Carbonic Anhydrase Activity and CO₂-Transfer Resistance in Zn-Deficient Rice Leaves

Abstract

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO₂ fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO₂-assimilation process increased from 10% to 21%. This change led to a reduced CO₂ concentration at the site of CO₂ fixation, resulting in an increased gradient of CO₂ between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO₂ from the stomatal cavity to the site of CO₂ fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.

CA (EC 4.2.1.1) catalyzes the reversible conversion of CO₂ to HCO₃, which can be dissolved more easily, and has been recognized as an important enzyme that is closely associated with photosynthesis. However, the application of inhibitors of CA to intact chloroplasts did not lower the rate of photosynthesis (Swader and Jacobson, 1972; Jacobson et al., 1975), suggesting that CA is not essential for the photosynthetic assimilation of CO_2. It has also been reported that inactivation of CA mRNA by the introduction of antisense RNA had no significant negative effect on the photosynthetic assimilation of CO₂ by tobacco leaves (Majeau et al., 1994; Price et al., 1994; Williams et al., 1996). In spite of experimental data showing the absence of an association of CA with photosynthesis, researchers showed that CA increased the CO₂ concentration at the site of CO₂ fixation in chloroplasts by 15 to 20 μL L⁻¹, and that CA played a role in facilitating CO₂ diffusion through the use of ¹³C and ¹⁸O in CO₂ (Price et al., 1994; Williams et al., 1996).

In a previous study we tried to separate the r_m into the r_r and the r_c by measuring the δ¹³C, and we succeeded in estimating the magnitude of the r_r from the intercellular space of mesophyll cells to the site of CO₂ fixation in the chloroplasts (Sasaki et al., 1996). The purpose of the present study was to elucidate the relationship between the activity of CA and each component of mesophyll resistance in an attempt to determine the mechanism that explains why CA activity is not related to photosynthetic performance.

CA is a Zn-containing enzyme, so Zn is essential for its catalytic activity (Bar-Akiva and Lavon, 1969; Silverman, 1991). Guliev et al. (1992) reported that removal of Zn from CA in vitro resulted in the irreversible loss of catalytic activity. Several authors have also reported that CA activity can be specifically inhibited by Zn deficiency in some plants without any significant reduction in the rate of photosynthesis (Edwards and Mohamed, 1973; Randall and Bouma, 1973; Ohki, 1976, 1978). Therefore, the second goal of this study was to elucidate the mechanism of the specific inhibition of CA activity by Zn deficiency at the level of expression of CA mRNA.

MATERIALS AND METHODS

Cultivation of Plants

Seeds of rice (Oryza sativa L. cv Hatsunishiki) were sown on wet quartz sand. Four weeks later, seedlings were transplanted to 40-L polyethylene containers filled with culture solution prepared as described by Yoshida et al. (1976) at a density of 56 plants per container. The solution contained 1.425 mm NH₄NO₃, 0.323 mm NaH₂PO₄, 0.513 mm K₂SO₄, 0.998 mm CaCl₂, 0.998 mm MgSO₄, 0.009 mm MnCl₂, 0.075 μm (NH₄)₆Mo₇O₂₄, 0.019 mm H₃BO₃, 0.155 μm CuSO₄, and 0.036 mm FeCl₃. The plants were treated with 150 nm ZnCl₂ from 4 weeks after germination (+Zn), with 15 nm ZnCl₂ from 11 weeks after germination (±Zn), and without Zn (−Zn). Three containers were prepared for each treatment. The plants were grown in a naturally illuminated greenhouse that was air conditioned to give a day temperature of 30°C and a night temperature of 25°C.

