Abstract
As an approach to understanding the physiological role of chloroplast carbonic anhydrase (CA), this study reports on the production and preliminary physiological characterisation of transgenic tobacco (Nicotiana tabacum L.) plants where chloroplast CA levels have been specifically suppressed with an antisense construct directed against chloroplast CA mRNA. Primary transformants with CA levels as low as 2% of wild-type levels were recovered, together with intermediate plants with CA activities of about 20–50% of wild-type levels. Plants with even the lowest CA levels were not morphologically distinct from the wild-type plants. Segregation analysis of the low-CA character in plants grown from T1 selfed seed indicated that at least one of the low-CA plants appears to have two active inserts and that at least two of the intermediate-CA plants have one active insert. Analysis of CO2 gas exchange of a group of low-CA plants with around 2% levels of CA indicated that this large reduction in chloroplastic CA did not appear to cause a measurable alteration in net CO2 fixation at 350 μbar CO2 and an irradiance of 1000 μmol quanta·m−2·s−1. In addition, no significant differences in Rubisco activity, chlorophyll content, dry weight per unit leaf area, stomatal conductance or the ratio of intercellular to ambient CO2 partial pressure could be detected. However, the carbon isotope compositions of leaf dry matter were significantly lower (0.85%o) for low-CA plants than for wildtype plants. This corresponds to a 15-μbar reduction in the CO2 partial pressure at the sites of carboxylation. The difference, which was confirmed by concurrent measurement of discrimination with gas exchange, would reduce the CO2 assimilation rate by 4.4%, a difference that could not be readily determined by gas-exchange techniques given the inherent variability found in tobacco. A 98% reduction in CA activity dramatically reduced the 18O discrimination in CO2 passing over the leaf, consistent with a marked reduction in the ratio of hydrations to carboxylations. We conclude that a reduction in chloroplastic CA activity of two orders of magnitude does not produce a major limitation on photosynthesis at atmospheric CO2 levels, but that normal activities of the enzyme appear to play a role in facilitated transfer of CO2 within the chloroplast, producing a marginal improvement in the efficiency of photosynthesis in C3 plants.
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Abbreviations
- CA:
-
carbonic anhydrase
- Chl:
-
chlorophyll
- pa :
-
ambient CO2 partial pressure
- pc :
-
CO2 partial pressure in the chloroplast stroma
- pi :
-
CO2 partial pressure in the intercellular air spaces
- Rubisco:
-
ribulose bisphosphate carboxylase-oxygenase
- WT:
-
wild type
- Δ :
-
carbon isotope discrimination
References
Badger, M.R., Price, G.D. (1989) Carbonic anhydrase associated with the cyanobacterium Synechococcus PCC7942. Plant Physiol 89, 51–60
Badger, M.R., Price, G.D. (1992) The CO2 concentrating mechanism in cyanobacteria and microalgae (Mini-Review). Physiol. Plant. 84, 606–615
Brugnoli, E., Hubick, K.T. von Caemmerer, S., Wong, S.C., Farquhar, G.D. (1988) Correlation between the isotopic discrimination in leaf starch and sugars of C3 plants and the ratio of intracellular and atmospheric partial pressure of carbon dioxide. Plant Physiol. 88, 1418–1424
Burnell, J.N., Gibbs, M.J., Mason, J.G. (1990) Spinach chloroplast carbonic anhydrase. Nucleotide sequence analysis of cDNA. Plant Physiol. 92, 37–40
Cowan, I. R. (1986) Economics of carbon fixation in higher plants. In: On the economy of plant form and function, pp. 133–170, Givnish, T.J. ed. Cambridge University Press, Cambridge
