The catalytic properties of hybrid rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source rubiscos and can support tobacco growth

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL ^S) produced tobacco^Rst transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L ^sS^t). The tobacco^Rst plants required CO ₂ (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K_m, for CO ₂ and CO₂/O₂ selectivity of the L ^sS^t enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L^sS^t content were sufficient, tobacco^Rst plants grew to maturity without CO₂ supplementation. When grown under a high pCO₂, the tobacco ^Rst seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L^sS^t content in tobacco^Rst leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO₂ and growth illumination CO₂ assimilation in mature tobacco^Rst leaves remained limited by Rubisco activity and its rate (approximately 11 μmol m^-2 s^-1) was half that of tobacco controls. ³⁵S-methionine labeling showed the stability of assembled L^sS^t was similar to tobacco Rubisco and measurements of light transient CO₂ assimilation rates showed L^sS^t was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco^Rst growth primarily stem from reduced rbcL^S mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L ^sS^t synthesis.