Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthetic CO2 assimilation

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 microbars CO2 and an irradiance of 1000 micromole 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 ppt) for low-CA plants than for wildtype plants. This corresponds to a 15-microbar 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 CO2 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.