Edwards, G.E., Mohamed, A.K. (1973) Reduction in carbonic anhydrase activity in zinc deficient leaves of Phaseolus vulgaris L. Crop Sci. 13, 351–354
Evans, J.R., Sharkey, T.D., Berry, J.A., Farquhar, G.D. (1986) Carbon isotope discrimination measured concurrently with gas exchange to investigate CO2 diffusion in leaves of higher plants. Aust. J. Plant Physiol. 13, 281–293
Everson, R.G. (1970) Carbonic anhydrase and CO2 fixation in intact chloroplasts. Phytochemistry 9, 25–32
Farquhar, G.D., von Caemmerer, S. (1982) Modelling of photosynthetic response to environmental conditions. In: Encyclopedia of plant physiology, vol. 12B: Water relations and photosynthetic productivity, pp. 549–587, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer, Berlin Heidelberg New York
Farquhar, G.D., Lloyd, J., Taylor, J.A., Flanagan, L.B., Syvertsen, J.P., Hubick, K.T., Wong, S.C., Ehleringer, J.R. (1993) Vegetation effects on the isotope composition of oxygen in atmospheric CO2. Nature 363, 439–443
Fett, J.P., Coleman, J.R. (1993) Two different cDNAs encode carbonic anhydrase in Arabidopsis thaliana. Plant Mol. Biol., in press
Graham, D., Reed, M.I., Patterson, B.D., Hockley, D.G. (1984) Chemical properties, distribution and physiology of plant and algal carbonic anhydrases. Ann. N.Y. Acad. Sci. 429, 222–237
Hatch, M.D., Burnell, J.N. (1990) Carbonic anhydrase activity in leaves and its role in the first step of C4 photosynthesis. Plant Physiol. 93, 825–828
Herrera-Estrella, L., Simpson, J. (1988) Foreign gene expression in plants. In: Plant molecular biology: A practical approach, pp. 131–160, Shaw, C.H., ed. IRL Press, Oxford
Hubick, K.T. Farquhar, G.D., Shorter, R. (1986) Correlation between water-use efficiency and carbon isotope discrimination in diverse peanut (Arachis) germplasm. Aust. J. Plant Physiol. 13, 803–816
Hudson, G.S., Evans, J.R., von Caemmerer, S., Arvidsson, Y.B.C., Andrews, T.J. (1992) Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants. Plant Physiol. 98, 294–302
Jacobson, B.S., Fong, F., Heath, R.L. (1975) Carbonic anhydrase of spinach. Studies on its location, inhibition and physiological function. Plant Physiol. 55, 468–474
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987) GUS fusions: β-glucuonidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907
Kachru, R.B., Anderson, L. (1974) Chloroplast and cytosolplasmic enzymes. V. Pea-leaf carbonic anhydrase. Planta 118, 235–240
Laisk, A., Oja, V., Kiirats, O., Raschke, K., Heber, U. (1989) The state of the photosynthetic apparatus in leaves as analysed by rapid gas exchange and optical methods: the pH of the chloroplast stroma and activation of enzymes in vivo. Planta 177, 350–358
Lloyd, J., Syvertsen, J.P., Kriedemann, P.E., Farquhar, G.D. (1992) Low conductances for CO2 diffusion from stomata to the sites of carboxylation in leaves of woody species. Plant Cell Environ. 15, 873–899
Magid, E., Turbeck, B.O. (1968) The rates of the spontaneous hydration of CO2 and the reciprocal reaction in neutral aqueous solutions between 0° and 38°. Biochim. Biophys. Acta 165, 515–524
Majeau, N., Coleman, J.R. (1992) Nucleotide sequence of a complementary DNA encoding tobacco chloroplastic carbonic anhydrase. Plant Physiol. 100, 1077–1078
Majeau, N., Coleman, J.R. (1993) Co-ordination of carbonic anhydrase and Rubisco expression in Pea. Plant Physiol., in press
Makino, A., Sahashita, H., Hidema, J., Mae, T., Ojima, K., Osmond, C. (1992) Distinctive responses of ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase in wheat leaves to nitrogen nutrition and their possible relationships to CO2transfer resistance. Plant Physiol. 100, 1737–1743