Determination of Zn Content

The leaf area of the uppermost fully expanded leaf on the main stem was measured for 6 plants from each container (18 plants for each treatment) 13 weeks after germination, and these leaves were dried in an electric dryer. The dry weight was determined and the specific leaf weight was calculated. Samples of 0.1 g dry weight were digested overnight in 5.0 mL of concentrated HNO₃ and 0.3 mL of 40% (w/v) HF in a 50-mL Teflon beaker, and wet ashed on the heater the following day. The Zn content was determined with an inductively coupled plasma atomic spectrometer (SPS-7000A, Seiko Instruments, Tokyo, Japan).

Measurement of the Gas-Exchange Rate

Leaf photosynthesis and transpiration were measured simultaneously for the uppermost fully expanded leaf on the main stem for 6 plants from each container (18 plants for each treatment) 13 weeks after germination using a handmade gas-exchange system with an IR gas analyzer (ZAP-AZ012, Fuji Electric Co. Ltd., Tokyo, Japan) and a humidity sensor (HMP111Y, Visala, Helsinki, Finland). A leaf (approximately 8–12 cm²) was clamped in the leaf chamber with a water jacket providing background cooling, and maintained under controlled conditions of temperature, light, humidity, and CO₂ concentration. The measurements were made at a CO₂ concentration of 340 μL L⁻¹, an irradiance greater than 1400 μmol m⁻² s⁻¹ photon flux density under artificial light, and a leaf temperature of 30°C ± 3°C, after preillumination for 60 min to obtain steady-state readings of photosynthesis and transpiration.

Measurement of ¹³C Values of Soluble Sugars

Ten plants in each container were placed in darkness at 12 pm to starve the leaves of photosynthetic products. The next morning the leaves were exposed to artificial light at an irradiance greater than 1400 μmol m⁻² s⁻¹ photon flux density from 9 am to 12 pm in a 72-m³ laboratory ventilated at 2160 m³ h⁻¹, and were then cut off from the plant. No one entered the laboratory during the exposure and there was no difference between the values of the isotopic composition of CO₂ in the air in this room and outside. The accumulation of soluble sugars was conducted for three treatments at the same time. The soluble sugars were extracted and ¹³C was determined with a mass spectrometer (MAT-252, Finnigan MAT, San Jose, CA) as reported previously (Sasaki et al., 1996).

¹³C Determination

¹³C of soluble sugars was determined with a mass spectrometer according to the method of Sasaki et al. (1996). Δ can be expressed by the following equation (Hubick et al., 1986):

1

where δ_a and δ_p are the relative concentrations of ¹³C in the atmospheric air and in the photosynthetic products, respectively. The δ_a was determined as −8.0‰ ± 0.1‰ from the actual measurement of the air in our laboratory, and δ_p was determined by the mass-spectrometric method with soluble sugars extracted from the leaves. The Δ obtained was inserted into Equation 2 (Sasaki et al., 1996):

2

where 4.4 is the discrimination coefficient in the CO₂-diffusion process through stomata in C₃ plants, 1.1 is the discrimination coefficient in the CO₂-dissolution process in water at 25°C, 0.7 is the discrimination coefficient in the CO₂-diffusion process in the liquid phase at 25°C, and 29 is the discrimination coefficient in the CO₂-carboxylation process by Rubisco. Carbon discrimination also occurred in the processes of dark respiration and photorespiration; however, we assumed the value of these factors to be zero because according to published reports the extent is almost negligible (Farquhar et al., 1982; Evans et al., 1986). [CO₂]_atm was maintained at about 340 μL L⁻¹ in this experiment and [CO₂]_stc was obtained from the measurement of photosynthesis and transpiration. Therefore, [CO₂]_cht can be obtained from Equation 2 after inserting the value of Δ determined from Equation 1.

