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 antisense-suppressed 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.

Figure 1

Comparison of triacylglycerol content (A), total CA activity (B), and relative CA expression (C) in embryos excised from cotton bolls at 25 and 40 DPA. The relative amounts of TAG in cell-free homogenates were estimated by scanning densitometry (National Institutes of Health Image) of thin-layer chromatography (TLC)-fractionated lipid classes in comparison with triolein standards. Total CA activity was determined electrometrically in cell free homogenates. Poly(A⁺) RNA was isolated from cotton embryos and the ratios of CA to actin transcripts were evaluated by northern-blot analyses. The results depicted here are representative of replicate experiments.

Figure 2

Effects of different CA inhibitors in homogenates of cotton embryos (34 DPA). The graph shows the percent remaining CA activity when inhibited with 1 and 10 mm sulfanilamide, acetazolamide, and ethoxyzolamide. Values shown represent the means and sds from three separate experiments. CA activity with dimethyl sulfoxide (DMSO) alone in the amounts required to dissolve the above inhibitors was 98.6% ± 4.0% of that without.

Figure 3

Time-dependent incorporation of [¹⁴C]acetate into cottonseed total lipids. Embryos (30–38 DPA) were pre-incubated with DMSO (control) or different concentrations of CA inhibitors. Both 1 and 10 mm concentrations of ethoxyzolamide were incubated for 30 min before the addition of radiolabeled [¹⁴C]acetate. Total lipids were extracted at 10, 15, 20, and 30 min after the addition of the radiolabeled acetate. Incorporation of [¹⁴C]acetate into lipids was not linear after 30 min. Aliquots were used to quantify the incorporation of [¹⁴C]acetate into embryos by liquid scintillation counting. Data points represent mean and sd of three independent experiments for 10 and 30 min. Lines are plotted from linear regression analyses (Prism version 3.02, GraphPad Software, San Diego) of the data with r² = 0.97 for controls, r² = 0.97 for 1 mm ethoxyzolamide, and r² = 0.94 for 10 mm ethoxyzolamide. Rates estimated from linear regression analyses were 12.36 ± 0.71, 9.42 ± 0.52, and 6.15 ± 0.53 pmol acetate min⁻¹ mg⁻¹ protein, respectively. For reference, the rate of acetate incorporation into embryos not pre-incubated with DMSO was 12.44 ± 0.62 pmol acetate min⁻¹ mg⁻¹ protein.

Figure 4

Comparison of embryo protein synthesis in vivo (A) and ACCase activities in vitro in the absence or presence of CA inhibitors. Embryos (34 DPA) were pre-incubated with ethoxyzolamide and acetazolamide 30 min before the addition of [³⁵S]-Met. After 1 h of incubation, the incorporation of [³⁵S]-Met into trichloroacetic acid (TCA)-precipitated protein was quantified by liquid scintillation counting. ACCase activity was assayed in cell free homogenates of 34-DPA embryos. The embryo homogenates were pre-incubated with DMSO (control), 1 mm ethoxyzolamide, 10 mm ethoxyzolamide, or 10 mm acetazolamide for 30 min before assays. Values shown represent the mean and sd from three independent experiments. With DMSO added (the controls shown above), total protein synthesis was 92% ± 9% and ACCase activity was 94% ± 3%, respectively, of that without DMSO.

Figure 5

Incorporation of [¹⁴C]acetate into total lipids of tobacco cv xanthi cell suspensions. Cells were pre-incubated with DMSO (control), 1 mm ethoxyzolamide, or 10 mm ethoxyzolamide for 30 min before the addition of radiolabeled [¹⁴C]acetate. Total lipids were extracted at 1, 2, and 4 h after the addition of the radiolabeled acetate. Data points represent mean and sd of three independent experiments. Lines are drawn from linear regression analyses of the data with r² = 0.98 for controls, r² = 0.98 for 1 mm ethoxyzolamide, and r² = 0.88 for 10 mm ethoxyzolamide. Rates estimated from linear regression analyses were 453.4 ± 42.4, 300.7 ± 31.4, and 159.4 ± 41.3 pmol acetate h⁻¹ mg⁻¹ protein, respectively. For reference, the rate of acetate incorporation into tobacco cell suspensions not pre-incubated with DMSO was 461.3 ± 11.5 pmol acetate h⁻¹ mg⁻¹ protein.

Figure 6

Incorporation of [¹⁴C]acetate into total lipids of chloroplasts isolated from tobacco leaves. Total lipids were extracted at 5 min, 10 min, 30 min, 1 h, 2 h, and 4 h after the addition of radiolabeled acetate (A). CA activity determined in leaves of antisense CA (A and B)-suppressed transgenic and WT plants (B). Data points represent mean and range of duplicate samples within a single experiment. Similar trends were observed in replicate experiments. Lines represent linear regression analyses of the data with r² = 0.95 for controls, r² = 0.98 for antisense plant A (AS-A), and r² = 0.97 for antisense plant B (AS-B). Rates estimated from linear regression analyses were 70.3 ± 35.3, 39.9 ± 1.6, and 41.5 ± 2.3 pmol acetate min⁻¹ mg⁻¹ chlorophyll, respectively.