Plant Cell Reports

, Volume 38, Issue 2, pp 147–159 | Cite as

Expression of seven carbonic anhydrases in red alga Gracilariopsis chorda and their subcellular localization in a heterologous system, Arabidopsis thaliana

  • Md. Abdur Razzak
  • JunMo Lee
  • Dong Wook Lee
  • Jeong Hee Kim
  • Hwan Su Yoon
  • Inhwan HwangEmail author
Original Article


Key message

Red alga, Gracilariopsis chorda, contains seven carbonic anhydrases that can be grouped into α-, β- and γ-classes.


Carbonic anhydrases (CAHs) are metalloenzymes that catalyze the reversible hydration of CO2. These enzymes are present in all living organisms and play roles in various cellular processes, including photosynthesis. In this study, we identified seven CAH genes (GcCAHs) from the genome sequence of the red alga Gracilariopsis chorda and characterized them at the molecular, cellular and biochemical levels. Based on sequence analysis, these seven isoforms were categorized into four α-class, one β-class, and two γ-class isoforms. RNA sequencing revealed that of the seven CAHs isoforms, six genes were expressed in G. chorda in light at room temperature. In silico analysis revealed that these seven isoforms localized to multiple subcellular locations such as the ER, mitochondria and cytosol. When expressed as green fluorescent protein fusions in protoplasts of Arabidopsis thaliana leaf cells, these seven isoforms showed multiple localization patterns. The four α-class GcCAHs with an N-terminal hydrophobic leader sequence localized to the ER and two of them were further targeted to the vacuole. GcCAHβ1 with no noticeable signal sequence localized to the cytosol. The two γ-class GcCAHs also localized to the cytosol, despite the presence of a predicted presequence. Based on these results, we propose that the red alga G. chorda also employs multiple CAH isoforms for various cellular processes such as photosynthesis.


Carbonic anhydrase Subcellular localization CO2 Phylogenetic tree Gracilariopsis chorda Red algae 



This work was supported by grants from the National Research Foundation of Korea, Ministry of Science and ICT (No. 2016R1E1A1A02922014), and from the Collaborative Genome Program of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) (20180430). Dong Wook Lee was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ01335801), Rural Development Administration, Republic of Korea.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

299_2018_2356_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1115 KB)
299_2018_2356_MOESM2_ESM.pdf (612 kb)
Supplementary material 2 (PDF 611 KB)
299_2018_2356_MOESM3_ESM.pdf (568 kb)
Supplementary material 3 (PDF 567 KB)
299_2018_2356_MOESM4_ESM.xlsx (44 kb)
Supplementary material 4 (XLSX 43 KB)
299_2018_2356_MOESM5_ESM.docx (15 kb)
Supplementary material 5 (DOCX 14 KB)
299_2018_2356_MOESM6_ESM.docx (13 kb)
Supplementary material 6 (DOCX 13 KB)