Calculation of Stomatal Transfer and Fixation Resistances

The r_s and the r_m were calculated using the following equations:

3

4

where e_i and e_a are the intercellular and atmospheric vapor pressures, respectively, Tr is transpiration, LPS is leaf photosynthesis, and Γ is the CO₂-compensation point. The factor 1.56 is the ratio of the diffusivities of water vapor and CO₂ in air. If [CO₂]_cht is obtained from Equation 2, the r_r from the stomatal cavity to the site of CO₂ fixation and the r_c can be obtained using the following equations:

5

6

Determination of CA Activity

CA activity was measured in the uppermost fully expanded leaf on the main stem for 6 plants from each container (18 plants for each treatment). The detached leaves, which had been illuminated for 1 h at 1400 μmol m⁻² s⁻¹ photon flux density, were ground with a buffered solution (pH 8.3) that contained 50 mm barbital-H₂SO₄, 5 mm DTT, and 0.2% (w/v) PVP. The homogenate was centrifuged at 12,000g for 2 min, and the supernatant was used for the determination of CA activity, according to the method of Sasaki et al. (1996).

Determination of Rubisco Content and Activity

The content of Rubisco was determined in the uppermost fully expanded leaf on the main stem for six plants from each container (18 plants for each treatment). The leaves were excised immediately after the measurement of leaf photosynthesis and stored at −80°C in a freezer before the determinations. The content of Rubisco was determined by the method of Sasaki et al. (1996). The activity of Rubisco was measured in terms of the initial activity at 30°C according to the method described by Usuda (1985) for the uppermost fully expanded leaf on the main stem for 6 plants from each container (18 plants for each treatment) after illumination for 1 h at 1400 μmol m⁻² s⁻¹ photon flux density.

Quantitation of Soluble Protein

The amount of soluble protein in the supernatant prepared for the measurement of the content of Rubisco was determined as described by Lowry et al. (1951) using BSA as the standard.

Quantitation of Chlorophyll

The amount of chlorophyll in the homogenate prepared for the measurement of Rubisco was determined for 6 plants from each container (18 plants for each treatment) as described by Schmid (1971).

Isolation of RNA and Northern-Blot Hybridization

Total RNA was isolated from 0.2 g of frozen sample as described by Fromm et al. (1985). Total RNA (20 μg per lane) was fractionated on a 1.15% agarose gel that contained 1.85% (v/v) formaldehyde, transferred to a nylon membrane (Hybond-N, Amersham), and allowed to hybridize with radiolabeled DNA probes. After hybridization the blot was washed twice in 2× SSC (SSC contains 0.15 m NaCl and 0.015 m trisodium citrate) containing 0.1% SDS at 42°C for 5 min, and then washed again in 0.2× SSC containing 0.1% SDS at 45°C for 1 h. The signals attributable to the radiolabeled probe were visualized and quantified with a Bio-Imaging Analyzer (BAS 2000, Fujix, Tokyo, Japan). A 0.64-kb fragment of cDNA for rice chloroplastic CA (S. Suzuki and J.N. Burnell, unpublished data; accession no. U08404) and a full-length clone of the rice Rubisco small subunit (Matsuoka et al., 1988; accession no. D00644) were used as the probes.

RESULTS

Levels of Zn and Enzymes and Rates of Photosynthesis

The Zn contents per unit leaf area of the −Zn and ±Zn plants were as low as 0.19 and 0.23 mg m⁻², respectively, compared with 0.51 mg m⁻² in the +Zn plants (Table I). In spite of the great reduction in the Zn content of leaves, we observed no change in specific leaf weight or in chlorophyll content, which is considered to be a critical indicator of Zn deficiency. Therefore, −Zn and ±Zn plants were only moderately stressed. In contrast, although the CA activity in −Zn plants decreased dramatically to as little as 14% of that in +Zn plants, the Rubisco activity decreased only to 89% of that in +Zn plants. The levels of soluble protein and Rubisco increased slightly. Moreover, little change in the rate of photosynthesis was observed in the leaves with a reduced Zn content. These results indicated that Zn deficiency resulted in the specific inhibition of CA activity.

Table I.

