The Catalytic Properties of Hybrid Rubisco Comprising Tobacco Small and Sunflower Large Subunits Mirror the Kinetically Equivalent Source Rubiscos and Can Support Tobacco Growth

Abstract

Abstract Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcLS) produced tobaccoRst transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (LsSt). The tobaccoRst plants required CO2 (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K  m, for CO2 and CO2/O2 selectivity of the LsSt enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf LsSt content were sufficient, tobaccoRst plants grew to maturity without CO2 supplementation. When grown under a high pCO2, the tobaccoRst 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 LsSt content in tobaccoRst 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 tobaccoRst 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 LsSt was similar to tobacco Rubisco and measurements of light transient CO2 assimilation rates showed LsSt was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobaccoRst growth primarily stem from reduced rbcLS mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted LsSt synthesis.