernández declares that she has no conflict of interest. Michael Y. Roleda declares that he has no conflict of interest. Ralf Rautenberger declares that he has no conflict of interest. Catriona L. Hurd declares that she has no conflict of interest.
This article does not contain any studies with animals performed by any of the authors.
- Bi Y, Zhou Z (2016) Absorption and transport of inorganic carbon in kelps with emphasis on Saccharina japonica. Appl Photosynth New Progress, Dr Mohammad Najafpour (ed), InTech, https://doi.org/10.5772/62297
- Flores-Moya A, Gómez I, Viñegla B, Altamirano M, Pérez-Rodríguez E, Maestre C, Caballero RM, Figueroa F (1998) Effects of solar radiation on the endemic Mediterranean red alga Rissoella verruculosa: photosynthetic performance, pigment content and the activities of enzymes related to nutrient uptake. New Phytol 139:673–683CrossRefGoogle Scholar
- Graham M, Vásquez J, Buschmann A (2007) Global ecology of the giant kelp Macrocystis: from ecotypes to ecosystems. Oceanogr Mar Biol Annu Rev 45:39–88Google Scholar
- IPCC (2013) Climate change 2013: the physical science basis. In: Stocker TF, Qin D, Plattner GK, Tignor M, Allen SK, Boschung J, Nauels A, Xia Y, Bex V, Midgley PM (eds) Contribution of Working Group I to the fifth assessment report (AR5) of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, p 1535Google Scholar
- Miyachi S, Tsuzuki M, Avramova ST (1983) Utilization modes of inorganic carbon for photosynthesis in various species of Chlorella. Plant Cell Physiol 24:441–451Google Scholar
- Olischläger M, Wiencke C (2013) Ocean acidifi cation alleviates low-temperature effects on growth and photosynthesis of the red alga Neosiphonia harveyi (Rhodophyta). J Exp Bot 18(5):587–597Google Scholar
- Ramazanov M, Semenenko VE (1988) Content of the CO2-dependent form of carbonic anhydrase as a function of light intensity and photosynthesis. Sov Plant Physiol 35:340–344Google Scholar
- Sültemeyer DF (1998) Carbonic anhydrase in eukaryotic algae: characteristics, regulation, and possible function during photosynthesis. Can J Bot 76:962–972Google Scholar
- The Royal Society (2005) Ocean acidification due to increasing atmospheric carbon dioxide. Policy document 12/05 Royal Society, London. The Clyvedon Press Ltd, CardiffGoogle Scholar
- Thomas TE, Harrison PJ (1988) A comparison of in vitro and in vivo nitrate reductase assays in three intertidal seaweeds. Bot Mar 31:101–107Google Scholar
- van Hille RP (2001) Biological generation of reactive alkaline species and their application in a sustainable bioprocess for the remediation of acid metal contaminated wastewaters. Ph.D. Dissertation, Rhodes University, South AfricaGoogle Scholar
- Waygood ER (1955) Carbonic anhydrase (plant and animal). In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 2. Academic Press, New York, pp 836–846Google Scholar
- Yue GF, Wang JX, Wang JF (2001) Inorganic carbon acquisition by juvenile sporophyte of Laminarials (L. japonica × L. longissima). Oceanologia et Limnologia Sinica 32(6):647–652Google Scholar