Effects of Zn deficiency on Zn content, photosynthesis, δ¹³C value, chlorophyll content, and levels of CA and Rubisco in fully expanded leaves

Parameter	+Zn	±Zn	−Zn
Zn content (mg m−2)	0.51  ± 0.05	0.23  ± 0.03 (45)	0.19  ± 0.04 (37)
Specific leaf wt (g m−2)	19.3  ± 2.0	20.2  ± 2.6 (105)	19.9  ± 3.6 (103)
Chlorophyll content (g m−2)	0.59  ± 0.09	0.49  ± 0.15 (82)	0.45  ± 0.14 (78)
Photosynthesis (μmol CO2 m−2 s−1)	22.9  ± 1.7	19.7  ± 3.6 (86)	20.4  ± 2.3 (89)
Soluble protein (g m−2)	6.5  ± 0.3	7.7  ± 0.3 (118)	7.5  ± 0.2 (116)
CA activity (× 103 units m−2)	473  ± 57	126  ± 23 (27)	68  ± 36 (14)
Rubisco content (g m−2)	3.5  ± 0.3	4.3  ± 0.3 (126)	4.8  ± 0.4 (139)
Rubisco activity (μmol CO2 m−2 s−1)	29.4  ± 2.6	23.5  ± 4.3 (80)	26.3  ± 5.2 (89)
δ13C value (‰)	−25.83  ± 0.24	−24.05  ± 1.27	−23.55  ± 1.91

Values are expressed as means ± se; n = 18 except for the δ¹³C value (n = 3). Numbers in parentheses are percentages relative to values for +Zn plants.

Stomatal Transfer and Fixation Resistance

To clarify the contribution of CA activity to the assimilation process, we calculated the r_c and the r_r by measuring leaf photosynthesis and ¹³C in the soluble sugars extracted from the leaves (Table II). Although no significant difference was found in r_c and r_s in leaves of +Zn, −Zn, and ±Zn plants, we observed a 2.3-fold increase in r_r, which increased from 1.2 mol⁻¹ CO₂ m² s in +Zn plants to 2.7 mol⁻¹ CO₂ m² s in −Zn plants. This corresponded to an increase from 10% to 21% when r_r was calculated as a percentage of total resistance. This indicates that the Zn deficiency affected only the CO₂-transfer step in the CO₂-assimilation process.

Table II.

Effects of Zn deficiency on CO²-diffusion resistance

Resistance	+Zn	±Zn	−Zn
	mol−1 CO2 m2 s
Stomatal	4.7  ± 0.5 (40)	5.7  ± 0.6 (42)	5.2  ± 0.5 (40)
Mesophyll	7.0  ± 0.5 (60)	8.0  ± 1.6 (58)	8.0  ± 1.2 (60)
CO2 transfer	1.2  ± 0.3 (10)	2.2  ± 0.9 (16)	2.7  ± 0.7 (21)
CO2 fixation	5.9  ± 0.4 (50)	5.8  ± 0.8 (42)	5.3  ± 0.6 (40)

Values are expressed as means ± se; n = 18 for stomatal and mesophyll resistances and n = 3 for CO₂-transfer and CO₂-fixation resistances. Numbers in parentheses are percentages relative to the total resistance.

The Concentration of CO₂ in Leaves

In all leaves examined [CO₂]_stc was maintained at about 220 μL L⁻¹. In contrast, [CO₂]_cht in +Zn leaves was 195 ± 4 μL L⁻¹, and [CO₂]_cht decreased with a reduction in the Zn content of leaves to 171 ± 6 and 166 ± 5 μL L⁻¹ in ±Zn and −Zn leaves, respectively. The gradient of the CO₂ concentration between the stomatal cavity and the site of CO₂ fixation increased from 29 to 59 μL L⁻¹ (Fig. 1). The resistance in CO₂ flux with Zn deficiency and our findings support the hypothesis that CA plays a role in facilitating the supply of CO₂ to the sites of carboxylation.

Figure 1.

[CO₂]_stc and [CO₂]_cht in plants with three different levels of Zn. Values are expressed as means ± se. See text for details.