  1. Aebi M (2013) N-linked protein glycosylation in the ER. Biochi Biophys Acta Mol Cell Res 1833:2430–2437. CrossRefGoogle Scholar
  2. Ahn G, Kim H, Kim DH et al (2017) SH3P2 plays a crucial role at the step of membrane tubulation during cell plate formation in plants. Plant Cell. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Alber BE, Ferry JG (1994) A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proc Natl Acad Sci USA 91:6909–6913. CrossRefPubMedGoogle Scholar
  4. Arthurs GJ, Sudhakar M (2008) Carbon dioxide transport. Updat Anaesth 24:26–29. CrossRefGoogle Scholar
  5. Atkinson N, Feike D, Mackinder LM et al (2016) Introducing an algal carbon-concentrating mechanism into higher plants: Location and incorporation of key components. Plant Biotechnol J 14:1302–1315. CrossRefPubMedGoogle Scholar
  6. Burén S, Ortega-Villasante C, Blanco-Rivero A et al (2011) Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana. PLoS One. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cardol P (2005) The mitochondrial oxidative phosphorylation proteome of Chlamydomonas reinhardtii deduced from the genome sequencing project. Plant Physiol 137:447–459. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Coleman JE (1967) Mechanism of action of carbonic anhydrase. J Biochem 242:5212–5219Google Scholar
  9. De Marchis F, Bellucci M, Pompa A (2013) Unconventional pathways of secretory plant proteins from the endoplasmic reticulum to the vacuole bypassing the Golgi complex. Plant Signal Behav 8:1–5. CrossRefGoogle Scholar
  10. Di Fiore A, Alterio V, Monti SM et al (2015) Thermostable carbonic anhydrases in biotechnological applications. Int J Mol Sci 16:15456–15480. CrossRefPubMedPubMedCentralGoogle Scholar
  11. DiMario RJ, Quebedeaux JC, Longstreth DJ et al (2016) The cytoplasmic carbonic anhydrases β CA2 and β CA4 are required for optimal plant growth at low CO2. Plant Physiol 171:280–293. CrossRefPubMedPubMedCentralGoogle Scholar
  12. DiMario RJ, Clayton H, Mukherjee A et al (2017) Plant carbonic anhydrases: structures, locations, evolution, and physiological roles. Mol Plant 10:30–46. CrossRefPubMedPubMedCentralGoogle Scholar
  13. DiMario RJ, Machingura MC, Waldrop GL, Moroney JV (2018) The many types of carbonic anhydrases in photosynthetic organisms. Plant Sci 268:11–17. CrossRefPubMedGoogle Scholar
  14. Dionisio-Sese ML, Fukuzawa H, Miyachi S (1990) Light-induced carbonic anhydrase expression in Chlamydomonas reinhardtii. Plant Physiol 94:1103–1110. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Eriksson M, Karlsson J, Ramazanov Z et al (1996) Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 93:12031–12034. CrossRefPubMedGoogle Scholar
  16. Fabre N, Reiter IM, Becuwe-Linka N, Genty B, Rumeau D (2007) Characterization and expression analysis of genes encoding alpha and beta carbonic anhydrases in Arabidopsis. Plant Cell Environ 30:617–629. CrossRefPubMedGoogle Scholar
  17. Faye L, Daniell H (2006) Novel pathways for glycoprotein import into chloroplasts. Plant Biotechnol J 4:275–279. CrossRefPubMedGoogle Scholar
  18. Ferry JG (2010) The γ class of carbonic anhydrases. Biochim Biophys Acta 1804:374–381. CrossRefGoogle Scholar
  19. Flouri T, Izquierdo-Carrasco F, Darriba D et al (2015) The phylogenetic likelihood library. Syst Biol 64:356–362. CrossRefPubMedGoogle Scholar
  20. Fujiwara S, Fukuzawa H, Tachiki A, Miyachi S (1990) Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:9779–9783CrossRefGoogle Scholar
  21. Fujiwara S, Ishida N, Tsuzuki M (1996) Circadian expression of the carbonic anhydrase gene, Cah1, in Chlamydomonas reinhardtii. Plant Mol Biol 32:745–749. CrossRefPubMedGoogle Scholar
  22. Gee CW, Niyogi KK (2017) The carbonic anhydrase CAH1 is an essential component of the carbon-concentrating mechanism in Nannochloropsis oceanica. Proc Natl Acad Sci U S A 114:4537–4542. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Harada H, Nakatsuma D, Ishida M, Matsuda Y (2005) Regulation of the expression of intracellular b-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum. Plant Physiol 139:1041–1050. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hiltonen T, Björkbacka H, Forsman C et al (1998) Intracellular beta-carbonic anhydrase of the unicellular green alga Coccomyxa. Cloning of the cDNA and characterization of the functional enzyme overexpressed in Escherichia coli. Plant Physiol 117:1341–1349. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hwang I (2008) Sorting and anterograde trafficking at the Golgi apparatus. Plant Physiol 148:673–683. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jacob A, Verpoorte, Mehta S, John T (1967) Esterase activities of human carbonic anhydrases B and C. J Biol Chem 242:4221–4229Google Scholar
  27. Jin JB, Kim YA, Kim SJ et al (2001) A new dynamin-like protein, ADL6, is involved in trafficking from the trans-golgi network to the central vacuole in Arabidopsis. Plant Cell 13:1511–1526. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kang H, Park Y, Lee Y, Yoo YJ, Hwang I (2018) Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants. Sci Rep 8:4612. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Karlsson J, Hiltonen T, Husic HD et al (1995) Intracellular carbonic anhydrase of Chlamydomonas reinhardtii. Plant Physiol 109:533–539. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298. CrossRefPubMedGoogle Scholar
  31. Kim DH, Hwang I (2013) Direct targeting of proteins from the cytosol to organelles: the ER versus endosymbiotic organelles. Traffic 14:613–621. CrossRefPubMedGoogle Scholar
  32. Koontz L (2014) TCA precipitation. Methods Enzymol 541:3–10. CrossRefPubMedGoogle Scholar
