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

Robert Edward Sharwood , Susanne von Caemmerer , Pal Maliga , and Spencer Michael Whitney ^*

Molecular Plant Physiology group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia; Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA and Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA

^* Corresponding author; email: spencer.whitney{at}anu.edu.au.

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubiscolarge subunit gene (rbcL) with that from sunflower (rbcL^S) producedtobacco^Rst transformants that produced a hybrid Rubisco consistingof sunflower large and tobacco small subunits (L^sS^t). The tobacco^Rstplants required CO₂ (0.5% v/v) supplementation to grow autotrophicallyfrom seed despite the substrate saturated carboxylation rate,Michaelis constant for CO₂ and CO₂/O₂ selectivity of the L^sS^tenzyme mirroring the kinetically equivalent tobacco and sunflowerRubiscos. Consequently, at the onset of exponential growth whenthe source strength and leaf L^sS^t content were sufficient, tobacco^Rstplants grew to maturity without CO₂-supplementation. When grownunder a high pCO_2, the tobacco^Rst seedlings grew slower thantobacco and exhibited unique growth phenotypes: juvenile plantsformed clusters of 10-20 structurally simple oblanceolate leaves,developed multiple apical meristems and the mature leaves displayedmarginal curling and dimpling. Depending on developmental stage,the L^sS^t content in tobacco^Rst leaves was 4 to 7-fold less thantobacco and gas exchange coupled with chlorophyll fluorescenceshowed that at 2 mbar pCO₂ and growth illumination CO₂-assimilationin mature tobacco^Rst leaves remained limited by Rubisco activityand its rate ( $~$ 11 µmol·m^-2·s^-1) was half thatof tobacco controls. ³⁵S-methionine labeling showed the stabilityof assembled L^sS^t was similar to tobacco Rubisco and measurementsof light transient CO₂-assimilation rates showed L^sS^t was adequatelyregulated by tobacco Rubisco activase. We conclude limitationsto tobacco^Rst growth primarily stem from reduced rbcL^S mRNAlevels and the translation and/or assembly of sunflower largewith the tobacco small subunits that restricted L^sS^t synthesis.