Northern-Blot Analysis

The results of northern-blot analysis of mRNAs for CA and Rubisco are shown in Figure 2. The level of the mRNA for CA decreased with the reduction in Zn content of the leaf, decreasing to 26% (±Zn) and 13% (−Zn) of that in control plants (+Zn). In contrast, the level of the mRNA for the small subunit of Rubisco showed no consistent trend. It seems likely that the reduction in CA activity was caused not by deactivation of the enzyme but, rather, by a decrease in the expression of the CA mRNA caused by Zn deficiency.

Figure 2.

Northern-blot analysis of the expression of mRNAs for CA and the small subunit of Rubisco in plants with three different levels of Zn. Total RNA, 20 μg per lane, was separated on 1.5% agarose containing formaldehyde, transferred to a nylon membrane, and hybridized to radiolabeled DNA probes. Each level of Zn included three lanes and each lane derived from one container. Levels of mRNAs in +Zn plants are set at 100%. Values are expressed as means ± se. See text for details. Ssu, Small subunit of Rubisco.

DISCUSSION

CA has been recognized as an important enzyme that is closely associated with photosynthesis. In C₃ plants CA activity is located mainly in the mesophyll chloroplast, with much smaller activity in the cytosol. Chloroplastic CA activity was reported as 87% of total cellular activity in potato leaves (Rumeau et al., 1996), and from 86% to 95% in wheat, spinach, Moricandia arvensis, and Mesembryanthemum crystallinum (Tsuzuki et al., 1985). Recently, cytosolic and chloroplastic CA isoenzymes were isolated in Arabidopsis and potato, and a polyclonal antibody hybridized to two kinds of CA (Fett and Coleman, 1994; Rumeau et al., 1996). The hypothesis that an alternative processing occurred during transit-peptide removal has been proposed by Johansson and Forsman (1992). Furthermore, two Arabidopsis cDNA clones encoding putative cytosolic and chloroplastic CAs were isolated, and comparison of the predicted amino acid sequences indicated very similar genes (Fett and Coleman, 1994). In our experiment CA activity in the crude extract was measured and cDNA for rice chloroplastic CA was used as a probe for the northern-blot analysis. Considering the localization, percentage, and homology, although the CA activity and mRNA expression for CA in our study could not be distinguished from those for cytosolic and chloroplastic CA, they were considered to consist mainly of chloroplastic activity and mRNA expression for chloroplastic CA, respectively.

The main purpose of this study was to analyze the relationship between the activity of CA and each component of mesophyll resistance by using plants with reduced CA activity as a consequence of Zn deficiency. Major reductions in CA activity to 27% and 14% in ±Zn and −Zn plants, respectively, of that in +Zn plants were observed without any change in r_s or r_c. Therefore, we were able to detect a correlation between the transfer of CO₂ from the stomatal cavity to the site of CO₂ fixation and CA activity. It appeared that r_r increased with the reduction in CA activity (Tables I and II). Although [CO₂]_stc was maintained at about 220 μL L⁻¹ during photosynthesis, [CO₂]_cht decreased with the reduction in CA activity, and the gradient of the concentration of CO₂ between the stomatal cavity and the site of CO₂ fixation increased from 29 to 59 μL L⁻¹ with the reduction in CA activity (Fig. 1). With antisense RNA for CA a great reduction in the levels of CA to as little as 2% of wild-type levels caused an increase in the gradient of the CO₂ concentration of about 15 μmol mol⁻¹ (Price et al., 1994; Williams et al., 1996), which is similar to our results. Consequently, we can conclude that a reduction in CA activity tampers with the transfer of CO₂ from the stomatal cavity to the site of Rubisco and causes a low [CO₂]_cht. CA is mostly localized in chloroplasts in C₃ plants. Therefore, our findings support the hypothesis that CA has a role in facilitating the supply of CO₂ to the sites of carboxylation within the chloroplast.

