Biochemical and Molecular Inhibition of Plastidial Carbonic Anhydrase Reduces the Incorporation of Acetate into Lipids in Cotton Embryos and Tobacco Cell Suspensions and Leaves

Abstract

Two cDNAs encoding functional carbonic anhydrase (CA) enzymes were recently isolated from a non-photosynthetic, cotyledon library of cotton (Gossypium hirsutum) seedlings with putative plastid-targeting sequences (GenBank accession nos. AF132854 and AF132855). Relative CA transcript abundance and enzyme activity increased 9 and 15 times, respectively, in cotton embryos during the maximum period of reserve oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [ 14 C]acetate incorporation into total lipids in cotton embryos in vivo, and in embryo plastids in vitro, suggesting a role for CA in plastid lipid biosynthesis. CA inhibitors did not affect acetyl-coenzyme A carboxylase activity or total storage protein synthesis. Similar results were obtained for two other plant systems: cell suspensions (and isolated plastids therefrom) of tobacco (Nicotiana tabacum), and chloroplasts isolated from leaves of transgenic CA antisensesuppressed tobacco plants (5% of wild-type CA activity). In addition, tobacco cell suspensions treated with the CA inhibitor ethoxyzolamide showed a substantial loss of CO 2 compared with controls. The rate of [ 14 C]acetate incorporation into lipid in cell suspensions was reduced by limiting external [CO 2 ] (scrubbed air), and this rate was further reduced in the presence of ethoxyzolamide. Together, these results indicate that a reduction of CA activity (biochemical or molecular inhibition) impacts the rate of plant lipid biosynthesis from acetate, perhaps by impairing the ability of CA to efficiently "trap" inorganic carbon inside plastids for utilization by acetyl-coenzyme A carboxylase and the fatty acid synthesis machinery.Carbonic anhydrase (CA, EC 4.2.1.1) is a zinccontaining metalloenzyme that catalyzes the reversible hydration of CO 2 to HCO 3 Ϫ . The widespread abundance of CA isoforms in plants, animals, and microorganisms suggest that this enzyme has many diverse roles in biological processes. CA plays a critical role in biological systems because CO 2 gas is the membrane permeable form of inorganic carbon for cells, and, in general, the uncatalyzed interconversion between HCO 3 Ϫ and CO 2 is slow when compared with the required rate in living cells (Badger and Price, 1994).In photosynthetic organisms, one generally accepted physiological role of CA is to provide sufficient levels of inorganic carbon as part of a CO 2 -concentrating mechanism for improved photosynthetic efficiency. In Chlamydomonas reinhardtii, Badger and Price (1992) suggested that chloroplastic CA plays a role in photosynthetic carbon assimilation by converting accumulated pools of HCO 3 Ϫ to CO 2 , which is the substrate for Rubisco. Moroney et al. (1985) revealed that the reduction of periplasmic CA activity by using CAspecific inhibitors significantly reduced the efficiency of external inorganic carbon utilization for photosynthesis. Like green algae, CA in cyanobacteria plays an important role in the CO 2 -concentrating mechanism and in photosynthesis (Badger an...

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

“…A number of studies have provided evidence indicating nonphotosynthetic roles for plastidial b-CAs, including involvement in lipogenesis in both photosynthetic and nonphotosynthetic tissues by supplying bicarbonate to acetyl-CoA carboxylase (Hoang et al, 1999;Hoang and Chapman, 2002) and in plant defense by acting as a salicylic acid-binding protein (Slaymaker et al, 2002). Wang et al (2009) have suggested that these two roles may be linked, as evidence exists for plant defense pathways being regulated by lipidbased signals (Nandi et al, 2004;Chandra-Shekara et al, 2007).…”

“…However, CA antisense and gene knockout studies have shown no significant impairment in CO 2 assimilation in mature plant leaves (Majeau et al, 1994;Price et al, 1994;Williams et al, 1996;Ferreira et al, 2008). Other work has suggested that chloroplastic b-CAs may have nonphotosynthetic roles, appearing to be involved in lipid biosynthesis (Hoang and Chapman, 2002) and disease resistance (Slaymaker et al, 2002;Restrepo et al, 2005;Wang et al, 2009) in C 3 plants. Interestingly, levels of a chloroplastic b-CA and a cytosolic b-CA increased during greening of Arabidopsis seedlings, and while levels of transcripts encoding the cytosolic enzyme mirrored those of the protein, transcript abundance for the chloroplastic enzyme did not change (Wang et al, 2006).…”

The Cytoplasmic Carbonic Anhydrases βCA2 and βCA4 Are Required for Optimal Plant Growth at Low CO2

“…In Gossypium hirsutum, it has been shown that CAs are involved in lipid biosynthesis, as the CA-catalyzed formation of HCO 3 2 in the chloroplast can be used by plastidal acetyl CoA carboxylase in the first step of fatty acid biosynthesis. Using the CA inhibitor ethoxyzolamide in cotton embryos decreased radiolabeled 14 C incorporation into total lipids (Hoang and Chapman, 2002). Also, tobacco (Nicotiana tabacum) cell suspensions incubated with ethoxyzolamide and tobacco CA antisense lines show lower levels of 14 C in total lipids (Hoang and Chapman, 2002).…”

“…Using the CA inhibitor ethoxyzolamide in cotton embryos decreased radiolabeled 14 C incorporation into total lipids (Hoang and Chapman, 2002). Also, tobacco (Nicotiana tabacum) cell suspensions incubated with ethoxyzolamide and tobacco CA antisense lines show lower levels of 14 C in total lipids (Hoang and Chapman, 2002).…”

“…The abundance of CAs in plant leaves as well as their various subcellular locations suggest that CAs may play multiple roles in plant metabolism, notably fatty acid synthesis, amino acid biosynthesis, and photosynthesis (Hatch and Burnell, 1990;Badger and Price, 1994;Fett and Coleman, 1994;Raven and Newman, 1994;Hoang and Chapman, 2002). Clearly, any metabolic reaction that produces or consumes CO 2 or HCO 3 2 has the potential to be affected by CA activity.…”