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...

Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-grown cotton (Gossypium hirsutum L.) seedling cDNA library. Nucleotide sequence analysis revealed two different CA isoforms designated GhCA1 and GhCA2. The encoded polypeptides possess N-terminal serine/threonine-rich regions indicative of plastid transit peptides, and approximately 80% sequence identity to other plant plastidial beta-CAs. The GhCA1 cDNA encodes a nearly complete preprotein of 323 amino acids with a molecular mass of 34.9 kDa and a predicted mature protein of 224 amino acids with a molecular mass of 24.3 kDa. Eleven nucleotide differences within ORFs of GhCA1 and GhCA2 result in 5 conservative amino acid substitutions. The 3' GhCA2 untranslated region contains five additional substitutions and one single nucleotide addition. GhCA1 clones, nearly full-length or with 70% of the transit peptide deleted, were expressed as LacZ alpha fusion proteins in E. coli. Lysates of these strains contained 9-fold higher levels of CA activity as compared to untransformed controls and this activity was inhibited by CA-specific inhibitors. Sulfanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombinant CA activity approximately 50%, 65%, and 75%, respectively. In plant tissue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively. Although CA activity was bighest in extracts of mature cotton leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in the cotyledons of dark-grown cotton seedlings. Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated seeds, suggesting a role for CA in postgerminative growth.

Recently, plastidial carbonic anhydrase (CA) cDNA clones encoding functional carbonic anhydrase enzymes were isolated from a 48 h dark-grown cotton seedling (cotyledons) cDNA library (Hoang et al., Plant Cell Physiol. 40: 1999). Here we examined the levels of relative transcript abundance and enzyme activities in cotyledons at different developmental stages and under different environmental conditions (i.e. altering CO2 and light conditions), during post-germinative seedling growth. Relative CA transcript levels and total CA enzyme activity in cotyledons of cotton seedlings increased from 18 h to 72 h of post-germinative growth in the dark, although somewhat later than the glyoxylate cycle enzyme, MS. When 24 h old seedlings were exposed to light for an additional 24 h, CA activity in greening cotyledons increased about 2-fold (compared with controls kept in the dark), whereas relative CA transcript levels were essentially the same. Removal of seed coats from cotyledons of 24 h old seedlings dramatically increased relative CA transcript abundance (measured 24 h later) in the dark, but did not influence CA enzyme activity. Manipulation of external CO2 environments (zero, ambient, or high) modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activity. On the other hand, regardless of the external CO2 conditions, cotyledons of seedlings exposed to light exhibited increased CA activity, concomitant with increased Rubisco activity and increased chlorophyll content. Collectively, our data suggest that steady-state levels of CA and rbcS transcripts are increased in response to environmental CO2 conditions, while CA (and Rubisco) enzyme activities are likely modulated at the post-transcriptional level following exposure of seedlings to light, and in parallel with development of functional chloroplasts.