Carbonic Anhydrase Activity in Leaves and Its Role in the First Step of C₄ Photosynthesis

Abstract

In C4 plants carbonic anhydrase catalyzes the critical first step of C4 photosynthesis, the hydration of CO2 to bicarbonate. The maximum activity of this enzyme in C4 leaf extracts, measured by H production with saturating CO2 and extrapolated to 250C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C3 leaf extracts. However, the calculated effective activity of this enzyme at in vivo C02 concentrations was apparently just sufficient to prevent the rate of conversion of CO2 to HC03-from limiting C4 photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.The initial carboxylation reaction of C4 photosynthesis, catalyzed by PEP2 carboxylase, utilizes bicarbonate rather than CO2 as the inorganic carbon substrate (9). To sustain this process, atmospheric CO2 entering mesophyll cells must be rapidly converted to HCO3-and this reaction should rightly be regarded as the first step in C4 photosynthesis. Consistent with this concept is the fact that the CA of C4 leaves is very largely or exclusively confined to the cytosol of mesophyll cells (4, 6, 8), where PEP carboxylase is also located. In spite of this apparently critical role of CA in C4 photosynthesis, the quantitative aspects ofthe operation ofthis enzyme have been largely neglected. The implicit assumption has been that the CA is present in a substantial excess in C4 and also C3 leaves. Hence, most studies have focused on the physical and kinetic properties of the enzyme (10).In this paper we report on the maximum activity of CA in leaf extracts of C4 and C3 plants expressed in units which allow comparison with photosynthetic activity. An error in the leaf activities reported in an earlier study (4) MATERIALS AND METHODS MaterialsPlants were grown in soil in a naturally illuminated glasshouse maintained between 20 and 30°C. Biochemicals were obtained from either Sigma Chemical Co. or BoehringerMannheim (Australia). Preparation of Leaf ExtractsAbout 0.5 g of leaf tissue was vigorously ground for about 20 s in a chilled mortar with 1 mL of either 40 mM barbitone-KOH or 40 mm Hepes-KOH (pH 8.0), each containing 10 mM dithiothreitol. After adding an additional 1 mL of the buffer mixture the homogenate was filtered through Miracloth. A sample of this filtrate was used to determine the Chl content of the extract (1), and the remainder was diluted in the blending buffer as required (usually 1 to 10 diluted) and stored at 0°C prior to being assayed.Assay of Carbonic Anhydrase CA was assayed by two different procedures. In the more conventional assay, the rate of CO2 hydration was measured at 0°C by following the change ofpH traced on chart recorder. Reactions contained 25 mm barbitone-KOH buffer (pH 8.2), in a final volume of 1 mL.

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

“…The HCO 3 Ϫ that is utilized by ACCase to form malonyl-CoA is released as CO 2 by subsequent reactions of the fatty acid synthase complex. It is possible that an enzymatic hydration of CO 2 at this point increases the efficiency of CO 2 utilization in plastids, a concept that is similar to the conservation of CO 2 in mesophyll cells of C 4 plants by the cooperative action of CA and phosphoenolpyruvate carboxylase (Hatch and Burnell, 1990). In fact, CA in plastids may interact specifically with ACCase and enzymes of the fatty acid synthase complex to efficiently "channel" carbon into fatty acid (Roughan and Ohlrogge, 1996), although a direct interaction remains to be shown.…”

“…Also, Maeda et al (2000) identified a gene (CmpA) that encodes a substrate-binding protein that can specifically bind to HCO 3 Ϫ in cyanobacteria, which may further aid in the diffusion of HCO 3 Ϫ and elevation of CO 2 in the carboxysome. In C 4 plants, CA is localized to the cytosol of mesophyll cells, where it supplies HCO 3 Ϫ to phosphoenolpyruvate carboxylase (Burnell and Hatch, 1988;Hatch and Burnell, 1990), and calculations show that without CA, the rate of photosynthesis is reduced. In fact, a recent study with transgenic Flaveria bidentis clearly supported a role for CA in the assimilation of CO 2 in C 4 plants (Ludwig et al, 1998).…”

Carbonic anhydrases in plants and algae

“…The mesophyll CA is thought to play a crucial role in C4 photosynthesis by providing HCO 3 -for PEP carboxylase (Hatch & Burnell 1990). In fact, the presence of CA in C4 plants has been suggested to accelerate the rate of photosynthesis in C4 plants 10 4 -fold over what it would be if this enzyme were absent .…”

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

“…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.…”

“…CA activity may play an important role in C 4 photosynthesis, as the majority of carbon fixed by phosphoenolpyruvate carboxylase (PEPC) that moves through the C 4 cycle initially passes through a CAcatalyzed reaction (Hatch and Burnell, 1990;Badger and Price, 1994). CA antisense constructs, which reduce the activity of cytosolic CA in Flaveria bidentis mesophyll cells by at least 70%, lead to diminished rates of photosynthesis, although CA levels must be severely reduced in order to see effects on photosynthesis rates, due to the high enzymatic activity of CA (von Caemmerer et al, 2004).…”