  33. Lee DW, Hwang I (2011) Transient expression and analysis of chloroplast proteins in Arabidopsis protoplasts. Methods Mol Biol 774:59–71. CrossRefPubMedGoogle Scholar
  34. Lee MH, Hwang I (2014) Adaptor proteins in protein trafficking between endomembrane compartments in plants. J Plant Biol 57:265–273. CrossRefGoogle Scholar
  35. Lee J, Lee H, Kim J et al (2011a) Both the hydrophobicity and a positively charged region flanking the C-terminal region of the transmembrane domain of signal-anchored proteins play critical roles in determining their targeting specificity to the endoplasmic reticulum or endosymbiotic org. Plant cell 23:1588–1607. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lee MH, Jung C, Lee J et al (2011b) An Arabidopsis prenylated Rab Acceptor 1 isoform, AtPRA1.B6, displays differential inhibitory effects on anterograde trafficking of proteins at the endoplasmic reticulum. Plant Physiol 157:645–658. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Lee JM, Yang EC, Graf L et al (2018a) Analysis of the draft genome of the red seaweed Gracilariopsis chorda provides insights into genome size evolution in Rhodophyta. Mol Biol Evol 35:1869–1886. CrossRefPubMedGoogle Scholar
  38. Lee DW, Yoo YJ, Razzak MA, Hwang I (2018b) Prolines in transit peptides are crucial for efficient preprotein translocation into chloroplasts. Plant physiol 176:663–677. CrossRefPubMedGoogle Scholar
  39. Lindskog S (1997) Structure and mechanism of carbonic anhydrase. Pharmacol Ther 74:1–20. CrossRefGoogle Scholar
  40. Liu S, Bugos RC, Dharmasiri N, Su WW (2000) Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures. J Biotechnol 87:1–16CrossRefGoogle Scholar
  41. MacAuley SR, Zimmerman SA, Apolinario EE et al (2009) The archetype γ-class carbonic anhydrase (cam) contains iron when synthesized in vivo. Biochemistry 48:817–819. CrossRefPubMedGoogle Scholar
  42. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2016) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200–D203. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Minh BQ, Nguyen MAT, Von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 30:1188–1195. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Mitra M (2004) Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 135:173–182. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Moroney JV, Bartlett SG, Samuelsson G (2001) Carbonic anhydrases in plants and algae: invited review. Plant Cell Environ 24:141–153. CrossRefGoogle Scholar
  46. Moroney JV, Ma Y, Frey WD et al (2011) The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles. Photosynth Res 109:133–149. CrossRefGoogle Scholar
  47. Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:174–268. CrossRefGoogle Scholar
  48. Parisi G, Perales M, Fornasari MS et al (2004) Gamma carbonic anhydrases in plant mitochondria. Plant Mol Biol 55:193–207. CrossRefPubMedGoogle Scholar
  49. Park Y, Xu Z-Y, Kim SY et al (2016) Spatial regulation of ABCG25, an ABA exporter, is an important component of the mechanism controlling cellular ABA levels. Plant Cell 28:2528–2544. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Razzak MA, Lee DW, Yoo YJ, Hwang I (2017) Evolution of rubisco complex small subunit transit peptides from algae to plants. Sci Rep 7:1–11. CrossRefGoogle Scholar
  52. Smith KS, Ferry JG (2000) Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24:335–366. CrossRefGoogle Scholar
  53. Sohn EJ (2003) Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell 15:1057–1070. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Supuran CT (2013) Carbonic anhydrases. Bio Med Chem 21:1377–1378. CrossRefGoogle Scholar
  55. Supuran CT, Capasso C (2017) An overview of the bacterial carbonic anhydrases. Metabolites. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Takeuchi M, Ueda T, Sato K et al (2000) A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant J 23:517–525. CrossRefPubMedGoogle Scholar
  57. Villarejo A, Burén S, Larsson S et al (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol 7:1124–1131. CrossRefGoogle Scholar
  58. Wang Q, Fristedt R, Yu X et al (2012) The γ-carbonic anhydrase subcomplex of mitochondrial complex I is essential for development and important for photomorphogenesis of Arabidopsis. Plant physiol 160:1373–1383. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Wyszynski FJ, Hesketh AR, Bibb MJ, Davis BG (2010) Dissecting tunicamycin biosynthesis by genome mining: cloning and heterologous expression of a minimal gene cluster. Chem Sci 1:581. CrossRefGoogle Scholar
  60. Xiang L, Etxeberria E, Van Den Ende W (2013) Vacuolar protein sorting mechanisms in plants. FEBS J 280:979–993. CrossRefPubMedGoogle Scholar
  61. Ynalvez R, Xiao Y, Ward AS et al (2008) Identification and characterization of two closely related beta-carbonic anhydrases from Chlamydomonas reinhardtii. Physiol plant 133:15–26. CrossRefPubMedGoogle Scholar
  62. Zimmerman SA, Ferry JG (2008) The beta and gamma classes of carbonic anhydrase. Curr Pharm Des 14:716–721CrossRefGoogle Scholar
  63. Zimmerman SA, Tomb JF, Ferry JG (2010) Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the γ class of carbonic anhydrases. J Bacteriol 192:1353–1360. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangSouth Korea
  2. 2.Department of Biological SciencesSungkyunkwan UniversitySuwonSouth Korea
  3. 3.Department of Biochemistry and Molecular Biology, College of DentistryKyung Hee UniversitySeoulSouth Korea
  4. 4.Department of Life and Nanopharmaceutical Sciences, Graduate SchoolKyung Hee UniversitySeoulSouth Korea
  5. 5.Department of Life SciencesPohang University of Science and TechnologyPohangSouth Korea

Personalised recommendations