The considerable reduction in CA activity was accompanied by the reduction in Zn content, but we observed no major change in the rate of photosynthesis. In the antisense analysis, rates of CO₂ assimilation were also unaffected by low levels of CA in chloroplasts in transgenic tobacco plants (Majeau et al., 1994; Price et al., 1994). Such observations might be explained by the smaller contribution of the CO₂-transfer process than the stomatal or CO₂-fixation processes (Sasaki et al., 1996). Although [CO₂]_cht decreased with a reduction in the activity of CA from 195 to 166 μL L⁻¹, we could not observe any marked decrease in the rate of photosynthesis as in [CO₂]_cht. If the CO₂-compensation point and other factors were constant, it is possible that a 29 μL L⁻¹ difference in [CO₂]_cht would cause an 18% to 20% decline in the rate of photosynthesis. We found that the rate of photosynthesis decreased only to 89% of that in +Zn plants. Although the initial activity of Rubisco was similar to the rate of photosynthesis, the Rubisco content unexpectedly increased slightly, from 3.5 to 4.8 g m⁻², with a reduction in the Zn content of leaves (the increase of Rubisco content has not been shown in other experiments on Zn deficiency). We suggest that the absence of any additional decrease in photosynthesis might be explained by the increase in Rubisco content.

Zn is necessary for catalysis by CA (Bar-Akiva and Lavon, 1969; Silverman, 1991). In this study expression of the mRNA for CA decreased to 34% (±Zn) and 13% (−Zn) of that in control plants. This decrease resembled that in the CA activity in these plants with the reduction in the Zn content of leaves (Table I; Fig. 2). Therefore, we suggest that the reduction in CA activity attributable to Zn deficiency was a result not of failure to activate CA but of a decrease in the level of CA. Although modification of transcript abundance does not always indicate transcriptional regulation, it is possible that a feedback system balances the amount of CA synthesized with the available Zn in the cell.

Some studies on the levels of CA in intact leaves have suggested that coordinated regulation of the expression of CA and Rubisco might occur under some growth conditions, e.g. with changes in the supply of nitrogen (Makino et al., 1992) and in the level of CO₂ (Porter and Grodzinski, 1984; Peet et al., 1986). Previously, we suggested that CA activity, most of which is found in the chloroplast, changes in association with changes in Rubisco activity in the chloroplast (Sasaki et al., 1996). Hudson et al. (1992) reported that CA activity changed in association with Rubisco activity in transgenic tobacco to which antisense Rubisco mRNA had been introduced. The activity ratio of CA to Rubisco is reportedly maintained in spite of differences in the levels of Rubisco and CA among cultivars and among leaf nitrogen contents in pea and wheat (Makino et al., 1992; Majeau and Coleman, 1994). Although such observations suggest the coordinated regulation of levels of CA and Rubisco, this relationship can easily be disrupted in low-CA plants (which are unusual), such as those in this study (Table I), which were induced by antisense RNA (Price et al., 1994). It seems likely that the level of CA is influenced by the level of Rubisco, but not vice versa, and although many studies show coordinated changes in CA and Rubisco, it appears that CA plays a role in facilitating the supply of CO₂ to sites of carboxylation.

Abbreviations:

CA

carbonic anhydrase

[CO₂]_atm

CO₂ concentration in the atmospheric air

[CO₂]_cht

CO₂ concentration at the site of CO₂ fixation in the chloroplast

[CO₂]_stc

CO₂ concentration in the stomatal cavity

Δ

carbon isotope discrimination by the plant

δ_a

relative concentration of ¹³C in the atmospheric air

δ_p

relative concentration of ¹³C in the photosynthetic products

δ¹³C

relative concentration of ¹³C in total carbon atoms

r_c

CO₂-fixation resistance

r_m

mesophyll CO₂ resistance

r_r

CO₂-transfer resistance

r_s

stomatal CO₂ resistance