Mead, D.A., Skorupa, E.S., Kemper, B. (1986) Single-stranded DNA “blue” T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1, 67–74
Nagel, R., Elliott, A., Masel, A. (1990) Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol. Lett. 67, 325–326
Oja, V., Laisk, A., Heber, U. (1986) Light induced alkalization of the chloroplast stroma in vivo as estimated from the CO2 capacity of intact sunflower leaves. Biochim. Biophys. Acta 849, 355–365
Peet, M.M., Huber, S.C., Patterson, D.T. (1986) Acclimation to high CO2 in monoecious cucumber. II. Carbon exchange rates, enzyme activities, and starch and nutrient conditions. Plant Physiol. 80, 63–67
Pocker, Y., Ng, J.S.Y. (1973) Plant carbonic anhydrases and the carbon dioxide hydration kinetics. Biochemistry 12, 5127–5134
Poincelot, R.P. (1979) Carbonic anhydrase. In: Gibbs, M., Latzko, E. eds. Photosynthesis II: Photosynthetic carbon metabolism and related processes, pp. 230–238, Springer, Berlin
Porra, R.J., Thompson, W.A., Kreidemann, P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four solvents: verification of the concentration of chlorophyll standards by absorption spectroscopy. Biochim. Biophys. Acta 975, 384–394
Porter, M.A., Grodzinski, B. (1984) Acclimation to high CO2 in bean: carbonic anhydrase and ribulose bisphosphate carboxylase. Plant Physiol. 74, 413–416
Price, G.D., Coleman, J.R., Badger, M.R. (1992) Association of carbonic anhydrase activity with carboxysomes isolated from the cyanobacterium, Synechococcus PCC7942. Plant Physiol. 100, 784–793
Randall, P.J., Bouma, D. (1973) Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach. Plant Physiol. 52, 229–232
Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Shure, M., Wessler, S., Fedoroff, N. (1983) Molecular identification and isolation of the Waxy locus in maize. Cell 35, 225–233
Swader, J.A., Jacobson, B.S. (1972) Acetazolamide inhibition of photosystem II in isolated spinach chloroplasts. Phytochemistry 11, 65–70
Tsuzuki, M., Miyachi, S., Edwards, G.E. (1985) Localization of carbonic anhydrase in mesophyll cells of terrestrial C3 plants in relation to CO2 assimilation. Plant Cell Physiol. 26, 881–891
von Caemmerer, S., Evans, J.R. (1991) Determination of the average partial pressure of CO2 in chloroplasts from leaves of several C3 plants. Aust. J. Plant Physiol. 18, 287–305
von Caemmerer, S., Farquhar, G.D. (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 376–387
von Caemmerer, S., Evans, J.R., Hudson, G.S., Arvidsson, Y.B.C., Setchell, B.A., Andrews, T.J. (1992) Photosynthesis in transgenic tobacco plants with reduced Rubisco contents. In: Research in photosynthesis, vol. IV, pp. 595–602, Murata, N. ed. Kluwer Academic Publishers, The Netherlands
Werden, K., Heldt, H.W. (1972) Accumulation of bicarbonate in intact chloroplasts following a pH gradient. Biochim. Biophys. Acta 283, 430–441
Wilbur, K.M., Anderson, N.G. (1948) Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176, 147–154
Yu, J-W., Woo, K.C. (1988) Glutamine transport and the role of the glutamine translocator in chloroplasts. Plant Physiol. 88, 1048–1054
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We thank Dr. John R. Coleman (Department of Botany, University of Toronto, Canada) for making his manuscript available to us prior to acceptance and Barbara Setchell for help with plant maintenance. G.D.P. acknowledges support from a QE II Research Fellowship (Australian Research Council).
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Price, G.D., von Caemmerer, S., Evans, J.R. et al. Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthetic CO2 assimilation. Planta 193, 331–340 (1994). https://doi.org/10.1007/BF00201810
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DOI: https://doi.org/10.1007/BF00201810