Functional integration of LCIB, a component of the algal carbon-concentrating mechanism, enhances carbon assimilation, nitrogen-use efficiency, and biomass in tobacco

Engineering carbon-concentrating mechanisms (CCMs) into C3 crops is a promising strategy to improve photosynthetic efficiency by increasing the availability of CO2 near the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the enzyme responsible for carbon fixation. The biophysical CCM of the green alga Chlamydomonas reinhardtii includes stromal limiting CO2-inducible protein B (LCIB) protein, which contributes to inorganic carbon retention. Here, we accumulated LCIB in the stroma of tobacco chloroplasts to evaluate its effect on carbon assimilation and plant productivity. LCIB was exclusively localized in the stroma and remained biologically active, leading to a ~3-fold increase in total carbonic anhydrase activity and a lower apparent CO2 compensation point (-10%). This resulted in a significantly 12% higher net CO2 assimilation rate under ambient conditions, reflecting an increase in photochemical performance including a higher electron transport rate and effective quantum yield of PSII (+14%). Consistent with improved assimilation, LCIB lines accumulated higher levels of soluble sugars and end-of-day starch. Untargeted metabolomics revealed widespread increases in the levels of amino acids and tricarboxylic acid cycle intermediates, suggesting enhanced integration of fixed carbon into nitrogen metabolism. LCIB lines also accumulated twice as much biomass as wild-type counterparts during early development, resulting in up to 19% more fresh weight (FW) and 15.4% more dry weight (DW) at the end of vegetative growth. These lines also maintained higher chlorophyll levels and elevated nitrate reductase activity under limiting nitrogen conditions, and accumulated substantially more shoot (+ 79% FW; + 55% DW) and root (61% FW; 46% DW) biomass while C/N ratios remained similar to wild-type plants. Together, these findings demonstrate that stromal expression of the algal CCM protein LCIB enhances photosynthetic carbon assimilation, supports coordinated carbon–nitrogen metabolism, and improves biomass accumulation in a C3 plant without reconstructing a complete CCM. The targeted enhancement of stromal inorganic carbon retention is therefore a useful incremental strategy to improve C3 photosynthetic performance.