The CO2 transfer conductance in leaves quantifies the ease with which CO2 can diffuse from sub-stomatal cavities to sites of carboxylation within the chloroplast. The aim of this work was to test the hypothesis that the CO2 transfer conductance is proportional to the surface area of chloroplasts exposed to intercellular airspaces. We compared two genotypes, wild-type and transgenic tobacco, that had been transformed with an antisense gene directed at the mRNA of the Rubisco small subunit. Transgenic tobacco had lower rates of CO2 assimilation than wild-type but similar chlorophyll contents. Leaf anatomy was altered by growing plants in two different environments: a high daily irradiance in a growth cabinet (12 h photoperiod of 1 mmol quanta m-2 s-1) and a sunlit glasshouse. The growth cabinet gave at least twice the daily irradiance compared to the glasshouse. The CO2 transfer conductance was calculated from combined measurements of gas exchange and carbon isotope discrimination measured in 2% oxygen. Following gas exchange measurement, leaves were sampled for biochemical and anatomical measure- ment. In transgenic tobacco plants, Rubisco content was 35% of that found in the wild-type tobacco, the CO2 assimilation rate was 50% of the wild-type rate and the chlorophyll content was unaltered. While leaf mass per unit leaf area of transgenic tobacco was 82% of that of the wild-type, differences in leaf thickness and surface area of mesophyll cells exposed to intercellular airspace per unit leaf area (Smes) were small (92 and 87% of wild-type, respectively). Leaves grown in the growth cabinet under high daily irradiance were thicker (63%), had a greater Smes (41%) due to the development of thicker palisade tissue, had higher photosynthetic capacity (27%) and contained more chlorophyll (58%) and Rubisco (77%), than leaves from plants grown in the glasshouse. Irrespective of genotype or growth environment, CO2 transfer conductance varied in proportion to surface area of chloroplasts exposed to intercellular airspaces. While the method for calculating CO2 transfer conductance could not distinguish between limitations due to the gas or liquid phases, there was no reduction in CO2 transfer conductance associated with more closely packed cells, thicker leaves, nor with increasing chloroplast thickness in tobacco.

Carbon isotope discrimination (Δ) and leaf gas-exchange were measured simultaneously for a number of C4 species. Linear relationships were found between A and the ratio of intercellular to ambient partial pressures of CO2, pI/pa. These data were used to estimate the fraction of CO2 released by C4-acid decarboxylation in the bundle sheath, which subsequently leaks out to the mesophyll. We define this fraction as the leakiness of the system and it is also a measure of the extent to which phosphoenolpyruvate (PEP) carboxylations exceed ribulose 1,5-bisphosphate (RuBP) carboxylations. For Sorghum bicolor and Amaranthus edulis, leakiness was estimated at 0.2 and was constant over a wide range of irradiances (between 480 and 1600 μmol quanta m-2 s-1), intercellular CO2 pressures (between 30 and 350 μbar) and leaf temperatures (from 21�C to 34�C). At irradiances less than 240 μmol quanta m-2 s-1, leakiness appeared to increase. For a number of dicotyledonous and monocotyledonous species, of the various C4-decarboxylation types, leakiness was also estimated at 0.2. Contrary to expectation, amongst the 11 species examined, those with suberised lamellae did not show lower values of leakiness than those without suberised lamellae. For one NAD-ME and one PCK monocot, the estimates of leakiness were significantly higher at 0.30 and 0.25, respectively. Long-term discrimination (assessed from carbon isotope composition of leaf dry matter) did not correlate well with these short- term measures of discrimination. We suggest that this may be due to differences between species in fractionations occurring after photosynthesis.

Tobacco plants with reduced amounts and activities of both the chloroplast cytochrome b6/f and ATP synthase complexes have been produced using antisense RNA techniques. Antisense constructs were generated from tobacco cDNA clones coding for the Rieske FeS protein and the δ subunit of the b6/f and ATP synthase complexes respectively. Transformants with altered activities were selected using pulse-modulated fluorescence measurements. The b6/ftransformants showed high levels of steady-state fluorescence and reduced levels of both photochemical and non-photochemical quenching. In striking contrast, the ATP synthase transformants showed low levels of steady-state fluorescence and greatly increased levels of non-photochemical quenching. Transformants with a range of suppression were isolated for both constructs, in some cases with photosynthesis reduced to less than 10% of wild-type values. The most severely affected transformants showed extremely slow growth and in some cases they were unable to grow and produce seed. Progeny from the R1 seed from several cytochrome b6/f transformants have been analysed and show segregation of phenotypes ranging from intermediate to severe in repression. Intermediate and severe phenotype plants showed a reduction in Rieske FeS mRNA of more than 90% while FeS polypeptide was reduced to 60 and 86% of wild type. There was a strong correlation between photosynthesis at air and Rieske FeS polypeptide content in the antisense plants suggesting that the cytochrome b6/f complex was a major determinant of photosynthetic rate under these conditions. Photoinhibition studies of FeS antisense plants showed that there was a reduced activity of the xanthophyll cycle in the most severe plants, consistent with a reduction in the transthylakoid pH gradient and a lowered non-photochemical quenching. Preliminary studies of the ATPδ antisense plants showed that they also had reduced levels of mRNA and ATPδ polypeptide.